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ATLAS AND PRINCIPLES

mec, EP RIOLOC’:

AND TEXT-BOOK OF

SPECIAL BACTERIOLOGIC DIAGNOSIS

BY
PROF. DR. K. B. LEHMANN
Director of the Hygienic Institute in Wiirzburg
AND

R. O. NEUMANN, DR. PHIL. and MED.

Assistant in the Hygienic Institute in Wiirzburg

AUTHORIZED TRANSLATION FROM THE SECOND
ENLARGED AND REVISED GERMAN EDITION

EDITED BY

GEORGE H. WEAVER, M.D.
Assistant Professor of Pathology, Rush Medical College, Chicago

PART I—TEXT

PHILADELPHIA AND LONDON

W. B. Mahl Sane & COMPANY
BOOKSELLERS TIONERS
VANS . ob UU,
435 XO

COPYRIGHT, I901, BY W. B. SAUNDERS & COMPANY

REGISTERED AT STATIONERS’ HALL, LONDON, ENGLAND

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EDITOR'S PREFACE.

THE scope and purpose of this work are sufficiently
stated in the authors’ preface. The need of such a work
has often been felt in directing the work of advanced
students especially, and it is with the hope of aiding them

_ that it has been undertaken to place the contents of this
work within their easy reach. Because of numerous
mistakes in the references in the original, all of those

which refer to Plates in the atlas have been verified or
corrected, and also as many of those which refer to the
literature as were accessible, A few references to original
articles in English have been inserted.

FROM THE
PREFACE TO THE FIRST EDITION.

Doubt, honestly arrived at and acknowledged, is better than apparent
certainty without a statement of those things upon which it depends.

For years my brother, J. F. Lehmann, the publisher
in Munich, has requested me to furnish him for his
**Medical Atlases’? one which would simplify bacterio-
logic diagnosis. After I had long refused to undertake
the vast labor which this would necessitate, a fortunate
circumstance in the summer of 1894 led me to accept the
plan. I discovered in Dr. R. Neumann, who was working
in bacteriology in my institute, so excellent a talent for
drawing and painting that I proposed to him that he
undertake the work with me. Whether we have solved
the problem remains for the critics te decide. It seems
to me that the plates, painted by Dr. Neumann with un-
tiring zeal under my continual supervision, and carefully
reproduced by the lithographer Fr. Reichhold in Munich,
are a useful addition to our means of teaching. With few
exceptions, the reproduction leaves little to be desired. At
least, we have had the satisfaction of finding the pictures of
great advantage in our own work and in that of numerous
gentlemen working in our institute. We carried out many
investigations regarding the method of illustrating before
selecting the one employed, which may be considered as
almost entirely satisfactory.

At the present time, when, properly, photography is so
much used for the objective representation of objects in the
natural sciences, especially those of bacteriology, many will

5

6 FROM THE PREFACE TO THE FIRST EDITION.

look with suspicion upon a painted bacteriologic atlas. We
hope, however, that the unprejudiced critic will concede
that for certain objects (stab, streak, and potato cultures)
a well-colored representation surpasses the best photo-
graph, and that for a second group of pictures (plate-
- colonies slightly magnified) a drawing, which can alone do
justice to the depth of the object, is at least equal to a pho-
tograph. We gladly acknowledge that for the representa-
tion of individuals magnified 1000 times photography is
the best method ; but there is now scarcely any doubt that
for the practical differential diagnosis of bacteria, only in
somewhat rare cases is the picture of the individual of
primary importance. We have, moreover, sought to take
advantage of the photographic method when the individ-
uals were to be represented, by comparing the splendid
photographs in the atlas of C. Frankel and R. Pfeiffer, and
also those in the literature (by Léffler, Heim, Roux, ete. ),
with our own preparations.

The choice of varieties for illustration was often very
difficult. To our great pleasure, we were able to present,
with the exception of about 4 per cent., only originals in
the atlas ; while, naturally, those required as supplements
to the text are more often copies. In the latter case the
original source is always given. Varieties important from
a medical standpoint, especially when they present any
visible characteristics, could scarcely be omitted; also,
almost all varieties pathogenic for animals are introduced.
Chromogenic, zymogenic, and saprogenic bacteria were
never, to our knowledge, so extensively represented be-
fore; nevertheless, in this portion a careful choice was
required. We acknowledge that some among those selected
might have been omitted, and others chosen.

The text is divided into a general part, which I have
prepared alone, and a special part, in which I have re-
ceived the constant cooperation of Dr. Neumann.

The general part furnishes a condensed survey of the
principal properties of bacteria so far as they are of prac-
tical value, especially so far as they are of diagnostic aid.
It is assumed that the reader has mastered the ordinary
elements of bacteriologic technic, but at the request of the
publisher we have appended a short list of media rules for

FROM THE PREFACE TO THE FIRST EDITION. 7

stains, etc., and constant reference is made to them. More
complete information in these matters is furnished by the
well-known works of C. Frankel, Giinther, Htippe, and
in especially painstaking minuteness by the exhaustive
work of Heim: “Lehrbuch der bacteriologischen Unter-
suchung und Diagnostik.’’

The special part attempts to give, so far as possible in a
natural botanical arrangement, a complete description of
the important varieties, with constant reference to less im-
portant ones which for any reason are worthy of notice.
Those which we have described in detail we have also
thoroughly investigated, thus supplying many previous
omissions.‘ A great part of the related species have been
studied so far as time, strength, and opportunity allowed.

Of new ‘‘species,’’ we have introduced only a very few;
identical varieties described under various names we have
grouped together; and in many places we have directly
tried to build up a natural system. It was evidently im-
possible to offer anything complete or final in the treat-
ment of the non-pathogenic varieties.

Moreover, we are of the opinion that the advance of bac-
teriology, which we seek, especially the elucidation of the
questions of variability, relation, distribution in and out-
side of living organisms, etc., cannot be accomplished by
one or several, but only by systematic national—or, better,
international—cooperation of investigators under a grand
division of labor. One task for this cooperation would be to
so improve and remodel the present often unprecedentedly
arbitrary and unscientific nomenclature of fission-fungi
that it will not challenge the derision of every scientist.
(Compare Introduction to Special Part.)

Not infrequently our observations did not agree with
certain statements of various respected observers, but
we have always expressly acknowledged the same, and
especially have pointed out the contradictions and defects,
hoping thus to do service.

For an extensive reference to literature we have found no

1Tf this were conscientiously done by all editors of bacteriologic
works, there would be at least a partial elimination of the varieties
which are non-critically enumerated, absolutely insufficiently de-
seribed, and often repeated under different names.

8 FROM THE PREFACE TO THE FIRST EDITION.

room, but have only employed citations to facilitate detailed
studies, especially pointing out recent reviews with numer-
ous references. Every bacteriologic investigator will be un-
able to dispense with the aids which we have employed:
Centralblatt fir Bakteriologie und Parasitenkunde (Redak-
teur Uhlworm, Kassel, seit 1887), Baumgarten’s Jahres-
bericht tiber die pathogenen, und Koch’s Jahresbericht tiber
die zymogenen, etc., Organismen. By their comprehensive
index they quickly furnish a complete abstract of literature.
If we have been able to somewhat further the diagnosis
of bacteria, to lighten the task of the beginner, to indicate
the numerous difficulties of this work, which are partly un-
determined and too little appreciated, then we are rewarded
for the great labor which we have expended. We hope
especially to furnish the student in bacteriology a sub-
stantial aid, and to make it possible for him to better
appreciate what is seen and heard. We beg our critics
not to censure too strongly defects and mistakes, which
necessarily entered because of the enormous material.

Pror. Dr. K. B. LEHMANN.

PREFACE TO THE SECOND EDITION.

SoonER than we dared to hope, a large German edition
of this work has been exhausted; the English, Italian, and
Russian editions also have found a large sale. We accept
this as an indication of the practical value of the book.
With special pleasure we have observed in the numerous
reviews of the book that its reformative tendency in regard
to the grouping of varieties of bacteria, the strict division
of the system especially, the rational naming of bacteria,
etc., have found warm praise. The text-books of Heim

and Mez have accepted our nomenclature entirely or in

part. For many new names in Fltigge-Kruse’s work,
which appeared a few months after ours, according to the
rule of botanical systems, the priority remains with us.
Moreover, where we have found that properly selected
names, older than those which we chose in the first

_ edition, existed, we have naturally strictly adhered to the

rule of priority. We affirm with pleasure that, because of
our exact observations and of reliable statements in the
literature, the carefully championed view of the exceed-
ingly great variability of bacteria finds more and more
recognition, and that the authors who to-day describe
‘“new species’’ are in the main fewer, as is witnessed by
the intelligent views advanced by the collection of bac-
teriologists in New York in 1895 (C. B. xx, 450).

The opinion advanced from an esteemed source, that the

- constant emphasis of variability, of the limits of our knowl-

edge, and of the uncertainty of known methods, may some-

_ times discourage the beginner, may not be entirely un-

——.

founded. Yet we believe this absolute frankness to be an

advantage, even if thereby the dogmatic sharpness of the

statements should sometimes suffer. With beginners one
9

10 PREFACE TO THE SECOND EDITION.

may and must leave much unsaid in order not to confuse;
but ever so short a text-book can only claim the designa-
tion of science if the student can follow the author’s
thoughts. Besides, for the learner there is no greater
satisfaction, when he comes upon difficulties, than the
certain statement that, in a certain point, the imperfec-
tion of our knowledge, and not his inability, is the cause
of the difficulty. :

The fruitful labors of all investigators in the field of bac-
teriology made necessary a complete revision of the text in
both the general and special parts. In the general part the
discussion upon the causes of disease, disposition, and im-
munity is substantially extended. Beginning with page
119 is an exposition of the most important botanical points
of view which are important in classifying and properly
naming fission-fungi. In the special part, in fifty varieties
dependent upon autopsies, we have made additions and
improvements; about eighty varieties are newly intro-
duced. We have especially undertaken fundamentally
new work upon the causes of diphtheria and tuberculosis,
together. with the related varieties. It is hoped that the
value of the atlas is essentially increased by the introduc-
tion of nine new plates, which replace three old ones
(diphtheria and the allied bacteria, varieties related to
the tubercle bacillus, gonorrhea, and pest).

The literature of the past three years has been conscien-
tiously studied; many statements are substantiated, and
everything which seemed of importance in the publications
up to about June, 1899, is taken up. We hope that we
have made.a proper selection from the almost immeasur-
able material, which increases daily. Perfection, naturally,
we cannot expect: some small mistakes and oversights
could not be avoided. The division of the work was the
same as in the first edition.

K. B. LEHMANN.
R. O. NEUMANN.

CONTENTS.

Part I.—General Bacteriology.

(A) InTRopuUcTION To THE MorPHOLOGY OF BACTERIA........

(B)
(C)

Tue CHEMICAL COMPOSITION OF BACTERIA...............-
Rapipity oF INCREASE AND DuRATION oF LIFE oF Bac-
af ys Se ee a ee ee ee ee eee

(D) ConpiTions oF Lire oF BACTERIA..............20200005

(E)
(F)

.N utrient MRE PN ee Pe ce” actinnt She ott oe ph he Pa eh

Injury to Bacteria i Chemical Substances...........
Deficiency of Nourishment and Water................
. Relation to Oxygen and Other Gases.................
Influence of Temperature on the Life of Bacteria.......
Mechanical and Electrical Influences.................
Influence of Light and Rontgen Rays................
The Effects upon Bacterial Growth of the Presence of

SE SPMEREN ONS oink, os sk A AAS oO AE wore eee

2.90 MOF St G8 RO

I Re iv See in a wie ba we se ees hos oo 5 GRR
THe AcTIvITIES OF BaAcTERIA, ESPECIALLY IN REGARD TO
APPLICATION OF THE SAME TO DIAGNOSTIC PURPOSES...

Spammoonatical Activity. .2 66... 0 Det eee

PUNE ACLAVRDY 50 Secs kad wht ope ee eet CdR
MENTE IL OUIVIGY oe ogy aicca vic alate eueks bse eb ere Sumas
RUEm CA DEN VI YS oo ae Se ca a dk ev cee was
I. The Bacterial Ferments and the Changes Produced
REINS a RONEN cP OA a SS Ros Corel adits otk v8

II. The Chemical Activity of Bacterial Metabolism. .
SPR IMMCNL POGUCHOR - 2 iis Dl Oia Se ee
Formation of Ammonia and Fermentation of
a SE RP Ses ope are COO OP I ere Rem ete eat
. Formation of Complicated Basic Metabolic
WRIANEOE So eat Salt Wh ae ke is De eae
. Production of Complex ‘ Albuminous” Poison-
ous Metabolic Products (“Toxalbumins,”
Lee PERSE sOT Pen en ies ea) EN ee Da Een dee sade
. Hydrogen Sulphid (H,S) ..................
PS AUOOMGUION PTOCEBSOS «oo 5 sda so eiecen sues
. Aromatic Metabolic Products...............
CRMC “ELS OF: DREHS )..s S54 is le eevee ae

11

- wo Ne

CONT Or

12 CONTENTS.

PAGE
9. Putrefaction =...65.0) 6.5) Ate eee
10; - Nitrification \30055.. 233. . = 81
11. Transformation of Nitrites (and Nitrates) into
a Free Nitrogen (Denitrification)............. 82
{tho 12. Nitrogen Assimilation..................... 83
ect: 13. Formation of Acids and Alcohol from Carbo-
hydrates... 000.00... . os 2s 85

14. Gas-production from Carbohydrates and
Other Fermentable Bodies of the Fat Series... 89
15. Production of Acids from Alcohols and from

Other Organic Acids... 2... .. 4. -.n seu 91

5. The Pathogenic Action of Bacteria. (Pathogenesis, Pre-
disposition, Resistance, Immunity.).......... 92
1. How do Bacteria Act Pathogenically? .......... 92
2. Variation in the Virulence of Bacteria............ 94

3. Predisposition and Congenital Immunity (Resist-
ALCS) oa Dee e's os ee 96
4. Acquired Specific Immunity and Its Causes....... 98

(A) Poison-resistance (Specific Poison-immunity) 99

(B) Resistance to Bacteria (Specific Bacteria-im-
munity) . 026200555. 00s ae er 103
b. Appendix: . 2... 6. 5 hee est he oa 110

Part II.—Special Bacteriology.

(A) INTRODUCTION TO THE CLASSIFICATION OF Fisston-FuNGI. 115
I. The Fundamental Ideas of Botanical Classification

Applied to Fission-fungi......................05. 115
II. Nomenclature of Bacteria... ...... 0.2.6.0) 119

III. The Classification of Families and Species of Fission-
PUMP osc ees he Ge ok es oye wed ee a 122
Supplement I.—Actinomycetes................... 127

(B) Systematic DrEscrRIPTION OF THE Most ImMpoRTANT VARIE-
TIES OF BACTERIA (FISSION-FUNGI)............2-2++--- 129

Explanation of the Terms Here Employed in the Description of
Cultures of Bacteria’ <2 ot vices leis o et ek

Family I1—Coccacee. Spherical Bacteria ............. 133
1, Streptococcus sy 5. o5 6 vi ws vl 4 co ee 133
2: Sarcoma (yl, es i a wae eB ob a Ae Oe ee 151
3; Micrococeus ©. .0.05 SS Ea 6 eee (Sn 163
Family IJ.—Bacteriaceer. Rod Bacteria............... 193
4.. Bacteriam . 2 e) so02e. Osis ie a se Os 193
2. . Bache: 5525505 San oi sew cleo ee 304
Family III.—Spirillaces. Spiral Bacteria.............. 352
A. Vibrio ives Saeed Us se ee 353
2. Spire Pesos o5 ws v8 ss 2 ee ee 376

3...Bpirochsete foo.) eg os Oe hoe ee 381

CONTENTS. 43

PAGE
Apprenpix I.—Actinomycetes ........... cece cece eee eeees 383
Pe COPYNODACCETIUM {Loi ee eet cee eee cee rete eee 383
PME VOCODSCCEIIUID ioe ook se ellen ee aye e Sieeg me ele 419
MRURIEIESN YC os Seas sos C wie 8 hn Slee eae abe hse Seow ns 4°
Apprnprix IJ.—Higher Fission-fungi (Spaltalgen)...........
UT ay POW ri, Sarin an Ten P ae wore
RATAN sO ong Sacieie 6 Ss Apidos a ele ee nt wonton
I TUEREEN NS oy hace o-Sals cis Ais in Via ate ce sot tee ;
Se a SS acy ie ee ee A on Dar ae a Saree 5
Apprenprx III.—Notes upon Diseases, Insufficiently Elucidateu,
but Probably due to Bacteria. -.............-.--2200- 005 467
Apprenpix IV.—Essentials of Bacteriologic Technique........ 474
I. Microscopic Examination .............0.--.2-eees 474
mee tivation Of Bacteria. oo ss 6 ce le eee eee 482
Bereamsmnal . tx periments, . 2 oc it vo se sae eee e ee ee 489
Appenpix V.—Brief Guide to the Recognition of Bacteria,
ME ORIG re NY Sos aussie a Seema Aw ae Owe OS 490
re yo vs bade vase tse sivicccin s Vols eM be Pewee es 495

SS a a a arr Pee LW Ae Ae hh Oy ee Pe Table

ee

ae re
Soeae

See
er ae

EXPLANATION OF ABBREVIATIONS
EMPLOYED IN REFERENCES.

A. H. = Archiv fir Hygiene. Munchen. Oldenbourg since 1883.

A. G. A. = Arbeiten aus dem Kaiserlichen Gesundheitsamt. Berlin.
Springer since 1885.

A. K. = Arbeiten aus dem bakteriologischen Institut der techn.
Hochschule zu Karlsruhe. Edited by Prof. Dr. L. Klein and
Prof. W. Migula since 1894.

A. P. = Annales de |’Institut Pasteur. Paris. Masson since 1887.

C. B. = Centralblatt fir Bakteriologie und Parasitenkunde. Jena.
Fischer. Since 1894 it has been divided into two parts.

C. B. L. = Centralblatt fir die landwirtschaftlichen, phytopatholo-
gischen und zymotechnischen Anwendungen der Mikrobiologie.

H. R. = Hygienische Rundschau. Berlin. Since 1890.

Z. H. = Zeitschrift fir Hygiene. - Leipzig. Veit since 1886.

Fliigge = Fligge: Die Mikroorganismen. Third edition. Leipzig,
1896

Heim — Heim: Lehrbuch der Bakteriologie. Second edition. Stutt-
gart, 1890.

Kitt = Kitt: Bakterienkunde ftir Tierarzte. Third edition. Wien,
1896.
Zimmermann I and II = O. E. R. Zimmermann: Die Bacterien
unserer Trink- und Nutzwasser. Chemnitz, I, 1890; m, 1894.
Migula, Schiz. = Migula, Schizophyta. Separate reprint from “ Die
naturl. Pflanzenfamilien von Engler und Prantl.’’? Leipzig, 1896.

Migula, Sys. = Migula, System der Bakterien. Volume I, General
Part. Jena, 1897.

Eisenberg =— Bakteriologische Diagnostik von James Eisenberg.
Hamburg and Leipzig, 1891. Third edition.

Lafar — Lafar: Technische Mykologie. Volume i. Schizomyceten-
garungen. Jena, 1897.

Giinther = Einfihrung in das Studium der Bakteriologie. Fifth
edition. Leipzig, 1898.

Zopf = Die Spaltpilze. Breslau. Third edition.

The references to illustrations in the atlas are given thus: the
Plates with Arabic, the Figures with Latin numerals. Thus, 5, Vur
signifies Plate 5, Figure VIII. :

16

BACTERIOLOGY.

A. Introduction to the Morphology of
Bacteria.

By bacteria (spaltpilzen, schizomycetes of Nigeli)
we understand a very large group of lower vegetable
organisms, morphologically very simple and uniform, but
biologically extraordinarily differentiated, which are so
connected with both the lower alge! and fungi by tran-
sition forms that a sharp separation by an accurate defini-
tion is difficult. Arthur Meyer emphasizes the relationship
of the spore-forming varieties to the ascomycetes, in which
the spore-forming cells appear as asci. Indeed, bacteria
bear a great resemblance to the simple flagellata, which
are usually conceived as animals. ”

The following definition may at least serve the practical
requirements of experimental bacteriology.

Small unbranched ® cells, rarely moré than 2, hardly ever
8-5 pin thickness, almost * always without chlorophyl, spher-

1 Recently we have learned that the green lower algve also possess
parallel colorless forms, which can be obtained from them by cultures
(Beyerinck); compare also Ludwig, C. B. L. 11, 348.

* Compare Biitschli in Bronn’s Klassen des Tierreiches, Bd. 1, Abt.
tm, Mastigophora.

Regarding the branching forms nearly related to bacteria compare
p. 19.

* Practically, important bacteria with chlorophyl are unknown.
Yet the green tadpole bacillus (Kaulquappenbacillus) of J. Frenzel
must be recognized as a bacterium (Z. H. x1, 207). There is more
doubt as to the relation of Dangeard’s Eubacillus multisporus to the
bacteria (C. B. x, 745). L. Klein described colorless varieties with
bluish-green spores (C. B. vi, 440).

2 17

18 MORPHOLOGY.

wal, rod, thread, or spiral in form, with no organs except
flagella which are used for locomotion. Vegetative increase is
by transverse, very rarely by longitudinal diwwision. <A series
of varieties form roundish, endogenous resting spores ; in
others there have been, or asserted to have been, observed
conidia-like formations (arthrospores). Other means of
propagation have not been observed.

Bacteria occur, so far as we know, only in the following
forms, which were first perfectly named by H. Buchner:

Fig. 1.—Forms of bacteria (after Buchner).

Individual Growth Forms.

Spherical form (Kugelform) (a).

Oval form (Ovalform) (b): length, at most 2 < the width.

Short rods (Kurzstabchen) (c): length, 2-4 width.

Long rods (Langstiibchen) (d): length, 4-8 width.

Thread forms (Fadenform) (e).

Half screw (Halbschraube ) = comma (f): a very short segment of a
screw; at most, half the turn of a screw.

Short screw (Kurzschraube) (g): a short turn of a screw.

» 9

FP ee RUA I Pe EF i geen! ek

"me "» =

"i a sear’ Be a" ,

a tie ak

PSEUDODICHOTOMY. 19

serew (Langschraube) = spiral form (/): all screw forms possess
either steep or flat turns.
Spindle forms (Spindelform) (7).
Oval rods (Ovalstabchen) (x) are differentiated from the spindle form
by less tapering ends, from the oval form by their greater length =

2-4 & the width.

Clubbed form (Keulenform) (7).

Growth Groupings.
Diplococeus (Doppelkugel) (m) with barely perceptible separation:
)

- Biscuit form (x).

Streptococcus brevis (Kugelreihe) (0) up to 8 cocci ; with barely per-
ceptible separation : Torula form (p).

Streptococcus longus (Kugelfaden )(q), or, if bent, Rosary form (Rosen-
kranzform) (s); with barely perceptible separation : Torula threads (7).

Staphylococcus (Traubenform ) (¢). Diplobacillus (Doppelstabchen)
(uw). Jointed threads (giederfaden) (v).

Tetrad (Tetradenform) (w): plane grouping of 4, 8, 16, etc., cells.

Sarcina (Wirfelform) (~), cubical form : solid grouping of 8, 32,
etc., cells.

Branching, i. ¢., springing up of a side bud, was until
recently unknown in connection with bacteria, and it is,
at all events, rare. Besides in others, it is well established
in the so-called tubercle and diphtheria bacilli as a frequent
appearance, and thus it is demonstrated that here forms
occur which do not strictly belong to bacteria.

_Exceptionally, true branching appears to occur in other
yarieties. Heim mentions it in Bact. fluorescens. Vin-
cenzi (C. B. xtv, 149) appears to have observed the same
in tetanus, but in spite of special care, we have made no
similar observations.

Often pseudodichotomy is confused with branching and

£ dichotomy. According to Babés (Z. H. xx, 412), it occurs

not infrequently in the most typical bacteria, and consists
in this, that either the lower member of a thread grows past
the side of the upper member, or that, in a row of cocci,
the division of a coccus parallel to the direction of the
string suddenly creates the beginning of a second thread.
Stolz (C. B. xxiv, 337) has recently studied exhaustively

1 Some authors falsely designate this true branching as true dichot-

omy, but true dichotomy means, according to botanical usage, only
_ the division of the growing ends of threads into two equal twigs, and

it is not certainly known to occur in bacteria.

20 MORPHOLOGY.

and represented this abnormal division in streptococci. It
occurs very frequently ; indeed, we have often seen it.

Regarding the structure of the bacterial cell, much
has been recently written. I must limit myself to what
seems to me the most probable.

According to Alfred Fischer,’ the conditions are very
simple (Fig. 3): The bacteria consist of a cell-membrane,
a protoplasmic layer, and a central fluid. Regarding a
nucleus see below. In saline solutions (sodium chlorid,
potassium nitrate, etc.) there occurs, the more concentrated

l 0 J

a
1 LIA fees 6
Ard 2 3 Soe
| S& 2 ss Ss
S 2a 2253
a: SS See
9 Go 8g

oO

a
Fig. 2.—Pseudodichotomy : a, In bacilli; 6, in streptococci.

\8&¢ | ——Protoplasmic layer.
)——Membrane. ,
=| | 43——Spaces filled with cell-juice,

Fig. 3.—Bacillus oxalaticus Migula (after Migula).

the solution the more rapidly, through abstraction of
water, a ‘* plasmolysis,’’—7. ¢., a contraction of the mass
of protoplasm with partial separation from the cell-mem-
brane. Thus are explained many clear vacuoles which
occur in an ordinary cover-glass preparation of many bac-
. teria (for example, B. typhi), and which were formerly

1Untersuchungen tiber Bakterien, 1894. Berlin. Separatabdruck
aus den Jahrbichern fiir wissenschaftl. Botanik, xxv, Heft 1; and
Untersuchungen tiber den Bau der Cyanophyceen und Bakterien..,
Jena, 1897.

2 Frequently the drying on the cover-glass is sufficient to produce
a picture of plasmolysis.

SPOROGENIC GRANULES. 21

looked upon as spores. (Compare Fig. 4, a, b,c.) In
water, this shrinking rapidly disappears, and also under
the prolonged action of saline solution.

Migula (A. K., Bd. 1), simultaneously and independently, reaches
the same opinion as A. Fischer regarding the very large Bacillus oxal-
aticus, a spore-forming variety related to the hay bacillus. It happens
especially in this that in the pressing outward of the protoplasmic
layer, the central fluid space becomes distinct ; in dehydrating media
it becomes smaller; in water, larger.

While the botanists have hitherto sought in vain for a
true nucleus, Arthur Meyer would recognize it in small,
single, oval granules, staining with Ruthenium red and
potassium iodid (Flora, 1897, Band 84,185), Compare

oat

Se, |
2 ie

c
a, Spirillum undula. 0, Bacillus Solmsii. _¢, Vibrio cholere.
Fig. 4.—Plasmolysis (after A. Fischer).

also the hitherto scarcely studied observations of A. Wagner

(C. B. xxu, 483), according to which nuclei were easily
stained by primulin and hot Bordeaux red.

_ Inthe interior of bacterial cells there are found, after proper
staining, very many varieties of peculiar granules, which
Babes, who discovered them, named metachromatic
bodies (i. ¢., staining differently from the cell-body).
Ernst, the first accurate investigator of these bodies, called
them nuclei or sporogenic granules.

While I refer to the literature of Babés (Z. H. xx, 412), which is
rich in controversy, I give only the seductively clear view of one of the
latest investigators of the subject, R.Bunge. Bunge (Fort. der Med.,
XII, 813 and 853) distinguishes :

_ 1. Ernsi’s granules. They stain blackish-blue when treated with
warm Loffler’s methylene-blue, washed in water, and after-stained
with Bismarck brown. These granules are entirely absent in. many

22 MORPHOLOGY.

spore-forming varieties (anthrax, megatherium), and in others it can
be shown that they have nothing to do with spores. They are there-
fore granules of unknown rank.

2. Antecedents of spores (Bunge’s granules). Small granules, usually
occurring multiple in sporulating cells, not staining according to
Ernst’s method, but, on the contrary, in boiling Loffler’s solution.
They are best shown, after previous treatment of the dried preparation
with chromic acid, sodium peroxid, or hydrogen peroxid, according to
the ordinary spore staining. (See Technical Appendix.) The per-
fected spores are formed by the union of many small antecedents.

Bunge explains the controversy which has occurred as due to much
confusion regarding the two varieties of granules.

Regarding the cell-membrane, it is especially to be re-
marked that it often appears somewhat swollen, and not
sharply outlined from without. In many bacteria (‘*cap=
sule-bacteria”’ of authors) the thickening of the membrane
or its outer layer is so extreme that the bacteria appear

Bacterium pneumonize Bacillus anthracis Streptococcus lanceo-
(Friedlander). (Cohn). latus (Gamal. ).

Fig. 5.—Capsule-formation (schematic).

surrounded by a true mucous envelope or capsule, which
is distinguished by its slight staining property with anilin
dyes. It is an interesting fact that most of these capsule-
forming bacteria only form these envelopes if-growing either
in the animal body or upon very special media,—fluid
blood-serum, bronchial mucus, and also, according to Paul-
sen, upon milk.! Upon gelatin, agar, and potato, nothing

1 Whether exquisite capsule-formation always occurs on these nutri-
ent-media, appears undecided. Recently, also, various authors have
pointed out that capsule-like formations can be demonstrated in a
wider field in the bacterial kingdom. Johne has described a method
(see Technical Appendix) for anthrax by which the capsule can be

FLAGELLA. 23

| of these capsules appears. See also, in the special part,

Streptococcus involutus and mesenterioides. Extensive

‘review of literature by Binaghi, C. B. L. 1v, 919.

Characteristic unilateral thickenings or swellings of the bacterial
membrane are presented by Bact. pediculatum, which is described

_ as an uncommon cause of the ‘‘ frog-spawn disease ’’ of sugar factories

(Fig. 6).

Teh seated

6 bec * a eee

APal

ee eT

ing especially striking membrane thickening at the ends of
threads (club formation ), see those of actinomycetes.

The outer surface of bacteria is often perfectly smooth
and without appendages in the short bacilli, and almost
always in the spherical forms, but the longer rods and spiral
forms are usually provided with delicate single or multiple
flagella. These are often distributed over the whole body
of the bacterium, often form only a little bunch at one end,
and often there is found buta single polar flagellum. Bac-
teria with polar flagella, shortly before division, show a

Fig. 6.—Bact. pediculatum (after Koch and Hosius).

single flagellum or a bunch of flagella at each end. As A.
Fischer particularly showed, flagella are not of the nature of
retractile and extending pseudopodia, but true hair-like
outgrowths. For the demonstration of flagella it is neces-
sary to treat the bacteria with especially powerful staining

agents. In this process the capsules of the bacteria, which

remain unstained in the ordinary method, are stained, and
so the bacteria appear very much thickened. Occasionally
wide layers of capsule remain unstained and the flagella
are then set upon a narrow ring-like halo, separated from
the bacillus by a colorless zone (Zettnow, von Stécklin,
A. Fischer). Unfortunately, very many of the procedures

made plainly visible ; also in this manner distinct capsules are obtained
in B. megatherium, oxalaticus, etc. Babés has depicted capsules in

connection with the Streptococcus pyogenes, and we have ourselves

occasionally seen similar formations in the case of many bacteria.
Masses of bacteria which are united into mucous clumps by swelling
of the capsules (often a sign of death) are called ‘* zooglea.’’

24 MORPHOLOGY.

employed in staining lead to a shedding and degeneration
of the flagella, so that their faultless presentation is often
a difficult task. (See Technical Appendix. )

The following figures give a schematic view of the three
types of equipment of bacteria with flagella. Many pic-
tures of individual varieties are found in the atlas.

_ Incultures of bacteria with abundant flagella, there at times occurs,
as first pointed out by Loffler, a peculiar tuft-like formation of shed
or broken-off flagella, intertwined with each other.

The ability to form flagella may be completely lost for
generations—whether permanently we do not know. Com-
pare Micr. agilis, Sarcina mobilis (Lehmann and Neu-

Oe

Fig. 7.—Types of flagella: a, Vibrio cholerz, one flagellum at
the end—Monotrichia type ; }, Bact. syncyaneum, tuft of flagella at
the end, rarely at the side—Lophotrichia type ;c, Bact. vulgare,
flagella arranged all about—Peritrichia type.

mann). There have occurred non-motile forms, which
were found for months by various authors to be without
motion, which later acquired flagella and became actively
motile (Lehmann and Zierler). Compare Bac. implexus.
Deceptions may thus occur in that many varieties, in
spite of being provided with flagella, are only slightly or
not at all motile. Also, on the other hand, in many vari-
eties the staining of flagella is so difficult that without
anything more certain, a variety cannot be designated as
devoid of flagella because it does not move.

The ordinary vegetative multiplication of bacteria
occurs through transverse fission in the middle of slightly
(cocci) or considerably elongated bacterial cells. As a

—

SPORE-FORMATION. 25

rule, after division, the micro-organisms separate, but the

_ contrary also occurs in the case of bacterial groupings, as

where, for example, chains of cocci or bacilli are present.
Under definite conditions of nourishment there occurs in
bacteria, vibriones, and higher fission-fungi, the formation
of longer threads, which further may always again break
up into segments. According to all the recent observa-
tions, the division of the cells proceeds, from the outer pro-
toplasmic layer.

Although longitudinal growth with transverse fission is
the rule for the host of bacteria, still in certain families—
for example, sarcina—there occurs a regular division in
three main planes, and at least occasional division in two
planes at right angles to each other is observed in very dif-

ferent bacteria,—for example, in streptococci,—whereby

ALY

Fig. 8.—Arthrospores of the vibrio cholerz (after Hiippe).

there may be produced cells with four parts and forkings
of the chains. (Compare Fig. 2.)

From the ordinary vegetative multiplication, propagation
by spore-formation is to be distinguished. To-day there
are recognized: (1) Endospores—strongly refracting oval
or round bodies, situated in the interior of cells, which, as
a rule, possess considerable resistance to injurious agencies
(heat, drying, chemicals); and (2) arthrospores (De
Bary, Htippe)—~. e., bud-like constrictions of the ends of
the cells. Also these (Fig. 8) should be characterized by
increased resistance, yet recent authorities have never ac-
knowledged that the proof of resisting arthrospores has
succeeded without objection. (Compare Vib. cholere and
Strept. pyogenes. )

1 A longitudinal division in bacilli, although rare, is undoubtedly
observed (Babés, Z. H. xx, 412). Stellate division has been observed

by Metschnikoff in a sporulating organism named “ Pasteuria,’’ but
it scarcely belongs among the bacteria in the restricted sense.

26 MORPHOLOGY.

In the following, therefore, where the term spore is used,
only endogenous resting forms are to be understood.

The origin of endospores proceeds similarly, but not
identically, in the individual varieties. For the investiga-
tion of a certain variety as to spore-formation, asa rule we
employ an agar-streak or potato culture, which has been
grown at a temperature near the optimum for the variety.
After twelve, eighteen, twenty-four, thirty, thirty-six, etc. ,
hours portions of the unstained culture are examined in
water with a narrow diaphragm. If spherical or oval,
strongly refracting spores seem to be present, they must be
stained according to Neisser or Hauser. (Compare Tech-
nical Appendix.) For the more exact following of spore-
formation it is best to place a few bacilli in a drop of gela-
tin or agar, and, with the aid of a warming apparatus or
in a well-warmed room, to continuously observe and draw
definite individuals.

Motile varieties (according to Fischer) always become
quiet before sporulation, yet without shedding their fla-
gella. Many varieties grow into longer threads, at first
unjointed, before spores form. To these latter belongs the
anthrax bacillus, whose sporulation will here serve us as a
model. (Compare Plate 86, Figs. m1 and yi.)

There is first seen a delicate dusty cloudiness in the pre-
viously homogeneous bacteria; then, according to Bunge,
instead of these finest dust-like particles, a small number
of slightly coarser granules appear, which unite among
themselves until at regular intervals there lie small round
spores (36, v1), which gradually change into the oval,
strongly refracting, mature spores (36, 11).

When the spore-formation is complete, there is seen in
the thread of bacilli a delicate partition-wall between each
two adjacent spores (86, Iv). Spores are not matured in
all the segments, although the globular precursors may
have prepared the way for it. Indeed, some varieties,
on account of certain gradually introduced cultural conditions,
lose the property of producing mature spores, only
physiologically worthless antecedents being formed (Roux,
K. B. Lehmann).

According to Lud. Klein (C. B. vi, 440), spore-formation is quite
different in five varieties of anaerobic bacilli, mostly motile, discovered

“eel

MATURE SPORES. a7

_ in swamp-water and studied by him, but unfortunately they were not
_ cultivated in pure culture. (Bacillus De Baryanus, Solmsii, Peroni-

ella, macrosporus, limosus). His observations were as follows: With-
out loss of motion, the end of the bacillus swells somewhat and be-
comes faintly greenish. Now the whole content of the swollen spot

_ contracts into a shining spore of a bluish-green color and highly

shining.

Mature spores are arranged in the most important
varieties in the following manner (Fig. 9): The spore lies
inside of an unexpanded short bacterial cell (a), or at the
extreme end of a bacterial cell (¢), or inside of a spindle-
formed bacterial cell, bulging at its center (d), or, finally,
the spores occur in a row in a thread, formed from short
cells, each cell containing a spore (0b).

b

<>
2 ee’
| Fig. 9.—Types of spores.

The spores, before germination occurs, are usually free
(an exception occurs in spirillum endoparagogicum ), often
show an indistinct border, always lose their luster, become
somewhat thicker, and rarely also longer. Usually after
one, two, or three hours the spore membrane bursts and
the young rod, sometimes suddenly, sometimes slowly,
presses itself through the rent. The germination in an-
thrax is polar—i. ¢., the young rods leave the spore capsule
at or near the pole (Fig. 10). In other varieties (B.
subtilis, mycoides, megatherium) the escape of the rod is
equatorial (Fig. 10, a). Burchard describes also a bipolar
and oblique mode of escape. According to the observa-
tions of Bunge (Fort. der Med., x11, 813, 853), in both
the polar and equatorially germinating varieties, single or
many individuals always present an oblique outgrowth.

28 MORPHOLOGY.

This has been completely confirmed by myself and Dr.
Hirai in Bac. anthracis, Bac. gangrenosus pulpe, and
Astasia asterospora. From what we have seen, it appears
strange to us that Burchard (A. K. m, 1) found twenty-one
‘new species of spore-carrying bacilli, the spores of which
all germinated so differently and characteristically that
he held the occurrence of the spore germination (appear-
ance of spore, point of germination, thickening of spore
capsule, etc.) to be a certain diagnostic aid in differ-
entiating the variety. Until now we unfortunately have

Fig.10 a.—Equatorial germination of spores in Bac. subtilis.

not been in a position to confirm the statements upon more —
extensive material.

Regarding method, I may remark that spores are allowed ~
to dry in a thin layer on a cover-glass ; a drop of agar is
placed thereon and the hanging drop examined upon <a
warm stage.

In old cultures of bacteria there are found almost always
dead, often very strangely deformed, bacterial cells, invo-
lution and degeneration forms, of which Plate 36, Fig.
v, and Plate 51, Fig. tv, give an idea. These swollen.
bent, often entirely unrecognizable forms stain poork —

4

CHEMICAL COMPOSITION. 29

ordinary means. ‘The beginner will often mistake involu-
tion forms for contaminations. Plate cultures soon deter-
mine whether one or more forms of bacteria are present.

B. Chemical Composition of Bacteria.

Qualitatively considered, the bodies of bacteria! consist
in great part of water, salts, and albuminous bodies ;?
in lesser quantity are present extractive substances soluble
in alcohol, and others soluble in ether (triolein, tripal-
mitin, tristearin, lecithin, cholestearin). In tubercle
bacilli Aronson found in the ethereal extractive (25% of
the dry substances), besides free fatty acids, large quan-
tities of wax, whose alcohol differs from cholesterin. In
no variety of bacteria could E. Cramer find grape-sugar,
although many varieties (Bacillus butyricus, varieties of
leptothrix) contain starch-like masses, turning blue with
iodin. True cellulose was found by Dreyfuss in B. sub-
tilis and a bacillus resembling the B. coli; also, the Myco-
_ bacterium tuberculosis forms cellulose in the animal body.
- The vinegar-forming Bact. xylinum produces such a quan-
tity that visiting cards have been,made from it as a curi-
osity. From cultures of Myc. tuberculosis and a ‘‘ capsule
bacillus from water’’ resembling the B. pneumonie of
Friedlander, on the contrary, no cellulose was obtained,
but instead there was found abundant mucoid carbo-
hydrates, C,H,,O;, closely resembling hemicellulose.
(For literature, see Nishimura, A. H. xvi, 318, and xx1,
52). Scheibler (Chem. Centralbl. x1,181) has described the
mucus-like material of the Streptococcus mesenterioides as a

1H. Buchner has directed that the cell-contents (bacteria proto-
plasm) be obtained by trituration and the hydraulic press (3 to 500
_ atmospheres). Compare Hahn (C. B. xxIII, 86).

2 Albumin and salts can constitute as much as 98% of the dry bodies
of bacteria (Vibrio cholerze); on the contrary, as much as 12% of
carbohydrates may be present in the capsules. In bacterial albumin
-Hellmich recognized a globulin (Arch. f. exp. Pathol. u. Pharmak.
XXVI, 345). Most improbable appears the statement of Fermi that
he has grown nitrogen-free (!) micro-organisms (C. B. L. 1, 505).

30 CHEMICAL COMPOSITION.

carbohydrate, C,H, ,O;, “«dextran’’; Kramer has obtained
a similar substance from the capsules of the Bae. viscosus
sacchari. Up to this time, nuclein has not been obtained
from bacteria in any quantity; on the contrary, of the nu-
clein bases, xanthin, guanin, adenin have been obtained
in considerable quantities. A number of bacteria con-
tain sulphur granules, which have arisen from sulphur-
etted hydrogen (Beggiatoa thiothrix). Others, still con-
sidered as belonging to the bacteria by many authors,
deposit oxid of iron in their capsules, obtaining it from
water containing iron (cladothrix, crenothrix).

Regarding quantitative composition, the methodical

work of E. Cramer has added considerable light, though
up to the present exact statements are submitted regarding
only the B. prodigiosum, B. pneumoniez, and some related
organisms, and also a series of cultures of the Vibrio cholerz.
Compare E. Cramer, A. H. x1u, 71; xvi, 151, and xx,

. 167.
: The water content of a culture grown on solid nutrient
media, as also the amount of ash, depend in a very large
measure upon the composition of the medium.

For example, the B. prodigiosum contained, when grown upon po-
tato, 21.49% dry substance, 2.70% ash in the fresh substance; when
grown upon yellow turnip, 12.58 % dry substance, 1.31% ash ‘in the
fresh substance. Besides the concentration of the nutrient medium,
higher temperature and lessened age of the culture act to increase the dry
substance and the ash.

Also the dry substance of bacteria varies in its compo-
sition under the influence of the nutrient medium.

Thus, for example, the Bact. pneumoniz Fried. shows upon meat-
infusion agar containing
1% PEPTONE AND 5%

1% PEPTONE. GRAPE-SUGAR.
Min oN eo ee 71.7% 63.6%
Ethereal and alcoholic extractives 10.3% 22.71%
0 NR eR ear ae ea ae oe Che 13.94% 7.88%

Evident increase in the proportion of peptone in the
nutrient medium leads to an increase in the albumin ia
the bacteria, while increase of grape-sugar makes the body
substance poorer in albumin and increases the alcoholic
and ethereal extractives (Lyons; A. H. xxv, 30).

s.

CHEMICAL COMPOSITION. 31

Yet there is much greater difference in the dry substance

_ of the cholera vibriones, if they are grown at one time

upon soda bouillon rich in albumin, and again upon
Uschinsky’s medium, free of albumin. Cramer here found

_as follows (the figures are the average from observations

upon five cholera cultures):

ALBUMIN ASH
Cholera vibriones on soda-bouillon . . . . . 65% 31%
Cholera vibriones on Uschinsky’s solution . . 45 11

He found also in the latter case a considerable quantity
of non-nitrogenous bodies, a part of which may be thought
to be hydrocarbons (or fats).

For the analysis of the ash of bacteria, consult Cramer

-(A. H. xxvimt) and de Schweinitz and Marion Dorset (C.

B. xxu, 993). The latter found almost only phosphate
in the ash of tubercle bacilli.

Of importance for the classification, even though more in a critic-
ally negative sense, is the fact, discovered by Cramer, that closely re-
lated varieties, which, upon many nutrient media, present analogous slightly

varying composition, suddenly upon a new medium conduct themselves dif-
_ ferently. Most interesting in this respect is the behavior of five cul-

tures of cholera, which in soda bouillon furnished vibriones of almost
exactly the same composition, but upon Uschinsky’s + solution pre-

_ sented very variable composition.

SopA BOovUILLON. USCHINSKY’S SOLUTION.

Albumin.| Ash. | Total. || Albumin.| Ash. Total,

Cholera, old .. .| 65.12 | 31.55 | 96.67 || 48.13 | 7.14] 55.27
Cholera, HamburgI | 69.25 | 25.87 | 95.12 || 35.75 | 13.70 | 49.45
Cholera, Paris. . . | 62.25 | 32.80 | 95.05 || 65.63 | 9.37] '70.00

Cholera, Shanghai .| 64.25 | 33.87 / 98.12 || 47.50 | 11.64| 59.14
Cholera, Hamburg IT) 63.94 | 29.81 | 93.75 || 34.37 | 14.74| 49.11

This result shows again how dangerous it is to make a
separation of two varieties because of any single chem-
ical or biological reaction. In order to understand the
astonishing differences, it is only necessary to recognize

_ the ability of one of these varieties to form thick cell-

1Compare p. 33.

32 CONDITIONS OF BACTERIAL LIFE.

membranes from Uschinsky’s solution. How easy it
would be for an author to pronounce the cholera of Paris
among this number, as a distinct species, since, upon
Uschinsky’s medium, i contains almost double the amount
of albumin which the Hamburg cholera does.

Bacterial spores have not so far, to my knowleding been
closely studied. One may naturally expect a decreased
water-content, from the analogy to the spores of molds.

C. Rapidity of Increase and Duration of
the Life of Bacteria.

Under favorable conditions (see below) bacteria multi-
ply very rapidly; according to Buchner, the number of
cholera vibriones, under most favorable conditions, is
doubled in twenty minutes (C. B. 1, 1). Compare also
Ficker (C. B. xxi, 1059). °

The duration of the life of bacteria is theoretically un-
limited, since from each cell by division two new ones,
with unlimited possibilities of division, are produced.
Practically, however, in our cultures the case is quite dif-
ferent. As pointed out by Gotschlich and Weigang, in a
cholera culture (agar-streak) at 37°, even after twenty-four
hours the number of live germs is practically reduced, and
after forty-eight hours many bacteria are injured by their
own products (Z. H. xx, 376).

D. Conditions of Life of Bacteria.
1. NUTRIENT MEDIA.

While a number of bacteria have hitherto been met with
only in the human or animal organism as parasites, and
appear to us as obligate parasites (example, Spirochete
Obermeieri), yet most of the parasites can be grown upon _

<x
SS

4 NUTRIENT MEDIA. 33

3
'
.

artificial nutrient media, either readily (example, Bacte-
rium typhi) or with more difficulty (example, Micrococ-

cus gonorrheee). Of the inhabitants of the manimate

surroundings of man, 7%. e., the so-called saprophytes,

most are easily cultivated on artificial media, similar to

those employed for parasites, while others—as, for exam-
ple, saliva bacteria and certain water bacteria—offer great

_ or in part insurmountable difficulties in their cultivation.

All nutrient media for bacteria must be rich in water ;

_ the presence of salts, and sources for the supply of carbon

and nitrogen are indispensable. Most varieties of prac-
tical importance and all pathogenic varieties prefer a

-medium containing albumin which is faintly alkaline in

reaction.
In some cases the demands of the bacteria as regards

the composition of the nutrient medium are very different.
_ As shown by Mead Bolton, a number of water bacteria

(Bacillus aquatilis Fliigge and B. erythrosporus Fltigge)
are contented with water which has been twice sterilized

- in glass vessels (Z. H. 1, 76). Here an increase of the
_ bacteria must occur at the cost of traces of impurities, or
of the ammonia and CO, of the atmosphere.

Almost simultaneously Heraeus (Z. H. 1, 193) observed

_ a variety of bacterium, which thrived in water which con-
_ tained ammonium carbonate as the only source of carbon
_ and nitrogen, being free from every organic nutrient ma-
terial. Here, then, there occurred the elaboration of
_ living substance from simple materials, just as occurs

in the higher plants which work with chlorophyll aided by

sunlight. Htippe and Winogradsky have demonstrated
_ by extensive studies the truth and importance of this ob-

servation. It appears that the energy necessary for the
synthesis of albumin is obtained by oxidation of ammonia

- into nitric acid. Among the practically important bac-

teria, such unparticular ones are very few. Many allow

albumin to be absent from the medium and are content

with very simply composed nutrient solutions. Cultures

upon such fluids were formerly much employed, and more

recently Uschinsky has again experimented with simple
sputrient solutions, The solution of Uschinsky is as fol-
OWS;

34 CONDITIONS OF BACTERIAL LIFE.

Water, 1000. Magnesium sulphate, 0.2 to 0.4.
Glycerin, 30 to 40. Di-potassium phosphate, 3 to 2.5.
Sodium chlorid, 5 to 7. Ammonium lactate, 6 to 7.
Calcium chlorid, 0.1. Sodium asparaginate, 3 to 4.

Instead of this complicated solution, one may employ
many simpler ones; for example, such as is recommended
by Voges and C. Frinkel (Hyg. Rundaonas 1894, No.
17, 769), which is as follows:

bh i errata snl
Bodine CHOTA. 6090 be ele a aileska an 5 gm.
Neutral commercial sodium phosphate . . . 2 gm.
Ammonium lnotate (830s i a Sete aes 6 gm.
PBAPAIIR 6 oe Aye oO Eee ae 4 gm.

Upon this (although there is no sulphur in the nutrient
medium) the following grow:
VERY WELL: FEEBLY:
Bac. subtilis and mycoides, Mic. pyogenes a aureus,
Bact. syncyaneum, pyocyaneum, Streptococcus pyogenes,
coli, acidi lactici, pneumoniz, Bact. typhi,
mallei, vulgare, Bac. anthracis.
All vibriones.
Not AT ALL:
Bac. tetani,
Bact. murisepticum,
Bact. erysipelatos suum,
Bact. cuniculicida.

Even with the addition of those substances recommended
by Uschinsky, other varieties, as diphtheria! and tetanus,
did not grow luxuriantly, but, by adding 3% to 4% of gly-
cerin, the medium can be used for cultivating many vari-
eties, even the tubercle bacillus.

While cultures upon the simple nutrient media just de-
scribed possess a great theoretical interest, yet they have
been but little employed for diagnostic purposes.

Very much more use is found for meat-infusion, peptone-
gelatin, agar, and bouillon (each with or without the addition
of grape- or milk-sugar), also glycerin-agar, milk, and slices
of potato. (For preparation see Technical Appendix. )

We must always keep these on hand, since without them

1 Recently Uschinsky has apparently obtained upon his non-albu-
minous nutrient medium a good growth with the production of toxin
in the case of a certain culture of diphtheria,

REACTION OF THE NUTRIENT MEDIA. 35

rr Bi:

no differential diagnosis is possible, and no variety can be
considered regularly described which is not tested in its
relation to all these nutrient media (with the exception of
_glycerin-agar ).

More rarely the following nutrient media are employed:
Potato water, veal bouillon, fluid and coagulated blood serum,
serum-agar, ascites-agar, blood-smeared agar, meat, pieces of
bread, potato-pap, rice-pap, cooked or raw eggs. (See Tech-

nical Appendix. )

_ Uncooked, sterile organs of animals are actually poorer
nutrient media for most bacteria than when cooked (Liv-
_ingood, C. B. xxm, 980 and 1002). Studies upon nutrient
media containing liver, kidney, thymus, adrenal extract,
etc., have been carried out, but without giving anything
of practical importance. Literature: (Wroblewski, C. B.
| Xx, 528).

+i

2. REACTION OF THE NUTRIENT MEDIA,

As stated above, the great majority of bacteria, especially
_ the pathogenic, prefer a neutral or faintly alkaline nu-
_ trient medium, and formerly the advice was always given
to neutralize the nutrient medium with soda solution, em-
ploying sensitive litmus paper as the indicator—. e., to
add alkali until red litmus paper was turned faintly blue.
Every chemist knows that no accurate terminal reaction
- for the titration of nutrient media containing phosphates
is obtained with litmus; that, further, various litmus
_ papers influence the result ; and, finally, that the titration
is practically impossible with gaslight. As early as 1891,
_N. K. Schultz had therefore advocated phenolphthalein
- as an indicator in the titration of agar. He recommended
_ that 8-10 c.c. less of normal sodium hydroxid be added
_ than is required for complete neutralization with the indi-
_ ¢ator. Such a medium is found to be suited to many bac-
e - teria, yet there are others which demand a complete neu-
4 tralization (C. B. x, 52).
i Without having noticed this proposal, I came upon the
_ Same idea in 1892, during my investigations upon bread-
acids. Often since then, and exclusively since the autumn
:. _ of 1894, in my institute there has been employed as neutral

7.
; .
°

oe)

36 CONDITIONS OF BACTERIAL LIFE.

2) eee

gelatin (and agar) a medium which contains just so much»

sodium hydroxid as is required to produce a minimum
reddening of phenolphthalein. All the plates in this atlas
are prepared with the use of such nutrient media for such
cultures. This was done after the investigation of five
important bacteria had indicated to us that additions of
acids or alkalis did not improve the growth. Since then I

have had the great majority of the bacteria described in

our atlas systematically studied as to their ability to grow
on the following nutrient media, by Dr. Winkler (Dissert.
Wiirzburg, 1896):

1. On ‘‘neutral’’ agar, neutralized with normal soda
with the employment of phenolphthalein.

2. On ‘‘acid’’ agar—z. e., on neutral agar to which
was added 10 and 20 c.c. of normal sulphuric acid per
liter.

3. On a sort of alkaline agar—z. ¢., on neutral agar to
which was added 10 c.c. of normal alkali solution per liter.

The result, as indicated briefly in Table I (at end of
book), is that almost all bacteria grow well on these three
media.

In every case the nutrient media neutralized by means

of phenolphthalein as an indicator may be implicitly
employed as universal nutrient media; moreover, the
virulence of those varieties tested by us (Bac. anthracis,
Bact. coli, Bact. of mouse septicemia and chicken cholera)
is well preserved thereon.

This method has the advantage over other methods in
that it is easily carried out (compare Technical Appen-
dix), and that it represents a very exact point, namely
this, where all free acids and acid salts are changed into
neutral salts (mono-sodium phosphate into di-sodium
phosphate).

Other recommendations—Timpe C. B. xtv, 845; Heim,
Lehrbuch, p. 73; Deelemann (A. G. A. xin, 374)—appear
to have no advantage.

If an acid nutrient medium is to be employed, we think
it best to add 10-20 or 30 c.c. of normal acid to a medium
previously neutralized with phenolphthalein. According
to Winkler, the first degree of acidity is well borne by
almost all bacteria. According to the certainly not super-

are

a

F

INJURY TO BACTERIA BY CHEMICAL SUBSTANCES. 37

e Bficial reports of Schliiter (C. B. x1, 589), which were sub-

stantiated by subsequent publications, many bacteria bear

much higher proportions of acid; according to observa-
tions in our own institute, as high as 100 c.c. of normal

acid per liter.

Nutrient media coniaining sugar usually favor the pro-

duction of acid, which, according to Hellstrém, soon be-

comes so abundant that the micro-organisms are killed.
Acid nutrient media are to be used for yeasts and

_molds and whenever it is wished to isolate a new bac-
_terium from an acid nutrient substance. For counting

the germs in air, soil, water, milk, etc., a neutral medium

_ is always employed.

3. INJURY TO BACTERIA BY CHEMICAL
SUBSTANCES.

We have already learned that too large a proportion of

either acid or alkali? interferes with growth, or, if still

stronger, produces death. Most varied chemicals, in certain

concentrations, operate similarly. Those which are strongly
_ active are called antiseptics or disinfectants.

Usually, with Hiippe, the following grades of influence

are distinguished:

1. Growth is not interfered with,? but the pathogenic

_ or zymogenic functions are weakened : Weakening, at-

-.

tenuation.

2. The organisms are unable to increase, but are not
killed : Asepsis.

3. The vegetative forms of the micro-organisms are de-
stroyed, but not the resting forms: Antisepsis.

4. Vegetative forms and spores are both killed: Steril-
ization or Disinfection.

Fermi (C. B. xx11I, 208) has published an extensive table regard-
ing the sensitiveness of various micro-organisms to acids, alkalis, and
various poisons. Unfortunately, he gives the number of drops of
solutions of various percentages which inhibit the growth of bacteria

in 5e.c. of agar.

2 At times there occurs a transitory or permanent interference with

“ growth; in other cases briefly acting antiseptics, also heat, cold, etc.,

cause a retardation of subsequent growth without producing a weaken-

38 CONDITIONS OF BACTERIAL LIFE.

Since the diagnostic value of the test of the resistance
to chemicals plays only a modest réle—various hopes in
this direction remaining unfulfilled—this section must be
very brief.

To determine the minimal concentration of a chemical
poison which produces asepsis—i. ¢., prevents growth—the
following procedure is adopted:

A solution of the disinfectant—for example, 1%—is em-
ployed; and of this, 1, 0.5, 0.8, 0.1 ¢.c. is added to 10c.c.
of liquefied gelatin. This nutrient medium, containing
now 0.1, 0.05, 0.03, 0.01% of the disinfectant, is used
for stab, streak, and plate cultures. Inoculations may
also be made with material containing only spores (ma-
terial freed from all bacilli by heating half an hour at
70°), and thus it may be determined whether the spores
grow out in cultures.

Behring has devised the following practical method of
making these tests: A drop of fluid nutrient medium ,—
for example, serum,—infected with the organism to be
tested, is removed before the addition of the antiseptic
and suspended from the under side of a cover-glass on a
hollow-ground slide, sealing it with a little vaselin (Tech-
nical Appendix). Then by degrees there is added to the
tube of serum, increasing known quantities of the dis-
infectant. After each addition and thorough shaking a
drop culture is prepared. The growth in each drop can
be examined after being kept twenty-four or forty-eight
hours in the incubator.

If the concentration necessary for antisepsis is to be
determined, the organisms to be examined are grown in
bouillon, and to 10 c.c. of the bouillon, free from spores,
and filtered through asbestos to remove any clumps of
bacteria, various quantities of a solution of the disinfectant
of known strength are added. From each of the tubes
after one, five, ten, fifteen, thirty minutes, one hour, etc.,
a platinum loopful of the material is removed, and placed
in 10 c.c. of lukewarm liquefied gelatin, from which a
plate culture is prepared. If it is suspected that a trace
of the disinfectant carried in the drop renders the gelatin
aseptic and so leads to an apparent death of the micro-
organisms, the result may be controlled by inoculating

g
*

ee

INJURY TO BACTERIA BY CHEMICAL SUBSTANCES. 5

_ gelatin, to which a similar trace of the disinfectant has

S$

been added, with fresh material.

The disinfectant to be tested should always be dissolved
in water. (Compare Water, p. 40.) If, because of slight
solubility in water, the employment of alcohol in the prep-
aration of the stock solution is unavoidable, then special
control investigations are required to determine whether
the alcohol is injurious in its effects.

It is found, as well for asepsis as for antisepsis, that the

_ yalue of the disinfectant is usually much lower if one is

working with nutrient media rich in albumin than when
working with those poor in albumin.! Creolin (Pearson)
produces asepsis in bouillon in the proportion of 1: 15,000 to
1:5000, but in beef serum in 1:150 (Behring). Cholera
vibriones, in bouillon free from or containing 1% of pep-
tone, were killed by 0.01% HCl in half an hour, with the

_ addition of 2% peptone by not less than 0.04% HCl in

the same time. For descriptive purposes, the test will
usually be made in 1% peptone solution, if one does not
wish to employ one of the nutrient media free from albu-

_ min, described on page 34. In any case one will treat the
_ bacteria used for comparison exactly the same, and must

give in a paper the finer details employed in the investi-
gation. Little is known of varying resistance in bacteria
which are free of spores, because of race or nutrient media
(compare spores), but there are isolated statements in this
direction regarding staphylococci which, perhaps, may
point to as yet imperfectly understood resting forms
(Esmarch, Z. H. v, 67).

A combination of disinfectants may enhance the action;
the addition of acid (hydrochloric or tartaric acid) espe-
cially intensifies the action of sublimate, also of phenol and
cresol solutions. Moreover, the effect is more certain if
the germs are few than if they are abundant, and greater
at higher than at lower temperatures.

1 Phenol is an exception,

20 CONDITIONS OF BACTERIAL LIFE.

4, DEFICIENCY OF NOURISHMENT AND
WATER.

If bacteria which require substrata rich in nourishment
in order to thrive (including most pathogenic) are placed in
pure distilled water, they usually die rapidly—~. ¢., in an
hour. As Ficker (Z. H. xx1x, 1) pointed out, numerous
minute influences here become accentuated. Thus, traces
of nutrient material, even long standing in glass vessels,
especially boiling in glass vessels, suffice to lessen the
bactericidal action of distilled water. Jena glass is espe-
cially recommended for control investigations, since it gives
up almost nothing to distilled water. Dense suspensions
and virulent cultures are more slowly destroyed; the age
of the individuals is indifferent. In well-water (even if
sterilized) the duration of life is usually not more than
eight to fourteen days, and an increase is rare. Certainly,
in a series of cases, much longer duration of life is observed,
but here the conditions mentioned above, and until now
usually neglected, come into question. Leipzig tap-water,
which remains long in pipes, is strongly germicidal, but
after being boiled it loses a part of this action, ete. (Com-
pare Ficker, /. c., and Loffler: Das Wasser und die Mikroor-
ganismen, 1896.) Deficiency of water exerts a disturb-
ing influence upon bacterial growth. My pupil, Leo Wolf
(A. H. xxxiv, 200), found that upon various nutrient
media (agar, gelatin, pulverized meat, cake), with 70%
and more of water, growth was most vigorous, with 60% it
was often interfered with, with 50% it was usually slight,
and when only 40% of water was present there was often
no growth to be detected, macroscopically at least. On
the contrary, upon nutrient media (agar, gelatin, potato)
dried gradually at room temperature, the duration of
life is often astonishingly long, even when there are no
endospores present to be responsible for it. At times one
may observe that even after a year a horny contracted rem-
nant of a culture upon being placed in bouillon (and with
proper varieties kept in an incubator) yields a most beau-
tiful growth.

Regarding the duration of life of bacteria, when they ”
are spread upon glass and dried, the literature contains

e
-

r

-

RELATION TO OXYGEN AND OTHER GASES. 41

_many contradictory statements, from which it may be con-
eluded that much depends upon the special conditions

under which drying occurs. Ficker (Z. H. xxix, 1) has

discovered some laws especially for the cholera vibrio.

According to him, the following are of special significance :

1. Very thin smears were more quickly injured than thick clumps;
slight variations in the thickness of the smear are without significance.

2. Thin smears die more quickly in the exsiccator, and thick ones
in room atmosphere.

3. The lower the temperature, the better the drying is borne.

4. Virulent cultures are more resistant than non-virulent ones.

5. Variations of moisture (exsiccator and moist chamber) kill

especially rapidly (shown in typhoid and pest).

6. Older cultures were somewhat more resistant in an exsiccator.

Also regarding the variability in a moist chamber, Ficker has
obtained interesting results. Here old cholera cultures present enor-
mously greater resisting power than young ones. For example, in the
moist chamber thin smears of cultures seven to twenty-one days old

_ live about thirty to fifty days ; those three days old, fourteen days ;
_ those two days old, as long as seven days ; and those one day old, only

one to two days. Yet the old cultures contained no spores, and were
equally or more susceptible than the young ones to heat and chemicals.

(Compare Vib. chol. in special part.) A complete summary of pre-

vious results upon dried cholera, typhoid, diphtheria, and pest organ-
isms is given by Ficker, J. c.

5. RELATION TO OXYGEN AND OTHER GASES.

Because of their relation to oxygen, bacteria are usually
placed in three classes (Fliigge and Liborius):
I. Obligate aerobes. Growth occurs only when air is

_ admitted, and every limitation to the entrance of air injures
_ the growth. Free oxygen is especially required_for spore-
formation. With a pressure of three to four atmospheres

~

of pure oxygen, according to Chudiakow, growth of aerobic
bacteria ceases ; the lowest limit for aerobes was found to
be 5 to 10 mm. of air. According to Roger (C. B. xx,

_ 626), a pressure of 500 to 600 atmospheres of air does not
_ kill bacteria.

Il. Obligate anaerobes. Growth and spore-formation

~ occur only when oxygen is completely excluded.1 In this

2 ing the means of preparing anaerobic cultures consult the

~ Technical Appendix. Chudiakow found that the strictest anaerobes

’ Ao
pai

42 CONDITIONS OF BACTERIAL LIFE.

class belong Bacillus cedematis maligni, Bac. tetani, Bac.
chauveel (symptomatic anthrax), and numerous inhab-
itants of mud and soil. Upon exposure to free atmos- —
pheric oxygen, the vegetative forms of these bacteria die
in one or more hours ; the spores, on the contrary, are
more resistant to oxygen. (Compare Chudiakow, C. B. L.
tv, 389.) Since the principal source of energy is excluded
from the anaerobes which is at the command of aerobic
bacteria (oxidation of the absorbed nutrient material by
means of free oxygen), they are assigned to nutrient mate-
rials which are readily utilized,—as, for example, grape-
sugar,—which liberate energy (heat) by division into two
smaller molecules (for example, alcohol and CO,, or acetic
acid, or lactic acid). Therefore anaerobes are often grown
upon gelatin or agar containing 1% to 2% of glucose.
Upon such media the virulence and also the ability to
produce spores suffer, so that recently sodium sulphid
(compare p. 43) and formate of sodium have been em-
ployed as additions to the culture media.

III. Facultative aerobes and facultative anaerobes.!
The great majority of the bacteria usually grown by us
aerobically—including almost all pathogenic forms—will
tolerate a limitation in the amount of oxygen admitted
without being injured, moreover without interference with
their growth. | This is favorable to a life in many parts of
the animal body; for example, in the intestinal canal, where
oxygen is limited or absent. Chromogenesis is almost
always suspended by the exclusion of oxygen; on the con-
trary, toxic metabolic products are sometimes abundantly
elaborated in its absence (Htippe).

It is very important, as recent inquiry has shown, that
aerobic varieties of the anaerobic -bacteria also exist. Their

still grew with an air pressure of 5 mm.; three of the best-known
anaerobes he found still able to grow with the barometer at 20 to
40 mm. (C. B. L. Iv, 389).

1As emphasized by Beijerinck, it would be better to speak of ‘‘ tem-
porary ’’ anaerobic varieties, if it has not been demonstrated that cer-
tain varieties can grow permanently as well aerobically as anaerobically
(C. B. L. 111, 40). The demonstration produced by Pfeffer is also
interesting, that many aerobic bacteria can bind loosely considerable
amounts of oxygen, which they gradually release in spaces free from
oxygen (C. B. L. 1, 763).

ey

RELATION TO OXYGEN AND OTHER GASES. 43

origin is usually not known. (Compare special part, Bac.
tetani, Bac. chauveei. )

Not infrequently varieties are observed which show
more or less anaerobic growth when first isolated (growing

_ especially in the deep part of the agar stab), and which

later present a pure aerobic behavior—. e., distinct sur-

face growth and poor growth along the stab. Thus, one
may properly speak of an adaptation to a gas-mixture,

rich in, or free from, oxygen, and in this way may ex-
plain very many things.
These observations prove to the classifier that bacteria

can not be thus simply separated into two classes, the one

aerobic and the other anaerobic.
Recently the fact has been repeatedly demonstrated that

_ true anaerobic varieties also grow well without the exclu-

—!

sion of oxygen, if they are associated with many aerobic
varieties; also that for the aerobic growth of anaerobic vari-
eties, often nutrient media suffice if aerobic bacteria have
previously grown in them and have been killed with chloro-
form before the anaerobe is inoculated (Kedrowski,
Scholz). The synergetic aerobic variety evidently oper-
ates in part by consuming the oxygen, in part by pro-
ducing materials required by the anaerobic varieties.
Trenkmann recognized such a substance in sodium sul-
phid. Two drops of a 10% solution of sodium sulphid
render bouillon suitable for the growth of anaerobic varie-
ties without the exclusion of oxygen (C. B. xxi, 1088).

While, besides the obligate anaerobes, all facultative
anaerobes thrive well in nitrogen and hydrogen, they var
in their toleration of CO,. (Compare Table I, at end of
book. )

A large number do not thrive at all, and remain completely inhibited
in their growth until oxygen is again admitted; for example, B. an-
thracis, subtilis, and related varieties. It is established regarding
some varieties (anthrax, cholera) that most of the individuals are rap-
idly killed by CO,, while some germs exhibit energetic resistance, and
render a complete sterilization by CO, impossible. A second group
presents a restricted growth, especially if the test is carried on at in-
cubator temperature (staphylococci, streptococci), while a third group
is not injured atall: B. prodigiosum, B. acidi lactici, B. typhi. They
grow as well as in air; the liquefaction of gelatin is not interfered
with, but naturally, from the exclusion of oxygen, chromogenesis is
checked. Moreover, a mixture of 25% air and 75% CO, has no

44 CONDITIONS OF BACTERIAL LIFE.

apparent injurious influence upon fungi, which remain absolutely
sells in an atmosphere of pure CO, (C. Frankel, Z. H. v,

Sulphuretted hydrogen appears to be well borne by
anaerobes (see above); other varieties are very susceptible
to large quantities, as, for example, Bact. Pfltigeri (photo-
genic bacillus) (Lehmann and Tollhausen, C. B. v, 785).

6. INFLUENCE OF TEMPERATURE ON THE
LIFE OF BACTERIA.

Every variety of bacterium demands a certain tempera-
ture of the nutrient substratum. Vegetative bacterial
life is possible from 0° to 70°, some varieties thriving at
the upper and some at the lower extreme. For each va-
riety the minimum and maximum temperatures lie about
30° apart,+ and we may form a comprehensive classifica-
tion dependent upon the temperature required somewhat
as follows:

Psychrophilic bacteria: minimum at 0°, optimum at
15° to 20°, maximum at about 30°. Most water bacteria
belong here; for example, many phosphorescent bacteria
of the sea. (Compare Forster, C. B. xm, 431.)

Mesophilic bacteria : minimum at 10° to 15°, optimum
at 37°, maximum at about 45°. Here belong all varieties
pathogenic for man, since one condition for a pathogenic
action is an acclimatization to the body temperature.

The B. vulgatus connects this and the following group, as it still
grows well at 50°.

Thermophilic bacteria: minimum, 40° to 49°; opti-
mum, 50° to 55°; maximum, 60° to 70°. Here belong
many bacteria of the soil, and almost all spore-producing
bacilli of the family of B. mesentericus (Globig, Z. H. m1,
294).

More lately Lydia Rabinowitsch has somewhat more closely described
eight thermophilic facultative anaerobes, all of which are spore-produc-

1Bac. vulgatus thrives, to be sure, from 15° to 50°, a variety of
Globig also from 15° to 68°, but such wide intervals of favorable tem-
perature are very rare. Globig found the range of temperature at
which thermophilic varieties will develop to be very narrow; for ex-
ample, he could grow one variety only from 54° to 65°.

Te _—

:
_ tributed, especially in feces. She has not undertaken a comparison
with the varieties described by previous authors. Other varieties
_ were isolated by Oprescu (A. H. xxx11I, 164). Some of the varieties
_ isolated by Schillinger appear more as abnormally thermotolerant
_ than thermophilic ; they grow well at 66°, but better and with fer-
_ mentation at 37° (H. R., 1898, 568).

INFLUENCE OF TEMPERATURE. 45

ing, non-motile bacilli, whose optimum temperature lies at 60° to 70°,
but they still thrive at 34° to 44°, although slowly, and best in ana-
erobic agar cultures (Z. H. xx, 154). The varieties are widely dis-

By carefully raising and lowering the temperature, Dieu-

donné (C. B. xv1, 965) was able to increase the range of

temperature within which the anthrax bacillus could

_ thrive, at both its upper and lower limits. The anthrax

bacillus can gradually become adapted toa temperature of
42°. Pigeons, which, according to the hypothesis of many

_ authors, are fairly immune to ordinary anthrax because of

their higher body temperature (42°), die somewhat oftener

_ after inoculation with cultures adapted to higher tempera-

tures. Still more striking were the results when Dieu-
donné gradually acclimated anthrax bacilli to a tempera-

_ ture of 12°, and found they could still kill frogs kept at

12°.
Temperatures somewhat below the minimum limit the

: growth, but do not injure the variety concerned. Pe-

truschky has even kept bacteria in an ice-box (about

| 4° to 6°) as a means of preserving not only the life but

also the virulence of certain varieties which rapidly die at
higher temperatures. They are first-allowed to grow
for two days at 20° (streptococci, ete. ).

Also temperatures below zero injure bacteria, but do

so slowly and with a rapidity varying with different vari-
eties. Individual statements are given in the special part

regarding the most important pathogenic varieties.

If temperatures 5° to 10° above the optimum are
allowed to operate upon cultures, they are injured in
various ways; some show lessened intensity of growth, the
virulence and the power of fermentation are reduced, also

_the ability to form spores is gradually lost. Sometimes

the injury is more marked in one direction, sometimes in

~ another.

lf the maximum temperature is exceeded, the culture
dies, and for the psychrophilic varieties about 37° is quite

46 CONDITIONS OF BACTERIAL LIFE

rapidly fatal; for the mesophilic,! about 60° (Forster);
for the thermophilic, 75°. A temperature of 100° is not —
withstood by any bacterium free of spores for more than
a few minutes.

7. MECHANICAL AND ELECTRICAL
INFLUENCES.

In the first edition, at this point I reported the astonishing state-
ments of Meltzer, according to whom short, feeble shaking would’
operate favorably upon the growth of fluid cultures of bacteria, while
more prolonged and more vigorous shaking or long-continued very
feeble shaking would operate very unfavorably (Meltzer, Zeit. f.
Biol. xxx, p. 464).

Otto Appel, who, at my request, restudied the whole question, ar-
rived at entirely different results. _No shaking of longer or shorter
duration injured the bacterial growth, except where very severe agita-
tion and the addition of glass pearls caused direct mechanical lesions
of the bacteria. Slight shaking, such as cultures experience when
placed upon the foundations of strongly working steam-engines, was
without effect. (Further communications thereon are found in the
Archiv fiir Hygiene. )

Most of the previously observed effects of the electric current are
easily explained as due to the action of heat and electrolysis. Thiele
and Wolf demonstrated by means of new investigations, which are not
open to objection, that neither the passage of a constant or interrupted
current through a culture of bacteria, if electrolysis be avoided, nor
the placing of a culture in an interrupted current induction coil, in-
jures the bacteria (C. B. xxv, 650); 1. ¢., also the previous literature,
which in part contains remarkable assertions.

8. INFLUENCE OF LIGHT AND RONTGEN
RAYS.

The cultures of all bacteria are restricted in their growth
by direct sunlight. If the action is more prolonged, they
are subsequently less able to grow luxuriantly in the dark,
and there results a generation of weakened organisms,
shown, forexample, by incomplete liquefaction, slight pro-
duction of pigment, lessened virulence, etc., which only
regain their original properties after repeated transplanta-

1According to Sternberg the following die at 56°: Streptococcus
pyogenes, Bac. anthracis, Bact. mallei, and Vibrio cholere (Amer.
Jour. Med. Sciences, July, 1887, 146).

- INFLUENCE OF LIGHT AND RONTGEN RAYS. 47

tion on fresh medium in the dark.* With still longer action
of the direct sunlight the micro-organisms die.

Bact. putidum and Bact. prodigiosum were materially disturbed in
their ability to produce pigment and trimethylamin by direct sun-
light, in July and August in one-half hour and in November in one
and a half hours. They grew slowly and prodigiosum liquefied slowly
(Dieudonné). Death was produced in these organisms in one and a
half and two and a half hours.

Dieudonné (A. G. A. 1x, 405 and 537, also an extensive

review of literature) has found ultra-violet, violet, and blue
light to be very injurious, green but slightly, and red and
yellow not at all. On the contrary, Beck and Schultz

‘Se

(Z. H. xxi, 490), who employed better light-filters, gen-

erally observed no injury from the colored light obtained

from sunlight. This is to be explained by the slight in-

tensity of the light employed. Also, Beck and Schultz
deny that diffuse daylight works injury to bacteria (the
chromogenic function is lost in many varieties even when

_ grown in the dark), while Dieudonné asserts :

In diffuse daylight, in spring and summer in three and a half hours

_ and in winter in four and a half hours, there occurs interference with

_ growth, while in from five to six hours death is produced. Electric
are light of 900 candle-power checks growth in five hours and kills in

eight hours. Incandescent light injures growth in from seven to eight

hours, and kills in eleven hours. Similar results occur with Bact.
coli, typhi, and B. anthracis. Naturally, Dieudonné’s positive results

are not disproved by Beck and Schultz’s negative ones.

For testing the sensitiveness to light, thickly sown

_ gelatin or agar plates are exposed to diffuse or direct sun-
light, after the method of H. Buchner, a dark paper cross
_ being placed upon the illuminated side. To exclude the
~ action of heat,! one may first carry the light through a

ad
s-

.

+

z

at
a

_ posed bacteria there occurs a sharply outlined cross con-

sisting of the colonies in a clear field.
.

“4
rs

layer of water or alum a few centimeters in thickness.
After the light has acted for one-half, one, one and a half,
two, etc., hours, the plates are placed in a dark room, and

it is observed whether the bacteria develop only on the

part covered by the cross; in complete death of the ex-

1 The action of the heat is entirely without interest.

“aa

48 CONDITIONS OF BACTERIAL LIFE.

The action of the light seems to occur under the coopera-
tion of the oxygen of the air ; obligate anaerobes (tetanus)
“and facultative aerobes (B. coli), when oxygen is com-
pletely excluded, withstand the sunlight very well; for ex-
ample, B. coli withstood direct intense sunlight for four
hours. (Compare also Wesbrook, Journal of Pathology
and Bacteriology, Iv, 352.) :

Regarding the mechanism of the action of light, the
observations of Richardson and later of Dieudonné appear
important, if not furnishing a complete explanation. They
assert that in illuminated agar plates, and indeed only in
blue to ultra-violet light, in a short time (even after ten
minutes in direct sunlight) peroxid of hydrogen! appears.
For its demonstration one exposes to the light an agar
plate, half covered with dark paper, then pours over
the same a weak iodid of potassium paste, and upon
this a weak solution of ferrous sulphate; the illumi-
nated side becomes dark blue. (With gases that do not
contain oxygen there is no formation of H,O,, nor in-
jurious action from light.) This also explains what has
been often observed, that one may obtain a slight attenua-
tion of bacilli if they are inoculated upon agar plates that
have previously stood in the sunlight.2 Bacteria previ-
ously exposed to light develop especially badly upon media
that have been illuminated, much more so than upon good
media.

According to Rieder, strong Réntgen rays injure bac-
terial growth in a way similar to light (Mtinch. med.
Woch., 1898, No. 4, 101).

9. THE EFFECTS ON BACTERIAL GROWTH OF
OTHER BACTERIA.

Although it is the endeavor of every bacteriologist to
always obtain bacteria in pure culture, we must not forget
that in nature bacteria often occur in combination. If

1 With gelatin it is hours before H,O, can be recognized.

Also other decompositions of the nutrient medium by sunlight
may occasionally render difficult a subsequent growth of fungi; for
example, the origin of formic acid from tartaric acid (Duclaux).

a ‘ia
fon

t
.

&

SYMBIOSIS. 49

we examine water, milk, or the intestinal contents in
health or disease, we always find many varieties simulta-

= “neously present. This mixture certainly appears tous asa
pure accident, but upon closer study it is found that also
Sin the domain. of bacteriology there are sSynergists
_ (moutual or one-sided aid) and antagonists (mutually

injurious, or one to the other). Nencki speaks of symbi-

osis and enantobiosis.

Experimentally, Garré has demonstrated the antagonism by inocu-
lating various bacteria simultaneously in streaks upon gelatin plates
as parallel or intersecting lines. It then appears that many varieties
thrive but slightly or not at all if another variety grows in the
immediate neighborhood. The antagonism is very often only on one
side; for example, the Bact. putidum grows very well if inoculated
between closely placed, well-developed streaks of staphylococci; on

_ the contrary, the Micr. pyogenes does not grow if inoculated between

luxuriantly developed cultures of Bact. putidum, and if the two are
simultaneously inoculated in alternating streaks, the former grows
very slightly (Garré, ‘‘ Correspondenzbl. f.. Schweizer Aerzte,’’ 1887,
387).

Another way of showing antagonism is by preparing plates of gela-
tin or agar (for liquefying varieties), which, while liquid, are inocu-

lated with equal quantities of two different varieties of bacteria; often

only one variety will develop (Lewek, C. B. vit, 107).

A third way of carrying out the investigation is to inoculate simul-
taneously the same fluid nutrient medium with two varieties, and,
later, determine microscopically or by plates which is triumphant in
the battle. This is what is commonly observed when a cause of fer-

_ mentation is abundantly introduced into a suitable nutrient medium;

the contaminating bacteria are overgrown and sometimes perish.

From these observations the practical conclusion is

- reached that for determining the number of bacteria in a ma-

terial the colonies in the plates must not be very thick, and also

that for the isolation of definite varieties, thin plates ware neces-

it a 5 ee nie as 4 wok 0 ieee
a ney er ero

sary; for example, if one wishes to isolate the Bact.
Pfliigeri from abundant Bact. putidum. In an area of
several millimeters about each putidum colony no Bact.
Pfliigeri will grow (K. B. Lehmann).

Finally, in the animal body bacteria may counteract

each other as antagonists ; as Emmerich has pointed out,

animals infected with anthrax may be saved by subsequent

infection with Streptococcus pyogenes. Literature by

_Miihlmann (C. B. xv, 895).
Symbiosis of bacteria appears to be of more practical
4

importance, and of this the following examples may be
cited : :

1. Some bacteria thrive much better together with others
than when alone. Some anaerobes thrive even with the
admission of oxygen, if only certain aerobic varieties are
present. (Compare B. tetani. )

' 2. Certain chemical transformations—for example, the
breaking up of nitrite with liberation of free nitrogen—are
not accomplished by many bacteria alone, while two
varieties jointly may do so. This fact is well worth
considering when searching for the cause of certain decom-
positions. Always when the isolated varieties, acting singly,
operate partially or not at all, the effects of combinations must
be investigated.

3. In a similar manner it is observed that, for example,
the single individuals of a series of soil bacteria are not
pathogenic, while certain combinations, inoculated into
animals, make them sick.! (Liermann, C. B. vim, 364.)
This experience also demands that especial care be taken
in searching for the cause of a new or puzzling disease-pic-
ture. Many authors believe that cholera has its origin in
two germs, ‘‘ diblastic theory’’ (Nageli, Buchner).

4, Attenuated pathogenic varieties—for example, atten-
uated tetanus bacilli—may increase in virulence when cul-
tivated together with other bacteria; for example, Bact.
vulgare.

50 = FORMATION AND GERMINATION OF SPORES.

E. The Conditions of Spore-formation
and Spore-germination.

The extent of the formation of endogenous spores appears
hitherto to have been very insufficiently understood. Ex-
cept ina large group of important varieties of bacilli, re-
lated to the B. anthracis and the B. tetani, undoubted

1Not quite appropriate here is the experience that the metabolic
products of one variety of bacterium, under some circumstances, may
enhance the action of another variety; for example, the metabolic prod-
ucts of the Bact. vulgare the action of the tetanus bacillus.

oly

>

MEDIA, TEMPERATURE, AND OXYGEN. 51

- endogenous spores are known only in Sarcina pulmonum,
and the strange Spirillum endoparagogicum. !

As H. Buchner (C. B. vir, 1) pointed out, sporulation
occurs in suitable varieties when the nutrient medium be-
gins to be exhausted, therefore most rapidly on nutrient
media very poor in ‘nutrient materials.

On the contrary, a good nutrient medium not only
favors the growth of bacilli but also the formation of spores,
in so far as the vigorously growing bacilli also luxuriantly
and regularly sporulate (K. B. Lehmann and Osborne, A.
H. x1, 51); see especially also Stephanidis (A. H. xxxv, 1).
The crop of spores is exceedingly large. The quality (resist-
ance) of spores which are grown upon various nutrient

_ media was not found by Stephanidis to vary. For many

details consult Schreiber (C. B. xx, 353).

For sporulation a higher temperature is sometimes
(always?) required than for the vegetative growth. The
anthrax bacillus, for example, thrives at 13° to 14°, but
does not form spores below 18°.

All aerobic bacteria require, especially for spore-forma-
tion, the presence of oxygen; how the facultative anac-

robes conduct themselves in this respect is still to be

learned.

Obligate anaerobes only produce spores if oxygen is
excluded or, with the admission of oxygen, in mixed cul-
tures or in association with dead synergetic bacteria.

Spores never germinate in media in which they have
developed when they have been exhausted or rendered
detrimental by metabolic products. Only after transfer-
ring to fresh nutrient media does germination occur, ap-
pearing in one or more hours, and having the mOTEDO MES
peculiarities described on page 26.

Against all injuries spores are substantially more re-

1As it is important for our classification, we have carefully sought,

_ in a number of varieties generally considered as being free from spores,

to obtain spores as had been done by Migula (Sys. 1, 207) by means
of quince and marshmallow decoction. We never obtained a perfectly

undoubted result. With Bacterium janthinum alone we saw detached

pictures, which could be interpreted as spores, but we have not studied
their germination. Upon the common nutrient media we have not
once seen sporulation in a variety commonly known as not possessing

spores.

62 FORMATION AND GERMINATION OF SPORES.

sistant than the vegetative forms. They require no
nourishment and no water in order to retain their ability
to germinate after years and often decades.! They are
more indifferent to gases than the bacilli, the spores
of anaerobic varieties usually bearing free oxygen well. ?
Spores are obtained by carefully removing sporulating agar
streak cultures, and warming the emulsion, prepared with
a little water, to 70° for five minutes.

Very important is the resistance of spores to dry and
moist heat. Dry heat is especially well borne, a tempera-
ture of 100° being withstood by many spores for a long
time. In a moist condition, a temperature of 70° kills the
anthrax bacillus in one minute; on the contrary, anthrax
spores withstand this temperature for hours; even in boil-
ing water or live steam at 100° they die only after two to
five, or at times after seven to twelve, minutes. The vary-
ing resistance of different anthrax spores (v. Esmarch,
Z. H. v, p. 67; Stephanidis, A. H. xxxv, 1) appears to be
partly a race peculiarity, but very probably also the
nutrient medium, the temperature at which they were
produced, the degree of maturity, etc., exert an influence
upon the resistance. Very accurate investigations upon
these points are almost entirely lacking. We only know
from Percy Frankland that spores formed at 20° are more
resistant to light than those originating at incubator tem-
perature (C. B. xv, p. 101).

The resistance of spores is tested by hanging in the
boiling steam-chamber little sacks of tulle containing frag-
ments or little plates of glass upon which anthrax spores
have been dried, and from minute to minute a sack is re-
moved and the pieces of glass laid upon an agar plate,
which is then kept at incubator temperature. A better way
it seems to me is as follows: 1c.c. of an emulsion of spores
is placed in 20 ¢.c. of water, and after shaking well five

1 According to an observation of v. Esmarch, if anthrax spores are
kept a long time the virulence appears to be reduced before the power
to vegetate is affected.

2 Spores of malignant edema in garden earth were well preserved in
my institute for four years. On the contrary, very astonishingly,
tetanus spores dried upon threads and kept in the room were still alive
after two days, but dead after three days.

RESISTANCE OF SPORES. 53

samples of 2 c.c. each are removed and placed in reagent-
glasses of equal thinness, while in a sixth one are placed
2c.c. of water and a thermometer. All six glasses are
“now plunged in a large water-bath containing boiling
water, and after two minutes the thermometer in the con-
“trol tube reaches a maximum temperature (99° to 100°).
Two minutes later one removes the first sample, four min-
utes later the second, etc., cools them rapidly in cold
water, and utilizes 1 c.c. and 4c.c. of each sample in the
Salle of plates. For further details, see Stephanidis,
fae. XXXV, 1.

4 ‘The varying resistance of apparently identical anthrax
Bepores is of great practical importance: (1) in disinfection
<periments, which should be carried out with spores of
: own resistance; (2) in differential diagnosis, as it indi-
_ cates how very careful one must be in placing dependence
“upon a slight difference in the resistance of spores in de-
at ermining two species.

2 ~ Very extraordinary is the resistance of many varieties occurring in
hay and soil. Christen found, for example (C. B. xv, p. 498), that

ith compressed steam the resisting spores in soil were killed—
At 100° in more than 16 hours.

*€105°-110° in 2 to 4 hours.

415° S 30 to 60 minutes.
“€125°-130° ‘ 5 minutes and longer.
A352 ee 1 to 5 minutes.

+ 140° Me 1 minute.

The apparatus employed brought the objects te the desired elevation
_ of temperature very quickly.

: Also against chemical agents spores are very resistant;
thus, anthrax spores (v. Esmarch, J. ¢.) resist 5% carbolice
acid for at least two days, and in many cases as long as
forty days. Very resistant anthrax spores withstood a 1%
_ aqueous sublimate solution for three days, but the viru-
: _lence is lost in twenty hours. Such experiments are best
Eee with thin suspensions of spores in water, to which
e disinfectant is added, just as was pointed out above for
te sting the action of antiseptics upon bacteria (p. 38).

vin testing the resistance of spores to gases, they are best
ied upon pieces of glass, and the gas allowed to operate
in a dry and also in a moist chamber (compare p. 41).

cepts are also less injured by light than bacilli are. As

54 ACTIVITIES OF BACTERIA.

with bacilli, an atmosphere containing oxygen is necessary
in order for injury to occur. According to Dieudonné,
anthrax spores upon agar plates were dead after an expo-
sure to direct sunlight for three and a half hours (bacilli
in one and a half hours); if oxygen was excluded, an ex-
posure of nine hours produced no injury.

F. The Activities of Bacteria,

Especially in Regard to the Application of the
Same to Diagnostic Purposes.

The activities of bacteria in the test-tube may be des-
ignated! as: (1) mechanical, (2) optical, (3) thermal, —
(4) chemical. They will here be discussed in this order;
a fifth section will show how the activities of the bac-
teria enable them to become the causes of disease (patho-
genic action).

All the activities of a-given variety of bactertum are
especially dependent upon, (1) the momentary condition of
the bacterium; (2) the nutrient substratum; (3) the entrance
of air; (4) the temperature; (5) the ulwmination.

Since we have already stated what is most important re-
garding the influence of temperature and light, in the
following I must especially discuss the influence of the
nutrient medium and the admission of air on one side, and
the composition of the final culture on the other. The latter
point must always be made especially prominent, in order
to show, in the largest possible range, how very much the
activity of bacteria changes, according as they are examined
when in a full zymogenic, chromogenic, or pathogenic condition,
or in an attenuated state.

1It is self-evident that to-day a division of bacteria into zymo-
genic, saprogenic, chromogenic, and pathogenic is not acceptable.
Bact. coli causes, for example, in sugar-solutions powerful fermenta-
tion; on nutrient media rich in albumin it produces abundant indol
and sulphuretted hydrogen; it forms upon potato very often a rather
bright brownish-yellow colored layer, and is besides pathogenic for
animals and man; it therefore combines the properties of all four

groups.

MECHANICAL ACTIVITY. 55

1, MECHANICAL ACTIVITY.

Under the microscope we easily observe that many bac-
teria present pronounced inherent motion, and by study
it is found that almost all the motile varieties 1 possess
flagella by means of which they propel themselves. The
character of the motion is exceedingly variable; for ex-
ample, creeping (B. megatherium), wabbling (B. subtilis),
rolling, snake-like (vibriones) ; sometimes it is very slow,
and sometimes so rapid that any detailed observation can
searcely be made (B. typhi).

In many cases it is difficult to decide whether true

active motion is present, or whether the micro-organisms
present especially well-marked Brownian or molecular
motion—i. ¢., the dancing and tremor exhibited also by
finely divided inorganic particles. In such a case it is
recommended to try to render the flagella visible by stain-
ing (Technical Appendix), and also to examine the organ-
ism in a drop of 5% carbolic acid or 1: 1000 sublimate
solution, when, if the motion still persists, we have only
to do with molecular motion. Many varieties appear on
brief observation to be quiet, but on longer examination
single individuals are observed to exhibit positive motion.
It seems that the endowments with flagella and motility,
when once present, are for the most part reasonably con-
stant peculiarities. Many varieties do not always present
motility, it being absent, especially, om many media. Ac-
cording to A. Fischer, with faultlessly developed flagella
motion may be absent; for example, in Bac. subtilis on a
nutrient medium containing 2% to 4% of ammonium chlo-
rid. We have never observed spontaneous motion or fla-
gella in two different cultures of the Micrococcus agilis Ali-
Cohen, obtained from reliable sources and grown upon all
ordinary media. We have arrived at the conviction that
the same variety may occur either with or without flagella.
(Compare special part. )
Th. Smith has described a non-motile form of the mo-

1 Upon the actively motile Spirochete Obermeieri and the slowly
creeping Beggiatoa no flagella have thus far been demonstrated; there-

_ fore the motion is supposed to depend upon a narrow undulating

membrane attached to the organism.

3 ;

ACTIVITIES OF BACTERIA.

tile hog-cholera bacterium ; and motile pest cultures and
motile bacteria of septiceemia hemorrhagica have been de- —
scribed in isolated instances. Compare also what is said
in the special part regarding the Bac. implexus.

As first shown by Pfeffer, many chemical substances ac-
tively attract (positive chemotaxis) and others repel bac-
teria (negative chemotaxis). Oxygen is particularly at-
tractive for aerobic and repellent for anaerobic bacteria.
Like Beijerinck, one may obtain very beautiful chemotaxic
or aerotaxic figures in the following manner: An un-
sterilized pea or bean is placed in a test-tube which is three-
quarters full of sterile water. The bean gives off nutrient
materials by diffusion, which slowly extend upward. In ~
this weak nutrient material certain varieties of bacteriain- _
troduced with the bean develop at sharply defined hori-
zontal levels, which slowly extend toward the top. Cer-
tain varieties form several layers above one another. I
have had. these interesting statements verified by Mr.
Miodowski, and have substantially confirmed them, with
the exception that we found a bacterium related to the Bac.
mesentericus and the Bac. subtilis predominantly present,
instead of the non-sporulating Bac. perlibratus Beij., which
Beijerinck found to principally compose the layers. (Com-
pare Beijerinck, C. B. xiv, 827; C. B. L. my, 1;. and
Miodowski, Dissert. Wiirzburg, 1896.) In his second
work especially, Beijerinck has related a number of inter-
esting observations, but I am unable to enter into details
regarding them, nor upon the analogous studies of Jegu-
now (C. B. L. 1). .

Schenk has observed a positive thermotropism. If a
hanging drop is warmed at one point by a warm wire
(temperature difference of 8° to 10°), the bacteria strug-
gle toward it (C. B. xiv, 33).

oo a eee eye ae ee

2. OPTICAL ACTIVITY.

There are found, fairly widely distributed, especially
in media rich in salt (sea-water, Elbe, salt-fish), fission
fungi which emit light, of which a considerable number,
mostly bacteria and vibriones, have been studied. The
phosphorescence is a life-symptom of the bacteria and

OPTICAL ACTIVITY. 57

“does not depend upon oxidation of a photogenic substance
separated from the bacteria (K. B. Lehmann and Toll-
-hausen, C. B. v, 785). Everything that interferes with
the life of the bacteria, lessens it also ; cold makes the or-
ganisms rigid, and interrupts the phosphorescence while it
continues. High temperature, acids, chloroform, etc., dis-
turb the light-phenomenon momentarily. Living cultures
may always be obtained by inoculating from cultures that
emit light. The germ-free filtrate never gives light.
While no light is given off except when the bacteria are
alive, still the live bacteria do not necessarily emit light;
for example, in an atmosphere of CO,. Similarly, a mus-
cle can not contract except it is alive, but may be alive
“without contracting. (Compare also Suchsland, C. B. L.
Iv, 713.)

According to Beijerinck (C. B. vit, 616 and 651), all light-giv-
ing bacteria, which he places in a (physiologic) ‘‘ genus,’’ photobac=
_terium, require peptone and oxygen in order to emit light. Two of
_his varieties are satisfied with this ; the four others require, besides
peptone, also a source for carbon, which may also contain nitrogen.
_ As such, small quantities of sugars (dextrose, levulose, galactose, partly
maltose) and glycerin, as also asparagin, are suitable. A higher pro-
_ portion of sugar, because of marked fermentation and production of
acid, stops the emission of light in somecases. As for salts, 3% to4%
of sodium chlorid is favorable, magnesium chlorid appears to promote
the production of light still more, while sea-salt is best.

__ To preserve the photogenic function it is best to em-
ploy a gelatin nutrient medium, which is prepared from
an infusion of fish in sea-water (or artificial sea-water con-
taining 3% of sea-salt) with the addition of 1% of peptone,

1% glycerin, and 0.5% asparagin. But even on this me-
dium, if the transfer is infrequent, the ability to produce
light is soon lost, so that the cultures found in laborato-
‘ries are not usually photogenic. By repeated frequent
transfer to suitable media the photogenic property may
often be regained. I employ two salt herrings, cooked in
_a liter of water, and, after filtering, add 10% gelatin with-
out neutralization,

4

58 ACTIVITIES OF BACTERIA.

3. THERMIC ACTIVITY.

The production of warmth during the metabolism of
bacteria is absent from our ordinary cultures because of
their limited size; even luxuriantly growing, fermenting
fluid cultures betray no appreciable warmth to the hand.
On the contrary, it is undoubtedly true that the heat ex-
hibited by decomposing organic materials, when stored in
a moist condition, as tobacco, hay, manure, ete., depends,
at least in part, upon bacterial activity. With the high
temperature which thus occurs, the conjecture of Lydia
Rabinowitsch, that here the thermophilic bacteria are con-
cerned, seems very probable. Accurate investigations into
the causes of these high temperatures are still lacking. —
(Compare Rabinowitsch, Z. H. xx, 154.)

4, CHEMICAL ACTIVITY.

The chemical activities of bacteria, accompanied par-
tially by the production of light and always by a minimum
amount of heat, are to-day, in spite of the very numerous
and satisfactory investigations of the last twenty-five years,
only known in the roughest outlines. We often know
only the principal products, without being accurately in-
formed regarding the mechanism of their origin, the inter-
mediate products, or the bodies occurring in small quan-
tities.

The following three principal varieties of chemical
activity may be distinguished:

1. The bacteria elaborate their own body substance.
Regarding this the most important points have already
been discussed in the proper place.

2. The bacteria secrete ferments, intended to make the
nutrient medium in their neighborhood more suitable for
assimilation. The products which thus occur in the sur-
roundings of bacteria may be designated as metabolic
products.

3. The bacteria assimilate materials and elaborate others
which are true metabolic products. It is wrong in
principle to make a division into fermentation and
metabolic products, as is still sometimes attempted, be-

PROTEOLYTIC FERMENTS. 59

cause materials are only fermented after they are taken
into the bacterial cell. Fermentation products are meta-
bolic products produced under the influence of special
nutrition. (Compare p. 64.)

I. The Bacterial Ferments and the Changes Produced
by Them.

Under ferments in the restricted sense—enzymes—
(the custom of calling micro-organisms ‘‘ living ferments ”’
is passing into disuse) one understands certain chemical
_ bodies, which, in a minimal quantity and without being
thereby destroyed, are able to split up large quantities of
definite elaborate organic molecules into smaller, simpler,
more soluble, and more diffusible ones. }

We can only properly speak of chemical ferments after
the following properties have been demonstrated:

1. Fermentation continues in the presence of materials
_ which are surely bactericidal, but do not injure ferments;
for example, phenol, 3%; thymol, 0.1%; chloroform,
ether ; or

2. The power of producing fermentation is possessed by
the germ-free filtrate, obtained by passing cultures of the
bacteria through clay or porcelain cylinders ; or

3. This activity is possessed by the pulverized and ster-
ile ferment-preparation obtained from cultures.

Of the extraordinarily numerous details which have
been taught by Fermi’s 2 methodical and exhaustive stud-
ies, only the most important can here be given. All fer-
ments dialyze a little, like ordinary albuminous bodies,
through good parchment paper.

Proteolytic or albumin-dissolving enzymes are widely
distributed. The liquefaction of the glue in gelatin (closely
related to albumin chemically) is a sure indication of
_ the presence of a proteolytic ferment. Since the reaction
_ of the gelatin when dissolved is always or may be alka-
_ line, the liquefaction is not due to pepsin (which is active

: 1 This definition evidently does not apply to rennet ferment, which
_ coagulates milk.

Pas. xX, 1; xu, 240; C. B. x11, 713; C, B. L. 1, 462.

60 ACTIVITIES OF BACTERIA.

only with an acid reaction), but to a trypsin. The individ-
ual bacterial trypsins differ very much as regards resist-—
ance to heat (they withstand 55° to 70° moist heat for one
hour), susceptibility to injury by various acids, etc.
Some are active with a suitable acid reaction, but never
more so than with an alkaline reaction.

Much more feeble than the action upon glue, is that upon fibrin.
Fermi has employed the following method as the easiest and surest
way of proving the presence of even traces of proteolytic ferments :
Tubes of the same size are filled to an equal height with an unneutral-
ized 7% solution of gelatin in 1% aqueous solution of carbolic acid.
The solution to be tested for proteolytic ferment has 2% carbolic acid
added to it, and is placed in layers upon the solidified gelatin. The
tubes are kept at room temperature and observations are made by
means of a millimeter scale, as to how much the liquefaction of the
gelatin extends in the course of days and weeks. For qualitative ex-
amination, the upper layer may consist of 1 c.c. of a liquefied gelatin
culture, sterilized with carbolic acid.2 This material also suffices if
one wishes to test the influence of the nutrient medium on ferment
formation. One may also, by this method, compare the action of
various concentrations of different purely prepared bacterio-trypsins.
The lower the percentage of gelatin and the nearer the temperature
approaches that of the incubator, the more certainly will one observe
effects from traces of ferment. - In such critical cases the examination
is continued fourteen days, and it is determined whether the gelatin
remains fluid in the ice-box, while that in the control tube solidifies.

To demonstrate the formation of true peptone from the albu-
minous bodies one proceeds as follows:

The variety of bacterium to be tested is grown upon a fluid nutrient
medium, rich in albumin, but containing no peptone (blood-serum,
milk-serum, milk). After the culture is grown, all albuminous bodies
except peptone are precipitated by the addition of strong ammonium
sulphate (about 30 gm. to 20 ¢.c.). Milk and milk-serum may be
warmed to 60° to 80° and blood-serum to about 40°. The precipitate
is filtered off, and the filtrate cooled. A sample is made strongly alka-
line with potassium hydroxid, and 1% solution of copper sulphate added
drop by drop. A rose-red color indicates the presence of peptone. *
Fermi has shown, by similar methods, that no variety of bacterium pro-
duces true peptone.

1 Fermi found only a few bacterio-trypsins acting upon fibrin, and
none upon egg-albumin.

2 Naturally, a control test with 2% carbolic-water (ferment-free)
must never be omitted.

3 Through more recent investigations it is certainly known that some
albumoses besides peptone remain partly unprecipitated by ammonium
sul phate.

wv . in

PROTEOLYTIC FERMENTS. 61

_ The production of proteolytic ferments fluctuates
with many, perhaps with all, species in a greater degree
than one would be led to suppose from the ordinary
descriptions. Beijerinck found that one of two photo-
genic vibriones at first liquefied slowly, but that after
longer culture gelatin was always liquefied more rapidly;
_ the other showed exactly the opposite. The same was ob-
served by Katz in the Australian photogenic bacterium.
_ Max Gruber and Firtsch (A. H.. vit, 369) have studied
_ particularly closely liquefying cultures of Vibrio proteus,
_ but they have also reported similar experiences with the
cholera vibrio, Bact. vulgare, the Micrococcus pyogenes;
indeed, many observers have even seen liquefying Strepto-
- cocci pyogenes.

_ We have observed, also, in many varieties, that upon thin
_ plates single, distinctly visible, superficial colonies of the
same bacterium present such varying degrees of liquefac-
tion that a beginner could scarcely be convinced that sev-
eral varieties were not present.

It is very unfortunate that, through these observations, one of
the readiest applied diagnostic aids, the liquefaction of gela-
tin, has lost not a little in value.

The causes of the decrease and increase of liquefaction
under prolonged cultivation we ascribe to our artificial
nutrient media or to the influence of the metabolic
_ products of the micro-organisms, but without being able to
_ give anything more decisive.

_ Regarding the influence of nutrient media upon the for-
: "mation of trypsin in a culture and the liquefaction of gela-
- tin, the following facts are known:

1. Most circumstances which interfere with the growth

ai

Fe a al NPT ere) Ber

&

. Bitions upon favorable nutrient media (C. B. vi, 266).
_ 2. In hydrogen and nitrogen the liquefying facultative
_ anaerobes do not liquefy gelatin,’ while in CO,, if they

. 1A single exception is the B. prodigiosum; but if grape-sugar be
added to the gelatin, it also ceases to liquefy.
i

*
"

62 ACTIVITIES OF BACTERIA.

can thrive in it, the contrary is true.! (Compare Table I. )
Since the gases, according to Fermi, are without influence
on the activity of the ferment, they must influence the for-
mation of ferment. On the contrary, obligate anaerobes pre-
ponderately present most beautiful liquefaction of gelatin.

3. The addition of sugar does not interfere with the
growth, but does with the liquefaction of gelatin in the
case of many bacteria; for example, Bact. vulgare (Proteus
vulgaris) (Kuhn, A. H. xi, 40).

Auerbach has shown in my Institute (A. H. xxx1,
311) that sugar influences, in varying degree, the lique-
faction of gelatin by various bacteria. In the instances
examined, this checking was dependent upon the fact
that no proteolytic ferment was formed in media con-
taining sugar, and not that the sugar or acids formed from
it interfered with the action of the ferment.

4. In fluid non-albuminous nutrient media, contain-
ing glycerin but no sugar, only a few bacteria produce
proteolytic ferment; for example, B. prodigiosum and B.
pyocyaneum. Also in peptone bouillon the ferment forma-
tion appears less than in peptone bouillon gelatin (Fermi).

Upon albuminous nutrient media the liquefying bacteria
produce bitter-tasting metabolic products; for example,
from milk in the case of many varieties (Htippe).

An enumeration of the varieties forming trypsin
may be omitted, since they are characterized by their
liquefaction of gelatin. The other bacterial ferments
have been less thoroughly studied.

Diastatic ferments change starch into sugar. They
are recognized in this way: To a thin starch paste contain-
ing about 1% thymol, a culture containing 1% to 2%
thymol is added. After keeping it in the incubator from
six to eight hours, it is tested for sugar with Fehling’s
solution, when it is recognized by the reduction of the cop-
per salt (reddish-yellow precipitate). One may also ex-
amine directly potato infusion cultures of bacteria for
sugar, in which case the sugar is extracted by boiling with
alcohol and the extract evaporated to a syrup. It is then
dissolved in water and the reaction carried out.

1Jt is questionable whether, in these experiments, equal care was
always taken to absolutely exclude oxygen.

RENNET FERMENTS. 63

: _ According to Fermi, about one-third of the varieties investigated
e7 the ability to form such ferment, but only on albuminous
nutrient media (A. H. x1, 1, and C. B. x1, 713). It is produced by
bacilli of the subtilis group (anthrax, megatherium, Fitzianus, ete.),
the vibriones related to the cholera vibrio; besides, Micrococcus
tetragenus, Micrococcus mastitidis, Bact. janthinum, Bact. mallei,
Bact. pyogenes foetidum, Bact. phosphorescens, Bact. pneumoniz,
‘Bact. synxanthum, Bact. aceticum. The remainder do not elaborate
it or it is doubtful whether they do. Besides, all actinomyces and
_ Oosporeze (with the exception of the Oo. carnea) form such a ferment.
_ Most of these varieties afterward utilize the sugar further to form

acid, while others do not; for example, Bacillus subtilis.

Inverting ferments—i. ¢., such as convert cane-sugar
‘into grape-sugar—are rare, according to Fermi and Mon-
_tesano (C. B. L. 1, 482). Their presence is easily dem-
onstrated by mixing a 1% to 2% solution of cane-sugar
containing carbolic acid and the culture treated with 1%
earbolic acid, and after a few hours testing with Fehling’s
solution, and learning if it reduces the solution after stand-

ing, which cane-sugar admittedly does not. Control ex-
_ periments with a solution of cane-sugar alone are always
necessary. Fission fungi invertin can (always?) stand
100° for over an hour; it is also produced in non-albu-
_ minous nutrient media, if glycerin is added. As producers
_ of inverting ferments the above authors mention only:
Bacillus megatherium, B. kiliense, B. fluorescens lique-

faciens, B. vulgare, and Vibrio cholerze and Metschnikovii.

Efforts to find a ferment resembling emulsin have been
frustrated. The ‘‘ Micrococcus pyogenes tenuis’’ splits
_off benzaldehyd from amygdalin, but without the function
_ being separated from cell-life.

_ Rennet ferments—i. ¢., bodies which coagulate milk
of neutral or amphoteric reaction, unconnected with the
_ action of acid—are not lacking among the products of bac-
teria. For example, cultures of the Bact. prodigiosum, if
not too old, sterilized by heat at 55° to 60°, cause a solid
_ coagulation of sterile milk in one or a few days (Gorini,
©. B. xn, 666).

_ Thorough investigations regarding the distribution of
_ this ferment are unknown to me. We may suspect it in
all varieties which coagulate milk without being able to

_ form lactic acid from milk-sugar.

e

=
yr

(2
_ad

64 ACTIVITIES OF BACTERIA. ;

II. The Chemical Activity of Bacterial Metabolism.

As with ferment production, so also most other chem=
ical activities of bacteria depend in great measure upon
nutrient media. This is most striking when one observes
the growth of several varieties of bacteria upon albuminous
nutrient media, at one time without sugar and again
containing sugar. While in the first case, except for the
formation of pigments and some odoriferous substances,
there is scarcely any appreciable metabolism, in the second
case there often occur striking distinguishing changes char-
acterized by the development of gas and active production
of acid. Thus the organism produces ‘‘ fermentation ’’
in the medium containing sugar; in the other, none.

Because of the practical (and diagnostic) importance of ©
the ability to produce fermentation, there must first be ~

given a precise definition of the process.

The expression ‘‘ fermentation” is employed in the
literature with the most varying meanings.

1. Many authors call every typical decomposition caused
by bacteria ‘‘fermentation,’’? and speak, for example, of
the putrid fermentation of albuminous bodies.

2. Others limit the word ‘‘ fermentation ’’ to processes

Te

es wee a ee ae ee Pre

:
r
7
:
|

i

;

which are accompanied by evident gas bubbles. According —

to this definition, the liberation of nitrogen from saltpeter

is as much fermentation as the breaking up of milk-sugar —

by the Bact. acidi lactici.

3. Still others only speak of fermentation when there
occurs a breaking up of carbohydrates, with or without
gas production.

To me the expression ** fermentation ”’ is only applicable

when it is proved that an organism produces, along with or vn-—

stead of its other metabolic products, one or more special meta-
bolic product in striking amount; products of metabolism
which almost always arise from the merely swperficial splitting
up of an easily decomposed bacterial nutrient material (split-

ting fermentation). Oxidation fermentation is more rare
(compare below). The essential for fermentation 7s al-_

ways the presence of a special nutrient material, which the
fungus appropriates very easily, often rejecting materials more

§
L
;
7

:

:
:
3
4

if CHEMICAL ACTIVITY OF BACTERIAL METABOLISM. 65

‘difficult of utilization, which i would reduce in the absence of
fermentable objects.
___Every fermentation has the object of furnishing a store
of energy to the fermenting organism. This is attained
in the splitting fermentation in this way: |
In the interior of the bacterial cell the complicated
fermentable molecule is decomposed into smaller fragments,
and thus energy is set free. I will illustrate this as it
/ occurs in the common fermentations of sugar, where the
ease is very simple:
C.H,,0, = 20,H, + 2CO,
1 Grape-sugar = 2 Alcohol + 2 Carbon dioxid.

Or
CgH},0, = 2C;H,0s
1 Grape-sugar = 2 Lactic acid.
Or
CsH.0, = 30C,H,0,
1 Grape-sugar = 3 Acetic acid.

_ Organisms growing with oxygen excluded especially
utilize one of these sources of energy, as the source of
energy at the command of aerobic varieties, residing in
the oxidation of resorbed substances through absorbed
_ oxygen, is cut off. Therefore all anaerobic varieties are
endowed with the ability to cause active fermentation of
sugar, while many facultative anaerobes only cause fer-
mentation of nutrient media containing sugar when oxy-
gen is excluded.
As already mentioned on page 29, Buchner has dis-
covered that by expression under oreat weight a ferment
_ (zymase) can be obtained from the bodies of yeast-cells
which ferments sugar most intensely. These discoveries
_haye advanced considerably our understanding of the
utilization of sugar within the cells of the yeast fungus.
_ We now know that the breaking up of sugar and the re-
sulting gain of power originates simply ‘‘through the
_ vital process,’’ but we recognize, in the zymase, the special
means which the organism makes use of for this activity.
- Heretofore it has not been possible to obtain zymase or
other intracellular acting ferments from bacteria, yet, for
j the present, we may suppose that yeasts and bacteria are
; similar i in this respect,
| 5

it

we
ar
~ ‘

66 ACTIVITIES OF BACTERIA.

A counterpart to splitting fermentation is the rarer oxi-
dation fermentation, of which the most beautiful ex-
ample is the formation of acetic acid from alcohol. Here,
likewise, there occurs one-sided metabolic activity by the
acetic-acid fungus, which obtains a large supply of energy,
not through the splitting up of an easily decomposed sub-
stance, but through oxidation of resorbed alcohol. The
gain of energy is here dependent simply upon a partial de-
velopment and enhancement of the ordinary phenomena
in the nourishment of bacteria. }

According to this, fermentation products as well as all
other products of bacterial cells are metabolic products, and
a special separate treatment of fermentation is not de-
manded. On the contrary, it seems most suitable to ar-
range the discussion of the single bacterial products acord-
ing to whether they originate in nutrient media which
contain sugar, or are free. from it, and to add something
concerning such activities of bacteria as result in the
breaking up of salts of fatty acids, alcohols, ete.

1. Pigment Production.

The pigments have been but little studied chemically,
yet recently, through several pupils of Migula, at least a
provisional survey has become possible. | Compare
Schneider (A. K. 1, 201) and Thumm (A. K. 1, 291).

The red and yellow pigments which have been
thoroughly studied are almost all 2? insoluble in water, but
soluble in alcohol, ether, carbon bisulphid, benzol, and
chloroform. For the present they may be placed in two
groups:

(a) Pigments of the carotin group. Yellow, orange,
rose colored, with strong sulphuric acid becoming bluish-
green ; with caustic alkalis, orange to red. In some
cases the pigments, which often consist of a mixture of

1Certainly splitting fermentation is always a restricted, and only
under certain circumstances a highly developed, function.

2In marked contrast to this are the findings of M. Freund (C. B.
XVI, 640), according to which the red and yellow pigments produced
by four newly discovered bacteria were always soluble in water and
insoluble in alcohol and ether.

F PRODIGIOSIN PIGMENTS. 67
:
several pigments, present great variation. (See Schneider,
_A. K. 1, 201, regarding spectra and peculiarities.) They
are, however, closely related to the widely distributed lipo-
chromes (pigment substances of fats, yolk of egg, etc.) and
the carotin of yellow carrots. (Compare Leisenberg and
- Zopt, C. B. xu, 659. )
(6) Prodigiosin pigments. By prodigiosin I designate
the beautiful pigment of the Bact. prodigiosum and its
nearest relatives. It is soluble in ether as yellowish-
brown and in alcohol as garnet-red. It is turned yellow
by alkalis, violet-red by acids, and brownish-red by con-
_ centrated sulphuric acid. Zine and hydrochloric acid re-
duce the pigment to a colorless leuko-product. The spec-
troscopic behavior is very characteristic.

Violet pigments. In connection with the bacterium
violaceum, and also the Bacterium janthinum, there is pro-
_ duced, according to Schneider (verified by myself), a violet

pigment (janthin) which is insoluble in water, readily
_ soluble in alcohol, but insoluble in ether, benzol, and chloro-
form. If dry, it becomes yellow when treated with con-
centrated sulphuric acid and emerald-green when treated
_ withcaustic potash. In alcoholic solution all strong acids
_ and ammonia produce a green or bluish-green color. With
zine and sulphuric acid the color is destroyed (Schneider,
l. c¢.).
The beautiful blue pigment of the Bact. indigonaceum
Claessen was very incompletely examined by Claessen and
‘Schneider (/. c.). This pigment is not dissolved by ordi-
nary solvents. Hydrochloric acid gives a transitory blue,
turning to a yellowish-brown solution. Also, other acids
in dissolving it cause its decomposition. Caustic potash
turns the color bluish-green. I am unable to add any-
_ thing further.
Different from these is the blue pigment produced by
_ the Bacterium syncyaneum (blue milk), which I propose
_ tocall syncyanin. It is also entirely independent of the
_ bacterio-fluorescein forms (see below). This pigment was
_ pointed out by Thumm as very unstable; acids turn it
_ 8teel-blue, in weaker acids it is blue-black, neutral it is
black, alkaline it is brownish-black. For details see the
_ special part.

em

68 ACTIVITIES OF BACTERIA.

The fluorescent pigments which occur in the cultures

of very many bacteria are identical, according to recent
investigations by K. Thumm. The pigment which I

propose to call bacterio-fluorescein, when dry, is lemon-
yellow and amorphous. It is soluble in waterand dilute
aleohol, insoluble in strong alcohol, ether, and carbon
bisulphid. The aqueous solution, when concentrated, is
orange; when diluted, pale yellow. The solution, when
acid in reaction, presents no fluorescence; when neutral,
a blue; and when alkaline, a green fluorescence. In the
culture the fluorescence is at first blue, and later, be-
cause of the ammonia produced by the bacteria, becomes
green. The pigment is not sensitive to oxidizing agents.
Colorless antecedents (leuko-bodies) are not observed.
Phosphoric acid and magnesium appear to be essential for
the production of bacterio-fluorescein. (See also E. O.
Jordan, Botanical Gazette, xxvu, 19.—Eb.)

We have more exact knowledge concerning the beauti-
ful blue crystalline pigment, pyocyanin (C,,H,,N,0). It
can be easily extracted from cultures of the Bact. pyocy-

aneum with chloroform, and separated from the bacterio- —

fluorescein. Thumm has entirely overlooked it.

Black=-growing varieties of bacteria have been but little |
studied. According to Marpmann (C. B. L. tv, 21), the —

black color is usually (always) dependent upon a gran-
ular secretion of sulphid of iron. It is thus easily under-
stood why the ‘‘ pigment-production’’ stops upon trans-
ferring to nutrient media free of iron. The almost black
forms of the Bact. coeruleum are, however, certainly not
colored by sulphid of iron (Lehmann).

There have been many investigations regarding fluctua= —

tion of the chromogenic function. All possible in-

fluences which affect the growth of the bacteria unfavorably —
also -lessen the production of pigment. After con- —

tinued cultivation upon unfavorable media or at unfavor-
able temperature, etc., the chromogenic function of the
descendants may remain permanently reduced. Thus,
there occur, for instance, examples of the Bact. syncy-
aneum which no longer produce a trace of pigment in
agar and milk (compare Behr, C. B. vu, 485), but which
color potato darkly in the neighborhood of the culture.

FORMATION OF AMMONIA. 69

: Pigment production appears to be lessened by merely in-
uent transfers of the agar culture.

_ The Bact. prodigiosum at 37° forms no pigment; if
__ grown at this temperature for a long time with constant
_ transfers, the power of pigment formation is, even under
~ favorable circumstances, lost for many generations (Schot-

telius).
=. Very interesting experiences with pigment-forming
peultures of varieties which usually produce colorless
_ growths are scattered through the literature; for ex-
z . ample, Fawitzky regarding yellow to rusty-red colonies of
z. _ Streptococcus lanceolatus; Kruse and Pasquale on colored

: varieties of the Streptococcus pyogenes (Ziegler’s Beitrage,

__ x11); also the experience recently published by Kutscher,

_ according to which a pseudoglanders bacillus, when ob-
tained from the animal, in the primary culture upon
serum develops a bright orange-red growth, but after a few

_ transfers the red color is completely exchanged for a white
one (Z. H. xxi, 156). Perhaps still more important is
the observation, which is easily made, that, from some in-
ternal condition, at times colored and colorless colonies

of the same variety grow side by side upon a plate; for

_ example, in Bact. kiliense. R. O. Neumann has, by selec-

_ tion, grown from the Micr. pyogenes a aureus white, yel-

low, and red varieties (A. H. xxx, 1).

_ An analogue to this variation from internal causes is re-
lated by F. Hildebrand, who observed~in a stock of Iris

_ florentina, which always bore pale-blue flowers each year,

_ the sudden appearance of two flowers presenting dark

__ violet portions arranged in sectors (Ber. d. deutsch. bot.
_ Gesell., 1873, 476).

sa gate nee

_ 2. The Formation of Ammonia and Urea-fermentation.

_ According to Sommaruga (Z. H. x1, 273), aerobic bac-
teria growing in non-saccharine nutrient media always
form an alkali from albuminous bodies.

_ When sugar is present, most varieties, besides produc-
_ ing alkalis, form acids from the sugar. In this way is
_ explained the fact that many young cultures of bacteria at
_ the beginning are neutral or faintly acid in reaction because

70 ACTIVITIES OF BACTERIA.

of the small amount of sugar in the bouillon (originating
in the meat). When the sugar is exhausted, then alkali
production advances more strongly (Th. Smith).

The bodies causing the alkaline reaction, so far as known,
are ammonia (at times it may be smelt), amine, and am-
monium bases. To determine the amount of alkali formed,
one titrates single tubes which contain. 10 c.c. of peptone
bouillon, both uninoculated and one to fourteen days after
inoculation, with decinormal acid, using phenolphthalein
as indicator. The difference upon titration gives the in-
crease of alkali.

The following may be useful as an example of the pro-
duction of alkali by bacteria which, in the presence of
sugar, form acids energetically (for 100 ¢c.c. amounting to
5 ¢.c. to 7 ¢c.c. normalacid). One hundred c.c. of a nutrient
medium was employed, which contained a trace of meat-
sugar, and was originally exactly neutral with phenol-
phthalein.

AFTER FIVE AFTER TEN | AFTER FIFTEEN
Days. Days. Days.

Inoculated with Bac-| 0.1 normal 0.1 normal 0.25 normal
terium coli. alkali. alkali. acid.

A special instance of the production of an alkali by bac-
teria occurs in the transformation of urea into ammonium
carbonate, CO(NH,), + 2HO, =CO,(NH,)z.

In 1896 we stated that of sixty varieties tested, only the
Bact. vulgare, Bact. prodigiosum, and Bact. kiliense were
found able to decompose urea. Brodmeier (C. B. xv, p.
380) has investigated the urea-splitting property quantita-
tively with the Bact. vulgare, and: my pupil, Dr. Mann,
has recently done the same with the Micrococcus pyogenes
a aureus and y albus, with two forms of the colon bacillus,
and several sarcine. One hundred c.c. of filtered urine,
sterilized at 85°, after being ten days in the incubator
contained abundant NH,. Mann could not demonstrate
any action by the same culture of the Bact. prodigiosum,
which we had previously found to cause energetic fermen-
tation of urea. Thus, this property also is variable, and

COMPLICATED BASIC METABOLIC PRODUCTS. 71

“the contradictory results of authors with the Bact. coli

oat
<
iy

“(see special part) and the Micr. pyogenes are thus ex-

plained.
~ What are described in the literature as Micrococcus

- ureze Leube, Bacillus ures Leube, Bacillus ures liquefa-
- ciens Fliigge, can be partially identified as the Micr. pyo-
y genes y albus and Bacterium coli, but the descriptions of
_ these varieties allow of no accurate identification. The
_ urea-splitting function appears to occur occasionally in
_ yery many varieties. Warington (C. B. v1, 498), Burri,

ny 4
a aa

i oer me
Pa ee

Herfeldt and Stutzer (C. B. L. 1, 284) have described urea-
splitting varieties. Compare also the investigations of Mi-
~ quel (Ann. d. Micrographie, Bd. 1 u. f.), which are very
- interesting biologically, but which lose much in value be-

cause Miquel has elaborated a very singular nomenclature

which does not take into consideration the usual varieties..
- Miquel has observed varieties which are able to decom-

pose as much as 60 gm. of urea toa liter. He claims to
have isolated a special ferment, urase, which decomposes

urea.

The older literature can be found as given by Leube

_ (Virchow’s Archiv, Bd. c, p. 540); the newer, with the
_ method of determining ammonia (according to Schlésing),

_ by Mann.

_3. Formation of Complicated Basic Metabolic
Products.

Especially through the investigations of Brieger (Ueber

_ Ptomaine, Heft 1-11, Berlin, Hirschwald), besides ammo-

nia, a large number of basic, crystalline, nitrogenous

bodies are known as products of bacterial metabolism.
_ These bodies are usually called ptomains (zzdpa, putrefac-

_ tion) or putrefaction alkaloids.1_ They occur, so far as
_ Closely studied, mostly in the following groups:

1 For a long time the poisonous ptomains were called toxins, yet now

_ most authors call all bacterial poisons toxins, without reference to

_ their chemical constitution, and usually one understands the term to
__ inelude the ‘‘ albuminous-like ”’ bacterial poisons more especially.

72 ACTIVITIES OF BACTERIA.

1. Amins. Methylamin, di- and trimethylamin :

CH CH CH,
a
H H CH,

similarly, ethylamin, di- and triethylamin.
2
Ethylendiamin-i| 7} and its homologues dimethylethylendi-
C\ NH |
amin-putrescin, isomer of sepsin; pentamethylendiamin, called

cadaverin, etc. Of these the most poisonous is ethylendiamin.
2. Ammonium bases. The best known is

ges

CH

Cholin = Bilineurin = N—OH,
=

Nearly related are muscarin (C,H,,NO,), vinylcholin (C,H, ,NO),
neuridin (C,;H,,N,), ete.

:
'
:
d
7
.
:
‘
d
1

3. Pyridin derivatives. Derived from pyridin (C,H,N), the —

following .are especially found: Collidin (C,H,,N), parvolin
(C,H, ,N).
4. Indol (C,H,N) and skatol (C,H,N). Conipare page 79.

In addition the following are known : Amido=acids (leu-
cin, tyrosin, etc.) related to guanidin, C(NH)(NH,),,
and also numerous, insufficiently or feebly characteristic
bodies, whose enumeration here would be useless, since the
poisons among them are not now recognized as the essen-
tial disease poisons, as was the case in former years.

The isolation of these bodies can here be only hinted at. The method
of Brieger, which is most employed, is as follows: Brief boiling of the
culture or ‘‘ decomposed material ’’? rendered weakly acid with hydro-
chloric acid ; reduction of the filtrate to a syrup ; dissolve in 96% alco-
hol and free from impurities (especially traces of albumin) with lead
acetate ; removal of the lead ; concentration of the filtrate and precipi-
tation from this of the double salt of mercury of the ptomain by means
of an alcoholic sublimate solution. After removal of the alcohol by
heat and the mercury by hydrogen sulphid, there is produced the
characteristic double gold and platinum compound, whose crystalline
quality is an index of its purity. One may try to obtain directly the
crystalline hydrochlorate, and by the aid of caustic soda the free bases,
which are often fluid.

Some ptomains, like very many plant alkaloids, as
soon as set free~by caustic soda, are easily obtained in
aqueous solution with ether. Stil’ Brieger’s procedure is
widely useful, as it takes into consideration many bodies

POISONOUS METABOLIC PRODUCTS. 73

_ which do not dissolve in ether. For extensive review of
erature upon ptomains, see Jacquemart (C. B. 1x, 107).

, a Production of Complex «Albumin-like’’ Poisonous
Metabolic Products (‘* Toxalbumins,’’ Toxins).

In addition to the discussion of the relatively simply
_ formed, basic, less poisonous metabolic products of bac-
_ teria, we may speak, as briefly as possible, of the other
4 - bacterial poisons. From the standpoint of our present
_ knowledge, they may be divided into three classes:
2 1. Bacterioplasmin (Buchner). Compare Hahn,
_ Miinch. med. Wochenschr., 1897, No. 48, 1844. The ex-
‘ pressed juice (compare pp. 29 and 65) of bacteria contains,
in ordinary unchanged form, poisonous substances ; pres-
ent in the bacteria of cholera, typhoid, and tuberculosis;
absent in Micr. pyogenes and Bact. anthrax. Koch’s
new tuberculin, ‘‘T R,’’ is essentially also a plasmin.
2. Bacterioprotein (Buchner). Under this head are
_ placed albuminous bodies, unaltered by heat, which pro-
_ duce fever (pyrogenic), inflammation, and suppuration?
is (phlogogenic). They are obtained by ‘boiling for several
hours scraped potato-cultures with 0.5% caustic potash
(about 50 volumes KHO to 1 volume of bacterial sub-
stance). The protein may be precipitated from the clear
fluid obtained by filtering through paper, by carefully
_ rendering it feebly acid. The protein is filtered out, -
_ washed and dried, and before being employed is dis-
_ solved in a small quantity of weak soda solution.
The best-known protein is the ‘‘old’’ tuberculin of
_ Koch; also mallein belongs here. According to Buchner
_.and Rémer, all bacterial proteins act alike and not specif-
ically.
__ 3. “ Toxalbumins,” now usually called toxins. The
_ isolated statements of earlier investigators (Christmas,
Roux and Yersin, Hankin) were confirmed to a great ex-
_tent by the investigations of C. Frankel and Brieger (Berl.
_ klin. Wochenschr., 1890, 241 and 268), who found that

ir ak Sade Eads a Err

_ +Suppuration is best produced by bacterial and non-bacterial prod-
ucts if they slowly diffuse from a gelatin capsule into the subcutane-
_ Ous tissues (Poliakoff, C. B. xvii, p. 33).

agents which precipitate albumin are able to precipitate,
from the bouillon culture of many bacteria, amorphous
poisons, which possess intense and, indeed, almost always
specific action (like the living cultures). They call these
poisons toxalbumins, and compare them with the ‘‘ poison- »
ous albuminous bodies ’’ obtained from many plants (ricin
from Ricinus communis, abrin from Abrus precatorius,
etc.). Most investigators have regarded, and some still to-
day regard, the poisons as unstable albuminous bodies,
which have their origin in the bacterial cells. Often, also,
they are compared to snake poisons and enzymes. Like
these bodies, they possess a great sensitiveness to heat,
reagents, light, etc. -

The toxin may be obtained as a crude product by pre-
cipitating it with absolute alcohol or ammonium sulphate
from an old bouillon culture of the bacteria, which had
previously been freed from living organisms by a porcelain
filter,’ and concentrated in vacuum. If ammonium sul-
phate has been used, it must first be removed from the pre-
cipitate obtained upon filtering by dialysis with flowing
water in a parchment coil, and then the toxin precipitated
with absolute alcohol after renewed concentration in
vacuum. Recently we have learned that zine chlorid pre-
cipitates the bodies quantitatively, and from the precipitate
the toxins can be obtained by precipitation of the zine with
ammonium bicarbonate and ammonium phosphate. In
the filtrate the toxin is precipitated with ammonium sul-
phate. For detailed communications upon this, see Brieger
and Boer (Z. H. xx1, 259, and Deut. med. Wochenschr.,
1896, No. 49, 783).

From the first there were strong misgivings that these
‘“toxalbumins’’ were only bodies carried down by precipi-
tated albumins, but having nothing to do with albumins
proper.

In connection with tetanus poison it has been possible
for Brieger and Cohn (Z. H. xv, 1) to obtain from the
crude poison, under great precautionary measures, by
means of lead acetate and ammonia, a pure poison. This
presents with copper sulphate and caustic soda a faint violet

1 Many authors have recently proved that toxins are held back in
part by thick porcelain filters.

74 ACTIVITIES OF BACTERIA.

TOXINS. 75

color, but otherwise no reaction for albumin. It is free from
phosphorus and almost entirely free from sulphur. Thus
“it seems to be demonstrated that the tetanus poison is
not an albuminous body.
Also the cholera and diphtheria poisons are to-day rec-
ognized by Brieger and his pupils as non-albuminous, or
_ at least not as ‘‘ albuminous bodies’’ in the ordinary sense.
The statement of Uschinsky, also, that at least certain
_ diphtheria cultures form toxins in non-albuminous nutri-
ent media, is not tobe questioned. But in contrast to the
bacterial toxins the powerful plant-poisons, abrin and ricin,
which in their properties present many similarities to the
_ former, are said to be true albuminous bodies.
_ Regarding the other properties of these toxins, I will
_ give some of the recent statements, using the tetanus poi-
_ son as an example (Brieger and Cohn, /. ¢.). The toxin
_ does not diffuse through membrane, and consequently is
_ purified by dialysis (Fedoroff, C. B. xv1, 484). Watery
_ solutions are not coagulated by heat, but in time lose their
_ poisonous properties. The poisonous property is very
much injured by the addition of small amounts of acids
or alkalis to the solution and by the prolonged passage
_ through it of carbonic acid and sulphuretted hydrogen.
_ When dry, the toxin withstands 70° for a long time,
_ while higher temperatures destroy it rapidly. In a dry
condition, protected from light, air, and moisture, it is
_ slowly converted into an inert body. It keeps better if
; pod with absolute alcohol, anhydrous ether, and the
€.
The statement is interesting that very small quantities
of bile, pancreatic secretion, etc., suffice to destroy large
quantities of diphtheria and tetanus poison (Nencki and
Sieber, C. B. xxi, 880). According to Ransom, tetanus
_ poison, introduced by mouth, remains unabsorbed and
_ escapes from the body by the rectum (Deut. med.
-Wochenschr., 1898, No. 8, 117).
_ The toxicity of the purest tetanus poison obtainable is
_almost unbelievable. A mouse weighing 15 gm. is killed
_ by 0.00005 mg.; a man weighing 70 kilos, if equally sus-
_ ceptible, would be killed by 0.23 mg. Of strychnia, 30
to 100 mg. are required to cause death in man.

& oe a ae
Ph 6 7 , b

76 ACTIVITIES OF BACTERIA.

According to Courmont and Doyon, the toxins are not ready-formed
poisons, but the body, during the time of incubation, furnishes the
final poison through the development of a ferment. The blood and
juice of muscles from animals suffering from tetanus act more promptly
than the toxin. Similar results were obtained by Blumenthal (Deut.
med. Wochenschr., 1898, No. 12, 185), G. G. Brunner contests this
upon experimental grounds (C. B. xxIv, 184).

5. Sulphuretted Hydrogen.

Sulphuretted hydrogen is a widely distributed bac-
terial product. It may be simply demonstrated as fol-
lows: By means of the cotton stopper a strip of moist
lead-acetate paper is fastened in the neck of the culture-
tube, which is then closed with a rubber cap (made from
dark rubber, free from sulphur). Frequent observations
are necessary, as the paper, which at first is brownish
and later blackish, and often only faintly discolored,
still later fades out. One must not arrive at negative
conclusions too soon. As the most beautiful method
for demonstrating sulphuretted hydrogen, Ernst em-
ployed gelatin colored Madeira-yellow with sodium ferri-
tartrate (ferrum tartar. oxydat. [Merk] 0.5, aq. 50.0,
and Na,CO, added until reaction is alkaline). This is
turned black by H,S. Literature: Petri and Maassen
(A: G. A. vin, 818 and 490) and Rubner, Stagnitta-
Balistreri and Niemann (A. H. xv1, 53).

The sulphuretted hydrogen may be formed—

1. From albuminous bodies. (As is well known,
boiling separates H,S from egg-albumin.) This ability,
according to Petri and Maassen, appears especially in fluid
nutrient media rich in peptone (5% to 10% ) and free from
sugar, in connection with all the varieties studied, but in
most variable degrees. In bouillon free from peptone only
a few varieties (for example, Bact. vulgare) form H,§,
and in 1% peptone bouillon about 50% (Stagnitta-Balis-
treri). Among sixty varieties studied upon 2% peptone
bouillon we found twenty-eight—7. e., 47%—to be pro-
ducers of H,S. (See Table I, at end of book. )

2. From sulphur powder. All bacteria in nutrient media to
which pure sulphur powder is added form really larger amounts of
H,S than without such an addition. Petri and Maassen point to this
production of H,S as a result of the action of nascent hydrogen, pro-

REDUCTION PROCESSES. 17

by the bacteria; that is, they look. upon this production of H,S
s a demonstration of "the formation of nascent hydrogen.

_ 3. From hyposulphites and sulphites. Especially studied in
but also demonstrated in some bacteria by Petri and Maassen.
“4. From sulphates. Beijerinck (C. B. L., Bd. 1, 1) has proved
this practically important function for his motile obligate anaerobic
irillum desulfuricans, which has only slight morphologic character-

istics. With other bacteria it is rarely found developed.
Rubner has pointed out that with the Bact. vulgare the liberated

pence sulphur always suffices for the formation of H,S.
The presence of sugar in nutrient media only rarely diminishes or
“prevent the formation of H,S, even if the bacteria are able to actively
ferment sugar. The breaking up of the carbohydrates does not pro-
tect the albuminous bodies from decomposition. The presence of salt-
Tis i operates injuriously ; under these circumstances only a little
2» is formed, but abundant nitrite (Petri and Maassen). Exclu-
— of oxygen favors the formation of H,S. With the passage of air
ugh cultures of facultative anaerobes which produce H,S, the
Bainount of H,S formed is markedly reduced, and, instead, sulphates are

_ produced.

Some (probably many) bacteria which form H,S also
produce foul-smelling mercaptan, CH,— SH, which is
demonstrable by the green color that it imparts to the yel-
lowish-red isatin-sulphuric acid. One places upon the cul-
ee a tube, open at both ends, filled with glass
arls, which are moistened with a 1. 5% solution of isatin
in concentrated sulphuric acid. The presence of sugar in
the nutrient medium lessens or prevents the production of
-mercaptan (Rubner, A. H. xrx, 136).

‘
AG
#.
+,

Pe

_ 6. Reduction Processes.

(Reduction of pigments, nitrates, etc.)

¥ We have seen that generally the aerobic bacteria are
_ able to change powdered sulphur into H,§, for which nas-
cent hydrogen is necessary.

_ The following processes are, indeed, similar to, and in
_ part probably dependent upon, nascent hydrogen:

q 1. Reduction of the complex blue litmus-pigment of
methylene-blue and indigo to colorless leuko-products.
‘The part near the surface in contact with the air often
Shows no reduction, but only the deeper layer. By shak-
ing with air the color may return, but where there has
_ been simultaneous production of acid, the returning color

78 ACTIVITIES OF BACTERIA.

is red. The method of procedure is self-evident; nutrient
bouillon is the medium employed. According to Cahen,
all liquefying bacteria reduce litmus. It may be observed
very beautifully with, for example, the Bacillus fluorescens
liquefaciens. There are also non-liquefying varieties, as,
for example, Bact. coli, which present this characteristic.

2. Reduction of nitrates to nitrites and ammonia.
The first property seems to be possessed by bacteria very
widely, at least Petri and Maassen found pronounced ni-
trite production almost without exception in six varieties
grown. upon bouillon containing 2.5% to 5% peptone and
0.5% saltpeter; only once was ammonia alone found.
Rubner (A. H. xvi, 53) failed to find nitrite production
in isolated instances only; Warington found eighteen pro-
ducers of nitrite out of twenty-five varieties. According
to our observations, the addition of sugar did not interfere
with the process in the case of the Bact. coli, typhi, vul-
gare, Bac. anthracis, subtilis, and Vibrio cholere. After
five days upon 0.5% saltpeter, 1% peptone-bouillon, the
nitrite reaction was equally great with and without the
addition of 1% of grape-sugar.

The demonstration of nitrite is conducted as follows:
There is added to the nitrate bouillon—also to two uninoc-
ulated control tubes—after the tubes have been kept some
days in the incubator, some colorless iodid of potassium-
starch solution (thin starch paste with 0.5% iodid of potas-
sium) and a few drops of dilute sulphuric acid. The control
tubes remain colorless, or, at most, gradually become faint-
ly blue. If, however, nitrite is present, there occurs a deep
blue to (with an abundant amount of nitrite) a dark brown-
ish-red color. Small amounts of nitrite are demonstrated
with metaphenylendiamin and dilute sulphuric acid (yel-
lowish-brown color) or (most distinctly) with a mixture of
sulphanilic acid and naphthylamin (red color). Compare
Dieudonné (A. G. A. x1, 508).

The demonstration of ammonia by the addition of
Nessler’s reagent is only allowable with inorganic and
sugar-free nutrient media. In bouillon, almost immedi-
ately there occurs a reduction of Nessler’s reagent to black
mercurous oxid. Strips of paper wet with the reagent
may be suspended above bouillon cultures, or the latter

AROMATIC METABOLIC PRODUCTS. 79

y be distilled after adding MgO and the distillate
cated with Nessler’s reagent. Yellow to reddish-brown
‘eolor indicates ammonia. Control experiments must
always be made.

7. Aromatic Metabolic Products.

Often, as the result of the action of very many varieties

‘of bacteria, there arise from albumin aromatic bodies, of

which indol, skatol, phenol, and tyrosin are best known.

-Methodical investigations are at hand regarding the occur-

‘rence of only indol and phenol, since these bodies are

easily recognized.

Demonstration of Indol.—There is added to the bouillon

-eulture, which is preferably not less than eight days old
and prepared without the addition of sugar, about one-
half its volume of 10% sulphuric acid. If, now, on

“warming to about 80° a rose or bluish-red color at once

appears, then both indol and nitrite are present. The

‘nitroso-indol reaction just described requires both these

bodies to be present for its success. With cholera and

‘most other vibriones, at times also with diphtheria, the

demonstration may be made (‘‘ cholera-red reaction ’’).

Usually the addition of sulphuric acid is not sufficient, and
it is necessary to add also a little nitrite. This may be

_added if, upon warming without the nitrite, no reaction,

or only a doubtful one, is obtained. One adds 1 c.c. to 2

¢.c. of a 0.05% solution of sodium nitrite until the maxi-

mum reaction is obtained. Addition of a stronger nitrite

‘solution colors the fluid brownish-yellow, and entirely

prevents the demonstration of indol.

Demonstration of Phenol.—The culture in non-saccharine
bouillon receives the addition of about one-fifth its volume
of hydrochloric acid and is then distilled. The distillate

gives a flocculent precipitate when treated with bromin

_ water. If carefully neutralized with calcium carbonate,

the addition of neutral very dilute chlorid of iron gives a

or eect color.

' Insixty varieties examined, we found (see Table I) indol

production twenty-three times. Our findings accord well
With the statements of Levandowsky (Deutsch. med.

80 ACTIVITIES OF BACTERIA. ’

Wochenschr., 1890, No. 51, 1186). The following are pro-
ducers of indol: The colon group as a whole, glanders, —
diphtheria, proteus, and most vibriones. With the excep-
tion of the vibriones, according to Levandowsky, those
mentioned as producers of indol also produce phenol.
We have demonstrated phenol production only in Bacte-
rium coli and vulgare, and have found only traces of
phenol in five-days’-old cultures. }

8. Decomposition of Fats.

Pure melted butter is no nutrient medium for bacteria. —
Rancid changes in butter depend upon (1) a pure chem-
ical decomposition of butter through the action of oxygen
under the influence of sunlight (Duclaux, Ritsert); (2) a
lactic or butyric acid fermentation of the milk-sugar pres-—

ent in the butter. Compare v. Klecki (C. B. xv, 354).
Finally, fat is also appropriated by bacteria with produc-—
tion of acids if it is mixed with gelatin as a nutrient me-
dium. vy. Sommaruga (Z. H. xvii, 441).

9. Putrefaction. (Supplement to 1 to 7.)

By putrefaction the laity understand every decomposi-
tion brought about by bacteria, accompanied by the
formation of foul-smelling substances.

The scientific view is, that the albuminous bodies and
their relatives (glue, albuminoid substances) are the sub-
stratum for putrefaction, being often first peptonized and
then further broken up. .

Typical putrefaction occurs only with a deficient or lim- —
ited supply of oxygen. Active passage of air through a
putrefying culture of bacteria—an occurrence which never
takes place in natural putrefaction—modifies the putrefac-
tive powers most actively. This is accomplished (1) bio-
logically, by killing or checking the growth of the anaerobic
putrefactive bacteria, and (2) by the influence of oxygen
upon the products or intermediate products of the aerobic
and facultative anaerobic bacteria. Finally, itis probable
that the same bacteria from the very start produce differ-_
ent putrefactive products when grown anaerobically from
those produced under aerobic conditions,

+.
*

NITRIFICATION. 81

_ As products of putrefaction we find those given in the
preceding sections:! Albumoses, ammonia and amine,
Jeucin, tyrosin and other amido-bodies, oxyfatty acids,
- indol, skatol, phenol ; then, sulphuretted hydrogen, mer-
captan, carbonic acid, hydrogen, and finally marsh-gas.
In the decomposition of different nutrient media by
yarious fungi, the metabolic products just enumerated are
found, as a rule, only in part and in most variable com-
pbinations, so that putrefaction can scarcely be defined
more exactly by chemical aids than is possible by the
senses. Iam, therefore, of the opinion that it is best to
_ employ the expression ‘‘ putrefaction’’ only in the general .
sense of the laity to indicate every foul-smelling decom-
position of albuminous bodies. (Compare Kuhn, A. H.
xu, 40.)

10. Nitrification.

_ According to Heraeus (Z. H. 1, 193), the ability to form
nitrite, at least in traces, from NH _ is widely distributed. ?
Most investigators, however, agree in stating that nitrite
production from NH, exclusively, or at least prepon-
derantly, is dependent upon an organism, possessing slight
_ morphologic characteristics, which Winogradsky, in his
original work, designated nitrosomonas. For more detailed
- description see special part.

_ Winogradsky has described somewhat more completely
_ the organism which forms nitrates from nitrites, and which
he calls ‘‘nitrobacter’’ (compare special part). Both
organisms are alike in that they grow only upon poor
nutrient materials—on mixtures of inorganic salts or agar
and mixtures of salts without peptone or sugar—and do
not grow on any of our ordinary nutrient media. The
contradictory statements of Stutzer and his pupils are
_ generally considered incorrect. Both organisms are widely

_ +The assertion is often made that the albuminous bodies are first
“peptonized in every putrefaction, but since Bact. vulgare, B. Zenkeri,
and Bact. putidum are generally recognized as ‘‘ causes of putrefac-
tion,’”’ but never liquefy gelatin, it is not proper to speak of peptoni-
zation of albumin as being always present in putrefaction.

_ ?Rullmann calls attention to the nitrite present in laboratory air,
which may easily cause mistakes (C. B. L. v, 212).
6

a.
-_——

distributed in the soil, in meadows often the nitrite pro-
ducers alone, in cultivated soil usually both. Both
organisms possess the greatest theoretic interest, since
out of inorganic nitrogen and carbonic acid (both free CO,
and Na,CO,; or the presence of a bicarbonate is necessary )
they are able to build up their body substances, 7. ¢.,
albumin, without the aid of chlorophyll, which the higher
plants require. 3 :

P. F. Richter (C. B. xvi, p. 129) often observed marked nitrite
reaction in urine freshly obtained with a catheter. From one specimen
of urine he isolated a medium-sized coccus, which produced very in-
tense nitrite reaction in fresh urine in twenty minutes. It also re-
duced nitrate to nitrite.

82 ACTIVITIES OF BACTERIA.

11. Transformation of Nitrites (and Nitrates) into
Free Nitrogen (Denitrification).

An entire series of organisms, which are widely distrib-
uted in dung, straw, field-soil, and filthy water, set free
gaseous nitrogen from nitrites (denitrification). Many are
able simultaneously to transform nitrate into nitrite, also
alone to set free gaseous nitrogen from nitrates (for ex-
ample, Bacterium Stutzeri, B. pyocyaheum—compare
special part), while others require synergetic bacteria to
change the nitrate into nitrite (for example, Bact. denitri-
ficans). Compare Burri and Stutzer (C. B. L. 1, 257, 350,
392, 422); Weissenberg (A. H. xxx, 274).

For the demonstration of the denitrifying action of bac-
teria, there is added to a liter of ordinary bouillon 2.5 gm.
of sodium nitrate or, better,—since only thus are all deni-
trifying varieties recognized,—sodium nitrite.

As was first completely demonstrated in my institute by
Weissenberg (Burri and Stutzer had made some similar
observations), the reduction of nitrite to nitrogen is much
promoted by the exclusion of oxygen, and is markedly or
completely inhibited by very free entrance of oxygen
(growth in shallow layers of fluids or with air passing
through). The organisms thus break up the nitrite to ob-
tain oxygen, and there thus originate considerable quan-
tities of NaOH or Na,COz, so that the fluid becomes
strongly alkaline.

ASSIMILATION OF NITROGEN. ~ 88

The test for denitrification is best made with fermenta-
tion tubes (p. 90), as the entrance of oxygen is here inter-
fered with; still, usually test-tube cultures suffice. There
occurs an abundant production of gas, which is not ab-
sorbed by KHO (not CO,), nor by KHO and pyrogallic
acid (not 0), and does not burn (not H or hydrocarbons),
therefore is nitrogen.

According to Stoklasa, the denitrifying action of bacteria
is most pronounced in nutrient materials which, like de-

cayed vegetable matter, straw, and manure, contain abund-
antly the pentose, xylose (C;H,,0;). Recent litera-
ture upon denitrifying varieties: H. Jensen (C. B. L. tv,
401), Kiinnemann (C. B. L. tv, 906). Here are also
described further denitrifying bacteria: Bact. agile Amp.
and Gar., Bacillus Schirokikhi Jensen, Bact. filefaciens Jen-
sen, Bact. centropunctatum Jensen, Bact. Hartlebii Jen-
sen, Bact. nitrovarum Jensen, Vibrio denitrificans Sev.
Most do not liquefy gelatin and are also able to break
up nitrate without the aid of synergetic bacteria. The
practical significance of the denitrifying organisms is
very great. They rob the soil, manure, etc., of the ni-
trates and nitrites which are so necessary for the nourish-
ment of plants, and so are powerful enemies of agricul-
ture.

12. Assimilation of Nitrogen.

While, according to our present knowledge, none of the
higher families of plants are able to assimilate nitrogen
from the air, this property occurs in one variety of bacter-
ium, Bacillus radicicola Beyerinck. This bacterium oc-
curs in the small root-tubercles of various leguminous
plants? (peas, clover, etc.), and may be cultivated from
them. It grows poorly or not at all upon the usual nutri-
ent media, but well upon an infusion of pea leaves, to
which is added 7% gelatin,0.25% asparagin,and 0.5% cane-
sugar. It does not liquefy gelatin, does not form spores

1 According to Severin (C. B. L. 111, 504), there are yet many more
denitrifying varieties ; for example, Bacillus subtilis (or closely re-
lated varieties), Bacterium indicum, and a coccus.

? Regarding the tubercles of the alder which assimilate nitrogen,
and their inclusion of fungi, see Hiltner (C. B. L. 1, 97)

84 ACTIVITIES OF BACTERIA.

and is non-motile. From the various families of legumi- —

noses various forms of bacteria are obtained, whose mor-
phologic differences, so far as generally definitely proved,
appear very slight. Yet every race of bacterium is espe-
cially adapted to one family of leguminose, and not every
race is able to cause tubercle formation on every family of
leguminose. There are, however, ‘‘ neutral’’ tubercle-
forming bacteria free in the soil, especially adapted to no
family of leguminose and able to produce tubercle forma-
tion in very different families of leguminoses: !

With the aid of these root-tubercles which are due to
the immigration of the root-bacteria, the leguminose are
capable of thriving upon relatively sterile soil very poor
in nitrogen. The bacteria increase in the tubercle and
assume bizarre forms, forked, star-shaped, ete., then die
out and are absorbed, the plant thus receiving the benefit
of the nitrogenous contents of the tubercle. If one ploughs
the luxuriantly growing legumines (lupines) into sandy
soil (green manuring), the latter is generally so enriched
with nitrogen that plants can now thrive that are depen-
dent upon the store of nitrogen in the soil (wheat, etc. ).
Consult Stutzer regarding the question of formation of
tubercles (C. B. L. 1, 68; 11, 650 and 665).

Mazé (A. P., 1897, No. 1, 44) has undoubtedly shown
that nitrogen is also assimilated by pure cultures of bacteria
(infusion of beans, 2% cane-sugar, 1% chlorid of sodium,
15% gelatin) accompanied by great consumption of sugar,
and also in fluids poor in nitrogen containing 2.6% of sugar.
The free passage of air over the culture is very important.

For a long time cultures of the bacteria of legume-tubercles have
been sold for the fertilization of soils poor in bacteria and nitrogen,
but with sufficient mineral constituents, under the name ‘‘ nitragen.’
(Compare Wollny, Vierte Sitzung des bayer. Landwirtschaftsrats,
1898, Heft 11.) Recently a preparation known as ‘‘ alinit’’ has been
advocated to increase the yield of wheat. It is a dried potato prepara-
tion, which is impregnated with very abundant spores of the so-called
Bacillus Ellenbachensis, identical, according to Lauck, with the
Bacillus subtilis, or, more properly, according to Stoklasa, with the

1 Regarding this point, lately defended especially by Nobbe and his
pupils, it must be mentioned that other authors, as, for example, Gon-
nermann, maintain a specific difference for the various tubercle-produc-
ing bacteria. As we have made no original investigations in the
matter, we desist from further details.

j
|
i
:
i

ee ee a a a

FORMATION OF ACIDS AND ALCOHOL. 85

Bac. megatherium. According to the latter, this fertilizer acts as
follows: The Bacilli mycoides and megatherium, in the first place,
in a nutrient medium poor in nitrogen store up atmospheric nitrogen
in the soil; in the second place, they change the nitrate into ammo-
nia and so protect it against the denitrifying organisms. Stoklasa
C. B. L. tv, 817). Lauck saw practically no value in alinit
te B. L. v, 20); Stoklasa asserts very good results by many ob-
servers (C. B. L. v, 350).

Recently the question has been advanced as to whether nitrogen-
assimilating organisms are not more widely distributed. Winogradsky
has shown in the case of a Cl. Pasteurianum, closely related to the
Clostridium butyricum, that it utilizes the energy obtained by the
fermentation of sugar in the building up of organic substances from
atmospheric nitrogen. Similar observations have been made with
molds and a spirillum.

13. Formation of Acids and Alcohol from Carbo-
hydrates.

As pointed out by Theobald Smith (C. B. xvim, 1),
the formation of free acids is only possible upon nutrient
media containing sugar. The acid formation in ordi-
nary bouillon only occurs because of the presence of grape-
sugar (very small amount originating in the meat).! <Ac-
cording to Smith, all obligate or facultative anaerobes form
acids from sugar, while the strict aerobic varieties either -
do not at all or only so slowly that the acid formation is
concealed by a corresponding production of alkali. Be-
fore knowing of this investigation we had determined that
all those varieties represented in the atlas which we exam-
ined (about sixty) produced more or less free permanent
acids in peptone bouillon containing 1% grape-sugar (com-
pare Table I). In connection with the production of
acids, perceptible gas-formation was either present or ab-
sent. Intense acid production may cause the death of
cultures (for example, Bact. coli, Bact. vulgare, etc. ).
Hellstr6m recently showed that sugar, since it strongly
favored the production of acid, especially in poor nutrient
media (bouillon without peptone), can shorten enormous-
ly the duration of the life of cultures. In peptone-free

* According to Th. Smith, 75% of commercial beef contains signifi-
cant amounts of sugar (up to 0.3%). Regarding the removal of this
sugar, el the Technical Appendix under the preparation of nutri-

ent media.

86 ACTIVITIES OF BACTERIA.

bouillon, 0.1% of sugar sufficed to kill the cholera vibrio —

in a few ‘days, 0.2% the Bact. typhi, and 0.8% most bae-
teria. In solutions rich in peptone the sugar produced
less harm.

Since by many varieties the acid-production with the —
reduction of sugar is very rapid and intense, one designates

this metabolism, brought about at the expense of the
carbohydrates, as fermentation (compare p. 64). Be-
cause not rarely gas is produced in abundance, this desig-
nation also seems proper to the laity.

If, after the sugar is exhausted, the quantity of acids

produced is not such as to kill the bacteria, then in the

nutrient medium, now free of sugar, other metabolic pro- —

cesses occur and the acids are neutralized and the reaction
becomes even alkaline.

Among the acids produced (besides the carbonic acid,
to be spoken of under ‘‘ gas-formation’’) the most impor-
tant, so far as we know, is lactic acid; almost always

there are at least traces of formic acid, acetic acid, pro- —

pionic acid, butyric acid, and also not rarely some ethyl
alcohol, aldehyd, or acetone. More rarely lactic acid is
absent and only the other acids are produced.

For obtaining and separating the acids one proceeds
somewhat as follows: One-half liter of peptone bouillon

containing from 2% to 5% of grape- or milk-sugar is placed _

in liter flasks, and about 10 gm. of carbonate of calcium
added to each. The acids produced unite with the calcium
carbonate as soluble salts, and carbonic acid escapes. The
reaction of the fluid remains neutral, and that is the prin-

cipal thing; a strong acid reaction would prematurely —

hinder further growth of the bacteria.

When growth ceases (after eight to fourteen days), the —

insoluble carbonate is filtered off, and from the fluid, with
neutral reaction, the alcohol, aldehyd, and acetone, etc.,
present are removed by distillation, thus reducing very
much the amount of fluid. The three mentioned sub-
stances are tested for together by Lieben’s iodoform re-
action. To the slightly warm fluid in a test-tube are added
five to six drops of a pure 10% aqueous solution of caustic
potash ; then drop by drop a weak solution of iodid of
potassium is added until a brown color appears, and the

FORMATION OF ACIDS AND ALCOHOL. 87

latter is again dissipated by a drop of potash. The pres-
ence of iodoform is proved by the characteristic odor
and, microscopically, by the small six-sided iodoform
plates. For the differentiation of alcohol, aldehyd, and
acetone, consult Vortmann, Analyse organ. Stoffe, 1891.
Then one acidifies strongly with phosphoric acid, and
with the aid of a current of steam distils off the volatile
acids. The distillation must be long continued, as the
complete separation of the volatile acids is difficult. The
non-volatile lactic acid (together with some succinic acid)
remains behind and is separated by repeated shaking with
pure ether, the ether then being distilled off.

The lactic acid obtained is always ethylidenlactic acid,
-CH,.CHOH.COOH, which occurs in two stereoisomeric
forms: (1) dextrorotatory with levorotatory zinc salts ;
(2) levorotatory with dextrorotatory zinc salts. If, asis
frequently the case, almost equal molecules of levorotatory
and dextrorotatory lactic acid are present, then the mix-
ture is optically inactive and is the so-called ‘‘ fermenta-
tion lactic acid.”’ I believe that often both lactic acids
originate from sugar, but that many bacteria use up one
acid exclusively or principally, while others appropriate
the other acid. Thus may occur now a uniform mixture
of both acids, now one acid exclusively or preponderantly.

Since Schardinger (Mitt. f. Chem. x1, 545) first discov-
_ ered the previously unknown levorotatory lactic acid as a
product of a short bacillus from water, many investiga-
tions have been made, especially by the pupils of Nencki
and Rubner, regarding the lactic acids formed by different
varieties of bacteria, with the hope of utilizing the results
in differential diagnosis.

For the methods for determining which lactic acid is
present, consult Nencki (C. B. rx, 305) and Gosio (A. H.
xxi, 114). They have to do with the determination of
polarization and the water-content of the zine salt.

88 ACTIVITIES OF BACTERIA.

The most important results of the investigations are :

DEXTRORO-
INACTIVE TATORY
Lactic Acip, | Lactic Actp
(PARALACTIC).

LEVOROTA-
TORY
Lactic Acip,

PM OORE 5 ia: 9 ea ee +

Bab? typi oi saris . Be
Microe. acidi paralactici we
Vibrio cholerz (Calcutta)
Vibrio cholerze (Massina)
Vibrio Metschnikovi. . .
Vibrio danubicus ....
Vibrio ‘‘ Wernicke’’, 1, 1,
Pr eid Sea okt calrases so
Vibrio ‘‘Dunbar”’. . . .
Vibrio proteus .....
Vibrio Weibel .....
Vibrio Bonhoff bB. . ...
Vibrio berolinensis
Vibrio aquatilis. ....
Vibrio tyrogenes . .. . \ pe

Vibrio Bonhoffa. .... ;

While at present these results are not of much value,
yet a continuance of these theoretically interesting studies
is desirable. (Compare special part, under Vibrio choleree
and Bact. coli. )

Various bacteria—often, however, insufficiently studied
morphologically or biologically—are able to produce buty-=
ric acid, butyl alcohol, or both from carbohydrates.

For a review of these varieties see Baier (C. B. L. 1,
17). Compare in special part: Bac. butyricus Hiippe,
Bac. butyricus Botkin, Clostridium butyricum, ete.

ee Le ee fo

In connection with the fermentation of sugar, de=-—

composition of cellulose may be mentioned as caused by
various bacteria. It occurs especially in the gastric and
intestinal contents of herbivora, and also in quagmire,
and forms marsh-gas as its striking product.

1 The statements regarding the coli group are from Nencki (C. B.
IX, 305); regarding the typhoid, from Blachstein ; regarding the
cholera group, from Kuprianow (A. H. xix, 283, 291) and Gosio (A.
H. xxi, 114).

PRODUCTION OF GAS. 89

_ Unfortunately the fermentation of cellulose by bacteria
is insufficiently studied. So much seems certain, that at
least one anaerobic variety converts cellulose into
marsh-gas and carbonic acid. Yet Van Senus main-
tains that the anaerobic ‘‘ Bacillus amylobacter,’’ iso-
lated by him, attacks cellulose only in symbiosis with
another small bacillus. (Compare the résumé by Her-
feld, C. B. L. 1, 74, 114, and also the special part. )

Fig. 11.—Bacterium coli upon sugar-agar, after twelve, twenty-
four, and forty-eight hours.

14. Gas-production from Carbohydrates and Other
Fermentable Bodies of the Fatty Series.

The only gas eventually arising in visible quantity!
in nutrient media which contain no sugar is nitrogen
(compare page 82).

~

eo ee

1Sulphuretted hydrogen and ammonia can scarcely occur in visible
Quantity.

ea Mall

ae

90 ACTIVITIES OF BACTERIA.

If sugar is broken up energetically by bacteria, gas- —
formation may be absent, only lactic or acetic acid
being produced (for example, Bac. typhi on grape-
sugar), but very often an enormous production of gas —
occurs, especially if air is excluded. About one-third
of the vigorous acid-forming varieties produce abundant —
gas, which consists always of carbonic acid, with a
constant admixture, according to Smith (C. B. xvi, 1), ~
of hydrogen. Marsh-gas appears to be rarely formed
(aside from the bacteria causing fermentation of cellu-
lose). Compare in special part: Bact. brassice acide.

To determine whether gas is formed, the shake-culture
in 1% grape-sugar agar is very useful (Fig. 11). After

AUD

T
|

= eT a eee ee

HAAN

—- eS

Fig. 12.—Fermentation tube.

twenty-four hours (if incubator temperature is available, —
often after six to twelve hours) the agar is beset by gas —
bubbles or cleft by numerous deep holes and cracks. If it
is desirable to collect and measure the gas, to investigate
the curve of the intensity of gas production, or to analyze —
it, the gas is best collected after the method of Th. Smith in
a fermentation tube, such as has been long employed in
physiologic and pathologic chemistry (Fig. 12). |
The tubes, which preferably have the same form, are —
filled with 1% grape-sugar peptone-bouillon (without air-
bubbles) and sterilized in the steam sterilizer.
After inoculating with a platinum loop, they are kept

io]

PRODUCTION OF ACIDS FROM ALCOHOLS, ETC. 91.

in the incubator and the following observations made
(Th. Smith):
1. If cloudiness occurs only in the open limb, then one
is dealing with an aerobic variety; if only the fluid in the
closed limb becomes cloudy, while that in the bulb re-
_ mains clear, then an anaerobic variety is present.

2. One notes the daily gas-formation by an ink mark,
and, if the tubes are graduated, in four to six days, after the
gas production has ceased, the percentage of gas formed on
each day can be determined.

3. A rough analysis of the gas is made. With this
object in view, after indicating the quantity of gas pro-
duced by means of a mark, the open bulb is completely
filled with 10% solution of caustic soda and closed tightly
with the thumb. The fluid is shaken thoroughly with
the gas and allowed to flow back and forth from bulb to
closed branch and the reverse several times. Finally, the
gas is allowed to again rise in the closed limb, and after
removing the thumb, the new volume of gas is read. The
part removed consists of CO,; that remaining consists of
nitrogen, hydrogen, and marsh-gas. For quantitative
analysis of these gases it is best to employ Hempel’s gas
pipet. (Compare Cl. Winkler, Lehrbuch der techn. Gas-
analyse, Freiberg, 1892.) The principle of the method is
that hydrogen mixed with oxygen carried over red-hot pal-
ladium asbestos is converted into water, thus disappear-
ing; marsh-gas in a red-hot platinum capillary is burned
up to carbonic acid, and as such is determined; what is
left is nitrogen. With some practice the investigation is

easy and accurate.

15. Production of Acids from Alcohols and from Other
Organic Acids.

_ ‘The transformation, in weak solutions, of ethyl alcohol,

under energetic consumption of oxygen, into acetic acid
by the Bact. aceti or its nearest relatives has long been
known (compare p. 66 and special part):

CH, + 0, = CH, + H,0
CH,OH COOH

92 ACTIVITIES OF BACTERIA.

Also higher alcohols: Glycerin, dulcite, and mannite are
changed into acids; glycerin as constantly as sugar is
(v. Sommaruga, Z. H. xv, 291).

Finally, numerous results have been obtained regarding
the transformation of acids of the fatty series (or their
salts) into other fatty acids by bacteria. Unfortunately,
the earlier observations were made without the employ-_
ment of such pure cultures as satisfy the present demands.
Usually lactate, malate, tartrate, citrate, and glycerate
of calcium were employed, while almost always mixtures
of acids were obtained as a result of the activity of the
bacteria. Among these, butyric, propionic, valerianic,
and acetic acid play the principal réle; often also there
occur succinic acid, ethyl alcohol, and more rarely formic
acid. Of gases, carbonic acid and hydrogen occur
especially. ;

Such investigations were formerly carried out particularly by Fitz,
and more recently they have been performed on an extensive scale
with undoubtedly pure cultures and with interesting results by
P. Frankland.

Here only two illustrations are given: Pasteur found that anaerobie

’

bacteria converted lactate of calcium into the butyrate:
2(CH, — CHOH — COO),Ca =

Lactate of calcium.

CO,Ca ++ 3CO, + 4H, + (CH, — CH, — CH, — COO)Ca

Butyrate of calcium,

According to P. Frankland, the Bacillus sethaceticus Fitz forms from
the glycerate of calcium, (CH,0H-CHOH-COO),Ca, ethyl alcohol,
acetic acid, carbonic acid, and hydrogen. = |

5. THE PATHOGENIC ACTION OF BACTERIA
(PATHOGENESIS, PREDISPOSITION,
RESISTANCE, IMMUNITY).

I. How Do Bacteria Act Pathogenically ?

If micro-organisms enter the tissues or blood of an
animal, there occurs an infection if, at the same time—

1. The micro-organisms can remain alive and increase
within the host. é |

2. If the micro-organisms produce substances which are
injurious to the host.

F | PATHOGENESIS. 93

_ Theoretically, the blood and organs of healthy animals

‘contain no germs, yet we must accept the fact that often

solitary streptococci, tubercle bacilli, etc., are present in

the healthy body, circulating in the lymph- and blood-

stream and becoming fixed at loci minoris resistentie,

from which places they extend further. Perez found, in

a systematic examination of healthy bodies, that only the

lymph-glands contain bacteria, but here the bacterial flora

was very rich.! In dead animals after sixteen to twenty

hours at room temperature or after five to six hours in the

incubator, bacteria are found in the blood and organs

(Trombetta), having for the greater part wandered from

the intestine. In the most frequent artificial method of
infection, 7. ¢., subcutaneous injection, the bacteria are

absorbed through the lymph-stream, and in part are held

back in the lymph-glands, weakened in their virulence
and even killed ; but if the organisms are strongly ‘‘ path-

ogenic,’’ they resist total destruction, and, on the contrary,

begin to increase a few hours after entrance. Regarding
the method of destruction of bacteria, compare page 97.
_ Wherever we inspect the character of the pathogenic
action of bacteria, they are found to operate through chem-

ical substances which they produce or which are produced
from them in the animal body. Thus far we are able to
understand the action of only those bacteria which in cul-

tures form poisonous substances, by means of which we
an also reproduce the characteristic picture of the disease
more or less accurately. Bacteria of this class are: Bae. °
tetani and Corynebact. diphtheriz, Streptococcus pyo-
genes, Micrococce. pyogenes, Vibrio cholere, etc. There
has already (p. 73) been given a sketch of what we know
chemically about these poisonous substances.

_ On the contrary, the means for an explanation upon a
‘chemical basis are almost entirely absent as yet in a series
of important infectious diseases, among which are, for ex-
ample, anthrax (Conradi, Z. H. xxxi, 287) and swine

: *It may be mentioned here that often a considerable number of mi-
-¢ro-organisms are also rapidly secreted through the bile and urine with-
out any perceptible injury to the organ being demonstrable ( Bied] and
‘Kraus, Z. H. xxvi, 353), yet many authors assume, in these cases, at

least microscopic rupture of vessels (Opitz, Z. H. xx1x, 505).

q

94 ACTIVITIES OF BACTERIA,

erysipelas. The filtrates through porcelain from the most.
virulent cultures are without effect ; cultures carefully de-
vitalized by short heating or brief action of chloroform,
when injected produce only the general effects of protein

(fever), and still in these diseases poisoning by the meta
bolic products of bacteria is certainly present.

It is to be noted as an interesting finding that Petri and.
Maassen (A. G. A. vit, 818) could demonstrate sulphuret-
hemoglobin lines in the fresh blood and edematous fluid
of pigs sick with swine erysipelas,—an indication that
at least sulphuretted hydrogen poisoning is concerned a
the death of the animal. Alsoin malignant edema a similar
demonstration is successful. Hoffa has tried to conceive
of rabbit septicemia as poisoning by methylguanidin (Lan-
genbeck’s Archiv, 1889, p. 273). Emmerich and Tsuboi
(Miinch. med. Wochenschr. , 1893, No. 25, 473)—but cer-
tainly without receiving any support—attempt to explain
cholera as poisoning by nitrite.

Certainly these explanations possess great interest, but.
often they do not suffice, for at least there occur, besides
the just mentioned poisoning processes, also ’ specific
changes in the blood and tissues of animals, which are
proved, among other things, by the generation of specific
protective substances (‘+ anti-bodies’’).

‘
a

II. Variation in the Virulence of Bacteria.

The virulence of bacteria is just as variable as are all
the other functions (chromogenesis,. fermentation, ete. ).
It is best preserved by continual inoculation of the bactal
ria from one susceptible animal to another. It may also.
be accomplished in many varieties by rather frequent
transfer (about monthly ) from one artificial nutrient me-
dium to another, but it is best to pass the bacteria through
an animal from time to time. On the contrary, the viru-
lence usually suffers if, with infrequent transfer, the cul-
ture remains long in contact with its accumulated meta-—
bolic products. |

Reduction of virulence is not difficult to bring about:

(a) By growing at a somewhat too high temperature anthrax be-—
comes completely avirulent—at 42.5° in three to four weeks, at 47° after —

J :

VARIATION IN VIRULENCE. 95

‘several hours, and at 50° to 53° in a few minutes. By a proper regu-

lation of the reduction through the action of heat the anthrax bacillus
ean be attenuated to such a degree that it will kill only mice, or mice
and guinea-pigs, or, besides these, also rabbits. Also spores (symp-
tomatic anthrax) may be attenuated by means of dry heat or short
careful steam disinfection (Kitt).

(6) By growth upon unfavorable nutrient media. An addition of
phenol (;4,) and of bichromate of potassium (0.04% to 0.02%) te nutri-
ent media is employed to attenuate the anthrax bacillus, and iodin
trichlorid the diphtheria bacillus. Growth on media containing sugar
always, in time, lowers the virulence (Levy).

(ce) By the action of sunlight, compressed oxygen, ete.

(d) By repeated passage through unsuitable animals. Swine erysip-

elas becomes much less virulent after repeated passage through rabbits,

the variola organism (although not a bacterium) after passing through
the cow.

It is much more difficult to restore a heightened viru-
lence to attenuated bacteria. In general it is true that
the more rapidly the attenuation is accomplished, the
more rapidly will the virulence be spontaneously restored.

Varieties which have gradually (spontaneously, 7. ¢., _
from the action of their metabolic products) suffered loss

‘in virulence may have their virulence again increased

in most cases, but not always, in one of the following
ways:

1. Growing in bouillon to which has been added ascitic fluid
(streptococci, diphtheria bacilli). (Compare von Dungeren, C. B. xrx,
137, and special part. )

2. By first inoculating especially susceptible animals,—i. e., very
young animals of the susceptible variety,—and if these succumb, trans-
ferring the causative agent directly with the blood of this animal to
an older, more resisting example of the susceptible species, and later to
still more resisting species of animals. Each passage through the ani-
mal strengthens the virulence, until a certain maximum is reached.
Compare also Knorr’s experience with the Streptococcus pyogenes.

3. By first infecting susceptible animals with large quantities of
fresh bouillon cultures of the variety concerned, the simultaneously
introduced metabolic products cooperate to heighten the disposition
of the animal for the injected organisms.

4. Together with the bacteria (especially staphylococci and strep-

_ tococci) there is injected a large quantity of the metabolic products of
_ Bact. vulgare. The action is explained as under 3.

5. By the injection, together with the weakened bacterium, also of
another which in itself is entirely harmless; for example, with the Ba-

_ cillus cedematis maligni or Bacillus anthracis, the Bact. prodigiosum.

¥

6. The injection of the culture mixed with an injurious substance
not of bacterial origin; for example, lactic acid. With the Bac.

_ @dematis maligni increased pathogenic properties have thus been ob-

.
.
a

96 ACTIVITIES OF BACTERIA.

served, due to a local lowering of the bacteria-resisting property of the?
inoculated animal at the point of the injection.

III. Predisposition and Congenital Immunity
(Resistance).

The susceptibility of various species and single individ
uals to various infectious diseases from birth is striking
and not easily explained. .

In the first place, certain species of animals are naturally
absolutely immune! to certain infectious agents; for
example, man to murrain, cattle to glanders, all experi-
mental animals to sy philis, malaria, and gonorrhea.

Other diseases occur at least only rarely and with diffi--
culty in certain varieties of animals; for example,
anthrax in certain races of pigeons, rats, and sheep. .
Here there exists a relative immunity. Relative immu-
nity is usually proportionate to the strength and maturity —
of the animal. All kinds,of injury (hunger, cold, over-
exertion, incorporation of certain poisons) lessen the im-
munity,—considerably increase the disposition,—so that
a large number of animals, weakened in this way, succumb ~
to a subsequent infection. |

It is, therefore, necessary, with every newly isolated
variety of bacterium of which one desires to ascertain the -
pathogenic action, to include in the test the most varied
animals, if the first chosen animal gives a negative result.
Our principal experimental animals are: White house
mice, white rats, guinea-pigs, rabbits, chickens, pigeons, —
and, for special purposes, monkeys. More rarely we oan
ploy gray house mice, rats, field mice, gophers, dogs, cats,
cows, sheep, pigs, and horses. The most desirable ex-
perimental animal is the guinea-pig, but it requires <a
care. It is recommended by its convenient size, docility,
and limited consumption of food. Animal diseases are
usually much easier to investigate and explain absolutely —
than human diseases, because the susceptible experi-— :

1 It is especially noteworthy that very closely related varieties often |
conduct themselves very differently in this regard ; as, for example,
the glanders bacillus easily infects field mice, but not house mice ; ia
anthrax bacillus kills house mice with almost absolute certainty, but —
is not pathogenic for the rat, etc.

| a

a.
ee
rm

£ CAUSE OF CONGENITAL IMMUNITY. 97

mental animal is always at command. In difficult cases
experiments in infection have also many times been carried
out on man.

The causes of congenital immunity (resistance,
Buchner) lie in protective arrangements of the organism,
regarding which I cannot here speak exhaustively. Only
so much is given as is fairly in accord with all the facts,
corresponding to the views formulated by Buchner as a
compromise to the various opposed opinions. Upon in-
yasion of a resisting organism by pathogenic germs, a part
is destroyed by pre-existing protective substances (alexins)
which are in solution in the serum (originating from leu-
kocytes); another part is destroyed by alexins, which are

produced from leukocytes (also from other tissues even-
tually) under the influence of the bacteria. 1

In the body the leukocytes appear to be able to live
while supplying these secretions; in the test-tube certainly
it is only possible to obtain bactericidal substances from
leukocytes with certainty through injury (freezing, distilled

‘water, foreign serum, toxins) (Schattenfroh, A. H. xxxv,

135). Theserum acts more strongly upon pathogenic than
upon non-pathogenic varieties (Leclef); also spores may

_be destroyed.

Part of the organisms destroyed by the alexins are sup-

“plementarily taken up by leukocytes, but, moreover, it is
undoubtedly true that at least some organisms are, while

living, devoured by leukocytes. Metschnikoff and his

_ pupils, moreover, insist (without, in recent times, contest-

ing the significance of the alexins) that the latter phe-
nomenon (phagocytosis) is of the highest import in im-
munity. Denys (C. B. xxiv, 685) has directly shown
that pathogenic streptococci are scarcely at all, while, on

the contrary, non-pathogenic ones are very rapidly de-
_youred by leukocytes; the absence of phagocytosis here

ee ee eee ee

_ only concerns the pathogenic streptococci.

So far the alexins have not been isolated; they are very

* Recently, however, Sawtchenko and Schattenfroh have commu-
-nicated investigations, "from which it follows that, at least in some

_ Gases, the poly- and mono-nuclear leukocytes contain no alexin, in

_ spite ‘of strong bactericidal action of the serum (Schattenfroh, Miineh,

ed. Wochensehr., 1898, No. 12, 353),

7

Pad
4
a

ak)

98 ACTIVITIES OF BACTERIA.

unstable, and are rendered inactive by a temperature of —
50° or by the action of sunlight. q
Recently statements regarding heat-resisting sub-
stances which are injurious to bacteria, and which ated
from alexins, have multiplied (Léwit, Bail). These ap-
pear to belong to the nuclein compounds, ? and are particu-—
larly produced from lymph-glands. |
Besides the bactericidal alexins, frequently agglutinin
and antitoxic substances (for example, against diphtheria)
are demonstrable in healthy individuals as congenital.
An increase of the congenital resistance to various
infectious diseases has been sought and obtained in many —
ways. Favorable effects sometimes against one, some-
times against several infectious diseases have been obtained -
by a number of investigators by injecting animals with
thymus extract, spermin, abrin (poisonous albuminous
bodies from the paternoster pea), papain (albumin-dis-
solving ferment from papaw-tree); also cinnamic acid, tri-_
chlorid of iodin, carbonate of sodium, ? ete.
Recently there has been discovered a protective action —
against tetanus and botulism through injections of brain
substance (Wassermann and Takaki, Kempner), and against —
typhoid through injection of spleen substance (Aujeszky). —

IV. Acquired Specific Immunity and Its Causes.

There is a Sharp contrast to this heightened resistance,
according to most authors, and the specific immunity —
against a definite disease, which originates when an ~
attack of the disease is acquired and recovered from ~
spontaneously or when it follows purposeful injection
(active immunity): |

1H. Kossel has shown that less than a 0.5% solution of albumin pre-
cipitating nucleinic acid (obtained from calves’ leukocytes) has strong
bactericidal action. Yet the nucleinice acid could scarcely explain the
alexin action. Previously (1893) Vaughan and MacClintock recog-
nized nuclein as destructive to germs.

According to Fodor, an increase in the alkalinity of the blood —
is accompanied by an increased resistance to many bacteria, but, —
according to Fodor and Rigler, also every introduction of toxin is fol-
lowed by a decrease, and every introduction of antitoxin by an in-
crease, in alkalinity (C. B. Xx1, 134). |

; aE

POISON RESISTANCE. 99

1. With naturally or artificially attenuated infectious
agents of similar kind ; or

2. With devitalized cultures of the micro-organisms
concerned.

So far we know of two entirely different causes for this
immunity.

A.—Poison Resistance (Specific Poison Immunity).

After recovery from a series of infectious diseases (diph-
theria, tetanus, botulism) which have this in common,
that during their course very active poisons are produced
by the bacteria, there are found in the blood, and espe-
cially in the serum, characteristic substances which are
actively antagonistic to the poisons (antitoxins), The
antitoxic serum protects as well if it is used before the
introduction of the poisonous culture (it immunizes
passively ) as if it is injected at the time of, or subsequent
to, the infection (it heals). It operates as well against
the introduction of the toxins of the concerned bacteria as
(in larger doses) against the introduction of living cul-
tures.
As yet little is known regarding antitoxins, but they
are more resistant than the alexins to injurious influ-
ences. Thus the tetanus antitoxin bears very well
without being destroyed: a temperature of 60°, also 70°
to 80° for a shorter time, the action of sunlight (yellow
better than blue rays), and putrefaction. Brieger and
Ehrlich have obtained diphtheria antitoxin from the milk
of goats, immune to diphtheria, in solid form,—whether
it is an albuminous body or adheres to albuminous
bodies, isstillunknown. The antitoxins are best separated
by means of zinc chlorid, but so far can not be freed
from the final traces of zinc (Brieger and Boer, Z. H.
XxI, 266).
_ Regarding the action of antitoxins and toxins upon each
_ other the following is now known. As Behring and Kita-
sato supposed, a toxin solution in a test-tube with a suffi-
‘cient quantity of antitoxin added is completely inactive,
because toxin and antitoxin mutually neutralize each

other (somewhat as base and acid). Buchner explained

a

100 ACTIVITIES OF BACTERIA.

this neutralization as only apparent, and maintained that
both antitoxin and toxin continue together, but mutually
hide each other because they influence the same animal
organism in opposite ways, somewhat like atropin and
muscarin. Now Buchner (Mtinch. med. Wochenschr.,

1899, 523), especially since the work of Knorr (Z. H. XI,
407), has accepted essentially the views of Behring. The.
principal proofs for these views are: (1) Dilute solutions
of toxin and antitoxin neutralize each other more slowly
than concentrated ones (in higher dilutions many chemi--
cal reactions occur slowly). (2) Equalized toxin an
antitoxin mixtures after heating or long storage remain.
non-toxic, while toxin is much more resistant than anti-
toxin. (3) Animals into which are injected subcutane-
ously corresponding mixtures of toxin and antitoxin, after
two hours show no antitoxin in the blood, although the:
toxin is much more rapidly absorbed. Finally, it is very”
noticeable that ricin (poisonous albuminous body from
ricinus seeds) and antiricin act also antagonistically

upon dead bodies in vitro (diluted blood) as if they neu-
tralized each other ; the antiricin increases the coagulating
action of ricin (Ehriich, Fort. der Med., 1897, 41). Simi-
larly rattlesnake poison (cobra toxin) in vitro dissolves
red corpuscles, while cobra antitoxin hinders this action. —
Stephens-Meyers (London Lancet, Mar. 5, 1898, 644). :

As to opposing statements : Take an animal that is pro-
tected against a mixture of toxin and antitoxin, but nol
also protected against two, four, six, or ten times the quan-
tity of the mixture (Bomstein and others). Cobbett and |
Kanthack assert as their positive opinion, at least in the

case of diphtheria, that, if the toxin has been completely

neutralized, even ten times the quantity of the mixture —
could be tolerated (C. B. xxrv, 129), but, naturally, if
the mixture contains a slight excess of toxin, ten times
the quantity would be injurious.

Reference must be made to the incompletely understood.
fact that to a neutralizing mixture of toxin and antitoxin”
a very large quantity of toxin must be added before slight”
toxic action results from its injection, but that also very”
large quantities of antitoxin are required to again over-
come this weak toxic action.

:
=

ANTITOXIN. 101

_ For the explanation of the origin and action of an-
titoxin in the human body, Ehrlich first expressed the
‘idea that the antitoxins are exactly identical with those
constituent parts of the poison-susceptible cells which are
injured by the poison. The antitoxins are the ‘ toxo=-
phoric side chains” or, more simply, according to Blumen-
thal, the ‘* toxin-binding group ”’ of the toxin-susceptible
albumin molecule.
The recent experiences of Wassermann, Behring, Blu-
-menthal, Metschnikoff and Maria, and especially of Knorr
_ (Minch. med. Wochenschr., 1898, Nos. 11 and 12), accord
yery well with this explanation. Thus, what occurs in the
case of tetanus may be presented somewhat as follows: —
If one introduces tetanus poison into an animal suscept-
ible to tetanus (guinea-pig), after a time the poison disap-
pears from the blood and becomes insolubly fixed by the
chemical constituents of the ganglia of the spinal cord,
and is therefore no more obtainable from the spinal cord.
_ The binding of the poison by the spinal cord leaves it dis-
eased ; the poison-binding cells become functionally inca-
_pacitated. Wassermann could directly demonstrate that
_ the spinal cord (and brain) can bind tetanus poison, since
_he showed that mixtures of tetanus toxin and emulsion
of spinal cord are non-toxic. It is further interesting that
_ the cord of animals susceptible to tetanus alone possesses -
_ this property, the cord of hens, which are immune to teta-
nus, not at all. The side chains are absent here, and the
hen is insusceptible to tetanus for the same reason that its
cord is without effect upon the toxins. On the contrary, the
cords of animals dying from tetanus contain sufficient anti-
toxin to protect other animals from the disease. This is no
_ objection, if the first animal died when a certain part of
its spinal cells were poisoned through the union with toxin
and long before all the poison-binding affinities of its
spinal cord were satisfied.
_ Knorr has shown the identity of the antitoxin and the
poison-fixing substance of the spinal cord by demonstrat-
_ ing that both possess the same susceptibility to injurious
_ influences.
_ Where, by careful repeated poisoning of an animal until
_ the surplus supply of antitoxin is dissolved in the serum,

—

102 ACTIVITIES OF BACTERIA.

the phenomena may be represented, according to Ehrlich,
somewhat as follows:- If, with the first cautious intro-
duction of a small amount of tetanus poison only a small
part of the toxophoric side chains is fixed, then only a -
few cells are injured, or many cells are so slightly injured —
that no disease symptoms occur. The side chains united
with toxin are thrown off and replaced by the cells. This
repair is always accomplished more rapidly, the more fre- —
quently and extensively the side chains are torn off by the
toxins. Finally, the cells produce more side chains than ~
can remain connected with them, and they are discharged
into the blood as antitoxin. Knorr finds this explanation —
of the facts unsatisfactory, and claims that, while in one
part of the diseased organism the antitoxin groups are
fixed by toxin, the unpoisoned cells containing antitoxin —
are stimulated to a larger production of this substance.

From this essential independence of antitoxin from the ©
chemical composition of the toxin one can understand that
a specificity of antitoxin does not strictly exist. For ex- —
ample, tetanus and rabies antitoxin also protect against —
cobra poison (Calmette, A. P. 1x, p. 225), and, according
to Tizzoni, sterile non-toxic cultures of pneumococcus in
rabbits’ blood protect against tetanus poison (C. B. xxty,
904). 3
The value of antitoxic sera is expressed as follows:

1. After Behring. He designates as a normal poison a
toxin solution of which 0.01 c¢.c. is sufficient to kill a
guinea-pig weighing 250 gm. within four days. The toxin
is always injected in 4 c.c. of water just beneath the skin.
Of this normal diphtheria toxin (DTN) 1 c.c. is sufficient
to kill one hundred guinea-pigs, each weighing 250 gm.,
or 25,000 gm. in weight of guinea-pigs. Behring ex-
presses this as follows:

The DTN has a working value of 25,000; it kills 25,000
times its weight of guinea-pigs. A toxin ten times as
strong is represented as DTN?°; a ten times weaker one, as
DTN
=40 |:

The quantity of antitoxin that is required to just pro-
tect 25,000 gm. weight of guinea-pigs from the minimal
fatal dose of poison is called one immunizing unit.

ng
= :
Cs
*

BACTERIA RESISTANCE. 103

If an immune serum contains in 1 c.c. one immunizing
unit (IE),—~. e., neutralizes 1 c.c. DT N,—then it represents

_ anormal antitoxin (DAN).

To determine the strength of an immune serum, 1| c.c.
of normal toxin is mixed with increasing quantities of the
serum ; the quantity of the serum which suffices to neu-
tralize it—for example, 0.1 c.c.—contains one immunizing
unit, or the serum contains 10 IE to the ¢.c., and is then

ten times DAN, and is represented thus, DAN?°.

To cure a sick man, usually 600 to 1800 IE are em-

_ ployed, which are contained in 2, 4, or 6 ¢c.c.; then it is a

DAN?®°° that is used.

Recently a dried DA has been produced, of which 1 gm.
contains as much as 5000 IE; of this about 0.125 gm.
suffices for a single healing dose.

2. After Ehrlich. Ehrlich has recently introduced in
the institute for testing serum, as a standard for deter-
mining values, a very durable dry antitoxin, which con-
tains 1700 IE in 1 gm. A test-toxin is prepared corre-
sponding to this antitoxin, and with this toxin the strength
of the unknown serum is titrated. For the crude estima-
tion of the working value of a serum, a toxin can be pre-
pared corresponding to a higher serum of guaranteed
strength of IE, and the unknown serum be titrated with
this toxin. Because of the numerous cautions to be ob-

served, serviceable results can be obtained only by experts.
Compare Ehrlich, Klin. Jahrbuch, Bd v1.

B. Bacteria Resistance (Specific Bacterial Immunity).

While the antitoxins antagonize the toxins of the bacte-

_ Yia in an active manner, they are able, according to older

observations not at all, according to more recent observa-
tions! only to a slight extent, to kill bacteria—i. ¢., to act as
bactericides. On the contrary, in a second group of infec-
tious diseases (typhoid, cholera, swine erysipelas) the
immunity depends upon the bactericidal action of the

1According to van de Velde (C. B. xxt1, 527), strong antidiphthe-
ria serum also possesses considerable bactericidal action; similar double
action is presented also by various other immune sera; for example,
antipyocyaneus serum. Compare also Emmerich and Low, p. 110.

104 ACTIVITIES OF BACTERIA.

sera. If an animal has recovered from an infection with one

of these diseases, or has received in a proper manner the

devitalized cause of the disease, then its body, especially
its blood, contains materials which even in high dilutions
possess intense and even specific destructive action upon
the micro-organisms concerned. Also in man suffering
from the infectious diseases such bactericidal bodies de-
velop in the blood; indeed, they are often present from the
eighth to the fourteenth day of the disease, and may per-
sist after recovery for weeks, months, or even years. An
active immunity of this kind has been successfully pro-

duced in man against cholera and pest. In these diseases -

a positive immunity is conferred by the injection of deyi-
talized culture masses (Ferran and Hafkine).

The bactericidal serum from animals actively immun-
ized to a high degree has been employed with good results
to produce passive immunity in other organisms in various
diseases (for example, swine erysipelas by Emmerich).
Usually there is injected, besides the serum, also the normal
or attenuated specific agent. ?

To obtain serum one proceeds as follows:

1. Collection of human serum, The finger is carefully
cleansed with soap and water, alcohol and ether, and a
small puncture is made with a strong needle on the front
and to the side at the end of the finger, so that three or
four large drops of blood are obtained. The blood is col-
lected in a U-shaped capillary tube, and the ends closed
with wax or sealing-wax. As soon as possible the sample
is centrifugated in a high-speed hand centrifuge, the wax
being previously removed and the open ends of the tube
being turned toward the center. The centrifugation is
continued for ten minutes. After it is completed each
limb of the tube contains about 1 to 1.5 cm. of clear serum.
A piece 1 cm. long is now measured off in each branch and,
after marking it lightly with a file scratch, the tube is

1In mouth and foot disease, for example, Léffler, for the purpose of
producing immunity, injected a mixture of serum from the recovered
animal together with the virulent contents of the vesicle of the sick
animal, Kolle, in immunizing against murrain, employs the simul-
taneous injection (in two different places) of serum from a recovered
cow, and virulent blood from the diseased animal.

e . AGGLUTINATION. 105
:
_ broken off ; thus two pieces 1 cm. long and filled with
clear serum are obtained. |
2. Collection of serum from animals. A rabbit is injected
with cultures of typhoid or cholera, devitalized by heat,
_ following the detailed directions given in connection with
typhoid and cholera. After about ten days the animal is
bled from the carotid and the serum separated by standing
in a tall cylinder in a cool place or by centrifugation.
The measurement of the serum and the diluting fluid is
carried out by means of uniformly wide capillary tubes,
which are marked off with ink-lines in lengths of a centi-
meter each. The relative and not the absolute quantities
of the materials are thus known. In the following, when
I speak of 1 cm. of serum, I mean that contained in a
piece of a capillary tube 1 cm. long.
The bacteria are always employed as a suspension in 0.5
e.c. of bouillon, one loopful! (2 mg.) of a twenty-four
hours’ agar culture (37°) being used in its preparation.
The serum is also diluted with bouillon.
For demonstrating the injurious action upon bacteria,
_ we have two methods: The demonstration of the specific
- agglutinating material according to Gruber and Durham,
tq the specific bactericidal material according to R. Pfeif-
er.
1. Demonstration of agglutinin, after Gruber and Durham
(Minch. med. Wochenschr., 1896, 206 and 285). If it
_ is desired to make a macroscopic demonstration (little used),
0.5 c.c. of the suspension of the bacteria is added to 0.5 c.c.
_ of serum diluted fifty times, and it is noted in a very nar-
row test-tube whether there occurs a visible clumping of the
bacteria with clearing up of the fluid. If after ten to fifteen
minutes, or at most one hour, no reaction occurs in the in-
cubator, the serum in a dilution of 1 : 50 is not active upon
the variety of bacteria tested, and we may repeat the test
with stronger concentrations. On the contrary, the ex-

1 The loopful is estimated by weighing the empty and full loop on
the needle.

__ #Tf one employs more bacteria, the action of the serum is weaker.
In order to obtain cultures that are readily broken up, the culture is
_ prepared upon agar that has been somewhat dried by being previously
_ kept in the incubator for twenty-four hours.

106 ACTIVITIES OF BACTERIA.

periment is repeated with more dilute serum if it results —

positively in the first instance.

More accurate than the macroscopic is the microscopic —
method, also suggested by Gruber and Durham. In the >

examination of human blood rarely is there more serum
at hand than is required for the microscopic examination.
After the serum has been obtained by centrifugation in
two glass capillaries 1 cm. in length (see above), the serum
from one tube is blown out into a cell by means of a fine

tube placed above it, and then has added to it bouillon from -

5 segments, each 10 cm. long, of a tube of similar size to that

which contained the serum. Ina hanging drop, by means of —
the immersion lens, it is observed whether agglutination —
of introduced bacteria occurs. This follows, if the reaction —

is strong, in a few seconds; if the action is weaker, in
from ten minutes to one hour. It is observed that the
organisms suffer a loss of motion, become somewhat
swollen (rarely seen) and cemented together in irregular
bunches and clumps. Single bacilli often remain longer
motile. If the reaction is not promptly positive, the
preparation is kept in the incubator and examined after
half an hour and one hour. Positive results after two
hours are not of much value. Control preparations with-
out serum, but with bouillon only, should always be pre-
pared by the beginner, so as not to mistake a sedimenta-
tion, etc., for agglutination. ?

If the action occurs with a dilution of 35, then itisa
positive reaction, and it can then be determined whether
TOD) FVD BOD) TOT ANd gy'gq are also active, the necessary
dilutions being prepared preferably by further dilutions of

the first sample. If no result occurs with the dilution of —

=, then the reserved centimeter tube of serum is diluted
>; and if still no reaction is obtained, the diagnosis is ab-
solutely negative. In general it is customary to attach no
value to reactions with higher concentrations than 75 to 35.
(Compare further under Bact. typhi and Vibrio cholere. )

The reaction is in a great degree specific (see below).

1 Cultures killed with chloroform vapor are likewise agglutinated;
also some non-motile varieties, as Streptococcus lanceolatus, Bacterium
pestis, and Bact. pneumoniz, have been caused to agglutinate by spe-
cific sera.

eo

— 2

a ee Oe

AGGLUTINATION. 107

- Virulent and avirulent cultures are alike affected; even the

expressed bacterial cell-juice and, moreover, the germ-free

filtered bouillon cultures are precipitated by specific im-

mune sera (Kraus, Wien. med. Pr., 1897, 608).
The paralyzed and clumped organisms are not dead, or

_ only partially so, for after twenty-four hours an active in-

crease of the organisms is often observed. Although the
clumps do not dissolve, and at most loosen up, the prepa-
ration swarms with actively motile forms. If one adds
new bacteria to a preparation in a state of agglutination,
they are not affected, the agglutinin having been consumed,
and with the addition of new serum agglutination again
occurs.

2.1 Demonstration of specific bactericidal ? bodies in immune
sera according to R. Pfeiffer. We will employ cholera as an
example. If one mixes a suspension of one loopful of
virulent cholera culture in 1 c.c. of bouillon with 0.01 c.c.
to 0.03 ¢.c.* of cholera-immune serum and injects the
mixture into the peritoneal cavity of a healthy guinea-pig,
he will observe there, besides paralysis and swelling, death,
granular degeneration, and, finally, solution of the intro-

duced germs. In this case a virulent culture must be

selected, since avirulent organisms, even without the addi-
tion of immune serum, die and are dissolved in the
peritoneal cavity. This reaction is specific to a high de-
gree (compare below). To make the examinations, peri-
toneal lymph is obtained with a capillary pipet through
a small opening in the abdominal wall, and examined
microscopically every ten minutes for about half an hour
to one hour to determine the fate of the bacteria. After
this time, if the reaction is positive, nothing more is to be

1A third ‘‘specific’’ serum reaction has been recommended by

-y. Dungern (C. B. xxiv, 710). A little of the serum from animals

which have passed through cholera and staphylococcus infection, and
anthrax, exerts a marked inhibitory action upon the liquefaction of
gelatin by a portion (1 ¢.c.) of a liquefied gelatin culture of the same
variety. Normal serum restrains it less; the interference with the
ferments of other varieties is slight.

2 C. Frankel has proposed the name ‘“‘ lysogenic material ’’ for that
which acts as a bactericide.

* Tf one draws 0.2 ¢.c. in a ¢apillary tube and divides the filled
length of tube into twenty parts, then each part represents 0.01 c.c.

108 ACTIVITIES OF BACTERIA.

seen of the vibriones except single granules, and these

:
a
are not always easily found, and the peritoneal contents |
-
serum at most causes in quantities of 0.1 c.c. and upward —

have become viscid, mucoid, and tenacious. If the re-
sult is negative, the peritoneal exudate after an hour con-
tains large numbers of actively motile vibriones. It is
recommended that a control test be made upon a second
animal with the same bacteria and normal serum. Normal

a very slight positive reaction—. e., it causes a few vibri- —

ones to undergo granular degeneration (compare R. Pfeiffer
and Kolle, C. B. xx, 129).

R. Pfeiffer and Marx (C. B. xx, 858) have shown

the places of origin of the bactericidal bodies to be the
spleen, and also the bone-marrow and lymph-glands,
which possess specific bactericidal action much earlier
than the blood. Rath (C. B. xxv, 549) could not make
the same demonstration regarding agglutinin.

According to Max Gruber and Bordet, the action under
1 and 2 does not differ in principle. Their extremely

) , ae :
"Sree ae ee ee

simple theory, recently confirmed in the essential points —

by Trumpp (A. H. xxxim, 70), is as follows:

In immune serum substances are present which cause —

the bacterial cells (especially their membranes) to

swell, thus, without killing them, interfering with their —

motion and causing them to stick together.1 In this

weakened condition of the bacteria, the alexins of the —
body act as powerful bactericides. According to Gruber —
and Trumpp, also, the bactericidal action depends upon ~
the combined effects of the agglutinin and the alexin, —

Trumpp proves this view by showing that also in vitro
bacteria which are swollen, paralyzed, and clumped
under the action of immune serum are killed by contact
with the fresh serum of healthy animals. Also, Land-
steiner’s investigations are in accord with this (C. B. xxm,
847). *

While this sounds so exceedingly simple, still there are
a series of observations which speak in favor of the view

1 According to Paltauf and Nicolle, the agglutination is to be —

explained by a cementing over of the bacteria by a precipitate which
is produced by the serum. (Compdre Kraus and Seng, Wien. klin.
Wochenschr., 1899, 1.)

AGGLUTINATION. 109

_ of Pfeiffer that there is an essential difference between the
. Be eegtutinating and the specific bactericidal materials.

1. There are sera which in definite dilutions-no longer
_ agglutinate, but yet act as bactericides in the peritoneal
_ cavity (R. Pfeiffer, Deutsch. med. Wochenschr., 1898, No.
31, 489).

9. Often an immune serum, from which all agglutinins
_ haye been abstracted by long luxuriant growth of the
- inoculated bacteria, which after sixteen hours are no
_ longer paralyzed (which, therefore, is devoid of all ag-
glutination), is still active in the peritoneal cavity.

___ The observations may, however, be in part explained by
_ the discovery of Emmerich and Low, that in the abdomi-
nal cavity the action of immune sera is very much in-

creased by the lack of oxygen (see p. 110).

The action of agglutinin and specific bactericidal sub-

_ stances is, like that of antitoxin, in a great measure spe-

cific. For the bactericidal action R. Pfeiffer has main-

_ tained absolute specificity ; also other authors, as Dunbar,
_ Sobernheim, Loffler, and Abel, arrived at results that speak
_ very much in favor of specificity (Compare typhoid, chol-

= era, etc. ).

ea

The agglutination phenomenon has been studied by very
many investigators, and the standpoint taken by its dis-
coverers has been confirmed as entirely correct. The action
of immune sera is strongest upon the variety against which
_ the immunity has been produced; less, but similar, against
_ related varieties (only in high concentration); and fails with
varieties that are not related.

Thus, for example, a serum from an animal which was
immunized against the Bact. typhi was active in a ciation
of shy upon Bact. typhi, and upon Bact. coli at #5

It is evident that this property can be of Aol ces
value.

1. If we have serum from an animal which is immun-
ized against true Bact. typhi, then it is employed to iden-
tify goubtiul bacteria as typhoid bacteria, if the serum
dilution of #; acts distinctly upon the bacteria to be diag-
nosticated, a not upon related bacteria ; for example,
Bact. coli.

2, If one has undoubted typhoid bacteria, one can as-

110 ACTIVITIES OF BACTERIA. 7

certain whether a man has had (Gruber and Durham) or ~

r
‘

still has (Vidal) typhoid fever, if it is demonstrated that

the serum from a blood specimen in a dilution of 4, causes —

marked agglutination of true typhoid bacilli, while it is
without effect upon closely related organisms (Bact. coli).
(For further details see special part. )

In conclusion, we may say that the essential separation
of immunity into antitoxic and bactericidal appears to-
day to be entirely warranted, but that in a series of cases
it is established that not infrequently antitoxic and bac-
tericidal immunity are both present. Brief reference was
made above to the fact that strong diphtheria antitoxin
has also some bactericidal action (van de Velde). Wasser-*
mann found that an animal protected against pyocyaneum
poison also tolerates the virulent Bact. pyocyaneum in
large doses, and other similar experiences are contained
in the literature (compare under Cholera).

APPENDIX.

According to investigations by Emmerich and Léw which —

have just appeared (end of May, 1899) (Z. H. xxx, 1),

the whole doctrine of the bactericidal action of the body —
fluids and the immunity depending thereon appears in a ~

surprisingly altered light.

In every old culture of bacteria, according to the —
authors, there are found. bacteriolytic, remarkably heat- —

resisting enzymes—. e., ferments, which are able to dis-
solve and kill bacteria, especially old cells. Agglutination
is only the first stage of the solution and depends, as Gru-
ber held, upon a swelling of the external membrane.
Thus, in old cultures there is always a sort of agglutina-
tion, and then a dying out of the bacterial cells occurs.
The enzymes are usually only slightly specific; the pyocy-
aneum enzyme (pyocyanase) is, for example, active against
anthrax. They operate much better if oxygen is excluded
than in its presence. Also, certain bacterial poisons—for
example, diphtheria toxins—are destroyed by the pyocy-
aneum enzyme.

APPENDIX. 111

If£an old culture or its ‘‘ metabolic products ”’ are intro-
duced into the body of animals, within them there occurs
-aunion of the zymase with the body albumin—immun-
proteidin (Emmerich). These immunproteidins have the
‘same solvent action upon bacteria as the bacteriolytic
enzymes, but are more durable and, above all, more capa-
ble of persisting in the blood. At least in some infectious
‘diseases the immunproteidins can be produced synthetic-
ally in vitro instead of in the animal body, and thus, ac-
cording to Emmerich and Low, materials may be produced
rapidly and cheaply which possess very high immunizing
power. The immunproteidins operate also much more
strongly anaerobically than aerobically. The difference
between the Gruber-Durham reaction (agglutination with-
out death) and the R. Pfeiffer reaction (death in the ab-
dominal cavity) is essentially dependent upon the follow-
‘ing: In the peritoneal cavity a scarcity of oxygen prevails
and the peristalsis mechanically disturbs the agglutination;
also, Emmerich and Low find the bactericidal action of
“normal blood to be dependent upon enzymes.
_ This mass of observations, which are most worthy of
“notice, is not to be overlooked to- -day, although there has
been no opportunity for substantiating them. If they
prove true, they render an essential revision of the whole
question of immunity necessary.

Summarized presentations regarding immunity or of the greater part
of the subject are : Buchner, H., Schutzimpfung, etc., in ‘‘ Handbuch
der Therapie,’’ Jena, 1897. Metschnikoff, ‘“‘Tmmunitat,’’ Jena, 1897.
Trumpp, A. H. xxx, 70. Dieudonné, ‘< Experimentelle und krit-
ische Beitrige zur Kenntnis deragelutinieren den Stoffe, ete.’? Habili-
tationsschrift. Wiirzburg, 1898. Dieudonné, ‘“Schutzimpfung und
‘Serumtherapie. ” Leipzig, zweite Aufl., 1899.

e OPART HL.
2CIAL BACTERIOLOGY. |

A. Introduction to the Classification of
Fission-fungi.

I. The Fundamental Ideas of Botanical Classification
Applied to Fission-fungi.

All individual plants which upon careful examination

are alike and transmit their characteristics to their descen-

dants are designated as representatives of a botanical vari-

_ ety (species).

The nomenclature of the animal and vegetable kingdoms
employed at present is founded upon the assumption that

_ avery definite number of varieties of plants (species) are

present upon our planet which can certainly be distin-
guished from each other by characteristics visible with more
or less ease, and which, through propagation, reproduce
themselves unaltered in all essential characteristics. A
number of such species possess certain common character-
istics and thus exhibit a certain close relationship,—these
species are placed together in a genus. As genus charac-
teristics it is only allowable in general to select actual
characteristics, usually those concerning the structure of
the organs of reproduction. Some genera consist of single
species, others include hundreds. A group of genera
forms a family.

In certain groups of the vegetable kingdom the actual
circumstances suit this scheme very well. The individu-
als can be divided easily into a number of sharply charac-
teristic varieties, not connected by any transition ; a num-
ber of varieties group themselves naturally into a genus,
and the genera constitute a natural, sharply defined family.

The conditions are nearly so in the case of the German
malvacese. The family is sharply characterized ; it con-

sists of four genera, and each genus includes from one to
115

116 CLASSIFICATION OF FISSION-FUNGI.

seven species, which are sharply differentiated from each
other. Such groups afford pleasure to the classifier.

It is entirely different with other groups. The family
rosaceze possesses very sharply differentiated genera, but
in three of these (rubus, potentilla, rosa) the multiplicity
of species is so great that scarcely two classifiers, in the
endeavor to bring order out of chaos, arrive at the same
classification. Essentially two exactly opposite methods
exist for the solution of this problem. According to the
first, one distinguishes every form which differs in any
way whatever by a name (consequently, for example,
every individual rose-bush !) and then arranges the count-
less forms thus obtained in the most natural manner pos-
sible. Or—and this is to-day generally preferred—a
number of the most striking and widely distributed forms
are selected as species, and the others are grouped as sub-
species, forms, varieties, and transitional forms of these
main species.

A strict classification of bacteria appears more difficult
than that of any other group in the vegetable kingdom
for the following reasons:

1. Bacteria, because of their minuteness and simple
structure, possess very few morphologic characteristics
suitable for classification.

—— =e!

Sa ee

2. The description of the individual varieties of bac- q

teria represented in the literature has been absolutely
insufficient ; even recently there has been much sinning
in this direction.

3. There are a great many rarely described varieties
of bacteria, which can no longer be obtained in cul-
ture, with which, therefore, there is no possibility of
comparison with an apparently new variety.

4. Quite a number of those describing ‘‘new’’ varie-
ties have taken no trouble to look over the contribu-
tions of their predecessors, but this, to be sure, is often
excusable because of the conditions represented under 2
and 3.

Still greater difficulties in the proper definition of —

species among bacteria lie in the extremely great vari-
ability of bacteria, so often referred to in the general part.
Cohn and Koch could easily show that Nageli, who had

a

VARIABILITY OF SPECIES. 117

first asserted this in a broad sense, was partially led to this
conclusion by inefficient methods. But also Cohn’s doc-

trine of the constancy of species, which for a long time

was most strongly advocated by Koch and his pupils, is
not to-day tenable in the old sense, for continued and
always more penetrating investigation has demonstrated
that almost all the properties of a well-defined spe-
cies are exceedingly variable. For example, we have
learned that upon various nutrient media the microscopic
forms vary throughout a wide range; that dwarf forms
occur; that the liquefaction of gelatin (p. 61) and for-
mation of pigment (p. 69), clouding of bouillon, pellicle
and sediment production, ability to produce fermentation
(p. 86) and pathogenic effects (p. 94) are exceedingly
variable quantities, which can vary from a maximum to
nil; even the ability of forming spores (p. 26) and, appa-

rently, the production of flagella and spontaneous motility

(p. 24) are properties that may be lost, although rarely.
This means that bacteria vary as remarkably as other
known plants, somewhat similarly to many cultivated
plants.

For many of these variations one may recognize the
cause in the influence of the nutrient medium, and speak
of them as adaptations to changed conditions of life, as
variations from external causes. Other observations, of
which we related a great many in the first edition (the
origin of organisms obtained upon plating a culture, which
are entirely different as regards liquefaction and color,
while the original culture had for many generations ap-
peared pure), can properly be explained as dependent upon
internal causes.

While we may deplore these facts from a didactic stand-
point, since they make the teaching and learning of bacte-
riologic science much more difficult, and not rarely also
made the solution of a concrete problem by the expert

_ impossible, still we must not overlook them if we would

a
Se

advance scientific bacteriology. It is possible that the
hope of those may be realized who expect that new inves-
tigations may disclose hitherto hidden diagnostic aids,

which, consequently, when applied may disclose the

longed-for constancy and sharp definition of species. _Un-

a

> +
a,
ae
-. 4

118 CLASSIFICATION OF FISSION-F UNGI. ’

fortunately, we hold the fulfilment of this hope most im- —
probable, and look for the simplification of our subject —
through approaching the question from a different point

of view and by an improved nomenclature.

In every species of bacterium which is closely studied, ©
there are found closely related forms that not rarely rep-—
resent to the unprejudiced unbroken links to other species.

I will recall only the discoveries which have been made —
regarding the streptococci, the colon group, the diphtheria
organisms, and the relatives of the cause of tuberculosis, —
which so long stood almost entirely isolated.

With this condition of things I have sought to apply to
bacteria, with the greatest possible care, the principles
which have been found satisfactory with the pleomorphic
phanerogams, with which I have worked for years. With
the principal varieties, which were completely described,
we have grouped related varieties without assigning to the>
latter the rank of varieties. We omit this, because we
must have made changes in the nomenclature, but espe-
cially because also the principal varieties are often separated
from each other by characteristics that would scarcely be con-
sidered as sufficient for the characterization of varieties in the
botany of higher plants. It is naturally almost impossible
to state exactly the grade of relationship between closely
standing varieties, and it often becomes a matter of taste
whether one states ‘‘ identical with the preceding variety ”’
or ‘‘ very closely related,’’ ete. We certainly believe it
belongs to the future to convert varieties of bacteria into
others, in a manner scarcely to be imagined to-day. The
forms of the Micrococcus pyogenes are convertible into
each other; the Bacterium pyocyaneum and Bacterium
fluorescens can, indeed, almost certainly be converted into
each other; and similar statements regarding typhus and
coli, diphtheria and pseudodiphtheria, etc., are always
still looked upon with skepticism, but the possibility, yes
even the probability, can scarcely be contested any more.

In spite of all the things which make a rational division
and classification of bacteria more than ever difficult, we
take the stand that it is absolutely essential to strive after
it, and that also for medical men the division of bacteria
into pathogenic and non-pathogenic, etc., as is still always

—
+

‘a

THE NOMENCLATURE OF BACTERIA. 119

done in text-books, has failed absolutely. We can un-
derstand and know the pathogenic varieties only if we

_ study simultaneously the non-pathogenic, from which the

=

oa

former have once originated and still always originate!
(see Pest).
The doctrine of the absolute constancy of bacteria,

_ which for ten years was almost a dogma, is now scarcely
_ at all seriously advocated.

II. The Nomenclature of Bacteria.
The nomenclature at present employed in bacteriologic

_ works written by medical men is characterized by a limit-

less arbitrariness and inconsistency. Since these nomen-
clators often possess absolutely no sentiment for their

_ arbitrariness, and the simple rules of scientific nomen-
_ clature are often entirely unknown to them, I allow

J ahd ke oat

ee ere ae nee Tee
_ ¥ ‘

myself to set down, as briefly as possible, the most essential

‘rules, which are, by international agreement, accepted by

all educated peoples, especially as they bear upon bacteri-
ology.

1. Every plant and also every fission-fungus belongs to
& species, every species to a genus, every genus toa family.

2. Following the precedent of Linné, every vegetable or
animal organism, therefore every variety of bacterium,
should have two Latin names: the first designating the
genus to which the concerned organism belongs, which
name is a substantive; the second indicating the variety
(species), and being an adjective (not two) or the genitive of
a substantive, only rarely a substantive in the nominative
ease. ‘Thus, in the genus bacillus belong the species Bac.
subtilis (hay bacillus), also the species Bac. anthracis
(anthrax bacillus), and Bac. megatherium.
3. Genera must only be founded upon important mor-
phologic characteristics; so-called ‘‘ biologic genera,’’ such
as photobacterium for all light-emitting bacteria, pyo-
bacterium for rods causing suppuration, etc., are only cal-
culated to produce confusion.

1 Tf the pathologist may, perhaps, say that the pathogenic bacteria
alone interest him, such a statement—as I have often heard—from the
mouth of a hygienist is almost beyond understanding.

120 CLASSIFICATION OF FISSION-FUNGI.

4. As designations for species many authors have used,
instead of one adjective or substantive, a plurality of ad-—
jectives, evidently with the object of furnishing a descrip-—
tion through the name: Bac. rosettaceus metalloides,
Staphylococcus pyogenes aureus, Bacillus pyogenes foeti-
dus, Bacillus mesentericus panis viscosi I and 1. This
effort can be understood, but it has been abandoned as —
entirely impractical by all descriptive naturalists since —
Linné. The name of the species should indicate only the
variety wnequivocally; the characterization belongs to the
description. It does no harm if two or more organisms —
possess names that mean the same, if they do not sound ~
alike. Besides, a Micrococcus albus, also a Micr. niveus,
albissimus, candicans, and purus are entirely right; the
description must give more exactly the kind of differences —
existing between these white cocci.

5. Improperly (i. e., contrary to the binomial rule)
formed names may be replaced. We have done this with
the greatest consideration for the existing name whenever
possible.1_ We have not changed names like Bae. acidi
lactici, because acidum lacticum represents a single idea,
and names like Sempervivum Reginze Amalie, Pedicularis
Friderici Augusti, Trigonella Foenum greecum, Pedicularis
Sceptrum carolinum have remained, although certainly not —
convenient, still uncontested. Varieties which we have not —
studied more closely or which in our opinion should be
suppressed, have not been renamed; on the contrary, Mez
has conducted this renaming in the widest extent in a
most acceptable manner.

6. If names are properly formed in the binomial man-
ner and correctly published, then they must not be changed
by the author himself, much less by others, even if subse-
quently another name appears better. Also, the reason
that the name is philologically incorrect or not beautiful
gives no occasion for change. Even if, for example, it was

1 We regret that we had to do this also in the case of a number of
convenient and very familiar names; for example, those of Fliigge.
Unfortunately, also, Kruse has formed a large number of new names
contrary to rule. Our names have the priority over his, because pub-
lished about two months earlier, but they are to be preferred besides,
in so far as Kruse’s are formed contrary to rule.

THE NOMENCLATURE OF BACTERIA. 121

literally more correct to call the genera which we call
“mycobacterium”? tuberculomyces,’’ such a _ propo-
sition is absolutely unallowable. Renaming is only re-
quired if the name given was employed earlier with another
signification. Thus, Cohn founded upon a certain organ-
ism the new genus streptothrix, without knowing that
Corda, about thirty years previously, had conferred this

name upon a fungus that is totally different from his.
His new variety must, therefore, receive a new genus name,
which he who first observed Cohn’s oversight is justified
in establishing.

7. It happens that an author differs from his predeces-
sor regarding the bounds of the genera, that therefore he
transfers a species from one genus into another, pre-exist-
ing or newly formed by himself. This is permissible; sézll,
the designation of the species must not be changed. So we had
the right, when we broke up the very large genus bacillus,
following the suggestion of Htippe, into the two genera,
bacillus and bacterium, to rename a number of varieties
(for example, Bacillus pyocyaneus being renamed Bac-
terium pyocyaneum), but we did not have a right (how-
ever much the name pyocyaneum was disliked) to rename

_ the organism Bacterium cceruleo-viride or Bacterium Ges-
sardi or anything else.

8. The author who names a genus places his name after
it. We speak of the Bacillus Cohn, and mean the genus
bacillus as Cohn established it ; of the Vibrio Ehrenberg
emend. Léffler, and mean the genus vibrio as established
by Ehrenberg and afterward more accurately described by
Loffler.

9. Whoever discovers a new species or names one not
previously named lege artis, gives it a genus and a species
name, and places his name after the latter. Fliigge, who
first named a large number of bacteria, gave, for example,

_ the name Bacillus pyocyaneus Fliigge to the long-known

_ cause of bluish-green suppuration.

_ 10. When one places a species in a new genus he puts

_ hisown name after the new name, thus, Bacterium pyocy-

_ aneum Lehmann and Neumann, but it is always to be rec-

- ommended to add, in parentheses, the name of the author

_ who first named the species. Therefore we always write,

=

eo

122 CLASSIFICATION OF FISSION-FUNGI.

where it does not become too cumbersome (in titles, ete. ),
Bacterium pyocyaneum (Fliigge) Lehmann and Neumann.

While we desire that all names which express the sys-
tematic position of the variety of bacterium shall conform
to the general rules of nomenclature, still we believe that
names currently used in bacteriologic literature, as gono-
coccus, pneumococcus, staphylococcus, tubercle bacillus,
diphtheria bacillus, can be still used, but as so-called ordi-
nary names. Thus also the strictest botanist, if not speak-
ing in a strictly systematic sense, often speaks of the oak
instead of quercus, and strawberry instead of fragaria. We
must, however, strictly avoid smuggling into the literature
as names of genera such names as gonococcus, etc.

III. The Formation of the Families and Genera of
Fission-fungi.

The families of the fission-fungi are given fairly uni-
formly by the more recent investigators. Here a better
division does not seem possible at present ; on the con-
trary, regarding the genera, the comprehension is most
variable. The simplest and most natural division is that
of Fliigge (retained by Kruse in Fliigge, third edition),
which so properly includes the genera micrococcus (strep-
tococcus), sarcina, bacillus, and spirillum, but without
rejecting energetically such genera as staphylococcus, or
separating the causes of diphtheria and tuberculosis. A
more copious selection of genera is made by Htippe, still
more by Migula, and the most extensive by A. Fischer.
After mature deliberation we have followed Fliigge most
closely as to the coccacee and bacteriaceze, on the other
hand, the works of Léffler and Migula as to the spirillacee.

I. Family Coccaceze Zopf, emend. Migula. Spherical
Bacteria.

Cells, when free, are perfectly globular ;! division in one,
two, or three directions of space, in which each spherical
cell divides into halves, quarters, or eighths of a sphere,

1 Unfortunately this applies, only imperfectly to the Strept. lan-
ceolatus and Micrococcus gonorrheeze.

FAMILY COCCACE ZOPF. 123

which again grow out into perfect spheres. Endospores
and flagella very rare. Before division the cells may be
one and a half times as long as broad, faint staining then
revealing an unstained line of division.

1. The cells divide (almost) only in one direction of
space at right angles to the direction of growth, so that if
the products of division remain attached, they form (es-

pecially in bouillon) shorter or longer rosary-like chains,
the chains often consisting of distinct pairs of cocci. Under
certain circumstances there are only (or largely) pairs of
cocci instead of chains. Streptococcus Billroth.

_ 2. The cells regularly divide, at least on the most suitable

_mutrient medium (hay decoction), in three directions of

Space, ? and remain united in larger or smaller cubical fam-

ily groups. Sarcina Goodsir.

3. The cells divide irregularly in various directions, so
that there occur single cocci, single groups of from two to
four cells, and, finally and. preponderantly, irregular
_ grouped bunches. Here belong all forms that do not
-appear as undoubted streptococci or sarcinee. Micrococcus
_ Cohn.

_ The recognition of these three genera of cocci is largely
artificial, and there occur perfect transitions.
_ The genus Staphylococcus Ogston has no botanical

_ rights, for the property of forming ‘‘ grape-like’”’ clusters is

" possessed at times by all varieties described to-day as mi-
_ crococci. The name staphylococcus does not primarily

- designate any ‘‘new’’ genus. Ogston found (microscop-
_ ieally) two forms of micrococci in pus (without cultivating
them), grape cocci and chain cocci, and designated them
the well-chosen names of Staphylococcus and Strepto-
-coceus (Billroth). Rosenbach later cultivated the varieties

*

1 Here belongs Leuconostoc Cienc., which is only astreptococeus
with at times enormously thick capsules ‘(see below). Also part of the
_ “‘diplococci’’ are naturally included here.

? The varieties which, by division in two planes at right angles to
~ each other, form flat groups, and which are described by authors as
_ pediococcus, merista, merismopedia, we leave among the micro-
= ~ cocci. Since even the é ‘genus’? Sarcina is separated with difficulty,
_ we do not recognize the need for the genera planococcus and planosarcina
_ of Migula, which are founded upon one or two flagellated varieties,
_— as the formation of flagella varies (see below).

x
bt
ie ol

124 CLASSIFICATION OF FISSION-FUNGI.

which Ogston had seen, and gave the name Staphylo-
coccus to the bunched cocci, which we may to-day employ
as the ordinary name for species of micrococci causing sup-
puration, and which we will use, but it must be dropped
from the botanical classification.

II. Family Bacteriacez Zopf, emend. Migula (Bacil- ©

lacez A. Fischer). Rod Bacteria.

Cells at least one and a half but usually from two to six
times as long as broad, straight or somewhat bent in one ~
plane only, never spiral,? at times forming long true or ap- —

parent threads. Division (almost) always is at right

angles to the long axis, after elongation of the rod; with ~
or without flagella; with or without endospores. The —

varieties in which spores are wanting sometimes form
arthrospores, according to many authors. Yet it is not pos-

sible to turn these ‘‘arthrospores’’ to account diagnost- —

ically, and they are entirely denied by many investigators.

1. Without endogenous spores, alleged to often have
arthrospores. Rods usually less than 0.8 to 1 thick.
Bacterium? Cohn, emend. Htippe.

2. With endospores. Rods often more than 1 z thick.
Bacillus Cohn, emend. Htippe.

- Cohn in his classification laid more value upon growth
into long threads (which, according to him, is character-
istic of bacilli) than upon the property of spore-formation;
he, however, often emphasizes the fact that most bacilli
produce endogenous spores.

The fact that, through certain injurious influences, spore-
formation may be lost is no valid objection to the classifi-
cation, since in most cases also typical bacilli without
spores are recognizable or may be conjectured. It is more
unfortunate that there appear to be varieties of bacilli
which at least are related to varieties in which spores never
form; for example, Bacillus erythrosporus. There always

’ Unfortunately it must be said tha “never spiral’’ is really un- —

true, since, for example, in the case of anthrax, Bac. Zopfii, ete., tuft-
like ‘loops occur that cannot possibly be in one plane.

? Here belongs the genus Proteus Hauser.

-“——

FAMILY SPIRILLACEX MIGULA. 125

TSS

‘appears to us to be less possible objection to the method of
‘division adopted by us than to the other.

Critical Remarks Regarding Other Classifications of the
Bacteriacez.

The following subdivision of the genus bacillus appears to us of
little value :

Spore centrally located without a bulging of the vegetative cell.
Bacillus in a strict sense.

_ Spore centrally located with bulging of the vegetative cell. Clos-
tridium Prazmowski.

_ Spore located at the pole without a bulging of the balance of the
vegetative cell. Paraplectrum A. Fischer.

There occur various transitions in the same species, for example,
the Bac. cedematis maligni, and even, according to recent investigators,
_all anaerobes sometimes present clostridium, sometimes paraplectrum
forms.

- In the effort to build a genus classification upon the flagella,
-Migula?* has arrived at the following often unnatural classification, in
the more extensive application of which new complications are to be
feared :

1. Cells without organs of locomotion, often with endospores.
Bacterium Cohn, emend. Migula.

2. Cells with motile organs distributed over the whole body, often
with endospores. Bacillus Cohn, emend. Migula.

3. Cells with polar organs of locomotion, endospore formation
more rare. Pseudomonas Migula.

Thus in one genus are located Bac. anthracis, Bact. cuniculicida,
and Streptococcus lanceolatus (!); in another, Bact. typhi and Bae.
subtilis. This is contrary to all natural relationship !

The classification of the bacteriaceze by A. Fischer is logically con-
structed and clearly stated. He divides the bacteriaceze into not less
than four genera without and twelve with spores, which are differen-
tiated by the number and location of the flagella and also according to
the form of the rods containing spores. Because of the great varia-

bility of these properties, this too schematic classification has won few
friends. Many varieties can as well be placed in one genusas another.
We desist, therefore, from giving this method of classification.

Ill. Family Spirillacee Migula. Screw Bacteria.

Vegetative bodies are unicellular, sinuously or spirally
curved and twisted, more or less elongated; division
always at right angles to long axis; cells often united in

1 Even if Migula desired to classify the bacteriaceze according to

motility, the old names of Davaine —bacterium for motile and
_bacteridium for non-motile varieties—certainly demanded rehabili-
tation.

126 CLASSIFICATION OF FISSION-FUNGI.

short chains of a few links, very often in pairs; usually
actively motile because of flagella located at the ends.
Endospores known in only two varieties.

1. Cells short, slightly bent, rigid, comma-like, ay
times attached in a screw-like manner, always one (excep-
tionally two) flagellum at the end. According to Hiippe, |
they possess arthrospores. Vibrio! O, F. Miller, emend.
Loffler.

2. Cells long, spirally bent, like a conkaueane rigid, usu-
ally with a polar bunch of flagella formed of many long
principal and several short accessory ones. In the Spir.
sputigenum Miller the bunch of flagella is not at the end,
but on the side. Spirillum? Ehrenb., emend. Léffler.

3. Cells consist of flexible, long, spiral, coiling threads.
Flagella unknown. Locomotion by means of an undulat-—
ing membrane is suspected. Spirochzte Ehrenb. |

|
|

bt
—— wise”

In a strict sense the causes of glanders, diphtheria,
leprosy, and actinomycosis do not belong among the
fission-fungi. It is generally acknowledged to-day that
they must either be designated as fission-fungi, which form
a connecting-link to the higher fungi (hyphomyeetes), or

1 Migula, with Schroter, called the group which is now almost uni-—
versally designated as vibrio, microspira—a designation that is unnec-
essary if we accept the definition of vibrio suggested by Lofiler.
Moreover, Schroéter’s definition of spirillum and microspira does not
suit the known properties of the varieties therein included. For the
few non-motile (without flagella) rigid vibriones Migula has intro-—
duced the name Spirosoma Migula. !

2 Here belong such forms as the Spirillum endoparagogicum ©
Sor., described by Sorokin and which he once found in a hollow tree in ~
Kasan. This remarkable typically spiral-shaped organism formed
typical endospores, which germinate while still within the spirillum,
and so offer characteristic pictures (C. B. 1, 466). The organism ap-
pears to connect the spirillaceze and the bacilli. According to Praz-
mowski, the Vibrio rugula possesses a spore causing swelling of the
end where it is located. Spore-formation has not been described in~
other vibriones. We know nothing regarding the flagella of this
vibrio rugula, which resembles the Bac. cedematis maligni. Moreover,
Zettnow expressly contradicts the idea that the vibrio rugula forms
spores.

ACTINOMYCETES. 127

candidly as low leet hn rig 1 as was done for the first
time in the first edition of this book, in 1896. Kruse has
placed the actinomyces, together with its nearest relatives,
in a family of hyphomycetes, ‘‘streptotrichex,’’ while he
still speaks of a Bacillus tuberculosis, etc. Recently,
Lachner-Sandoval has introduced the name actinomycetes
to designate the group of ‘‘fission-fungi closely related to
the hyphomycetes’’ (as we had designated them in the
first edition), and until we have something better it
answers for practical purposes.

SUPPLEMENT I.

Actinomycetes (Lachner-Sandoval).

_ Delicately threaded organisms, free of chlorophyll, with
true branching, in part very abundantly ramifying myce-
lium, partly with the formation of conidia. Young cul-
tures often present only unbranched rods resembling bac-
teria, which can in no way be differentiated from ordinary
‘fission-fungi. According to many authors there is a ten-
dency to the formation of clubs. or knobs at the ends of
the threads.

1. Microscopic: Slender often somewhat bent rods, often
with a tendency to a clubbed swelling of the ends, branches
rarely observed in young cultures, easily broken off, and
often difficult to find also in old cultures. Always non-
motile; never conidia.

a. Rods stain interruptedly (striped) with weak stain-
‘ing-solutions, since the organism is composed of parts with
different staining properties. Not stained by the method

__ 4As hyphomycetes there have been designated for a long time in
botany a large number of threaded fungi, of which nothing is known
except threads and non-sexual spores that are upon threads or
special carriers. The group is constantly growing smaller, as many
earlier ‘‘hyphomycetes’’ have become known as members of the
sharply characterized groups of fungi (ascomycetes, zygomycetes,
_basidiomycetes). The actinomycetes appear to form an entire nat-
ural group of the ‘‘ hyphomycetes.’’

128 CLASSIFICATION OF FISSION-FUNGLI.

for the tubercle bacillus. Clubbed, wedge-shaped, and
pointed rods frequent. Corynebacterium L. and N.

&. Rods stain with usual staining-solutions with diffi-
culty or generally not at all. Stain by the tubercle bacillus
method, 7. ¢., it is acid resisting. Clubbed swelling of the
ends in cultures rare, in tissues somewhat more often.
Mycobacterium L. and N.!

2. Mycelial threads, long, thin, extended, or winding,
without dividing partitions, with delicate sheaths and true
branches. Many species separate from the air-hyphe
rows of short spores (conidia), which, whitish and mold-
like, project upward above the solid nutrient substratum;
in connection with other species, conidia-formation is
unknown. Not stained by tubercle bacillus method.
Motility sometimes absent, sometimes present. Almost
all varieties emit a musty odor. Actinomyces Harz.

We have determined to follow the example of Gasparini
and designate this genus as actinomyces. Streptothrix,
as these varieties, together with others, are called by
Kruse, is a name given by Corda in 1839 to a certain
mold-like organism which Cohn, because of an over-
sight, in 1875 introduced a second time into the literature.
Cladothrix, which many authors to-day call these varieties
(compare Giinther), is the designation for an entirely dif-
ferent pseudodichotomous plant (see Supplement 1m). In
the first edition we accepted, with Sauvageau and Radais,
the old designation of Wallroth, oospora, but since Lach-

1 Since we proposed this name in the first edition, we have seen that
Metschnikoff ( Virchow’s Archiv, 113, p. 70, 1888), who first recognized
the peculiar position of the tubercle bacillus as opposed to the other
then known bacteria, in a work ‘‘ Regarding the Phagocytic Réle of
the Tubercular Giant Cell,’’ has said: ‘‘ If one considers that in the
perfected stage the tubercle bacteria have grown into (although short)
threads, and also differ from other analogous forms (except the lepra
bacteria) in the possession of a very dense envelope, then perhaps it
will be easier to accept the designation Sclerothrix for the genus,
and Sclerothrix Kochii for the species of the tubercle bacterium.’’
We should have immediately accepted these names if we had known
of them, but believe that according to the rules of botanical nomen-
clature our names should now stand, since Metschnikoff only made a
conditional proposal, did not accurately define his new genus, and never
made any use of the new name himself, while we have ourselves
already established a name.

TRANSITION FORMS. 129

ner-Sandoval (Dissert. Strassburg, 1898) has convinced
us that the true oospora varieties are much larger although
similarly constructed organisms, we also, with this author,
consider the name actinomyces (Harz) at present the
most correct.

Some varieties of wide practical importance, closely
related to bacteria, but reminding one very strongly of
true algi (oscillaria), have been included under Supple-
~ment IT.

lf we cast a glance over this system, we can not deny
that the families and genera are often connected by tran-
sition varieties ; we recall only the following: The border
between the coccaceze and bacteriaceze is obliterated by
oval and lance-formed(!) cocci and certain extremely
short bacilli (compare, in the special part, Micr. meli-
tensis, Bacterium Fraenkelii); between streptococcus and
micrococcus, micrococcus and sarcina, it is often un-
safe to distinguish. In the cycle of growth of many
bacilli twisted forms occur ; flagella and endospores occur
in such various forms that it would lead to an entirely
unnatural grouping if the attempt were made to found
a classification that depended in part upon the flagella
or endospores.

The Bacterium Fraenkelii Hashimoto, for which we are indebted to
the kindness of the authors, unfortunately died before we could study
it. Upon solid nutrient media the organism forms short rods with
polar flagella; upon fluid media, on the contrary, it forms quite long
chains of cocci and occasionally sarcina forms. Thus it connects the
coccaceze with the bacteriaceze, as does the Micr. melitensis, and shows,
as we have indicated above in other examples, that sarcina forms occur

as growth forms in cocci and that the presence of flagella.is also vari-
able. (See Hashimoto, Z. H. xxxX1, 85.)

B. Systematic Description of the Most
Important Varieties of Fission-fungi.

INTRODUCTORY REMARKS TO THE SYSTEM-
ATIC PART, ABBREVIATIONS, ETC.

1. We have described about eighty species as completely and ex-
haustively as possible, several hundred are briefly described, and many
9 ;

130 IMPORTANT VARIETIES OF FISSION-FUNGI.

varieties which we are not acquainted with in detail are briefly referred
to in the connection where they belong.

2. The colonies, slightly magnified, are described and drawn with
closed diaphragm, and so placed that the peripheral portions are sharply
visible.

3. For the drawings and descriptions plates with a medium number
of colonies, 60 to 100, were always employed. Usually the smaller
colonies were selected.

4. All statements, unless otherwise qualified, regarding the growth
upon gelatin apply at a temperature of 22°, upon agar at 37°.

5. When nothing particular is said regarding the color and consist-
ency in the description of the agar streak culture, and of the surface
growth in the agar stab culture, they are the same as upon the agar
plate. ;

6. Regarding the formation of pigment, odoriferous, gustative,
and other metabolic products nothing has been said unless special
investigations have been made upon the same.

7. Our original purpose of treating exhaustively the resistance of
all important varieties to injurious influences has been abandoned as
being too far-reaching. This decision was also partially dependent
upon the fact that the statements of authors often deviate so widely.
Therefore we have restricted ourselves to making complete statements
regarding some varieties (Micr. pyogenes, Strept. pyogenes, Strept.
lanceolatus, Bac. anthracis, Bact. typhi, Corynebact. diphtheriz, My-
cobact. tuberculosis, Vibrio cholere).

8. References to the illustrations in the atlas are always
given thus: Plates with Arabic numerals, figures with Roman.
Thus, 5, VIII, signifies figure VIII in Plate 5.

The introductory remarks of the separate sections, coccaceze, bac-
teriaceze, spirillaceze, are also to be heeded.

Statement of the Terms Employed by Us in the
Description of Cultures of Bacteria.

I. STAB CULTURES.
A. Not liquefying.
1. Stab canal:
(a) Thread-like: Uniform growth without anything especially
characteristic.
(2) Smooth.
(8) Rough.
(b) Nodular: The stab canal is beset with larger or smaller tuber-
cles, points or teeth.
(ec) Hairy : The stab canal is beset with delicate longer or shorter un-
divided spurs, which are (a) parallel, (3) curled, (y) matted.
(@) Branched: The stab canal is beset with dividing outgrowths.
(e) Beaded: Thestab canal consists of small roundish or round con-
nected colonies.
(f) Band-like: Growth as a small band, produced by making the
stab canal with a loop.

re - “wy

DESCRIPTION OF CULTURES OF BACTERIA. 131

2. Surface growth:

Here the same applies as to the non-liquefying superficial colonies in
the plate. r

B. Liquefying.

(a) Fixed form of liquefaction, if the zone of liquefaction following
the stab becomes larger, but assumes substantially no other form than
at the beginning.

1. Tube shaped: Slowly, weak, and small.
2. Stocking shaped: Sack-shaped, rapid, strongly, at times with
scalloping of the walls.
3. Vesiculated: Bubbles are formed and confined in the depth.
(b) Variable form of liquefaction.
I. Initial stage:

1. Saucer shaped.

2. Funnel shaped.

3. Flattened funnel shaped.

1. Advanced stage:

1. Cylindrical: The liquefaction extends more above and soon
reaches the glass, and then extends, witha horizontal limit-
ing surface, downward.

2. Funnel shaped: The liquefaction extends more uniformly
from the culture. The funnel shape is preserved still in
later stages. Often the second form is succeeded by the
first.

Il. STREAK CULTURES.
A. Surface growth: The same designations apply as to the sur-
face cultures upon plates.
| B. Water of condensation.
a) Clear, with or without sediment. ,
b) Cloudy, with poorly defined sediment.
ce) Pellicle on surface.

Ill. BourLLon CULTURES.
A. Fluid:
(a) Clear.
b) Cloudy.
ce) Syrupy, gelatinous.
B. Sediment :
(a) Cloudy.
(b) Flocculent, if upon shaking it rises as a twisted column, and
can be homogeneously distributed.
(c) Sandy, if it lies steadily at the bottom and, upon shaking, is
distributed as small fragments.
IV. Porato CULTURES.
The same designations apply as to the streak and plate cultures.
_ Y. PLATE CULTURES.
A. Without liquefaction.
(a) Form: F
1. Point-like, when the dimensions are very slight.

132 IMPORTANT VARIETIES OF FISSION-FUNGI.

Round, circular.

. Oval.

POE WP

Curled, coiled.

(b) Elevation :
1. Flat.
2. Veil-like.
3. Wavy.
4. Reticulated.
5. Terraced.

(<) Optical peculiarity of surface :

1. Moistly shining, highest
degree of luster.

Greasy.

. Faintly shining.

Dull.

Consistency :

Veil-like.

Membranous.

. Leathery.

Tenacious.

mm 09 9

=
gq
wa

(e

Entire.
Rough.
Smooth.
Dentate.

. Lobulated.
Scalloped.

Internal structure :
Homogeneous (without
structure).
In zones.
Radially striped.
Radially wrinkled.
Finely dotted.
Coarsely dotted.
. Granular.
. Coarsely granular.

B. With liquefaction.

(a) Form:
1. Saucer-shaped depression.
2. Pocket-shaped depression.
(b) Appearance :
1. Liquefied medium clear—

GOT G9 30 eal ol a

=
DW Tw wR pA

. Roundish, not perfectly circular.

DID

DW AFAN

Dat ON

Whetstone shaped, pointed nt both ends.

. Elevated.

Nail-head.
Drop-like.

. Corniform.

. Finely granular.
. Transparent.
. Iridescent, pearly.

Opaque.
Chalky.

. Slimy.

. Cartilaginous.
. Friable.

. Butter-like.

Peculiarity of border, Snes i magnified with microscope :
ed.

. Short-haired.

. Long-haired.

. Curly.
. Matted.

. Finely lobulated, mulberry-

like.

. Coarsely lobulated, scaly.
. Irregularly spotted.

. Grained.

. Curly.

. Crumbly.

. Matted.

(a) With compact original colony.
(8) With original colony disintegrating.

2. Diffusely cloudy.

a’

F COCCACEZ. SPHERICAL BACTERIA. 138

f.

pepecial Introductory Remarks Concerning the Coc-
cacee. Spherical Bacteria.

1. Since almost all the varieties presented, with the

_ exception of the Micr. gonorrhcez, stain with the ordinary

anilin dyes and by Gram’s method, we usually state

nothing regarding the staining properties, except when
they can not be stained by Gram’s method.

2. When no mention is made of flagella and spores, they are
absent.

3. No mention is made of the intense stain with watery

_ solutions of anilin dyes, which occurs with all varieties,
and a similar statement would have to be always repeated.

_ It isstrongly recommended, when it is desirable to obtain

_ the cement substance between the bacterial cells unstained
(capsules), to employ a dilute aqueous solution of anilin dyes,
or after staining with stronger solutions to employ dilute
acetic acid as a decolorizing agent, or to use Gram’s
method. This is obligatory in the case of sarcine and
diplococci in order to render the line of fission in dividing
cocci visible, etc. (An exception is the gonococcus. )

4, Since all varieties of the genus micrococcus not infre-
quently occur as diplococci, tetrads, and short chains, we
have only said anything regarding the grouping when there
as something special to notice.

5. For an exhaustive discussion upon suppuration and
the part played by micro-organisms in the same, see Kurt
‘Miller, C. B. xv, 735, and Poliakoff, C.-B. xvin, 33.

FAMILY I—COCCACEAE. SPHERICAL
BACTERIA,

Family diagnosis and genera scheme, see page 122.

1. Streptococcus (Billroth).

The cells divide only in one direction of space at right
angles to the direction of growth, so that if the multiply-
ing cells remain attached to each other, shorter or longer
rosary-like chains are formed. Often the chain appears
to be be built up from distinct pairs. Chains are formed
with most constancy in bouillon ; upon gelatin and agar,

134 IMPORTANT VARIETIES OF FISSION-FUNGI.

as also in the animal body, very often no chains occur. It
is therefore always desirable to prepare bouillon cultures of
any variety in which there 1s a suspicion of a streptococcus be-
fore arriving at any conclusion. It is not unusual to find
single members in a streptococcus chain of somewhat
larger dimensions than the rest, but otherwise exactly re-
sembling the other members of the chain. It is, at least,
so far not certain that the cells contain arthrospores, as
many authors believe.

Key to the Recognition of the Most Important
Varieties of Streptococci.

I. Strings of cocci upon all nutrient media (also upon those con-
taining grape- and cane-sugar), without thick capsules; at most, with
delicate capsules. ;

(A) Do not grow as a yellow ‘‘creamy layer’’ upon sheep- and
calf-serum, and microscopically are without wide capsules.

(a) Cocci. spherical or, when dividing, hemispherical, capsules al-
most always absent.

1. Gelatin liquefied slowly or not at all. Cells 0.6 u to 1 wv; long
or short chains ; often thrive better anaerobically ; slight growth
on all nutrient media ; pathogenic or non-pathogenic. Strept.
pyogenes Rosenbach,’ page 135.

2. Gelatin rapidly liquefied in tube form; cells very minute (0.2 u
to 0.4 ~); forms long chains, and grows poorly upon potato,
agar, and serum. According to Escherich (‘‘ Die Darmbak-
terien des Sauglings,’’ Stuttgart, 1886, p. 77), it is constantly
present in the feces of carnivora. Not pathogenic for guinea-
pigs. Strept. coli gracilis Escherich. Strept. gracilis
(Escherich) Lehm. and Neum.

(8) Cocei more or less lance-shaped, capsules usually absent in arti-
ficial media, but never in animal body. Upon gelatin, poor growth
and no liquefaction. Strept. lanceolatus Gamaleia,? page 143

(B) Form a yellow creamy layer upon fluid sheep- and calf-serum.
Microscopically from these nutrient media they have wide unstained
capsules. Strept. involutus Kurth, page 149.

II. Chains of cocci upon grape- and cane-sugar nutrient media with
thick gelatinous capsules, which may be ten times as thick on all sides
as the chain of cocci. Upon other nutrient media it is not differ-

1 The streptococci scorn every exact method of division. That |

given here, while apparently a convenient and accurate scheme of
division, suffers very much in the closer description of varieties from
peculiarities, transition forms, etc.

2 Compare also Strept. intracellularis (Weichselbaum) Lehm. and
Neum., page 148.

STREPTOCOCCUS PYOGENES. 135

entiated microscopically from Group I. Strept. mesenterioides
Migula, page 150.

Streptococcus Pyogenes (Rosenbach’).
(Plate 1.)

Synonyms. — Strept. erysipelatos Fehleisen, Strept.
puerperalis Arloing, Strept. articulorum Fltigge, Strept.
pyogenes malignus Fliigge, Strept. septicus Nic, Strept.
scarlatinosus Klein (compare also pp. 140 and 141).

Ordinary Names.—Chain coccus, string of pearls coc-
cus.

Most Important Literature.—Rosenbach (‘‘ Mikro-
organismen bei den Wundinfektionskrankheiten des
Menschen,’’ 1884). Fehleisen (‘‘ Aetiologie des Erysi-
pels,’’ Berlin, 1883). v. Lingelsheim (Z. H. x, 331; xu,
308). Kurth (A. G. A. vu, 389). Behring (C. B. xu,
192). Knorr (Z. H. x1, 1893, 427). Pasquale (‘‘ Zieg-
ler’s Beitrage,’’ x11, 433,—extensive list of literature).
Marmorek (‘‘ Wiener med. Wochenschr.,’’ 1895, 1346).
Koch and Petruschky (Z. H. xxi, 477). Widal and
Besancon (C. B. xx, 240).

Microscopic Appearance. —The characteristic chain
growth presents itself especially in fluid cultures (bouil-
lon). Upon solid nutrient media and in the animal body
the chains are often very short or the arrangement ex-
tremely irregular (1, 1x, x).

Upon close observation of faintly stained preparations the individ-
uals of the chain usually consist of two hemispheres, which are con-
nected with each other and the adjacent member of the chain by a
colorless mass. More rarely definite mucoid capsules are seen about
the chains (compare Babés, Z. H. xx, 412).

Staining Properties.—As usual and well by Gram’s
method.

Relation to Oxygen.—Facultative anaerobe, some-
times better aerobically, sometimes better anaerobically.

Requirements as to Temperature and Nutrient

1 Since all efforts to divide the streptococcus pyogenes into several
sharply differentiated varieties must be recognized as failures, because
connecting transition forms between the subvarieties occur, so we
shall treat the variety as a unit, and at the end will add something
regarding its forms.

136 IMPORTANT VARIETIES OF FISSION-FUNGI.

Media.—Growth rather slow, best at 37°. Above 47° no
growth (Arloing).

They also grow more slowly but more luxuriantly upon feebly acid
nutrient media (hydrochloric acid, tartaric acid). Grow more slowly
but with greater vitality at 23° than at 37°. Especially good growth
occurs in exhausted cholera or pyocyaneum bouillon either after or
without filtration (Turré, C. B. xvii, 865).

Gelatin Plates. — (a) Natural size: Very small,
whitish, roundish, flat, rarely slightly elevated colonies,
which do not grow perceptibly after a longer time (1, v).

(6) Magnified fifty times. Superficial: Roundish colonies
with smooth border (1, vu e), but may present also
wavy, scalloped, serrated, as well as fringed and torn
forms (1, v1 e). Color is gray to yellowish, structure
delicately punctate to finely granular, usually transparent.
Deep lying: Roundish to whetstone-shaped, rough or
smooth border, somewhat more coarsely punctate than the
superficial (1, vit i; vii).

Gelatin Stab.—Stab: At first thread-shaped ; after a
short time there appear numerous small nodules in the
stab (1,11). Surface growth is like that in the gelatin
plate. ?

Gelatin Streak.—Narrow, beautiful, delicate growth
along the streak, beset at the borders with little nodules.

Agar Plates.—(a) Natural size: As on gelatin plates.

(b) Magnified fifty times. Superficial: Spherical colonies
with delicately punctate edge, transparent, grayish-yellow,
at first very delicately punctated, later (fourteen days) at
times granular ; frequently there is a distinct appearance
of lobulation (1, vite). Deep lying: Smaller and some-
what darker (1, vim i).

Glycerin-ascites-agar.—Colonies distinctly more lux-
uriant. From the periphery of the superficial colonies
there often extend outward numerous shorter or longer coil-
ing chains, so that the colony appears not unlike a young
anthrax colony. Also, the granulation in the interior of
the colonies is somewhat more marked than upon agar.

1Liquefaction, according to German authors, is very rare. Pane
saw the Strept. pyogenes from human abscesses at a temperature above
24° produce regularly liquefaction of gelatin which he had so pre-
pared artificially that it was first melted at 30° (C. B. xvI, 228).

|

&§ STREPTOCOCCUS PYOGENES. 137

|

Agar Stab.—Stab: Thread-like, later sometimes gran-

ular (1, m1). Surface growth: Very delicate growth,
transparent, gray, irregular, unimportant. Atypically,
the growth may be much more vigorous, with whitish-

gray color and smooth wavy border (1, Iv). Similar also
on glycerin agar.

Agar Streak.—As on gelatin. Water of condensation:
Clear with slight whitish deposit.

Bouillon Culture.—Varies greatly in the different .
forms, from diffuse cloudiness to the formation of a com-
pact sediment with clear fluid (see p. 141).

Milk Culture.—Usually firmly coagulated in from four

to twenty-four hours.

Potato Culture.—Invisible growth, at times entirely

absent, rarely more luxuriant (compare p. 141).

Non-albuminous Medium.—Faint growth.
Vitality.—/n cultures usually only a few weeks. Ac-
cording to Petruschky, cultures on gelatin, grown for

_ forty-eight hours at 22°, if kept in an ice-box retain their

vitality and virulence for months. The Strept. pyogenes

_ belongs among the varieties that die quickly. Bouillon
- cultures, if oxygen is admitted, usually live only for weeks,
but in hydrogen for months.

Resistance to Drying.—Vitality and virulence are
retained several months, especially in dried pus.
Chemical Activities.— (a) Chromogenesis: Almost

| always without pigment production ; cultures were grown

by Kruse and Pasquale in Italy with yellowish-brown to

blood-red pigment. These were highly virulent, short-

chained forms obtained from cases of tuberculosis.

(6) No indol, little sulphuretted hydrogen.

(c) Acid production from carbohydrates in our cultures
was minimal ; no gas formation.

According to Sieber-Schoumoff, certain cultures (Strept. erysipelatos
and Strept. scarlatinz) produce levorotatory lactic acid, others (Strept.
pyogenes) inactive lactic acid from grape- and milk-sugar. All cul-
tures produce, besides, some volatile fatty acids, poisonous albumoses,

_ and of gases only carbonic acid, with the exception of the form found

+.)

in scarlatina, which also produces hydrogen.

Emmerling’s investigations (C. B. L. rv, 342) regarding the decom-
position of fibrin by streptococci under anaerobic conditions gave the
remarkable result that a solution of fibrin was effected. He found

1388 IMPORTANT VARIETIES OF FISSION-FUNGL

succinic, acetic, propionic, normal butyric, and caproic acid, methyl-
amin, trimethylamin, collidin, but no toxins. .

(d) Toxin production: Upon albuminous nutrient media
streptococci produce toxins, soluble in water and precip-
itated by alcohol. To collect them the cultures are killed
with chloroform or filtered through porcelain. Large
doses of the metabolic products cause suppuration and
fever, and even death. This appears beet to be only
the action of protein.

Occurrence.—(a) Outside the body: In soil, canal-
water, once in a well (Landmann, C. B. xty, 431), in
the air of operating rooms, ete.

(6) In the healthy body: In mouth, nasal cavities,
vagina, not rarely cervix uteri; at times, moreover, in a
virulent form.

(c) In diseased human organism: The streptococcus is
capable of causing a large number of diseases, namely, in-
flammation and suppuration in all parts of the body. It
causes especially often the following diseases : Erysipelas,
phlegmonous abscess,! lymphangitis, follicular angina,
bronchitis, impetigo contagiosa, cellular pneumonia (Fink-
ler), pyemia, septicemia, and puerperal fever. More
rarely, pleuritis, pericarditis, meningitis, enteritis,? ete.,
some cases of osteomyelitis, elephantiasis nostras (Sab-—
ouraud ).

Recently, Escherich with his pupils has emphasized the significance
of the streptococci in the diarrheas of children. The form isolated —
from such cases can not be imagined as a new sharply defined variety,
in spite of slight deviations, but belongs in the division of the Strept.
pyogenes or lanceolatus. Escherich, Th., ‘‘ Ueber Streptokokkenen-
teritis im Siiuglingsalter.’’ Separatabdruck aus Jahrbuch f. Kinder-
heilkunde, N. F., Bd. xLrx, 1899.

It is found in the blood and urine rather often, either —
with or without symptoms of a general disease.

The following also certainly depend upon Strept. pyo-

1In phlegmons and abscesses more often the staphylococcus (Micr.
pyogenes) is present, or a mixture of both.

2In the institute for infectious diseases in Berlin, Beck described a
case of streptococcus infection (intestine, blood, viscera) that caused
death in three days and presented during its course the typical picture
of Asiatic cholera (C. B. x1, 632). Compare Tavel, de Cérenville, —
ete. (C. B. xviil, 547).

be

|

i

‘genes infection : Some cases of nephritis, articular rheu-
matism, myelitis, and infantile paralysis. Mannaberg has

found it in fourteen cases of Bright’s disease (C. B. v, 93),
whether as primary cause is questionable.

The streptococcus plays an important réle in diphtheria,
searlatina, and phthisis. It accompanies the specific cause
of disease, and markedly influences the disease-picture,
especially the course of the fever (hectic fever is strepto-

- coccus fever) (Petruschky, Z. H. xvu, 59).

(d) In animals: As the cause of similar diseases (com-
pare, for example, Strept. equi, p. 142).

In the vaccine of cow-pox institutes it is not uncommon,
but usually possesses little virulence.

_ Experimental Observations Regarding Pathogenic
Action.—With living cultures. The virulence fluctu-
ates greatly; even freshly isolated organisms may be very
faintly virulent, and virulence for experimental animals
does not prove virulence for man ; with cultivation upon
the ordinary nutrient media the virulence is rapidly lost.
By repeated transmission through animals, a virulence
which was high at first may be much intensified. Mar-
morek obtained cultures of such virulence that 5,455 ¢.mm.
killed almost all, and jz 5455 ¢.mm. some, mice when
given subcutaneously—. e., quantities that contain only
relatively few germs.

_ The virulence is well preserved, according to Marmorek,
upon (1) two parts of human or horse serum and one part
of bouillon ; (2) one part of fluid from ascites or pleural
exudate and two parts of bouillon, even after keeping two

months in the incubator without transfer to fresh nutrient
media.

In general the most susceptible to the streptococcus
among animals are mice and rabbits; much less, dogs and
rats (Pansini). Streptococci are still better tolerated by
sheep and goats, and best by the horse and ass.

Knorr has ascertained the following principal points re-
garding the virulence: By repeated transmission through
mice an organism is obtained which is very pathogenic for
mice, but at the same time its virulence for rabbits was
gradually lost. This is a strong indication that one must
not found any species upon a specific virulence. The more

STREPTOCOCCUS PYOGENES. 139

140 IMPORTANT VARIETIES OF FISSION-FUNGI.

virulent a form is for a variety of animal, the more cer-
tainly it kills without suppuration, the latter being caused

only by feebly virulent forms.

Almost all the diseases enumerated above may be pro-
duced experimentally in animals; the result in experi-—

mental animals depends very largely upon the virulence
and amount of infectious material.

Also in man streptococci have been successfully inocu-

lated (erysipelas, phlegmon).

Immunity and Immunization.—If an animal resists an injection
of the metabolic products, and has after a time recovered from the
consecutive cachexia and loss of weight, then the dose may be increased

and gradually a high degree of immunity be obtained. Yet the state-_
ments of Marmorek are contested, when he claims that horses and asses —

may thus supply a serum which cures human sepsis (Petruschky,

Schenk). At any rate it has been shown, according to the investiga-—
tions of Denys and his pupils (C. B. xxtIv, 685), that the individual
_ varieties of streptococci yield a serum that is active only against the ~
particular variety employed in producing the immunity ; thus also
animals, in order to yield serum of therapeutic value, are to be treated —
with the most variable cultures possible of streptococci (‘‘ polyvalent —

serum ’’). Regarding the way in which the serum acts, compare page
97.

Special Methods for Demonstration.—Microscopic

form and staining by Gram’s method; agar plate in ineu-

bator; bouillon culture to obtain chains; animal inves-

tigation (mouse).

Forms and Subvarieties of the Strept. Pyogenes.

All efforts of authors to characterize sharply the forms 4

of the Strept. pyogenes as varieties, subvarieties, or

species, and to cover them with names are to be considered —
as failures. Countless transition forms and the enormous —
variability of all the properties make every classification —

appear insufficient. Even the separation from the Strept.
lanceolatus is not always possible. Pasquale (Ziegler’s
Beitriige, xm, 433), Lemoine (H. R., 1896, 892), Widal
and Besancon (H. R., 1896, 996), and Petruschky (H. R.,

1897, 77 2) have all come to analogous results from their -

minute studies.

Behring and his pupil v. Lingelsheim arrived at the

following useful? division:
1 There are found by many authors a ‘‘Strept. brevis’? without

ih i

; STREPTOCOCCUS PYOGENES. 141
- (a) In bouillon form short, slightly tortuous chains;
‘bouillon cloudy; gelatin is very slightly liquefied; signifi-
cant growth upon potato; growth even at 10° to 12°.
Virulence usually absent. Strept. brevis v. Lingels-
heim.
(6) In bouillon the streptococci form very tortuous,
long chains (forty and more members), which make up a
flocculent or slimy sediment, leaving the bouillon clear.
Gelatin always remains solid; visible growth on potato is
absent, virulence is usually great. No growth below 14°
to 16°. Strept. longus v. Lingelsheim.

The subdivision of the Strept. longus into the following varieties

_ (Behring) has now only a historical interest, since according to
Behring’s pupil, Knorr, the characteristics of these subvarieties, upon

repeated cultures, are variable, and so the identity of these subvarieties

ean be demonstrated: (1) Turbidus, with turbid bouillon culture;

tie

(2) viscosus, with clear bouillon culture and delicate sediment;

(3) conglomeratus, with clear bouillon and granular sediment.
The same was also found by Kruse and Pasquale (Ziegler’s Beitrage,
Xi, 1893, 433). Interesting but unsatisfactory is also Pasquale’s at-
tempt at a classification of streptococci (C. B. xv, 761). Also Babés

_ came to little sharp differentiation; for him, as for us, all forms (in-
_ eluding the Strept. lanceolatus) are connected by transition forms.

The findings of Waldvogel are interesting. Three times he ob-

tained, after inoculation with Strept. longus (the bouillon remained
_ clear and there was an insignificant granular sediment), from the heart’s |
_ blood of the inoculated mouse an organism forming chains with from

oe ee

four to six members, and causing a diffuse cloudiness of bouillon.
Upon potato both forms grew equally poorly. By growth in strongly
alkaline bouillon the long chain form could be transformed into one
producing a slizht diffuse cloudiness; and by growth in almost neutral
bouillon of the form producing turbidity a race was again obtained

_ which produced no flocculi in clear fluid and formed long chains.

- After such experiences more recent authors do not make

a division of the Strept. pyogenes into different forms, and

.: CO

prefer to designate the forms described by them as Strept.
pyogenes, the form being described in a few words. We
also believe this to be right. Compare also Zenoni, C. B.
xxi, 10, and the interesting studies of Seitz concerning

gelatin liquefaction, and a Strept. longus with slight liquefaction;

_ also occasionally a Strept. longus with a visible and a Strept. brevis

without a growth on potato. Marignac and d’Espine found Strept.

_ brevis which formed sediments in bouillon and did not cloud it.

Marbaix proved complete independence of the length of the chains

- and pathogenic quality.

142 IMPORTANT VARIETIES OF FISSION-FUNGI.

maststreptococci (Strept. aggregatus Seitz; C. B. xx,
854) from the mouth, which with their very marked vari-
ability still always belong in the group of the Strept. pyo-
genes.

Streptococcus equi (Kitt). Drusestreptococcus
(Schitz).

All the morphologic characteristics agree throughout with the
Strept. pyogenes, also the pathogenic effect fluctuates asin it. Details
concerning it by Cappelletti and Vivaldi (A. H. xxxiy,1). Also
in horses, as in man, streptococci cause pneumonias, the organisms
resembling sometimes the Strept. pyogenes, more often the Strept.
lanceolatus (compare Ligniéres-Alfort, C. B. xxi, 768). ‘* Druse ”’
(French ‘‘ gourme’’) is an inflammation of the upper air passages in
horses, with inflammatory disease of the adjacent lymph-glands, in
which not rarely abscesses form. The differentiation between glanders
and this disease is easy by microscopic examination and the positive
results of inoculation of house mice (Schiitz, C. B. v, 44).

Streptococcus agalactie (Adametz) — Strept. masti-
tidis sporadicz Guill., Strept. mast. epidemicze
Guill., Galtcoccus.

Morphologically sometimes a short, sometimes a long-chained Strept.
pyogenes. Cause of the ‘‘ gelbe Galt,’’ a sporadic or epidemic inflam-
mation of the udder of cows and goats. The milk becomes very scanty,
yellowish, beset with flocculent coagula and often gas-bubbles. The
form producing long chains is more virulent than the one occurring in
short chains. It is important that many cultures break up grape- and
milk-sugar energetically with gas-formation, according to Nencki, es-
pecially with the formation of dextrorotatory paralactic acid and car-
bonic acid (no hydrogen), traces of fatty acids, and alcohol. This fer-
mentation of the milk causes a low grade of cheese (inflated cheese).
The virulence and ability to cause fermentation vary in this organism
very much. (Compare Adametz, ‘‘ Milchzeitung,’’ 1893, and Zschokke,
C. B. xxii, 784.)

The Micr. acidi paralactici Nencki (C. B. vu, 130) and Strept.
acidi lactici Grotenfeldt (‘‘ Fortschritte der Medizin,’’ vi, 121) ap-
pear to be closely related; the latter forms no gas and thrives especially
anaerobically. Also similarly the Micr. Sornthalii Adametz (C. B. L.
1, 465), an organism fermentirg milk with intense production of gas
(CO, and H) and causing inflation of cheese, which in its cultural be-
havior upon gelatin plates reminds one somewhat of the Strept pyo-

genes. Instab cultures the growth is somewhat more profuse. Micro-

scopically, it is a round or oval coccus, either single or in short chains.
Kronig has described varieties of anaerobic non-pathogenic strepto-

Peery ae a ewe

STREPTOCOCCUS LANCEOLATUS. 1438

Henrici, which were not examined as to their effects upon sugar, milk,

potato, and animals (A. K. B., Heft 1, 1); and Strept. tyrogenus, albi-
us, magnus, granulaius, pallens, pallidus, Henrici,! which are only
differentiated by characteristics that are not very pronounced and are
ill to be tested as to their constancy (more or less granulation in
the plate cultures, character of cloudiness in bouillon, slightly
‘different adaptability to aerobic and anaerobic life). The Strept.
‘Stramineus Henrici, which grows as a straw-yellow, shining deposit,
“appears to differ more strongly.

‘Streptococcus lanceolatus? (Gamaleia). (A. P., 1888,
e ii, 440.)
4 (Plate 2.)

_ Synonyms.—Diplococcus pneumonie A. Frankel and
Weichselbaum, Dipl. of sputum septicemia A. Frinkel,
Meningococcus Foa, Pneumococcus Foa, Dipl. lanceolatus
‘sive lanceolatus capsulatus Foa and Bordoni-Uffreduzzi,
Bact pneumoniz Migula, Micr. pyogenes tenuis Rosenbach
aC. B. vu, 177).

_ Ordinary Names.—Capsule coccus of pneumonia,
‘pneumococcus, Frinkel’s pneumonia coccus.
Literature.—Exhaustive critical studies by Kruse and Pansini

‘(Z. H. xt, 279), Levy and Steinmetz (Arch. exp. Path., 1896, 89).
Literature by Schabad (C. B. xrx, 991).

Microscopic Appearance.—Arranged usually in pairs
or chains of from four to six members, roundish or—what
is especially characteristic—lancet-shaped (2, x). When
obtained from the animal body or when cultivated upon
sterilized sputum and tracheal mucus, or in fluid rabbit’s
serum, it usually presents a significant capsule, which may
be stained (p. 22, Fig. 5) (2, rx).

1 Here also belongs the Strept. cinereus Zimmermann (Bd. 11, 64),

‘obtained from tap-water, which is said to present somewhat more
prominent cultures on gelatin plates.

_ #Since the name Strept. pneumoniz is applied by Weichselbaum to
a Strept. pyogenes from cases of pneumonia, it would lead to con-
fusion if, following the rules of strictly botanical nomenclature, the
Dipl. pneumoniz was renamed simply the Strept. pneumonie. On
the contrary, the name Strept. lanceolatus is also very old (1888),
characteristic, and unmistakable,

i

144 IMPORTANT VARIETIES OF FISSION-FUNGI

Often single members present larger dimensions and the -
form of a club—. ¢., to a large sphere is attached a small,
thin neck-piece. These are not, however, resting forms.
(Compare Stolz, C. B. xxiv, 337, )

According to Kruse and Pangini and our own investiga-
tions, all transitions up to the Strept. pyogenes occur, so-
far as concerns the form of individuals and the structure
of chains. (Compare also Binaghi, ‘‘ Ueber einen Strept.
capsulatus,’ C. B. xxm, 278.)

Relation to Oxygen. —Facultative ahaereble

Intensity of Growth.—Grows fairly rapidly but not
luxuriantly at 37°. At ordinary temperature (22°) 7
slowly, and more often not at all.

Gelatin Plates.—(a) Natural size. Superficial: Round-
ish, dim, diffusely gray, transparent colonies, which after
four days have attained a diameter of from 1 mm. to 2mm,
Deep: Very small, roundish, whitish-gray (2, v). 3

(b) Magnified seventy times. Superficial: Circular or
roundish colonies with almost smooth border, colorless,
and delicately granular. They are often so delicate that,
with the narrowest diaphragm the periphery can hardly
be differentiated from the surrounding medium (2, vmI, e).
Deep: Round, sharply outlined, slightly more granular’
(2, vIu, 2). P

Gelatin Stab Culture.—Stab: At first thread- like, ee
resembling a string of pearls; growth faint. Superficial”
growth: Minimal, almost none (2,1). No liquefaction. —

Agar Plates. (a) Natural size: Like gelatin plates:
(2, v).

(b) Magnified fifty times. Superficial: Roundish, stall
even border, at times somewhat fringed, delicately
punctate, a little more compact than the gelatin culture,
colorless, perfectly transparent (2, v1). Deep: Roundish
or whetstone- shaped, almost even-bordered, opaque, gray

to grayish-black, more coarsely punctate than the super-
ficial (2, viz).
Agar Stab.—Stab: Thread-like, whitish-gray (2, mr).
* MacCallum and Hastings have described a liquefying form (analo-

gous to certain rare varieties of the Strept. pyogenes) as Micr. zymo-~
genes (C. B. xxv, 384).

:

mail

See one eT

STREPTOCOCCUS LANCEOLATUS. 145

ee

Surface growth: Very delicate, transparent growth, with
even border, faintly glistening (2, Iv).

_ Agar Streak.—Extremely delicate, transparent, gray-
ish-white, faintly glistening, often not sharply outlined
from the agar. Water of condensation clear, with very
little whitish sediment (2, 11).

Serum Culture.—Slimy, almost transparent growth.

Ascites-glycerin-agar.— More luxuriant cultures.
Those lying superficially are usually even-bordered, the
periphery somewhat padded, and throughout (especially

_ inold colonies) coarsely punctated to mulberry-like. They
then resemble old gonorrhea cultures or at times even very
young agar cultures of the colon bacillus.
Bouillon Culture.—Short, straight chains ; sediment
light and not holding together (Kurth).

Milk Culture.—Milk coagulated. This property, ac-
cording to Kruse and Pansini, is very rarely absent. In
the milk small amounts of acid are formed.

Potato Culture.—WNo growth.

_ Vitality in Cultures.—Very short duration of life

(often only a few days), and even a more rapid lessening
of virulence. In bouillon occurs the most luxuriant growth,
but it is least durable.

Resistance to Drying.—In dried blood as long as
forty-five days; in dried sputum as long as one hundred
and twenty to one hundred and forty days in diffuse light,

and nine to twelve hours in direct sunlight. Literature,
Germano, Z. H. xxv, 66.

Chemical Activities.—Fawitzky isolated three cul-
tures, which were able to produce a brick-red pigment
(best in bouillon). (Compare Strept. pyogenes.) Fil-
tered and devitalized unfiltered cultures contain toxins,
but in relatively small amount. In other respects it is
like the Strept. pyogenes.

Occurrence.—(a) Outside the organism: Not found.

(>) In healthy organisms: Often in saliva.

(c) In diseased human organism: One of the most im-
portant pathogenic varieties. In the most various inflamma-
tory processes, especially such as attack mucous and serous

_membranes, also not infrequently causing suppuration.
Especially frequent as the cause of croupous and catarrhal
10

146 IMPORTANT VARIETIES OF FISSION-FUNGI.

pneumonia, pleuritis, pericarditis, endocarditis, peritonitis,
otitis, meningitis, conjunctivitis, and ulcus serpens cornez.
More rarely as the cause of nephritis and perinephritis,

metritis, pyosalpinx, strumitis, parotitis, amygdalitis, —

arthritis,’ osteomyelitis, periostitis, abscesses, and general

sepsis. It may also cause erysipelas (Schiirmayer, C. B. —

xxi, 183). In many of these diseases the organism is
found not only locally, but also in the blood. Very often

other exciters of inflammation accompany and aid the

Strept. lanceolatus, which is always more difficult to cul-
tivate, so that if ordinary agar is employed for cultures,
staphylococci, etc., may alone be observed. Therefore
ascites-agar and similar media are to be preferred. The
Strept. lanceolatus escapes from the diseased person in the
milk and urine. 3

Regarding the participation of the Strept. lanceolatus in cerebro-

spinal meningitis, see under Strept. intracellularis, page 148.
Marchoux (A. P. XIII, 193) repeatedly found in soldiers, as a sequel
to pneumonia, a tendency to sleep (‘‘Schlafsucht,’’ maladie du som-

meil), and upon section there were changes in the cerebrospinal mem- —

branes with the Strept. lanceolatus present.

Experimental Observations Concerning Patho-

genic Effects.”—(a) In animals: Of animals, the rabbit —

and mouse are especially susceptible, the rat less so, and
guinea-pigs, sheep, dogs, and birds almost not at all.

The mouse dies in from twelve to twenty-four hours after subeutan-
eous infection of septicemia; spleen enlarged, eyelids glued together.
In the blood are large numbers of diplococci. In mice pneumonia
also can be produced by inhalation. Likewise in rabbits septicemia
with fever and swelling of the-spleen follows subcutaneous and more
rapidly intravenous inoculation with strongly virulent cultures; death

follows in forty-eight, twenty-four, twelve, or even five hours. At- —

tenuated cultures cause, according to the point of inoculation, pneu-
monia, pleuritis, peritonitis, ete. Honl especially recommends for

1 Here also belongs the excitant of chronic deforming inflamma-
tion of joints, described by v. Dungern and Schneider (Minch. med.
Wochenschr., 1898, No. 43, 1369).

2 The virulence is exceedingly variable and in the usual cultures it
is rapidly lost. For the preservation of the virulence of the Strept.
lanceolatus during about two months it was recommended, for ex-
ample, by Bordoni-Uffreduzzi to dry upon glass the blood of rabbits
which the infection had killed. Foa places such blood for twenty-four
hours in the incubator, and then preserves it in the cold.

vi ay pitta

toy”
¥
q 5
a
Py
¥
ri

STREPTOCOCCUS LANCEOLATUS. 147

diagnosis and for demonstration purposes the subcutaneous injection
of sputum in the rabbit’s ear; death follows after two to five days,
and the bacteria are found especially numerous and with typical cap-
sules in the edematous fluid, which is obtained by incision of the
doughy infiltration over the lower jaw (C. B. XXII, 274).

(6) In man: Subcutaneous injection of from 0.1 to
0.2 c.c. of virulent culture in seven men was without
important effect except local symptoms, some fever, and
headache.

Immunity and Immunization.—Mennes, whose care-
ful work (Z. H. xxv, 413) should be consulted in the origi-
nal, has recently obtained fairly active protective serum.
The action of the serum consisted in this, that it renders
the leukocytes of normal animals capable of devouring the
Strept. lanceolatus (phagocytosis). Encouraged by the
investigations upon animals (Emmerich and Fawitzky,
Foa, Klemperer), curative injections of the metabolic prod-
ucts and the serum of immunized animals have been
tried also upon man, but so far without indisputable prac-
tical results.

Special Culture Methods.—The Strept. lanceolatus is
most easily obtained by inoculating a mouse or rabbit
with fresh rusty sputum from croupous pneumonia, and
making cultures from the heart’s blood of the dead animal
upon ascites-agar plates. It is also often easily obtained
from an eye with ulcus serpens cornee by the preparation
of streak or plate cultures upon ascites-agar, and placing
them in the incubator.

Forms and Subvarieties of the Strept. lanceolatus.

We must frankly admit that a sharp separation of the
Strept. pyogenes from the Strept. lanceolatus seems to us,
as to many authors, to be impossible, if the typical form
of the Strept. lanceolatus is to be determined by capsules,
lancet-shaped individuals, and a tendency to form only
very short chains. Many investigators who have espe-
cially studied the Strept. lanceolatus have tried to set up
definite forms, which can scarcely be identified subse-
quently. Almost all of these divisions have consisted of
somewhat differently defined varieties, as is the case with
the Strept. pyogenes. (Compare Kruse and Pansini, Z. H.

148 IMPORTANT VARIETIES OF FISSION-FUNGI.

x1, 279; Pansini, Virchow’s Archiv, cxxm, 424; Banti,
C. B. 1x, 275; Foa.)

Streptococcus intracellularis. ( Weichselbaum. )
Lehm. and Neum.

(Plate 68, 111, Iv.)

Synonym.—Diplococcus intracellularis meningitidis Weichselbaum.

Literature.—Jager (Z. H. XIX, 351); Weichselbaum ( ‘‘ Fortschritte
der Medizin,’’ 1887, v, 573). Recent literature is comprehensively
reviewed by Kamen (C. B. xxiv, 545); in the latter place (as also in
the article by Jager) are found illustrations.

While a number of authors—for example, Bordoni-Uffreduzzi and
Foa; Paniénski (C. B. xvii, 651); Henke (C. B. x x11, 59)—have found
the Strept. lanceolatus to be the cause of cerebrospinal meningitis, and
others—for example, Bonome—have found the Strept. meningitidis
Bonome, which is closely related to the Strept. pyogenes, to be the
cause, still other authors, and especially Jager, have described as the
exciting agent, an organism which is indeed very closely related to the
Strept. lanceolatus, but which, it is said, can be clearly separated from
it. The statements of different authors diverge very widely as re-
gards details.

The cultures are often indistinguishable morphologically from the
Strept. lanceolatus, but they remain alive and capable of transplanta-
tion for a longer time (seventeen to forty-three days). Some authors
found growth to occur upon potato ; many obtained even strikingly
luxuriant, moist, yellowish-gray cultures upon glycerin-agar, resem-
bling the Micr. tetragenus (68, 111 and Iv) (Mayer, Munch. med.
Wochenschr., 1898, 1111), and we received such cultures from Jager
in December, 1896. C. Frankel cultivated, on the contrary, an ex-
ceedingly delicate growth, which only grew with certainty upon agar
smeared with blood. Such a culture we obtained from Kral.

The following points are asserted to be of diagnostic value: The
organisms, sometimes as diplococci and tetrads, sometimes as short
chains, lie oftentimes in groups within the pus-cells, especially also within
the cell nuclei. 'They possess more or less distinct capsules. Accord-
ing to the beautiful investigations of v. Hibler, the most variable path-
ogenic cocci and bacilli are found in the cells, so that this property is
not at all characteristic (C. B. x1x, 33). In smears from the pus and
from cultures, they sometimes stain well by Gram’s method, but more
often poorly, and in sections are not stained (alleged contrast to the
Strept. pyogenes and lanceolatus). In the chain form of the organism
it is said to be characteristic (Jager) that the individuals are so ar-
ranged that the line separating the diplococci extends in the direction
of the chain. But as Stolz has pointed out, exactly similar pictures
occur in typical Strept. lanceolatus and Strept. pyogenes (C. B. XXIv,
337). We found such pictures exquisitely shown in a streptococcus
growing in a putrid mixture. With this state of affairs it is difficult
to consider the Strept. intracellularis a single organism, since the forms

ee ee

ee.

STREPTOCOCCUS INVOLUTUS. 149

which at times resemble the Micr. gonorrhee, at times the Strept.
lanceolatus and pyogenes, and at times the Micr. tetragenus lack a
common chara« teristic. +

The organism is said tobe found only in the meningeal pus, nasal
mucus, sputum, and urine of men who are affected with epidemic
cerebrospinal meningitis. Recently, A. Schiff claims to have isolated
it from the nose of patients without meningitis (C. B. xxv, 437). C.
Frankel cultivated it from eyes apparently affected with diphtheria
(Z. H. xxxi, 221). Together with the Strept. intracellularis
there occur mixed infections by the Strept. pyogenes and Strept.
lanceolatus. Certainly at least a considerable portion of the cases of
cerebrospinal meningitis are caused by the Strept. lanceolatus alone.

Regarding the cerebrospinal meningitis of domestic animals, conflicting
statements are also encountered; here also it is possible that different
related infectious agents take part in the main epidemics. (Consult
Siedamgrotzky and Schlegel, C. B. xx, 694, and Schneidemuhl, C. B.
XXIII, 892.) It is interesting that Johne found in an epidemic disease
of horses an organism which Jager declared identical with the Strept.
intracellularis. The organism was pathogenic for guinea-pigs, horses,
and goats. (Consult Councilman, Mallory, and Wright, Amer. Jour.
of Med. Sciences, March, 1898. —Ep. )

Fig. 13.—Strept. involutus (from a photograph by Kurth); partly
schematic.

Streptococcus involutus (Kurth).

Bsn —Streptococcus of foot-and-mouth disease.

Literature by Kurth (A. G. A., Bd. vir, 1893, 439-465).

Upon gelatin, etc., indistinguishable from the Strept. pyogenes; on
the contrary, bouillon which is rendered diffusely cloudy by some cul-
tures, and only presents a sediment with others, often contains cells
of strikingly elongated, vesicular, spindle form. No spontaneous
motion.

Two especially striking characteristics are present in serum or serum
mixtures:

1. In fluid serwm or serum bouillon there develops in the upper
part of the tube a pale-yellow creamy layer, which upon microscopic
examination reminds one at first of anything else rather than micro-
organisms, but on further examination the following is learned:

1 The following is said to be characteristic: Agar cultures after five to
six transfers cease to grow, and the organism generally does not grow
on cow’s serum.

150 IMPORTANT VARIETIES OF FISSION-FUNGT.

The luxuriant, waxy, shining masses consist of dense zooglea of

streptococci which are surrounded by very extensive, enormously
swollen capsules, which do not stain with anilin dyes. The capsule
formation occurs best upon calves’ serum, but is also seen upon sheep’s
serum.

2. Upon plates prepared with 10 c.c. of agar, and 2 ¢.c. of serum,! both
warmed to 40°, there is found about each of the small pure growths a —
halo of strongly refracting granules, which are doubtless composed of
the same material as forms the capsules of the single cells. How these
spheres originate and whence they arise, Kurth is unable to say.

Kurth had already stated that the Micr. pyogenes and Micr. tetra-

genus can furnish similar pictures upon calves’ but not upon sheep’s —

serum. He found later that also streptococci not connected with —
foot-and-mouth disease, although rarely, furnish similar serum cul-
tures. The organism has nothing to do with foot-and-mouth disease
(see Appendix).

Streptococcus Mesenterioides (Cienkowski) Migula.

Synonym.—Leuconostoc mesenterioides Cienkowski.
Ordinary Name.—Frog-spawn fungus of sugar factories.

Literature.—Zopf and Liesenberg, ‘‘ Beitrage zur Physiol. uw.
Morph. niederer Organismen,’’ Heft. 1, Leipzig, 1892 ; C. B. x11, 659.

The organism grows upon nutrient media free of grape-
and cane-sugar, like the Strept. pyogenes ? microscopically

Fig. 14.—Strept. mesenterioides (after Zopf).

and macroscopically ; ina stab of gelatin containing grape-
or cane-sugar, on the contrary, it grows upon the surface
as a luxuriant deposit consisting of dense, whitish, jelly-
like clumps, which possess a ‘‘strong glassy luster at the

1 The serum must not be sterilized with chloroform, but only by
heat.

2 Liesenberg and Zopf call these forms Strept. mesenterioides var.
nuda.

) F | SARCINA. 151

t

summits,’’ and along the stab as luxuriant stalactite-like
‘masses. The colonies are at first cartilaginous, then moist,
and finally pap-like. Upon grape-sugar agar plates the
superficial colonies are warty and luxuriant and spread
like a wrinkled film ; the deep ones are at first smooth and
later sago-like warty balls.

Microscopically the form upon sugar media presents
as thick, gelatinous capsules (of dextran, compare

30

Pho gelatinous covering protects for fifteen minutes
against 75°. All the varieties of sugar ordinarily em-
ployed undergo fermentation, with the formation of gas
and acid. The fungus formerly was often the cause of
the very troublesome frog-spawn fermentation of sugar
solutions in sugar factories.

Leuconostoc lagerheimii Ludwig consists of small
(0.6 » to 0.8 #) cocci within thick capsules. It causes
alcoholic fermentation in the slimy secretion from oaks.
The organism is said to occur also without envelopes as
short rods with flagella (?).

ae Ca mG Ao SARE see 9 “ye Salt. ads F

fehl at ie lat ied re

2. Sarcina (Goodsir).

The cells divide (at least upon suitable nutrient media
—hay decoction, bouillon) in regular succession in three
directions of space, and remain grouped in larger or
smaller cubical families. } ;

The boundary of this genus is not sharp, although
the sarcina is held by many authors (Niageli!) as an
especially natural genus. Many varieties only produce
true cubical arrangement upon certain nutrient media, and
it appears that this property may also be acquired or lost
(compare Sarc. rosea). In the case of varieties with in-
complete packet formation there is always doubt whether
they belong to the sarcina or micrococcus. It is our con-
viction that the sarcina is connected with the micrococcus
by unbroken transition forms, and is only separated arbi-
trarily. Examples follow.

1We call eight cubically arranged cocci a packet ; cubical combina-
tions of packets, bales of packets ; irregular combinations, heaps of

packets.

’

152 IMPORTANT VARIETIES OF FISSION-FUNGI.

The synopsis of definitions and the descriptions may be
prefaced as follows: All the sarcine which we have in-

{

vestigated grow—to be sure, in part very imperfectly— —

also anaerobically, and then produce H,S, in from barely —

perceptible to large quantities. Aerobically, H.S is not
produced in 2% peptone bouillon by all, and in marked
quantity only by those where we expressly state it. A
minimal formation of indol occurs with all. In grape-
sugar bouillon, with few exceptions, only a little acid is

formed in six days (lactic acid), about 0.8 ¢.c. normal —
acid to 100 of bouillon. Many convert urea into carbonate ~

of ammonia.

It can not be doubted that sarcinee can cause cloudiness —
and souring of beer (Lindner). (Compare Schonfeld, —
C. B. L. Iv, 865.) These are said to originate especially —

from horse-manure.

All sarcinee stain well by Gram’s method. Beautiful —
pictures are also obtained by staining with a solution of ©
fuchsin and differentiating with acetic acid. It isimportant —
always to observe the fresh preparation ina hanging drop. —

One must guard against mistaking tetrads (or eight-celled
cubes) for single cells, which quite easily occurs, especially
with deep staining.

We have not made statements regarding the size of

sarcinee, since we here found especially varying results. It —
impresses one as if the cells often grew very large and then

in rapid succession divided into eight parts.

Endospores we have been unable to find except in Sare.
pulmonum Hauser.

Spontaneous movement has not been observed in any of
the sarcinee examined by us with the exception of the Sare.
pulmonum, but often strikingly marked molecular motion
was present, which continued in sublimate solution. The
Sare. mobilis Maurea, obtained from Kral, was always
non-motile and devoid of flagella.

In many varieties cultivation in fluid nutrient media
(hay decoction and bouillon) led to the formation of
packets and bunches of packets, which were otherwise
formed with difficulty or not at all. When no packets are
produced upon these nutrient media, one will seek them
in vain upon ‘solid nutrient media. The macroscopic ap-

P
’
‘
7
q

KEY TO RECOGNITION OF THE SARCIN2. 153

pearance of the bouillon cultures is of little value in differ-
entiating species, as it seems that most varieties finally
produce a more or less viscous or friable sediment in the
- clear bouillon, and that in the same variety the character
_ of this sediment varies. The precipitate either forms upon
the bottom or on the walls and bottom, without the bouillon
becoming cloudy ; or the precipitation is preceded by a
longer or shorter diffuse cloudiness of the bouillon. The
bouillon takes on in some varieties (Sare. alba), but not
always, a characteristic gummy, viscous quality.

The following presentation is dependent not only upon
our own studies, but upon the critical elaboration of the
material, which Dr. Stubenrath cultivated during about
two years under our direction, and upon which he has re-
ported in a monograph, ‘‘ Das Genus Sarcina,’’ Miinchen,
1897. The literature is there extensively presented.

Space does not allow us to enter more into particulars
concerning the uncritically described and very numerous
varieties of Henrici! and Gruber?. Stubenrath (J. ¢.) has
referred to the fact that those contributions, in a work
which does not at all consider the variation of bacteria,
have loaded us with many names, but that our knowledge
is scarcely advanced thereby.

Key to Recognition of the Sarcine.

I. WITHOUT PIGMENT PRODUCTION UPON AGAR AND GELATIN.
(a) Potato growth delicate, brownish-yellow from the first. Gela-
tin and agar growth, delicate, finely notched and wrinkled. Young
cultures motile, old cultures often with spores. Sarc. pulmonum
Virchow, page 155.
(b) Potato growth always remains white or grayish-white.

(a) Gelatin plate magnified sixty times; very finely granular;
limited liquefaction. No formation of large regular bales of
packets. Sarc. alba Zimmermann, page 160.

(8) Gelatin plate magnified sixty times; medium-sized granules;
liquefaction more rapid; formation of beautiful regular bales
of packets. Sarc. canescens Stubenrath, page 159.

Il. Upon AGAR AND GELATIN GRAYISH-YELLOW, GREENISH-YEL-

LOW TO CHROME-YELLOW.

(a) Gelatin plate magnified sixty times; very finely granular ;

oo “Beitrag zur Bakterienflora des Kiises’’? (A. K. Bd.
a1).
* Gruber, ‘‘ Die Arten der Gattung Sarcina’’ (A. K. Bd 1, 241).

a.

ce

154 IMPORTANT VARIETIES OF FISSION-FUNGI.

potato growth chrome-yellow, glistening ; no large regular bales of
packets formed. Sarc. flava de Bary, emend. Lehmann and Stuben-
rath, page 159.

(6) Gelatin plate magnified sixty times; medium-sized granules ;

beautiful regular bales of packets formed. This group contains transi- —

tions from flava to lutea, and from the yellow to the white forms.
(2) Potato growth ; at first dark gray, only later yellowish-brown.
Sarc. livido-lutescens Stubenrath, page 159.

(8) Potato growth ; from the beginning grayish-yellow, at other —

times very similar. Sarc. equi Stubenrath, page 158.
(y) Like Sare. equi, but motile from long flagella, sometimes
somewhat fluorescent. Sarc. mobilis Maurea, page 160.

(ec) Gelatin plates magnified sixty times are coarsely granular.
Formation of beautiful regular bales of packets ; potato growth, from
beginning, luxuriant lemon-yellow. Sarc. lutea Fliigge, emend.
Lehmann and Stubenrath, page 157.

III. Upon AGAR AND GELATIN ORANGE-YELLOW. Sarc. auran-
tiaca Fliigge, page 160.
IV. UpoN AGAR AND GELATIN BROWNISH TO BROWNISH-YELLOW.
(a) Agar streak succulent, broad, reddish-brown. Sarc. cervina
Stubenrath, page 162.
(b) Agar streak thin, finely notched, and furrowed, yellowish-
brown, transparent. Sarc. fulva Stubenrath, page 156.
V. Upon AGAR AND GELATIN BRIGHT ROSE-RED.
(a) Gelatin and agar streak rose-colored ; sarcina form observed only
upon hay decoction. Sarc. rosea Schroter, emend. Zimm., page 162.

(6) Gelatin and agar bright red ; sarcina form observed by us only ~

once upon hay decoction. Sarc. erythromyxa Krdl, page 162.

That it will always be possible to distinguish the
‘‘forms’’ presented in the key, we can not certainly claim,
since in spite of the observation during two years of very
numerous forms, we have reached no final judgment con-
_cerning the extent of variability and perhaps the occur-
rence of transition forms.

Leaving the chromogenesis out of account, we can cite
at least two striking examples of their variability (com-
pare Sarc. variabilis and mobilis); thus, the following
appears the natural relationship:

1. Sarcina flava,—therefrom is the white form, Sare.
alba.

2. Sarcina equi,—therefrom is the white form, Sare.
canescens.

Between equi and canescens Sare. livido-lutescens and
Sare. variabilis reestablish a connection.

The varieties Sare. flava, equi, and lutea form a series

ee Pee te | ee eee

SARCINA PULMONUM. bs iy

which the coarseness of the granules of the culture and
the size of the bales of packets continually increase;
entirely parallel with this is the series Sarc. alba, vari-
pets, and canescens. 4

o
N!
¢ Sarcina pulmonum (Virchow, Hauser).

(Plate 6, vI—x. )

2

__ Literature.—Hauser, ‘‘ Deut. Arch. f. klin. Med.,’’ xi11, 127 ; Stu-
_ benrath, monograph.

Microscopic Appearance.—Upon the various nutrient
_ media only small and not especially regular bales of
_ packets were formed.
_ Motility.—Young cultures exhibit exquisite waltzing
movement (Hauser) dependent, according to Job (Diss.
_ Wiirzburg, 1896), upon not very numerous, long, coiled
flagella. Older cultures, and quite often also young ones,
_ exhibit no motility.
_ Growth.—Very slow even at incubator temperature.
_ Gelatin Plates.—(a) Natural size: Extremely small,
_ roundish, yellowish-grayish-white, punctiform colonies.

(6) Magnified fifty times. Superficial: At first roundish,
smooth border; gray, almost opaque, not different from
the deep ones. After two to three weeks the peripheral
part is lost from sinking in of the colony, and it then ap-
pears torn, and (especially at the edge) transparent,
coarsely crummy. Packets are not to be made out; color
gray. Deep: Roundish, gray, opaque, without any visible
internal structure (6, vir).

Gelatin Stab.—At first thread-like, and only after a
long time crummy; gray to yellowish-gray. Surface
growth: After twenty days, 2mm. to 3 mm. wide, gray,
transparent, roundish, serrated, faintly shining. Later it
begins to sink in (6, v1).

1 We have not described a Sarc. ventriculi Goodsir because the
description given by Falkenhain (Arch. fiir exp. Path. u. Phar. x1x,
339), which was copied by Gruber, does not agree accurately with any of

_ our forms, and, as Oppler (Miinch. med. Wochenschr., 1894, No. 29,
570) first pointed out, the stomach contains a whole series of sarcinz.
_ (For details thereon, see Stubenrath. )

156 IMPORTANT VARIETIES OF FISSION-FUNGI.

Agar Plates.—(a) Natural size: Like gelatin oil
only somewhat whiter. 7
(6) Magnified fifty times. Superficial: Round, light toe
dark gray, periphery lighter, transparent; tetrads visible
as tiny crumbs. Deep: Roundish, dark, finely granular.
Agar Stab.—Stab : Thread-like, ‘later granular. Surface
growth: Grayish-white, shining, ‘slightly elevated; after
three weeks 4 mm. to 5 mm. in diameter. |
Agar Streak.—Restricted to the streak ; rather scanty
growth ; grayish-white, transparent, wavy, usually made
up of single crumbs. Water of condensation clear with ~
slight sediment (6, vil and 5, 11). :
Bouillon Culture.—Clear, little deposit, friable.
Milk Culture.—Milk very slowly becomes clear, with-
out preceding coagulation. |
Potato Culture.—Very poor growth; after three to
four weeks a growth 3 to 4 mm. wide, yellowish-gray to
brownish, shining, not sharply outlined from the potato
(6:-3x}.
Spores.—Typical, round spores first observed by
Hauser ; according to Hauser, they stain well.
Occurrence.—So far found only in the air passages of
men—for example, in cases of phthisis—apparently as
harmless settlers ; according to Hauser, are not pathogenic
for animals. .
The following appears very similarly (but always lacks
spores and flagella):

Sarcina fulva (Stubenrath).

In microscopic findings upon all nutrient media, in distribution and
consistency, liquefaction, etc., almost exactly like the preceding, but is
brownish-yellow to reddish-brown, and transparent upon agar and gelatin;
on the contrary, upon potato scarcely to be distinguished from Sare.
pulmonum. Bouillon becomes turbid, with tough crumbly sediment.
Grown with oxygen it forms some H, S, and rather abundant acid upon |
grape-sugar bouillon and milk. Upon. all nutrient media it forms
bunches and bales of packets, but of various sizes.

Cultivated in Wiirzburg many times from stomach contents and once
from preputial smegma ; a very striking and slowly growing variety.

Pe te te Sl eel

F
1
;
:
,

SARCINA LUTEA. 157

Sarcina lutea.! (Fliigge, emend. Lehmann and
Stubenrath).

_ Microscopic Appearance.—Upon nutrient media typi-
eal bales of packets.

Gelatin Plate.—(a) Natural size. Roundish, puncti-

form colonies, sulphur-yellow; after ten to twenty days,
sinking in (3, v). :
(6) Magnified fifty times. Superficial: Roundish, even-
bordered or almost smooth-edged colonies; pale yellow
with at first a finely granular and later (eight to ten days)
a more coarsely granular structure. After a very long
time the peripheral parts separate somewhat and, with
higher magnification, individual tetrads are seen (3, V1).
Deep lying: Roundish, dark yellow, even-bordered, finely
granular. |

Gelatin Stab.—Stab: Thread-like, with relatively few
coarse granules. Surface growth: Irregularly circular, with
‘a moist luster, somewhat elevated, sulphur, lemon-, or
even deep yellow. After ten to twelve days the superficial
ade sinks down. Liquefaction at first extends in a

unnel form and later as_a cylinder; however, we have
cultivated almost non-liquefying forms (38, 1).

Agar Plates.—(a) Natural size. Superficial: Round
or roundish, even-bordered, somewhat elevated; sulphur-
yellow, with a moist luster. Deep: Roundish to whet-
stone-shaped (3, vir). ;

(6) Magnified fifty times. Superficial: Roundish almost
even-bordered colonies; periphery delicately punctate;
peripheral zone transparent, pale yellowish, becoming
darker toward the center; finely to coarsely granular
(3, vil). Deep: Like those upon gelatin with coarser
granulation.

Agar Stab.—Stab: Thread-like, finely to coarsely gran-
ular, at times after a long while ray-like outgrowths ; yel-
low. Surface growth: Roundish, wavy, even border, some-

' Plate 6, Figs. I to v, illustrating the Micr. luteus Cohn, serve ex-
actly as well for the Sarc. lutea, except figure 111, where the bales of
packets are absent. Also Plate 3 would pass for the gelatin plate cul-
tures, except for the finely granular structure (3, VIII); a somewhat
lighter form (5, Iv).

158 IMPORTANT VARIETIES OF FISSION-FUNGI. —

what elevated, moist, of consistency of butter; sulphur- |
to chrome-yellow (38, U1).

Agar Streak.—Similar ; water of condensation clear ;
whitish-yellow precipitate (8, 11).

Bouillon Culture.—Clear ; abundant sediment.

Milk.—Coagulated after forty-eight hours.

Potato Culture.—Wavy surface growth, often much
elevated, shining, especially in old cultures having larger
or smaller elevations ; in young cultures with a moist lus-
ter, later dull, sulphur-, chrome- and more rarely grayish-
yellow, limited to the line of inoculation, only extending
a little more widely after a long time (3, Ix).

Chemical Activities.—In peptone-bouillon there is
formed some H,S and a trace of indol. The yellow pig-
ment is a lipochrome. In grape-sugar bouillon some acid
is formed. 3

Distribution.—Very common variety in the surround-
ings of men, especially in the air. In Wiirzburg every
plate from air contained it.

Remarks.—

The numerous forms isolated by Dr. Stubenrath which belong here,
we group under the following varieties :

(a) Typica (Lehmann and Stubenrath). The colony on gelatin ?
may be recognized upon the plate by a marked cleaving of the border, —
and even with progressing liquefaction of the gelatin the round form —
is not essentially changed.
- (8) Compacta (Lehmann and Stubenrath). The colonies on the
gelatin plate are very luxuriant, roundish, and so compact that a bor-—
der-zone can not be distinctly seen. As this form also causes almost —
no liquefaction of gelatin, the colonies lie upon the plate as a tough
film in the scarcely depressed gelatin.

(y) Diffluens (Lehmann and Stubenrath). This form shows upon ~
all nutrient media a very marked tendency to spread out. Upon gel-—
atin plates, which are liquefied quite rapidly, the colony spreads as a —
very much fissured, readily disintegrating mass.

x
-

Sarcina equi (Stubenrath).

In all respects similar to the Sare. lutea, but is differentiated: 4

1. By medium-sized granules, not coarse granules, in the gelatin
plate.

2. Less perfectly formed bales of packets.

3. More grayish-yellow color on all nutrient media; little lique-
faction.

Found repeatedly by Dr. Stubenrath in the urine of various horses —

f

-

4
j
:
’
:

SARCINA FLAVA. 159

“

-

he Coa:

Are
oa

in Wiirzburg. In cultures it remained constant for a year, the origi-

‘nal active liquefaction only being somewhat lessened. The three
following are subspecies or varieties:

Sarcina livido-lutescens (Stubenrath).

Like Sare. equi, but young potato cultures for ten days and more

are gray to reddish-gray; after twenty days they become brownish-

yellow in the center, and after a month throughout the entire culture.

- The constancy of this characteristic was observed for a year. Ina

case of enteritis it was grown abundantly from the stool by Dr.
Stubenrath.

Sarcina canescens (Stubenrath).

Differentiated from Sare. equi only by constant gray color and
somewhat coarser granulation (larger bales of packets) upon all
nutrient media (5, VIII).

Sarcina variabilis (Stubenrath).

This form, isolated from gastric contents, appears to us to be very
interesting. It is differentiated from the Sare. equi only by more
marked liquefaction of gelatin and by the property of furnishing on
the various nutrient media sometimes yellowish-gray, sometimes pure gray
colonies. Upon plates one often obtains gray and yellowish colonies
side by side, but this is alike repeated whether one inoculates from
gray or yellowish colonies.

Sarcina flava (de Bary, emend. Lehmann and
Stubenrath).
(Plate 3.)

Upon all nutrient media it is habitually very similar to the Sare.
lutea, being yellow to greenish-yellow. The principal difference lies
in the very finely granular gelatin plate colonies when magnified sixty
times. When magnified one thousand times, this fine granulation is
seen to depend upon very small bales and heaps of packets.1 We have
observed one form that is more luxuriant and distinctly liquefying, and
one that is more delicate, leaving the gelatin still solid after weeks,
growing feebly upon all nutrient media. It has been repeatedly culti-
vated from gastric contents.

1 The Sarc. flava, obtained from Kral, Dr. Stubenrath found to
form upon all fluid and solid nutrient media, usually only bunches of
cocci, rarely tetrads, and never true bales of packets.

160 IMPORTANT VARIETIES OF FISSION-FUNGI.

Sarcina alba (Zimmermann).

If one imagines the very feebly liquefying forms of Sare. flava with-
out formation of pigment, then one obtains the Sare. alba likewise with

variable liquefaction. The growths on the various nutrient media are _

white to grayish-white, usually very delicate. Microscopically this

variety is not distinguishable from Sare. flava, so that, when transition —

forms are found, they appear only as varieties.

Sarcina mobilis (Maurea).

The inoculation from an original culture sent to our institute by
Kral resembled our Sare. equi, very markedly in the color (grayish-
yellow), upon all the nutrient media and in its slow but always dis-
tinct liquefaction, yet the granulation in the gelatin plate cultures,
magnified sixty times, is still finer, somewhat like the Sare. flava,
midway between this and the Sarc. equi.

Now and then a yellowish-green fluorescence occurs upon agar and
gelatin, which we have observed in no other sarcina. Although the
granulation is fine, beautiful packets occur upon all nutrient media.
We were not able to see the spontaneous motion described by Maurea,

nor could we stain flagella. Our variety appeared to have lost the ability —

to produce flagella. KR. O. Neumann has grown a white and a yellow
culture. Migula, who has seen the flagella, produced a picture of
them. Sames has described and illustrated by photographs a gray
variety of sarcina, which is actively motile and provided with
numerous long flagella, obtained from dung-water (C. B. L. Iv, 664).
It may be called Sarc. fimentaria L. and N.

Sarcina aurantiaca (Flugge, Lindner). —
(Plate 4.)

Microscopic Appearance.— Beautiful bales and
bunches of packets upon all ordinary nutrient media.

Gelatin Plate.—(a) Natural size : Orange-yellow, small,
round, dot-like colonies, which soon sink into the gelatin.
After five to six days the peripheral part breaks up and
portions of the colony swim about in the plate-shaped
area of liquefaction. Thus the colony appears whitish
orange (4, v).

(b) Magnified fifty times. Superficial: At first round, —

almost even-bordered colonies, pale to deep yellow, struc-
tureless or finely granular. The shallow funnel-shaped
depression appears gray. Later the border of the colony
is broken, fringed, and wavy, and when magnified a hun-
dred times presents tetrads that are single or joined in

Pine

SARCINA AURANTIACA. 161

slumps. At this stage the peripheral zone is perfectly
transparent (4, vi). Deep: Like young superficial ones.
_ Gelatin Puncture.—The colony sinks in after thirty-
six hours, so that usually the gelatin presents the appear-
ance of a contracting blister. The stab-canal presents a
ie cl-chaped liquefaction, the wall being beset with fine
fragments of the colony. At the bottom of the funnel is
- orange sediment (4, 1).

Agar Plate.—(a) Natural size. Superficial: Round or
“roundish colonies, even-bordered, somewhat elevated,
orange witha moist luster. Deep: Roundish to whetstone-
‘shaped, similarly colored (4, vir).

(6) Magnified fifty times: Irregularly round; central zone
Opaque, brownish-green, toward the border lighter and
“more yellow, coarsely granular; with stronger magnifica-
tion individual tetrads are to be seen (4, vit).

Agar Stab.—Stab: Thread-like, coarsely granular.
Surface growth: Irregularly round, wavy, somewhat ele-
_yated, orange-yellow to orange-red, with a consistency like

‘ butter, shining moistly (4, Iv).

Agar Streak.—Like agar stab; water of condensation
‘clear ; yellowish sediment (4, 11).

Bouillon Culture.—Unevenly turbid, many single

floceuli, abundant sediment.

Milk Culture.—Milk is coagulated, and later the coag-
ulum is again liquefied.

Potato Culture.—Luxuriant growth,-with rough, wavy
border ; after a longer time distinctly elevated ; reddish-
orange, "especially i in old cultures, and then is usually dull
and irregular like a strawberry. In earlier stages it is yel-
lowish-orange and at times shining. Very similar to the
Mier. pyogenes aureus (4, 1x). (Compare also 8, rx.)

Chemical Activities.—The orange-yellow pigment is
alipochrome. In grape-sugar bouillon there is feeble acid
production. When grown aerobically upon nutrient media
et sugar, there is produced no H,§, but a trace of

‘indol.

Occurrence.— Outside the organism: Very common in
the air; almost upon every plate made from the air in
- Wiirzburg.

Related Varieties,—All orange-yellow sarcin, which
il

162 IMPORTANT VARIETIES OF FISSION-FUNGI.

were cultivated in our institute could be easily designated’ 3
as Sare. aurantiaca; moreover, we can not differentiate
Sarc. aurea Macé, Sarc. aurescens fusca and fusces=— '
cens Gruber from Gruber’s description.

ii i a isd

Sarcina cervina (Stubenrath).
(Plate 5, 1.)

Gelatin plate colonies, macroscopically, at first are whitish, after
four to five days pale brown, somewhat moist, slowly becoming sur- |
rounded by a zone of liquefaction. Magnified sixty times: with
coarsely granular projections, gradually breaking up at the edge into ~
coarsely granular, cloudy masses. Gelatin stab—superficial growth —
small, pale brown, very slowly sinking in. Stab—faint, thread-like,
finely granular. Agar plate—similar to that on gelatin. Agar
streak—broad, moist, elevated, yellowish-brown (5,1). Potato cul-—
ture —brownish-white. Magnified one thousand times, it is seen to-
consist of mostly irregular bales of packets, which appear a light
brownish color. This variety was once isolated from the gastric con-_
tents ina case of carcinoma.

4
Sarcina erythromyxa (Kral).
(Plate 5, 111.) ;

:

Literature.—Kral (list of the bacteria handed over); Mier. eryth-
romyxa Overbeck (Nov. Act. der Leop.-Carol, Bd. 55, No. 7, 1891). —
Good description by Zimmermann (I, 70).

Magnified one thousand times, usually only cocci, diploooadi: and ©
tetrads ; only once did we obtain upon hay decoction a beautiful for-—
mation of regular bales of packets.

Upon gelatin plates (natural size) the colonies are at first a lively —
greenish color, then beautiful carmine- to vermilion-red, and moist.
Magnified sixty times, almost. without granulation ; at the edge the
red colonies are usually transparent and finely notched. There is no
liquefaction. Gelatin stab, agar stab and streak, and potato cultures
gradually develop as an intensely red, shining, rather spare growth.
Upon milk a red growth forms on the surface, and the milk slowly be- —
comes clear without preceding coagulation. Bouillon becomes cloudy
with a coarse, crumbly sediment and at times a pellicle. Moderate —
production of acid on grape-sugar bouillon. '

—_—

Sarcina rosea. J. Schroter emend. Menge (B. vi, 596)
and Zimmermann (ii, 58).

The description of this organism (5, VI) coincides absolutely as re-—
gards its growth upon all nutrient media with that given for the Mier. —
roseus (p. 190); the illustrations in Plate 11 also are as good for the

bE MICROCOCCUS. 163

‘Sare. rosea. On the contrary, the culture we obtained from Kral, upon
agar, hay decoction, and urine, contained bales of packets.

3. Micrococcus (Cohn).

The cells divide irregularly in various directions and lie
singly, in pairs, in fours, or, finally and indeed mostly, in
irregularly bunched heaps. In this class are included all
‘cocci which are not undoubted streptococci or sarcine.

Key to the Determination of the Micrococci.

I. Does not grow upon any of the ordinary nutrient media aerobic-
ally or anaerobically ; on the contrary, grows upon human blood-
_ serum, agar smeared with blood, etc. Microscopically, pairs of kidney-
shaped cocci, connected by a usually broad, unstained cement line ;
round forms are more rare. Does not stain by Gram’s method. Never
found except in human body or its secretions. Micr. gonorrhceze
Neisser, page 164.

II. Poor growth upon the ordinary nutrient media and upon serum.

Besides cocci, often rod-forms (!) are found, which may be four times
as long as wide ; not stained by Gram’s method. Micr. melitensis
Bruce, page 168.

Ill. Upon the ordinary nutrient media a thick, white, abundant
growth, sometimes forming tetrads. In animal body always tetrads
with marked gelatinous capsules. Micr.tetragenus Gaffky, page 171.

IV. Grow upon ordinary nutrient media; always spherical ; no
tetrads in animal body.

A. Upon gelatin and agar, do not produce pigments (white to gray
varieties).

(a) Gelatin not liquefied ; colonies in plate roundish with no out-
growths. ‘

(a) Growth on gelatin and agar thick, pure white ; not patho-
genic ; arrangement irrregular.
3 “os aden rather large. Micr. candicans Fliigge, page
69.
2. Individuals very small. Micr. aquatilis Mead Bolton, page
71

171.
(8) Similar to a, but color yellowish-gray, not pure white. Micr.
Tosettaceus Zimmermann.
(y) Growth upon gelatin thinand iridescent. Micr.concentricus
Zimmermann, page 174.

__ (b) Gelatin not liquefied ; delicate white tendrils extend outward
from the deep colonies in gelatin plates and from the gelatin stab.
Micr. viticulosus Katz, page 174.

(ec) Gelatin liquefied ; plate and stab cultures without tendrils or
branches. Micr. pyogenes y albus (Rosenbach), L. and N.,! page 187.

+ Compare Micr. Freudenreichii Guillebeau, Micr. acidi lactis
Kriiger. ,

oe ‘
ee

164. IMPORTANT VARIETIES OF FISSION-FUNGL

(d) Gelatin liquefied ; colonies in plates with teeth or branches.
(a) Gelatin stab without branches. The funnel of liquefaction in
the gelatin plate cultures is surrounded after a few days with a
yellowish-white circle of ragged points and teeth. (Com :
also Micr. corallioides Zimmermann.) Micr. coronatus Fliigge,
page 175.
' (8) In gelatin stab are branches. The colonies in the gelati
plate present a circle of pretty rays. Micr, radiatus Fliigge,
page 176.
B. Upon gelatin and agar, sulphur-yellow to lemon-yellow pigment i
produced. * .
1. Colonies in gelatin coarsely granular ; liquefaction rapid. Mier.
luteus Cohn, emend. L. and N., page 176.
2. Colonies in gelatin finely ‘granular ; liquefaction rapid. Micr.
flavus Fluigge, L. and N., page 178.
3. Colonies in gelatin finely granular; no liquefaction. Micr.
sulfureus Zimmermann, page 178. ;
C. Upon gelatin and agar, formation of brownish-yellow pignee
Micr. badius, L. and N., page 178.
D. Upon gelatin and agar, orange-yellow to grayish-orange.
(a) Agar streak uniformly orange-yellow.
(a) Gelatin liquefied, pathogenic. Micr. pyogenes a aureus’
(Rosenbach) L. and N., page 181.
(8) Gelatin not liquefied ; found in air. Micr. aurantiacus Cohn,
page 189.
(b) Agar streak, mottled gray and orange. Micr. bicolor Zimmer-
mann, page 189.
E. Upon gelatin and agar, rose to crimson.
(a) Rose to cherry-red ; upon potato, slight growth. Micr.roseus”
(Bumm), L. and N., page 190. .
(b) Rose to cherry-red ; upon potato, broad, dry growth. Micr. |
cerasinus (List) L. and N., page 193.
(ec) Scarlet red. Micr. erythromyxa Zopf, page 193.
F. Upon gelatin and agar, cobalt blue. Micr. cyaneus (Schréter)
Cohn, page 193.

Micrococcus gonorrheeze (Neisser) (Fliigge).
(Plate 10.)

Synonyms.—Gonococcus (Neisser), Diplococcus gonor-—
rhee Bumm, Micrococcus Gonorrhcee Schroter. ,

Most Important Literature.—A. Neisser, ‘‘C. f. med. Wiss.,’’ 1879, —
497; Bumm, ‘‘ Der Mikroorganismus der gonorrh. Schleimhauter-
krankung,”’ ’ Wiesbaden, monograph, 1885; Wertheim, ‘‘ Archiv fur —
Gynakologie,’’ XLII, 1892, 1; 1; Wassermann, XXVII, 298. ‘Latest exhaus-—

1 Compare also the pathogenic Mier. ascoformans Johne, the Mier.
pyogenes f citreus (Passet), and the Micr, ochroleucus Prowe, which —
is said to form spores,

MICROCOCCUS GONORRHG@. 165

ive review of the literature by Fouleston, ‘‘ Transact. of the Inst. of
Prev. Med.,’’ Vol. 1, 1898.

__ Microscopic Appearance.—They usually occur as
pairs of organisms, somewhat kidney-shaped, united by a
Tenticular cement material which is often quite broad. A
pair is 0.8 » to 1.6 » long, and 0.6 4 to 0.8» broad
0, x).
‘ Staining Properties.— By the usual staining methods,
best with Léffler’s methylene-blue. It is not stained
_by Gram’s method, which is very important, as it differs
in this from almost all cocci. Recently many authors
“have claimed that gonococci at times stain by Gram’s
method. Weinrich (C. B. xxiv, 258), who discusses the
entire literature, maintains that prompt decolorization is
always obtained .if the preparations which are stained
_ with anilin- or carbol-gentian violet solution are brought
directly into Lugol’s solution without washing with water
and then into truly absolute alcohol. If one desires a con-
trast color in the cells, a weak aqueous solution of
Bismarck brown is employed after the cover-glass has
_ been brought from the absolute alcohol to water.
Relation to Oxygen.—Facultative anaerobe.
Requirements as Regards Temperature and Nutri-
ent Media.—Grows only at incubator temperature, best
at 36°. The extremes are from 25° to 39°. Growth on
all nutrient media very slight, and frequent transfer
is necessary to keep it alive. It is.one of the most
difficult varieties to keep permanently in culture. It is
remarkable that cultures die at room temperature in forty-
eight hours.

The growth of gonococci upon the ordinary nutrient
media is not to be undertaken.! Smears are to be made
upon one of the following nutrient media (3, 4, and 5 may

_ also be used for plates):

; 1. Ordinary nutrient agar, smeared over with human blood (from
the sterilized finger-tip of the investigator, Abel). To be recommended
as the simplest method.

2. Human blood-serum (from placenta or obtained by venesec-

: 1 The statements of Turré regarding the cultivation of the gonococ-
cus upon acid gelatin, the successful inoculation in the dog, and the
_ liquefaction of alkaline gelatin could be verified by no one.

166 IMPORTANT VARIETIES OF FISSION-FUNGI.
tion). The serum of animals is usually unsuitable, and upon it the — {
growth is always very slight (Bumm). q

3. We have (with Kiefer and Menge) obtained very good results —
with a nutrient medium, always prepared when used by mixing 2%
agar (with 1% peptone ‘and 5% glycerin), which has been xing 229
and cooled to 50°, with half its volume of ascites fluid or the fluid
from ovarian cysts (see Technical Appendix).

4. We have had no good results with a nutrient medium consisting
of urine and glycerin-agar, or with simple glycerin-agar.

5. Wassermann recommends the following as the best gonococcus
nutrient medium: 15 c.c. of swine serum, as free as possible from
hemoglobin, is placed in a small Erlenmeyer flask, diluted with 30 to
35 ¢.c. of water, and to this is added 2 to 3c.c. of glycerin and, finally,
0.8 to 0.9 gm. (about 2% ) of nutrose (casein-sodium phosphate). The —
whole is now mixed as thoroughly as possible by shaking and heated, —
with constant stirring, over a free flame to the boiling-point. The
previously turbid fluid becomes clear upon boiling, and may be
properly heated in the moist oven to render it sterile. The addition
of the nutrose prevents the precipitate from the serum. In the prep-
aration of cultures an equal quantity of 2% agar cooled to 50° is
poured into the flask, the two mixed and poured into a Petri dish.
As soon as it becomes solid, the nutrient medium is ready for use.
The cultures are the more luxuriant, the fresher the case of gonorrhea”
and the less it has been treated. Growth is favored by the admission
of air.

Plate Cultures.—(a) Natural size: Like the streak
culture.

(b) Magnified fifty times: The great delicacy of the
colonies is characteristic of the gonococcus. Upon blood-_
agar and serum-agar, as well as upon ascites glycerin-
agar, the colonies are transparent gray with a shade
of yellow, exceedingly delicate, scarcely at all, or only
very finely, granular. Often at the periphery the colony
is indistinguishable from the medium. They are very —
‘slightly elevated (10, mn, below; v, m). At this stage —
they are very similar to the Strept. lanceolatus. In older —
colonies the border, which was formerly smooth, becomes
partly wavy and irregular, the structure somewhat granu-
lar (10, 11), and eventually even moruloid (10, Iv), yet it
is always more delicate than the streptococcus. If an in-
oculation is made upon agar smeared with blood the col-
onies appear principally “at. the periphery of the streak,
like a cloud, or, upon becoming larger, press the blood
aside (10, It). The same happens if gonorrheal or blen-
norrheal pus is placed upon ascites-glycerin-agar. The

MICROCOCCUS GONORRHGZ. 167

ae s pushed aside forms septa, between which the colonies
develop. This is a very characteristic picture (10, v1).
_ Streak Culture.—Transparent gray deposit, perhaps
with a trace of dirty yellow, somewhat elevated especially
at the edge. It has an oily but not a moist gloss. It
| gives the impression as of mucus upon the surface, thus
- differing from the streak cultures of other delicately grow-
_ ing organisms, as the Strept. pyogenes or lanceolatus.
_ Toxins.—Upon nutrose-serum bouillon Wassermann obtained
vigorous cultures, which were still poisonous after being killed. The
' gonotoxin from the bodies of gonococci is very resistant to heat and
_ alcohol, kills mice, produces a doughy infiltration in rabbits and mice,
which often ends in necrosis. With large doses systemic effects occur
(compare Nicolaysen, C. B. xx11, 305). Gonotoxin injected sub-
cutaneously was without effect in chronic gonorrhea in man. The
marked reaction following the injection did not become less upon re-
peating the injection.

The gonotoxin explains the gonorrheal secretion. Also some points
in the history of chronic gonorrhea may be explained by the fact that
for a long time a few gonococci slowly multiply and die and keep up
a suppuration almost free of gonococci, but that they may increase
more actively after any injury, irritation, etc., of the tissue and an
acute exacerbation of the process, with abundant formation of toxin
and large numbers of gonococci, may develop.

Also, the filtrate from cultures of gonococci in ascites bouillon was

irritating, according to Schaffer, producing a suppuration upon the
urethral mucous membrane (C. B. XXIII, 708).

Distribution.—(a) Outside of the organism: Never, ex-
cept upon linen, towels, etc., soiled by those with the dis-
ease.

(b) In healthy organism: Never.

(¢) In diseased organism: In gonorrhea in the urethra
and prostate of men ; in the urethra, Bartholinian glands,
cervix uteri in women. Cause of vaginitis and urethritis
in young girls.

Besides these, in isolated cases it causes endometritis,
metritis, salpingitis, oophoritis, peritonitis, proctitis, ves-
ical catarrh, and probably also epididymitis. Cause of
blennorrhea neonatorum, rarely of diphtheritic conjunc-
tivitis in children (C. Frankel); the gonococcus also
causes, in adults, severe conjunctivitis, rarely rhinitis and
otitis. The gonococcus is often recognized as the cause of
arthritis, and more rarely of pleuritis and malignant endo-
carditis, abscesses, parotitis, periostitis, and bursitis. Now

168 IMPORTANT VARIETIES OF FISSION-FUNGL

and then these demonstrations are not entirely free from

objection. : .
As influencing the local infection, squamous epithelium _

is a better protection than cylindric epithelium. The

parasite gradually passes through the epithelium into the tf
connective tissue and causes an irritation and inflamma- —

tion there. No immunity follows recovery from the in- —

fection. .
In migrating to distant regions of the body, the gono-
coccus especially follows the lymph spaces and causes in-
flammations, which lead to fibrous proliferation (for ex-

ample, urethral stricture).

Experimental Pathologic Experiences.—Iln ani-
mals: The results of inoculation are always negative.
Large quantities of culture cause toxin inflammations with-
out increase of the cocci, just as is done by the toxins
alone.

In man: The production of gonorrhea and conjunctivitis
with pure cultures is easily accomplished.

Special Methods of Recognition.—The following
peculiarities are to be demonstrated : Diplococci, lying in
clumps in the leuokcytes about the nuclei, staining with
methylene-blue, and not by Gram’s method. Delicate
colonies in smears upon blood-agar and serum-agar. The
most positive control inoculation is upon the human
urethra.

Varieties related to the Micr. gonorrhez.

Several varieties have been partially studied by v. Bumm, which
may be mistaken for the Micr. gonorrhcee because of their microscopic
form. We will only mention them, as we have not studied them,
and will refer to the work of Bumm, already spoken of, for further
details.

Micrococcus albicans amplus.—Grows grayish-white upon gela-
tin, and is larger than the Mic. gonorrheee. Re

Diplococcus albicans tardissimus.—Microscopically is identified
morphologically with the Mier. gonorrhces, but grows upon gelatin,
although very slowly.

Micrococcus subflavus.—See under Micr. pyogenes.

; Micrococcus melitensis (Bruce).
Literature.—Durham, Jour. of Pathology, Vol. v, 1898, 377.

MICROCOCCUS CANDICANS. 169

Common Name.—Coccus of Malta fever.
- A-ssmall coceus; in fluids, especially in the incubator, it
- not rarely forms chains. Cultures at room temperature
- consist mostly of bacilli,t which are from two to four
_ times as long as they are broad. At body temperature
_ cultures of cocci again develop. Non-motile. Do not
_ stain by Gram’s method.
_ At 37° colonies grow slowly upon all nutrient media,
_ being white, hemispherical ; upon gelatin at room tem-
perature there is scarcely any growth.
Bouillon at first becomes cloudy, then presents a floccu-
- lent precipitate. Milk is not coagulated. Neither gas nor
_ acid is formed from sugar. There is usually an invisible
growth upon potato.

In man it causes Malta fever, also in monkeys after
_ cerebral injection. Rabbits and guinea-pigs may be in-
fected, guinea-pigs also intraperitoneally. The serum
causes agglutination of the cocci. The elimination of the
coccus with the urine, which may continue for months,
is interesting.

Micrococcus candicans (Fliigge).
(Plate 9, Iv-VIII. )

Microscopic Appearance.—Round cocci, lying singly
or in bunches, 1.2 » in diameter. Usually they present a
dividing line in the center (9, vu). >

Relation to Oxygen.—Grow well aerobically, and only
slightly in the lower parts of shake cultures.

Requirements as to Temperature and Nutrient
Media.—Grow at room and incubator temperatures and
upon all the usual nutrient media.

Gelatin Plates.—(a) Natural size: Round or roundish
colonies, after eight days at usual temperature being from
2mm. to 3 mm. in diameter, moistly shining, porcelain-
white, slightly elevated. Upon old plates there are always
found, besides flatly spreading colonies, those like grains
of sand or even conical elevations (9, v).

(6) Magnified fifty times. Superficial: . Round to round-

* Thus this organism lies between the families of the coccacee and

Tr1lacex,

170 IMPORTANT VARIETIES OF FISSION-FUNGI.

ish colonies, even-bordered, extremely delicately punctate,
at the periphery partially transparent, becoming opaque,
and yellowish-gray to black toward the center. Deep:
Roundish to whetstone-shaped, opaque, even-bordered,
dark (9, v1). :

Gelatin Stab.—Thread-like, granular, white. Surface —
growth: Wavy smooth border, somewhat elevated, shining
like porcelain, later somewhat dull, white, with consist-
ency of butter (9, Iv).

Agar Plates.—The colonies, when of the natural size
or magnified sixty times, are like those in gelatin plates,
except that they are somewhat more elevated and more
opaque. a

Agar Streak.—Slightly spreading, white, oily-looking —
growth, with a wavy, smooth border, and moderately
elevated. Water of condensation clear. White precipi-
tate (9, I).

Bouillon Culture.—Extremely cloudy with moderate
sediment; with some forms the bouillon remains clear, —
and there is formed a pellicle and sediment of greater
coherence.

Milk Culture.—Not coagulated in fourteen days, but it
becomes very feebly acid.

Potato Culture.—Thick, white, porcelain- like growth,
with an oily luster, much elevated, with a wavy border.
In time the neighborhood of the srowth i is discolored gray.
The growth of the same cultures upon old potatoes (March)
is much drier and more crumbly (9, viz).

Chemical Activities.—Does not liquefy gelatin, forms
no gas upon nutrient media containing sugar, and no indol
nor H,S.

Distribution.—(a) Outside the body: Very common in
air, water, milk; everywhere in Germany where it has
been looked for.

(6) In organism: Only epiphytic, for example, in pre-
putial smegma and human hairs.

Forms: We have isolated a Micr. candicans, which
differs only from the stock variety in liquefying gelatin
feebly.

Related Varieties.—The Staphylococcus cereus albus Passet only
differs from this variety in the smaller size of the individuals (perhaps

MICROCOCOCUS TETRAGENUS. 171

_ only a forma depauperata from long culture) (from 0.5 « to 0.8); other-
wise it corresponds in all particulars. According to Leube’s descrip-
tion (Virch. Arch. 100, p. 560), the Micr. urez is entirely identical
_ morphologically with the Micr. candicans (0.8 “); the colonies in gel-
atin plates at times present ‘sectorial cracks, old cultures have an in-
sipid, pasty smell. Any statement regarding the growth on potato is
lacking.

Microccocus aquatilis (Mead Bolton).

. We are not familiar with this organism. . It iscommon in the water
in Gottingen (Z. H. 1, 94), and is characterized by ‘‘ very small”’
individuals. The colonies in gelatin plates present something of
radial streaks and circular lines, so that rhomboid spaces occur. Fur-
ther characteristics are not given by Bolton. The organism is able to
grow in distilled water. According to Schroter’s insufficient descrip-
tion, it may perhaps be identical with the Micr, candidus Cohn.

Also the porcelain coccus of Escherich from the intestine (‘‘ Darm-
bakterien,’’ p. 90) appears similar ; it measures only 0.3 yu.

Micrococcus tetragenus (Koch and Gaffky).
(Plate 7.)

Synonyms.—Micr. tetragenus septicus Boutron, Micr.
tetragenus albus Boutron.

Principal Literatwre.—Koch and Gafiky, ‘‘ Mitteil. a. d. Gesundh.,”’
Bd. 1, 42; Langenbeck’s ‘‘ Archiv,’’ Bd. 28, 500 ; Boutron’s ‘‘ Thése
de Paris’’ contains a monograph upon the organism, Reference in C.
B. Xvi, 971 ; Teissier, ‘‘ Arch. de med. exp.,’’ vim, 14.

‘Microscopic Appearance.—Roundish or somewhat
oval cocci, usually lying in pairs or fours.! The size is
somewhat variable. Not infrequently one sees but little
characteristic cell arrangement in a microscopic prepara-
tion, made from aculture. Inthe animal and human body
the arrangement in tetrads is regular, and a rather thick
unstained gelatinous capsule surrounds the tetrad. In
sections stained by Gram’s method the capsule may be
counterstained with eosin.

Relation to Oxygen.—Grows well with oxygen, and
not so well without.

Requirements as to Temperature and Nutrient

?We have found, on one occasion, in old cultures in hay decoction

typical sarcina forms. Unfortunately the observation was not followed
further. Contamination is not excluded.

172 IMPORTANT VARIETIES OF FISSION-FUNGI.

Media.—Grows best at 37°, but also at room temperature,
upon all ordinary nutrient media.

Gelatin Plate.—(a) Natural size. Superficial: Small, —
irregularly shaped colonies, with even border, whitish,
slightly elevated, shining, moist. Deep: Uncharacteristic
(7, VII).

(6) Magnified fifty times. Superficial: Roundish colonies,
at first with perfectly even borders, later sinuously broken,
not unlike liquefying sarcina colonies.. With accurate
focusing the form of tetrads is recognizable in the gray,
transparent peripheral portion; toward the center the
colony is opaque, shaded gray. Deep: Irregularly formed,
smooth border, opaque, delicately to coarsely granular
(7, vm).

Gelatin Stab.—Stab : At first thread-like, later, in upper
part very granular, in lower part like a string of pearls,
white (7, 1). Surface growth: After ten days from 3mm.
to 4 mm. broad, irregularly round, partially lobulated,
much elevated in the center, like the head of a nail, moist,
pure white or somewhat yellowish, shining (7, m1).

Agar Plate.—The same as upon gelatin, only much
more luxuriant, opaque (7, VI). :

Agar Stab.—Sitab: Confluent, very rough, pure white.
In old cultures there often occur luxuriant outgrowths in
clumps (7, v). Surface growth: Irregularly round, sin-
uous or wavy. Much elevated, often with terrace-like
formation, pure white, with an oily luster, at times with
a suggestion of yellow (7, 1v). The agar streak corre-
sponds. Water of condensation clear, with white precipi-
tate (7, 1). |

Bouillon Culture.—Clear ; moderate precipitate, upon
shaking becoming distributed at first as flocculi and then
homogeneously.

Milk Culture.—After four days firmly coagulated, at
other times coagulation is absent. ;

Potato Culture.—Limited to the streak of the inocula-
tion, sharply outlined from the surroundings, but not
elevated. Border of the growth irregular and jagged,
pure white, dull, orfaintly shining. According to Gafiky,
thick, slimy, tenacious (7, x).

Chemical Activities.—It produces some acid upon

ad Ad Pict iad a A Ratti tes he

Pe a ee a ee ae

MICROCOCCUS TETRAGENUS. 173

_ grape-sugar bouillon, and a strikingly strong odor of glue

upon agar plates. It does not liquefy gelatin, nor does it

_ form H,S or indol upon 2% solution of peptone.

_ Distribution.—(a) Outside the organism: We have
never met it.

(b) In the healthy organism: In the mouth ; found by
Boutron in human milk.

(c) In diseased human organism: In pulmonary cavities
in phthisis (Gaffky); in abscesses.

(d) In animals: Found as cause of suppuration several
times (Karlinski, C. B. vir, 113).

Experimental Observations Regarding Pathogenic
Effects.—(a) Upon animals: In white mice it causes a
rapidly progressing septicemia. Guinea-pigs and white
rats are similarly susceptible. In rabbits there is usually
only a local affection (peritonitis, abscess, etc.). Gray
rats and gray mice are very resistant or even immune.

(6) In man: It has been demonstrated that the organ-
ism causes suppuration and not merely accompanies it
(Viquerat, Z. H. xviu, 411).

Special Methods of Detection.—Agar plates, micro-
scopic picture, experiment on the mouse. In bouillon
and hay decoction no packets of sarcina are formed.

Morphologically identical but not pathogenic is the
Micr. tetragenus albus Boutron. The Micr. tetra-
genus aureus Boutron is liquefying, non-pathogenic,
and was grown from human milk. _It was observed by
Boschi and Bellei (C. B. xxim, 856), after repeated
growth, to become colorless. They very properly con-
sider all these forms as only varieties of the Micr. tetra-
genus.

Related Varieties.

We are unacquainted with the Micr. tetragenus sub-
flavus, obtained by Besser from nasal mucus, which did
not grow upon gelatin, and was yellowish upon agar
(Ziegler’s ‘‘ Beitriige zur path. Anat.,’’ v1, 347).

We are unable to differentiate the Actinobacter poly-
morphus Duclaux by means of a culture from Kral.

The Micr. tetragenus mobilis ventriculi Mendoza
(C. B, v1, 566) is theoretically interesting. From the de-

Ageia

174. IMPORTANT VARIETIES OF FISSION-FUNGI.

scription it cannot be differentiated from the Mier. tetra-
genus Gafiky and Koch, except that it forms some skatol.
It possesses a very lively spontaneous motion, and consti-
tutes, until more material is at hand, the motile, presum-
ably flagellated, related form of the Micr. tetragenus (see
Micrococcus roseus).

Micrococcus rosettaceus (Zimmermann) (i, p. 72).

According to the description of Zimmermann it is almost identical
with the Micr. candicans, but upon gelatin it is grayish-white and upon
potato of a yellowish-gray color ; size from 0.7 to 1.0 u.

Micrococcus concentricus (Zimmermann) (i, p. 86).

Upon all nutrient media it forms only a thin, delicate, iridescent
growth, resembling somewhat the Bact. typhi. Upon gelatin plates
the border is irregular, and concentric zones are almost always seen on
gelatin. It never liquefies gelatin. Upon potato thin, yellowish-
gray, slimy growth. They are 0.9 » indiameter. Found by Zimmer-
mann in Chemnitz hydrant-water.

Micrococcus viticulosus (Katz).

This was, so far as we know, isolated only once by Katz in Fliigge’s
laboratory in Gottingen. In the gelatin stab and in deep colonies in
gelatin plates it forms delicate white tendrils. We know it only from
the description, according to which its cultures evidently have a great
similarity to those of the Bact. Zopfii, which we have represented in
Plates 29 and 30. Gelatin is not liquefied. The cocci are always oval,
being 1.2 u long and 1 » broad.

Micrococcus of Bitter Milk (Conn) (C. B. ix, 653).

Rather large coccus, non-chromogenic. Gelatin rapidly liquefied,
which, as well as bouillon, becomes very mucilaginous. Milk at first
is coagulated, then becomes clear and slimy. It tastes faintly acid, but
is very bitter.

Micrococcus Freudenreichii (Guillebeau).

Large cocci (2 “ and over in diameter), usually single, rarely (in
bouillon) arranged inchains. In milk gelatin, the colonies first appear
white, entire, finely granular; after two days rapid liquefaction occurs.
Agar culture is white. Potato culture sulphur-yellow to yellowish-
brown, at times delicate and at other times luxuriant. Bouillon first
becomes cloudy, then clear witha flocculent sediment. In sterile milk

:
+
f
i"
\

=
meee

MICROCOCCUS CORALLIOIDES. 175

there is formation of acid, early there is marked stickiness (tenacity'),

after a few days coagulation. Optimum 209°, Growth occurs from 11°
to 35°. It is perhaps a streptococcus.

a,
Micrococcus acidi lactis (Kriiger) (C. B. vii, 425,
464, 493).
' Oval coccus, forming diplococci and tetrads, from 1.0 “ to 1.5 in

diameter. It is a facultative anaerobe. Round, white colonies in
. with ragged border. Gelatinis liquefied. Gelatin stab. Stab:
: n

ular, white growth. Surface growth: White and later sinking
downward. From milk-sugar it forms lacticacid. Milk is coagulated
in five days at from 15° to 35°, then the albuminous bodies are pep-
tonized with the production of a sticky character and a pasty odor.

Micrococcus coronatus (Fliigge) (Ed. iii, p. 178).

Round cocci, from 0.8 “ to 1.64. Gelatin plate. Natural size: At
first small, white disks, which, when they are on the surface, have a
broad zone of liquefaction. At this stage, if magnified sixty times,
the colonies appear as gray, coarsely granular disks, with ragged bor-
ders, and later they break up into fragments and crumbs. The picture
of the natural-sized colonies later is quite changed; while a yellowish-
white, irregular clump lies at the bottom of the shallow funnel of
liquefaction, the clear funnel of liquid is surrounded by a zone of
sturdy, irregular points and outgrowths, which make the picture very
striking. The gelatin stab culture resembles that in the plate.

Agar plate : Deep colonies, round, white, almost opaque. Superficial,
at first round, then ragged, lobulated, wavy, luxuriantly developed.
Agar streak: Grayish-white, broad, jagged, somewhat dry. Potato
culture: Similar. Bouillon : Slightly cloudy with sediment. No in-
dol, a trace of H,S is formed. Milk becomes gelatinous in ten days ;
after fourteen days it is coagulated with a minimal acid reaction.

Found by Fliigge many times in examinations of air, by us in ex-
amination of smegma.

Micrococcus corallioides (Zimmermann) (ii, p. 72).

According to Zimmermann’s description it resembles the preceding,
yet is entirely different. Gelatin plate. Natural size: Colonies appear
as white, somewhat irregular masses, which after eighty hours form
outgrowths all around, so that finally they lie in the half liquefied
gelatin, with radiating, often branched, formations extending in all
directions. Magnified one hundred times the masses of bacteria appear
granular. Also the milk-white growth at the top of the gelatin stab
sends out indistinct outgrowths. Upon agar a broad, milk-white
growth, upon potato very little growth. Meat-infusion is uniformly
cloudy. Found by Zimmermann in water.

1 Weigmann’s Micr. of tenacious milk does not liquefy gelatin.

176 IMPORTANT VARIETIES OF FISSION-FUNGI.

Micrococcus radiatus (Fliigge) (Ed. iii, p. 179).
Micrococci of less than 1 4. _ The deep colonies in the gelatin plate, —
which are at first granular and Sharply outlined, when we | come to —
the surface become surrounded by @ Ting of pretty rays, w separate —
a little at the periphery, so that the colony is somewhat v
outlined. Later second and third rings of rays may develdp. ‘Im the
gelatin stab culture there is a pointed funnel of liquefaction. Onn
the lower part of the stab horizontal outgrowths radiate, so that the
stab appears as if feathered. We have not seen it; description is from
Fliigge. The color is described by Fliigge as white, with a yellowish-
green shimmer.

Micrococcus luteus (Lehm. and Neum.).
(Plate 6, I-v.)

Synonyms.—tThe insufficiently defined Micrococcus
luteus Cohn, designated by Schréter as Bacteridium luteum,
is not to be identified as a particular variety. We desig- —
nate the species to be described in this way, to express the
relation to the Sarcina lutea.

Microscopic Appearance.—Medium sized (0.4 » to
1.2 »), roundish cocci, often lying together in fours, often
only in pairs.

Relation to Oxygen.—In shake cultures strongly aero-
bic.

Requirements as to Temperature and Nutrient
Media.—Grows rapidly and luxuriantly at room and incu-
bator temperature upon all nutrient media.

Gelatin Plate.—(a) Natural size: Yellowish to yellow-
ish-white irregularly round colonies, after three days from
14 mm. to 2 mm. broad. In a short time they sink in,
without the form of the colonies being disturbed. Later
there follows a breaking up of the film into irregular gran-
ules and debris.

(b) Magnified fifty times. Superficial: Yellowish-gray to
grayish-brown, irregular, roundish colonies with wavy,
scalloped borders. In the periphery, at times individual
tetrads are plainly visible. The outer part is more trans-
parent than the central portion. The interior is uniformly
shaded in a gray color. Deep: Roundish to whetstone-
shaped, smooth border, finely granular, of the same color
as the superficial (6, Ir).

MICROCOCCUS LUTEUS. 177

Gelatin Stab.—Stab: Until liquefaction begins it is
granular. After two days liquefaction begins with a plate-
shaped depression, which later becomes cylindric. The
contents of the funnel are cloudy, greenish or yellowish-gray
6, 1).

ae Plate.—Both when of natural size and when
magnified fifty times, the colonies are like those in the
gelatin plate, only the granulation is finer. Sometimes
there are found thin, pale yellow, transparent deep colo-
nies, as much as 2 mm. broad, with a coarsely granular
or morulated structure.

Agar Stab.—Stab: Granular, yellow. Surface-growth :
Lemon-yellow, shining, roundish, with wavy border, some-
what elevated.

Agar Streak.—Corresponding to the stab. Water of
_ condensation clear, sediment yellowish.

Bouillon Culture.—Remains clear. The yellowish
precipitate is closely packed, rising up tenaciously only
with energetic shaking, and afterward becoming homoge-
neously divided.

Milk Culture.— After twenty days it is half coagulated.
Acid reaction.

Potato Culture.—Lemon-yellow to yellowish-green,
thin, faintly shining, with a wavy, irregular border, and
almost no elevation whatever. Sharply outlined from the
surroundings.

Related Varieties.

We consider this form identical with sarcina lutea—only
our form produces no sarcina groups, either upon solid
nutrient media, or in bouillon, or even in hay infusion.
Sarcina lutea would be its ‘‘ sarcina form.”’

The Streptococcus liquefaciens and Pediococcus flavus,! obtained
from Kral, are identical. The Strept. liquefaciens only produces a
cloudiness of bouillon and of the funnel of liquefied gelatin, and in old
agar streaks has a brownish-yellow shade—variations which are con-
stantly observed in Mier. pyogenes a aureus. From the description the
Micr. galbanatus Zimmermann is also identical, and, as we saw
subsequently, was found by Zimmermann to be identical with the

* Recently we have discovered beautiful packets of sarcina from old
hay infusion cultures of Pedioc. flavus. These were not so evident in
Strept. liquefaciens,

12

178 IMPORTANT VARIETIES OF FISSION-FUNGI.

Strept. liquefaciens Kral. One should ordinarily give the unambigu-
ous name of Zimmermann the preference over Micr. luteus, yet we
desired to indicate the analogy to the Sarc. lutea.

Micrococcus flavus (Fliigge). Lehm. and Neum.

Completely identical with the former, only it has finely granular
gelatin colonies and less tendency to the formation of tetrads. We
consider this form identical with the already described Sare. flava,
with which it corresponds, with the exception of the ability to produce
sarcina packets. We have obtained this organism as Staphylococcus ©
citreus from C. Frankel and as Sarc. flava from Prague. The latter —
was always without sarcina packets. We have been unable also to —
differentiate what we obtained as Micr. citreus agilis Menge (C. B.
xu, 49). It is devoid of flagella, very weakly liquefying, and non-
motile.

There appear to be transitions from the Micr. flavus to the Mier.
luteus.

Micrococcus sulfureus Zimm., Elaborated by Lehm.
and Neum.

We cover, provisionally, with this name all lemon-yellow as well as —
greenish to grayish-yellow cocci, which do not liquefy gelatin, of
which we have cultivated many from the air and water. They were —
all finely granular upon gelatin plates. We consider them as non-
liquefying forms of Micr. flavus L. and N.' Here also belongs the
Micr. sordidus Schroter.

Micrococcus sulfureus 7 tardigradus (Fliigge).
(Lehm. and Neum.)

Microccocus flavus tardigradus (Fliigge), page 178.

It is differentiated from the former only by very slow growth.
Found by Zimmermann in water. Only a variety of the former. Once
we found a Micr. sulfureus in the air whose superficial colonies pro-
duced sometimes no, sometimes very little, and again very active,
liquefaction, thus being a transition to the Micr. flavus.

Micrococcus badius (Lehm. and Neum.).

Medium-sized, round cocci, often united in tetrads, never showing
sarcina forms upon any nutrient medium. In the gelatin plate the
colonies appear as glue-brown, slightly elevated, transparent drops,
which when magnified sixty times appear entirely homogeneous or at

1 We have never found coarsely granular; non-liquefying forms re-
sembling the Micr. luteus.

most with concentric zones. Agar plate is similar. Gelatin stab: a
not very luxuriant, glue-brown, shining growth ; along the stab a del-
ieately granular growth. ‘Agar stab: Moist, transparent, glue-brown.
Gelatin is very slowly and slightly liquefied. Bouillon uniformly
cloudy. Upon potato there occurs a dark yellowish-brown, gelatinous
growth. Growth always slight. No growth in milk.
: It was obtained from Kral as Sare. lutea, and was not met by us
- elsewhere. It reminds one of the Sare. fulva Stubenrath.

MICROCOCCUS ASCOFORMANS. 179

Micrococcus ascoformans! (Johne).

- Synonyms.—Discomyces equi Rivolta, Micr. botryo-
genes Rabe, Botryomyces Bollinger, Botryococcus ascofor-
mans Kitt.

Literature.—Kitt (C. B. 111, 177), Schneidemiihl (C. B. xxtv, 271).

Fig. 15.—Ascococcus Billrothii Cohn (after F. Cohn).

It occurs in the tissue and pus of the pathologic forma-
tion, grouped like grains of sand, surrounded by a gelatin-
ous mass with a double-contoured, shining covering. In
cultures no capsule is formed, except that upon blood-serum
hartshorn-like plugs occur.
According to Johne’s description the cultures very much
resemble those of the Micr. luteus and flavus. The micro-
cocci are usually arranged in pairs or fours. Gelatin plate:

* The Micr. ascoformans recalls involuntarily an organism which
Cohn had described as Ascococcus Billrothii. It forms spherical or
lobulated colonies upon artificial nutrient media, which possess a thick,

_ gelatinous or cartilaginous capsule. A similar organism was described
by Hankin as Ascococcus cantabridgensis, obtained from the mouth of
astudent in Cambridge. The coccus quickly covers agar with a trans-
parent, slimy, very delicate covering of yellowish-white color, and

habe rather slowly in bouillon and gelatin. It is different from Asc.
illrothii in the oblong form of its individual groups and the less dis-
tinctly visible capsule.

180 IMPORTANT VARIETIES OF FISSION-FUNGI.

Macroscopically they appear as if sprinkled with grayish-
yellow pollen, with a fruit-like odor.* Magnified siaty
times: Round, sharply outlined colonies without special
characteristics. In the gelatin stab culture liquefaction
takes place slowly, with a cup-shaped depression. The
growth along the stab is white and thread-like. Upon
potato a hoartrost-like yellowish deposit with a fruit-like
odor. Upon agar the growth is scarcely perceptible. q
Kitt has expressed the belief that the organism is only a
special form of the Micr. pyogenes—which requires farthog
inv estigation.

It is pathogenic for guinea-pigs, sheep, goats, cows, |
swine, and especially for horses. It is found in thick, ©
cord-like or nodular connective-tissue growth, usually soft
ened in the center, in the perimysium, subcutis, spermatic
cord (after castration) and the retroperitoneal ‘connective.
tissue of horses. Besides, it is found in the lungs, udder,
lymph-glands, ear muscles, nasal mucous membrane and
bones. Recently cases have been described also where |
botryomycosis occurred in man (compare Schneidemihl, —
(; 3). ; :

Micrococcus pyogenes (Rosenbach)(Lehm.and Neum.).
(Plates 8 and 9, I-11.)

a Aureus (Rosenbach) Lehm and Neum.
& Citreus (Passet) < 6 7
y Albus (Rosenbach) ‘ c< 6

Synonyms.—Staphylococcus pyogenes aureus Rosen-—
bach, Staph. pyogenes albus Ros., Staph. pyogenes citreus —
Passet. ? :

Ordinary Names.—Grape coccus, pus coccus, simply —
‘* staphylococcus. ’’

se eS | ee fe be,

Principal Literature. — Rosenbach, ‘‘Mikroorganismen bei den
Wundinfektionskrankheiten des Menschen, ’? 1884; Passet, ‘‘ Aetio-
logie der eitrigen Phlegmone,’’ 1885 ; Garré, “Fortsch. d. Medic. Mi

1 Our Micr. luteus also possesses a sometimes agreeable sometimes —
disagreeable sweetish odor.

2 Compare also p. 187, regarding the Staphylococcus citreus Passet.

MICROCOCCUS PYOGENES. 181

1885, 11, 165; Liibbert, ‘‘ Biologische Untersuchungen tiber den Staph.
_pyog. aureus,’’ Wiirzburg, 1886.

Introductory Remarks.—For the comprehension of

the three forms given above as varieties of one form cer-

tain proof was hitherto lacking. R. O. Neumann (A. H.
xxx, 1) furnished it when he observed that in orange-
colored colonies sometimes lighter white or yellow sectors
appear (similar to those in Micr. bicolor), and by inocula-
tion from these, cultures are obtained which still more mark-
edly present the formation of paler sectors. By repeated
consistent transfers in this way white and yellow cul-

tures can be grown from orange-colored cultures, and even

a red culture could be obtained. These new cultures re-

“main in part permanent and in part revert to the original

form. Also consult Neumann concerning what was other-
wise known regarding the variations of this form.

Highly probable synonyms: Micrococcus liquefa-
ciens conjunctive Gombert, Eisenberg, 301; Micro-
coccus flavus conjunctive Gombert, Eisenberg, 302 ;

Staphylococcus salivarius pyogenes Biondi, Eisen-

berg, 309.

The effort of various authors to found a specific differentiation of
the three forms upon varying virulence is wrong. In the first place,
the fact that the golden-yellow form is distinguished by special
virulence (v. Tavel, Lannelongue, and Achard) is disputed. Levy

_ found that the more common form in Strassburg was the white

form, and it was just as pathogenic. In the second place, it is easily
shown experimentally that enormous reduction of virulence is entirely
independent of the color (compare page 185). Growth without oxy-
gen which increases the virulence lessens the production of pigment.

In the following the Micr. pyogenes 2 aureus only is
particularly described. Regarding the ? citreus and y
albus see page 187.

Microscopic Appearance.—Round, smaller or larger
cocci, on an average 0.8 », in pairs or singly, usually in
grape-like clusters. Often they have a small division
cleft (8, x and xr).

Relation to Oxygen.—Grow well aerobically and not
so well anaerobically.

Requirements as Regards Temperature and Nu-

_ trient Media.—Optimum at 37°, but grows well at room

temperature ; thrives upon all nutrient media, the pig-

au

182 IMPORTANT VARIETIES OF FISSION-FUNGI.

ment is developed most abundantly upon agar and po-
tato. )

Gelatin plate. (a) Natural size: Small, irregularly
roundish colonies of yellowish-white to yellow color. After
six days, 14 mm. in diameter. Old colonies are not much
larger. The colonies usually sink slowly into the medium
and become surrounded by a flat, plate-like zone of
liquefaction (8, vit). |

(6) Magnified seventy times. Superficial colonies: Round-
ish, faintly yellow to brown, with delicate, transparent
peripheral zone. Structure somewhat coarsely granular,
toward the periphery a little more finely granular (8, vm). .
Deep colonies: Roundish to whetstone-shaped, dark yellow
to brown, structure finely granular, border almost smooth.

Gelatin Stab.—Liquefaction along the line of punc-
ture after two to three days. The zone of liquefaction
is conical to bag-shaped, and later cylindric. The con-
tents of the cavity are grayish-white, cloudy in appear-
ance, and at the bottom a whitish to orange-yellow pig-
ment is deposited in little clumps. The intensity of the
liquefaction varies widely.

Agar Plate.—(a) Natural size: The superficial colonies
are round or roundish, orange-yellow, faintly shining,
evenly elevated, and as much as 4 mm. in diameter.
Deep: Roundish to whetstone-shaped, equally or more
deeply colored and never so large as the superficial (8, v).

(6) Magnified sixty times. Superficial colonies: Round,
almost or entirely even border, with transparent, delicately
-punctated peripheral zone, orange-yellow, toward the
center shaded to homogeneous gray, sometimes with a
more darkly colored ring near the periphery. Deep:
Colonies partly roundish, partly whetstone-shaped, dark
grayish-yellow, opaque, at periphery often somewhat more
coarsely granular. Often there are found in agar, broad,
pale yellowish, round, transparent colonies with the gran-
ulation more marked (8, v1).

Agar Stab.—Stab: Insignificant growth, at first thread-
like, later slightly granular. Surface growth: Roundish,
evenly elevated, with smooth, somewhat wavy border,
faintly shining, orange-yellow (8, m1).

Agar Streak.—Corresponds to growth in the stab.

MICROCOCCUS PYOGENES. 183

“Water of condensation cloudy. Precipitate whitish-

orange (8, 11).

Bouillon Culture.—Marked uniform cloudiness. On

the surface a delicate pellicle is formed. Sediment mod-

erate and upon agitation it breaks up into tiny flocculi.

_ In sugar bouillon the same.

_ Milk Culture.—<According to Passet’s and our own

_ observations it is gelatinous or firmly coagulated in from

one to eight days. According to Tavel it is flaky.

- Potato Culture.—Limited to the streak, at first whit-

ish, later pale orange-yellow, somewhat elevated and

erumbly, shining. Old cultures are wider, deep orange,

- and dry (8, rx).

’ Viability and Resistance.—(a) Jn body: Many cases

- in which the Micr. pyogenes has been found after very

_ long periods (from ten to thirty-five years), in areas

, (osteomyelitis) where they had been encapsulated during

” this time, appear to prove a long viability.
m= (6) In cultures : Very tenacious of life. Even after many
_ months are always alive, still there are no resting forms.

24 Resistance to—(a) Drying: According to Hagler, they

are alive after from fifty-six to one hundred days in dried

pus.
(6) Dry heat : According to Liibbert, they are killed in one
hour at 80°, and killed rapidly only at from 110° to 120°.
(c) Moist heat: Seventy degrees kills very quickly.
-(d) Cold: Are alive after sixty-six days in ice (Prud-
den).

. 3 (e) Disinfecting agents act rather slowly. In bouillon
cultures zs455 sublimate does not kill them within five
minutes.

_ Chemical Activities.—(a) Pigment production: Pro-

duces orange-yellow pigment of the carotin group (compare

p. 66), but only if oxygen is admitted. According to

Liibbert and F. Girtner, pigment production is greater, the

more oxygen is contained in the air.

._(b) Substances that are smelt or tasted: Agar cultures

smell like glue or spoiled yeast or paste (Becker, Passet).

(¢) Gas and acid formation from carbohydrates :

PNG here SEES

Whe CLE Ss Ts Bae PILE Pal AG Oe AS GOT BRR

aa

Rather abundant formation of acid from grape- and milk-sugar, but
no gas-formation. It forms from milk-sugar : lactic acid and volatile

184 IMPORTANT VARIETIES OF FISSION-FUNGI.

fatty acid ; from dextrose: lactic, acetic, and valerianic acids ; from
glycerin : lactic, isobutyric, valerianic, and propionic acids (Terni).

(d) Sulphuretted hydrogen: Rapid and abundant.

(e) Indol: Only a little. }

(f) Sometimes it produces a vigorous fermentation of
urea (Barlow, Mann). Other cultures are almost without
effect upon urea.

(g) Poisons: See under animal experiments.

Distribution.—(a) Outside the animal organism: In
milk, wash-water, dirty water (little in pure water and
soil), and.air it is widely distributed. The Micr. pyogenes
makes up 10% of the micro-organisms found in the air of
surgical operation room (compare Ullmann, Z. H. ry, 55).

(6) In healthy body: Upon the skin, especially of the
head, in the mouth and vagina, and not uncommonly in
the cervix uteri. Also found in the milk of healthy
women.

(c) In diseased human organism: All processes accom-
panied by suppuration or only inflammation in the various
regions of the body may be caused by staphylococci, and
are thus caused in a large percentage of cases. In other
cases they act together with the Strept. pyogenes, Strept.
lanceolatus, Bact. coli, Bact. typhi, etc. It must, how-
ever, always be firmly maintained that the last-named
organisms (as well as some others) may alone equally well
cause suppuration.

The following affections especially are often dependent
upon staphylococci : Acne of the sebaceous glands, sycosis
of the hair follicles, hydradenitis of the sweat glands,
pemphigus, ? phlegmon, furuncle, abscess, periostitis, osteo-
myelitis, septicopyemia. ?

They rarely cause erysipelas (Jordan). Fibrinous inflammation may
also be caused by them (Guthmann). Gradenigo and Maggiora ob-

served croupous inflammation of the nasal mucous membrane caused
by staphylococci (C. B. vii, 641).

1 Consult also page 188.

? Sahli has demonstrated it in the joints in a case of articular rheu-
matism. Singer has constantly cultivated staphylococci or (more rarely)
streptococci from the urine in seventeen cases of severe and mild artic-
ular rheumatism. The organisms were abundant during the disease
and disappeared with recovery (C. B. XVIII, 130).

i

MICROCOCCUS PYOGENES. 185

The following inflammations are also produced .by the Micr. pyo-

genes, but more rarely than by other varieties, as Strept. lanceolatus,
Btrept. pyogenes, etc. : pleuritis and pericarditis, pneumonia, hepa-
titis, etc.

(d) In diseased animal organism: Just as in man as the
cause of suppuration. Statements that animals have other

causes of suppuration than man are erroneous. It caused

_ an epidemic osteomyelitis in geese (Lucet) and a disease of

_ gudgeon (Charrin) in France.

Experimental Observations Regarding Pathogenic

_ Effects.—There is an extraordinary variation in the dis-

a

_ position of different apparently identical experimental ani-

mals, as also in the virulence of the micro-organisms them-
selves.

The disposition is greater in young, anemic and dia-
betic animals. The virulence of micro-organisms freshly
obtained from an animal or man is often considerable, but
sometimes even in this case it is slight. By growth upon
our artificial nutrient media it is sometimes rapidly

reduced and sometimes almost imperceptibly changed. By
repeated passage from animal to animal, in fatal doses,

_ the virulence. is increased for the concerned species
_ (Terni), also simultaneous inoculation with other bacteria

(Ortolani and De Blasi) or their metabolic products (for

example, the Bact. vulgare) increases the virulence. In
_ the same way repeated anaerobic cultivation heightens the

virulence. The intensity of the liquefaction of gelatin is

_ almost, but not entirely, parallel with the pathogenic
_ property.

With the most exalted virulence the staphylococcus

- causes no local suppuration, but a gelatinous edema, hem-
orrhages in thé kidneys, often inflammatory changes in

Po hey | Cee

a, | ‘
ee =

the cardiac valves and in the aorta. The relative suscep-
tibility of animals to infection with staphylococci is shown
in the following decreasing series: Horse, dog, man, cat-
tle, sheep, rabbits, guinea-pigs, mice. In the last-named
numerous bacteria are necessary for infection.
Subcutaneous injections cause abscesses, but a rather large

number of germs (according to Herman, 50,000,000 indi-
viduals equal 1 c.c. of culture) from a not highly viru-

lent culture is required to do this in rabbits.

bait
Ts 7

ae

i oy

186 IMPORTANT VARIETIES OF FISSION-FUNGL.

Intraperitoneally large quantities (13 ¢.c. and more)
are borne by rabbits ; infection of the pleura and surface
of the lung has often been undertaken.

Intravenous injection causes endocarditis, especially after
previous injury of the valves of the heart, and usually
also nephritis. The hyperemic kidney shows, macro-
scopically, yellowish, wedge-shaped areas in the cortex,
in which the straight urinary tubules are filled partly with
casts partly with cocci, and some are empty and com-
pressed. Cocci are found in the urine.

Injection into joints causes suppuration.

In man acne, furuncles, and phlegmons have been pro-
duced by rubbing into the skin.

Toxins, Immunity and Immunization.—The filtered
bouillon culture contains toxic substances which act in-
tensely. Injection of this into the peritoneal cavity of
dogs causes a sero-bloody peritonitis, eecchymoses in the
serosa and mucous membrane of the intestine, and death
from bloody diarrhea. By suitable modification of the
toxic property, quantity, etc., of the injected metabolic
products Kraft could produce all the forms of typical
peritonitis.

Subcutaneous injection of the filtered bouillon culture
produces all the processes, from a doughy swelling, which
disappears without suppuration, to typical suppuration,
even to fibrino-hemorrhagic necrotic inflammation. This
depends upon the virulence of the bacteria employed.

According to Viquerat (Z. f. B. xv, 487), the bouillon culture
contains no specific poisons, only pyogenic bodies, which are widely
distributed. —

After repeated injections of sterilized or filtered cultures
different authors have obtained entirely different results.
We quote only two examples:

Reichel observed a greater or less resistance to intraperitoneal in-
jection of the staphylococcus poisons in animals which he had injected
intraperitoneally for a long time at intervals of from two to five days
with filtered or otherwise sterilized bouillon or gelatin culture, and
even a relative immunity against the pus cocci. themselves. Nannotti
observed only chronic intoxication and no immunization in animals

_ which had received many injections of the metabolic products.

According to Rodet and Courmont, this contradiction is explained
by the simultaneous presence, but in varying proportion, of an immu-

MICROCOCCUS PYOGENES. 187

_ nizing and a predisposing substance, the former being precipitated and
> the latter soluble in alcohol. Tavel was, however, unable to produce
itaimunity with the alcoholic precipitate, the animals either dying

_ from chronic intoxication or succumbing to an additional injection

with virulent cocci.

The serwm of actively immunized animals has no noticeable effect
upon the Micr. pyogenes in vitro; it is also, so far, of scarcely any
_ practical value for producing passive immunity.

Special Culture Methods.—Isolation is accomplished
- most rapidly by means of agar plates at incubator tem-
perature. The potato culture is best for the study of the
chromogenesis. Milk cultures and animal investigations
are necessary.

Micrococcus pyogenes ; albus (Rosenbach).

In all respects like the Micr. pyogenes @ aureus. See
Plate 9, 1 and m1, and the remarks on page 181.

Here belongs the Micr. urez liquefaciens Fliigge (com-
pare page 71).

Micrococcus pyogenes £ citreus (Passet).

We have studied this organism only in a culture ob-
tained from C. Frinkel, and designated by him as identi-
cal with the Micr. flavus (page 178). It did not coagu-
late milk and produced a slow liquefaction of gelatin with
formation of air-bubbles. A Micr. pyogenes citreus is
said to exist, however, which corresponds entirely with
the Micr. pyogenes aureus except in the color. With this
the results of cultures by R. O. Neumann agree (page
181).

Varieties Closely Related to or Identical with the
Micrococcus pyogenes Ros. (Lehm. and Neum.).!

Micrococci in Variola.
Vanselow and Czaplewski ( Vierteljahrsschr. f. gerichtl. Med., 1899,

Heft 1) believed they had found an organism closely connected with
the variola process in what was previously named by Klebs the Micr.

1 The old names, Staphylococcus cereus flavus and Staph. ¢ereus
albus Passet, can not be sharply defined, and can well be dispensed
with. These varieties are rarely cultivated from pus and grow upon

188 IMPORTANT VARIETIES OF FISSION- FUNGI.

quadrigeminus Klebs. It was very like the Micr. pyogenes albus.
( However, it liquefied solidified blood-serum, which the typical Micr.
pyogenes is said not to do; its color is reddish, but in this property is
variable, as is to be expected.) They have already retracted this
hardly probable suspicion (C. B. xxv, 546).

Almost simultaneously Sanfelice and Malato (C. B. xxv, 641) have
reported that a coccus can be constantly cultivated from cases of variola,
which can not be differentiated morphologically from the Micr. a
aureus, but differs in its pathologic action from all other cultures of
Micr. pyogenes isolated by the authors. When injected into the cir-
culation, hyperemia of the skin and mucous membrane and sharply
outlined hemorrhages occur.

Regarding the much controverted ‘‘Cytoryctes variole’’ Guar-
nieris, of the group of protozoa, consult the literature in Galli-Valerio,
Kritische Uebersicht iiber den Zusammenhang der Variola mit Vaccine.
(C. B. xxv, 380 and 424).

Staphylococcus pemphigi neonatorum Almquist 4
(Z. H. x).

According to Strelitz (C. B. x11, 107), the Micr. pyogenes is itself
the cause of pemphigus, and besides being cultivated from pemphigus
vesicles, is able to reproduce the condition. Others obtained similar
results ; for example, Bodenstab (compare Vogel) found that four chil-
dren cared for by the same midwife developed pemphigus within two
weeks (C. B. XXI, 288).

Micrococcus biskra Heydenreich.

Cause of the Pende’s ulcer, tropical ulcer, Delhi boil, Clou de
? ?
Biskra, etc.)

According to the description of Heydenreich, it can not be differen-
tiated from the Micr. pyog. a aureus (C. B. v, 163). The statement
by Chantemesse (C. B. v, 221) that the gelatin is very slowly liquefied
also applies to many cultures of Micr. pyogenes. Chantemesse gives
as other points for differentiation from the Micr. pyogenes, its whitish
growth upon agar, and*its luxuriant, rapid, watery and orange-red

the surface in the gelatin stab as a faintly shining, waxy deposit with
a somewhat thick border. Both varieties are closely related to the
Micr. § citreus and y albus. They often pass as forms of these, but
are differentiated, according to the insufficient description at hand, by
absence of liquefaction and slight or no pathogenic quality. —

Without being able to show this interpretation to be incorrect, we
refer to our note (p. 170) that the Micr. cereus albus was found by us
to be identical with the Micr. candicans Fliigge, with the exception
of its smaller size. We are not familiar with the Micr. cereus flavus;
it may perhaps belong to Micr. sulfureus Zimmermann.

1 The Dipl. pemphigi acuti Demme, appears different. Is grown
only at incubator temperature (Cong. inn. Med. Wiesbaden, 1886).

MICROCOCCUS BICOLOR. 189

growth upon potato. These characteristics are not sufficient for sepa-
rating it, especially as Heydenreich does not describe his potato cul-
tures as essentially different from those of the Micr. pyogenes. Rapt-
_ schewsky declares (C. B. vi, 504) the Micr. biskra identical with the
_ Mier. pyogenes, and prefers to consider a streptococcus as the cause of
_ the disease.

Micrococcus of gangrenous mastitis in sheep, Nocard (A. P.1 417).
Staphylococcus hzemorrhagicus, E. Klein (C. B. xxu, 81).

De Jong’s Staphylococcus bovis is said to be different from the
Mier. pyogenes. Injected subcutaneously, intraperitoneally, and in-
travenously it is pathogenic for rabbits, dogs, and guinea-pigs.
Neither its white nor its yellow form liquefies gelatin, in spite of
luxuriant growth ; milk is not coagulated; in bouillon it forms a

' delicate, tenacious sediment.

The cause of a circumscribed falling of hair, without discolora-

. tion of the hair-bed and without a tendency to spread, is found,

according to Vaillard and Vincent, in a white liquefying coccus, 1
_ in diameter, which corresponds throughout, in its growth, to the

_ Mier. pyogenes y albus (A. P. Iv, 1890, 446).

7 (Literature by Hollborn, C. B. xvi11, 47, 108.)

ree peraien

Micrococcus bicolor (Zimmermann).

Round cocci from 1.2 4 to 1.6 4. Gelatin plate: At first yellowish,
_ succulent, elevated ; later, orange-yellow, slowly sinking, oily looking
_ golonies of round form; besides these there are others about the same,
_ but white in color. Magnified sixty times they are even-bordered and
faintly granular. Gelatin stab: Superficial growth is white, with a
slowly forming cup-shaped liquefaction. The growth along the stab
is thread-like. Agar plate is like gelatin, and also presents gray and
yellow colonies intermixed. Agar streak: Succulent, whitish or gray-
_ ish-yellow growth with orange-yellow islands and points. The surface
_ growth in the agar stab always presents more or less perfect gray and
_ orange sectors, from which it is often possible to_obtain pure gray or
__ pure orange-colored growths, but which in following generations again
produce the two colors. Bouillon becomes diffusely cloudy with mod-
erate, firm precipitate. Milk becomes a little acid and remains fluid.
Upon 2% peptone bouillon it forms a trace of H,S and indol. We
have obtained this organism, which was isolated by Zimmermann from
tap-water, from gastric contents. The Micr. cremoides Zimmer-
mann is very closely related to this. We were entirely unable to
differentiate the culture obtained by Zimmermann.

Also, the Micr. aurantiacus Cohn, which we obtained from Kral,
is distinguished only by the absence of liquefaction. We have also
obtained from it white, orange, and striped cultures, which pass from

one into the other.

=" At present we can give no other decisive characteristics of the Mier.
bicolor, awrantiacus, and even of the Micr. candicans as differing from

_ the Mier. pyogenes except the pathogenic action in animals and absence

of liquefaction.

190 IMPORTANT VARIETIES OF FISSION-FUNGI

Micr. roseus (Bumm) (Lehm. and Neum.).
( Plate 11.)

Synonyms.—Diplococcus roseus (Bumm) Fliigge.
See end of section.

Microscopic Appearance.—Round to irregularly
roundish cocci (from 0.6 » to 1.04), often with rather wide
line of division in the cocci (11, xr), at other times more
complete cocci lie together in pairs and small groups.

Motility is lacking. Compare page 192.

Relation to Oxygen, Nutrient Media, and Temper-
ature.—Grows slowly upon all nutrient media, best at
room temperature, also at 37°. In shake cultures it grows
only near the surface, the deep colonies only very slightly.
Pigment is only produced when air is admitted.

Gelatin Plate.—(a) Natural size: Superficial, irregu-
larly roundish, small, rose-red. After a long time they
become somewhat larger, evenly elevated, shining. The
deep colonies grow very little. After weeks the superficial
ones sink gradually into the gelatin.

(b) Magnified fifty times: Round or roundish colonies,
almost even borders, rather finely granular, colored pale
to rose-red. The deep appear the same, only they are
smaller (11, vm).

Gelatin Stab.—Stab: Thread-like. After several weeks
the gelatin begins to liquefy in a cylindric form. After
three months the growth has sunk in about 1 em. Sur-
face appearance: Roundish, sometimes lobed, rose-red
growth, which, later, on account of the liquefaction of
the gelatin, is almost entirely lost (11, 1).

Agar Plate.—(a) Natural size: Like gelatin.

(b) Magnified fifty times. Superficial: Round or round-
ish colonies with even or somewhat wavy border, yellowish
to red, from the most delicately punctated to coarsely gran-
ular (11, v), transparent, more intensely colored toward
the center. Deep: Roundish to whetstone-shaped, border
smooth or granular, finely to coarsely granular (11, v, v1),
opaque, darker than the superficial in color.

Agar Stab.—Stab canal: Thread-like, later granular
(11, 11). Surface growth: Roundish, evenly elevated, oily,
rose-red, of the consistency of butter (11, Iv).

MICROCOCCUS ROSEUS. 191

Agar Streak.—Growth spreads little, smooth border,
wavy. Water of condensation clear, reddish sediment
pc ii, 1).

Bouillon Culture.—Clear (only rarely more or less
cloudy). Sediment reddish, abundant, and coherent.

Milk Culture.—Usually unchanged.

Potato Culture.—Limited to streak, faint rose, with
oily luster, somewhat elevated, often surrounded by a whit-
ish, glistening zone (11, x).

Special Nutrient Media.—lIi the Micr. roseus is grown

upon the culture of a representative of the subtilis or

anthrax group its colonies grow considerably more lux-
uriantly and take on a more intense color (11, Ix).
(Doubtless on account of the alkalinity of the potato.)

Distribution.—(a) Outside the body: Very common
and widely distributed air-organism, scarcely ever absent
from a plate from the air in Wiirzburg.

_ (6) Inside the body: Not demonstrated.

a We have closely compared this fungus—which, I believe, was pri-
marily described from Wiirzburg as ‘‘rose-colored diplococcus’’ of
Bumm—with the following imported varieties :
1. Mier. agilis Ali-Cohen, isolated by Prof. Zimmermann in Chem-
nitz
4 2. Micrococcus agilis Ali-Cohen, hygienic institute in Berlin.
__ 3. Micrococcus roseus (author 2) from Prof. A. Fischer in Leipzig.
4. Micrococcus tetragenus ruber. From Kral in Prague.
5. Staphylococcus roseus Tavel. From Prof. Tavel in Bern.
_ 6, 7, 8, 9: Four air micrococci from Wiirzburg, which at first ap-
peared to differ somewhat upon the plates. 2
10. A red micrococcus from the stomach.
The result of these comparisons was that these ten organisms all
_ belong to the Micrococcus roseus,! of which we can distinguish two
_ fairly sharply separated varieties. 2

wre

REM

6 as

ey ea oe

bP rhinos

Pepe

1 According to the description, the Micr. cinnabareus Fliigge, cinna-
barinus Zimmermann, Micr. carneus Zimmermann, may also be in-
serted among the varieties differentiated by us. The ‘‘new micro-
coccus’’’ from red milk, recently described by Keferstein (C. B. x X1,
177), appears also very closely related. The Micr. latericius Freund
(C. B. XxX1, 834) appears somewhat different, yet the experiences ob-
tained in the study of the group of the Bact. prodigiosum remind us
to be cautious in the formation of new varieties.

ak etree a

«2 We have observed white, yellowish-red, rose-red, and carmine-
y _ red sectors upon agar in both varieties. They are connected by transi-
_ tion forms.

Se

192 IMPORTANT VARIETIES OF FISSION-FUNGI.

f
%
”

Micr. roseus (Lehm. and Neum.).

(a) Typus.—Agar streak, rose to carmine, more rarely whitish-— :
red. Streak upon the subtilis-potato (compare above), deep carmine- |
red. Milk unchanged, with beautiful rose-red precipitate. Here be- i
long the Micr. agilis of Zimmermann from Berlin and three of our
alr-cocci. cs

(8) Roseo-fulvus.—Agar streak, reddish-yellow to vermilion-red. —
Streak upon subtilis-potato, orange-red. Milk not coagulated, with —
yellowish-red cream layer and yellowish-red precipitate.

Here belong, according to our investigations, Micr. tetragenus ruber —
Kral, Mier. roseus A. Fischer, Staph. roseus Tavel, and one of our air- —
cocci ; perhaps also the Micr. fulvus Cohn, which is very ios ape
described.

But we must go a step further still. The Sarcina rosea ~
Schréter (compare p. 162) also stands in close relation —
to the described varieties. The Sare. rosea, obtained from
Kral (it belongs to the variety roseo-fulva), forms beautiful
sarcina balls upon fluid but not upon solid nutrient media,
but was otherwise not to be differentiated (compare p.
163). After we had kept our ten red cocci upon hay
decoction for a month, one of our red forms (from air)
produced typical sarcina packets, while the others were
only brought to produce tetrads.

Thus also, the Sarcina rosea may be thought of as the —
forma sarcinica of the Micrococcus roseus. The Mier.
corallioides Cantani (C. B. xxut, 309) is also very closely ~
related, according to the description of the author, but the
name ‘“ corallioides” (rectius ‘‘ corallioides”’) is already ©
given to another organism (p. 175). 3

Our point of view demands a special explanation regard-_
ing the interesting organism found by Ali-Cohen and ©
Zimmermann in water.

3

— —— =. =

Micr. agilis Ali-Cohen (C. B. vi, 33).

We have not seen spontaneous motion nor a flagellum,
either in the culture from Berlin or in the one from Kral,
in spite of all our pains, as growing upon slant of 5%
milk-sugar agar, upon sugar hay-decoction, bouillon, ete.,
employment of higher and lower temperatures, young»
and old cultures, etc. Neither culture is to be differ-
entiated from our Micr. roseus.

FAMILY BACTERIACEZ. 193

our Micr. agilis only as a Micr. roseus, which once possessed flagella
and then lost them.
We believe our observation is of primary significance in classifica-
tion, as many investigators consider the flagella as a very important
d constant differential aid. Migula has formed a genus planococcus
_ for the Micr. agilis ; without our observations we should have as-
: ted. But being in possession of this, it seems to us that our con-
f or is at present more natural than the other possible one, namely,
t the Planococcus agilis, because of the loss of its flagella, can no

1 be distinguished from the Micr. roseus, but that it still belongs
to a different genus.

: i: Micr. cerasinus (List.) (Lehm. and Neum.)

_ Micrococcus cerasinus siccus List. (Adametz, ‘‘ Bakterien der Trink-
und Nutzwisser ”).

Very small cocci of 0.3 u. Upon gelatin cherry-red, without lique-
- faction. ; Upon potato, dry, spreading deposit of cherry-red color.

Pigment insoluble in alcohol and ether; whether in water, we do not
_ know.

. Micr. erythromyxa (Overbeck).
Compare Sarcina erythromyxa, page 162. Sarcina formation seems
_ to be entirely absent at times.

Micr. cyaneus (Schroter) (Cohn).

Forms a cobalt-blue deposit, pigment soluble (!) in water, turns

_ red with acids, blue returns with alkalies. Schroter also described a
_ yariety of this, pseudocyanea, that at first produced verdigris-green
_ either remaining so or later becoming bluish-green to blue. So far
_ it has not been further described. Obtained from the air in Breslau.

ing the Micr. cyanogenus, consult Pammel and Combs (C. B.
PL. Il, 764).

Il, FAMILY BACTERIACEAE (ZOPF EMEND.
MIGULA).

(For diagnosis of family, see p. 124.)

1. Bacterium.1!

Cells at least one and a half, but usually from two to
_ six, times as long as broad, straight or bent in a plane

1 The ‘‘bacteria’’ of tuberculosis and diphtheria and those closely

_ related to them are to be looked for in Appendix I, Actinomycetes
eeomrare p. 127).
3

194 IMPORTANT VARIETIES OF FISSION-FUNGI.

(compare p. 124), sometimes forming long true or ap-
parent threads, with or without flagella. Always with-
out endospores; ;1 in single varieties “arthrospores are de-
scribed.

Many hundred spore-free, short rods haye been de- ~

scribed, and the need of arranging them in a natural sys-
tem, founded entirely upon morphologic peculiarities,

was strongly felt. The only characteristic which is ques-—
tionable is that of flagella, and we acknowledge that the
:

system founded by A. Fischer and Migula upon the
flagella appealed to us very favorably until we had our-

selves worked extensively with the staining of flagella. The

results of these extensive and careful studies were unfor-

tunately not of such a nature as to allow a classification -
founded upon the number and arrangement of the flagella ©
to appear practical. Especially the statements in the ©
literature regarding flagella are often inexact, and a num- ~

ber of inaccessible varieties could not be classified at all.
At times we observed that closely related varieties, as in
the colon group, occur which have either one flagellum or
many or no flagella. What appeared yet worse was that,
as in Bacterium violaceum, we found one form with

flagella on all sides ; another with only one or with one —

polar and one lateral flagellum. Migula found it to have
one polar flagellum.

In addition, there are the experiences which we have
encountered regarding the permanent loss of flagella in
Micrococcus agilis Ali-Cohen, Micrococcus agilis Menge,
and Sarcina mobilis, and regarding the acquiring of motil-
ity by the Bacillus implexus, and reported in this book.

a
4
a
4
‘
S
+
7
{

ot

If we ourselves have not observed similar occurrences in ~
any bacterium, we find the statement made by Germano ~
and Maurea, that they have twice seen non-motile cultures —

of the typhoid bacterium.

Finally, we feared to scare the beginner from making

1 Upon ordinary media (bouillon, gelatin, agar, potato) these varie- —
ties never possess spores. As already remarked, we were also unable ©
(with a doubtful exception in the Bact. violaceum) to observe spore- —
formation upon quince and marshmallow decoction in those previously —
considered as not forming spores. Migula seems to have been more ~

fortunate, but gives no particulars.

KEY TO GENUS BACTERIUM. 195

differentiations if we placed before him, as the first ques-
tion in the table of differentiation, the character and num-

‘ber of the flagella ; for if the staining of flagella is no
- spe ial art, yet it requires care and patience, and does not
yield regularly good results even to the expert.

_ We have therefore been required to select the appear-
ance of the cultures in plates and the production of pig-
pen as the important points in the separation of the
" bacteria, although we well know (and also always men-
- tion it) how easily the production of pigment is lost in
“some varieties. According to our conviction, however, at
"present, the proper definition of a Bact. violaceum, syn-
_ ¢yaneum, etc., which has become colorless would consti-
- tite an (almost) insurmountable difficulty, no matter

_ how one might construct the key for differentiation.

F
i.
Key to the Recognition of the Most Important Vari-
eties of the Genus Bacterium.

OUT OUTGROWTHS OR LONGER RADIATING PROCESSES, NO
BRANCHES IN GELATIN STAB.
(A) No growth upon ordinary nutrient media ; on the contrary,
3 a very delicate growth upon inorganic saline solutions. Forms
_ hitrate from nitrite, or nitrite from ammonia.
Forming nitrite from ammonia, Bact. nitrosomonas (Win.), L.
and N., page 200.
| Forming nitrite from nitrate, Bact. Bitrubscter (Win.), L. and N.,
page 200.
| (B) Scarcely any growth on ordinary media, but grows well
: ‘upon pea-leaf decoction containing cane- -sugar, gelatin, and
_ asparagin. Assimilates the nitrogen of the air. Grows in the
3 root-tubercles of leguminous plants.
Bact. radicicola, Beijerinck, page 83.
__(@) Upon the ordinary nutrient media (including serum and
_ glycerin- agar) only a very scanty growth. Delicate, drop-like
a

-

r FORMING UPON NUTRIENT MEDIA, ROUNDISH COLONIES, WITH-
¥

dé

colonies. Not stained by Gram’s method.
1. Small, thin, non-motile rods. ©
(a) For growth, the addition of a little blood is necessary. Bact.
influenzz (R. Pfeiffer), L. and N., page 202.

(6) Grow also without blood. Bact. egyptiacum (Koch-Weeks),
“ L. and N., page 204. Bact. tussis convulsive (Czapolew-
3 my). © L. and N, , page 205.

4 2. ee arranged in pairs. Bact. duplex (Morax), L. and N.,
page 206

196 IMPORTANT VARIETIES OF FISSION-FUNGI.

3. Chains of slender rods. Bact. ulceris cancrosi (Kruse), L. and
N., page 207.

(D) Grow well upon all ordinary nutrient media, especially
upon agar and gelatin.

I. Colonies and nutrient media remain colorless.

(A) Gelatin not liquefied, organisms without flagella, non-mo-
tile.
+. No visible gas formed from grape-sugar.!

1. Not stained by Gram’s method. When coming from the
animal body they show polar staining. Form abundant
acid from grape- and milk-sugar. . Milk often not coagu-
lated. Growth on potato usually poor, whitish-gray.
Bact. septicemiz hemorrhagice, Hiippe, page 208.2 —

2. Very similar to 1. Causes tuberculous-like changes in the
animal. Bact. pseudotuberculosis rodentium, L. and
N., page 213.

3. Very similar to 1; usually still more delicate. Tendency
to formation of involution forms upon chlorid of sodium
agar. Bact. pestis (Yersin-Kitasato), L. and N., page
213. |

4. Stains by Gram’s method. Grows poorly upon solid
nutrient media ; marked formation of acid from sugar ;
milk is coagulated. Bact. Giintheri, L. and N., page
223.

. Stains by Gram’s method. Abundant growth upon solid .
nutrient media. No formation of acid from milk-sugar. —

Milk becomes slimy. Bact. lactis viscosum (Adametz),

L. and N., page 230.

+-+. Evident gas-formation from grape-sugar ; closely related —
varieties.

1. Stains by Gram’s method. Marked fermentation of milk-—
sugar. Milk coagulated. Bact. acidi lactici, Hiippe,
page 220.

2. Does not stain by Gram’s method.

. Phosphorescence when oxygen is admitted. Bact. ©
phosphorescens, B. Fischer, page 231.

xx. No phosphorescence when oxygen is admitted (group |
of the Bact. pneumoniz Friedlander).

(a) Fermentation of milk-sugar with liberation of gas. —
Milk coagulated. Bact. aérogenes, Escherich, L.
and N., page 221.

(B) Fermentation of milk-sugar without liberation of
gas. Capsules formed in animal. Bact. pneu-—
moniz, Friedl., page 225. ;

(B) Gelatin not liquefied. Organisms motile, with many peri-—
trichous, rarely with one or a few polar flagella.
(a2) No fermentation of sugar with formation of gas. Milk not

See the remarks regarding our contradictory findings in connec-—
tion with Loffler’s swine plague.

2 See also Bact. heemorrhagicum (Kolb), L. and N,

ea Wh yy ey

e.g. ae

KEY TO GENUS BACTERIUM. 197

coagulated. No indol formation. Bact. typhi,’ Gaffky,
Eberth, page 232.

(8) Fermentation of grape-sugar with formation of gas. Milk-
sugar affected only slightly or not at all and without forma-
tion of gas. Milk not coagulated. Bact. cholere suum,
L. and N., page 252.

(y) Fermentation of grape-sugar with formation of gas, milk-
sugar scarcely at all affected. In growth it is between the
Bact. typhi and Bact. coli. Bact. icteroides (Sanarelli),
L. and N., page 256.

(6) Fermentation of grape- and milk-sugar with formation of
gas. Milk coagulated. Bact.coli (Escherich), L. and N.,
page 243.

(C) Gelatin not liquefied. Forms acetic acid , from alcohol.
More details in tables, pages 261 and 262. Acetic acid
bacteria.

(D) Gelatin liquefied, or consumed without visible liquefaction.
Organisms non-motile.

(a) Gelatin liquefied in a funnel form. Sugar fermented.
Abundant growth on potato. Optimum temperature about
25°. Agar is colored reddish-brown. Bact. discifor-
mans (Zimm.), L. and N., page 263.

(8) Gelatin consumed in a funnel form without perceptible
liquefaction. No growth on potato. Optimum tempera-
ture 12°. Agar not colored. Bact. salmonicida (Em-
merich and Weibel), L. and N., page 266.

(£) Gelatin not liquefied, only slowly drawn in. Spontaneously
motile from polar flagellum. Stains by Gram’s method.
Milk unchanged. Bact. canicule (Galli-Valerio), L. and
N., page 260.

(F) Gelatin liquefied. Organisms motile.

(a) Grape-sugar fermented. No branches sent out toward the
solid gelatin. Bact. punctatum (Zimm.?), L. and N.,
page 264. :

(8) Grape-sugarfermented. Branches sent out toward the solid
gelatin. Bact. vitulinum ( Weissenberg), L. and N., page
264.

II. Formation of a yellow (greenish-yellow to orange-yellow) pigment

in the cultures of the bacteria upon agar and gelatin. (Without fluores-

cent discoloration of the nutrient substratum. )

(A) Very small, thin, short rods ; upon gelatin and agar grow
slowly as a thin, intensely yellowish-green layer. Gelatin
very slowly liquefied. Possess a single flagellum. Bact.
turcosum (Zimm.), L. and N., page 267.

(B) Short rods of the dimensions of the Bact. coli.

(a) Without spontaneous movement.

1. Gelatin not liquefied.

*Compare Bact. typhi murium, page 258, and Bact. alcaligenes,
page 257.

? Compare also Bact. foetidum liquefaciens, cloace, agile, page 265.

198 IMPORTANT VARIETIES OF FISSION-FUNGI.

(c) Culture pale grayish-orange (cream). Bact. cremoides,
L. and N.,} page 267.

(8) Growth lemon-yellow. Bact. luteum (F1.), L. and N.,
page 268.

2. Gelatin slowly liquefied.

(a) Luxuriant lemon-yellow layer on gelatin. Agar and
gelatin colored red. Bact. erythrogenes (Groten-
felt), L. and N., page 268.

(8) Rather abundant lemon-yellow growth on gelatin.

Agar and gelatin colorless. Bact. helvolum (Zimm.), —

L. and N., page 268.
(y) Growth on gelatin at first white, then yellowish. Milk

slimy. Soapy smell. Bact. lactis saponacei, Weig-—

mann, page 269.

3. Gelatin rapidly liquefied. Growth upon gelatin very del-
icate. Little chromogenesis. Bact. nubilum (Frank-
land), L. and N., page 269.

(b) Spontaneous motility from polar flagellum. Gelatin lique-
fied, pale ocher-yellow sediment. Upon potato and nen
pale ochér-yellow deposit. Bact. ochraceum (Zimm.), L
and N., page 270.

(C’) Short rods to long threads. Cultures grayish-orange to pale
orange and brick-red. Never branches in the stab.

(a) Non-motile. Bact. fulvum (Zimmermann), L. and N., page

(d) Motile. Bact. chrysogloea Zopf., page 272.

_ III. Formation of a rose-red to a brown-red pigment upon agar and
gelatin. Especially beautiful chromogenesis upon potato. (For red-brown
and brick-red varieties, compare also Bact. fuscum and chrysoglea. )

(A) Stains by Gram’s method. Non-motile. Gelatin not lique-
fied. Bact. latericium (Adametz), L. and N., page 272.

(B) Does not stain by Gram’s method. Motile. Gelatin lique-
fied. Pigment rose to carmine-red, more rarely reddish-
yellow. Bact. prodigiosum (Ehrenberg), L. and N., page
272.

IV. Formation of a non-diffusible, violet or blue pigment in the cultures

upon agar, gelatin, and potato.

(A) Gelatin more or less rapidly liquefied. Formsa deep violet —

pigment, which is soluble in alcohol. Bact. violaceum,
Schroter, page 277.

(B) Gelatin ‘not liquefied. Pigment pale to deep indigo-blue,
insoluble. Bact. indigonaceum (Claessen), L. and N., page
280.

(C’) Gelatin slowly liquefied. Bluish-green, insoluble pigment,
especially marked on potato. Bact. ceruleum (Voges.), L
and N., page 280.

V. The pee of the bacteria are colorless or only slightly yellowish,
bluish, brownish, or greenish in color ; on the contrary, a yellowish-green to

1 For relatives and synonyms, see the text.

er |

atl a

id te ll it Be Nl,

KEY TO GENUS BACTERIUM. 199

bluish-green fluorescent pigment diffuses out from the culture,’ both in
gelatin and agar.
All varieties are provided with a single flagellum or a bunch of
flagella located at the end. The group consists of varieties very
closely related to each other, none of which forms gas from sugar.
_ According to Zimmermann, all fluorescent bacteria, when young,
stain by Gram’s method ; but according to our observations, they do
- not do so regularly.

: (A) Gelatin liquefied. Colonies in plate, after liquefaction begins,
: are surrounded by hairs.

(a) Intense production of pigment, usually bluish-green, upon
all nutrient media, also in milk and bouillon. Milk coag-
ulated with alkaline reaction ; then coagulum is dissolved.

4 Pathogenic for animals. Bact. pyocyaneum (Fliigge),
-- L. and N., page 281.

; (8) Pigment production less; in bouillon very slight. Milk
i not coagulated ; later it becomes clear and colored greenish-
> yellow. Bact. fluorescens (Fligge), L. and N., page

285. :
(B) Gelatin not liquefied. Colonies in plate, even-bordered,
wavy, reminding one of the Bact. coli.

(a) Growth on agar and gelatin, white or yellow. No formation
= of blue or brown pigment aside from the fluorescent mate-
: rial. Bact. putidum (Fliigge), L. and N., page 287.

(8) Besides the fluorescent pigment, there is also formed a
blue, deep blue, or dark brown pigment in varying amount.
Grape-sugar milk becomes blue to bluish-gray. Bact. syn-
cyaneum (Ehrenb.), L. and N., page 289.

VI. The bacterial growths are pale (white to brownish colored), and
through diffusion the surrounding nutrient medium is colored intensely
brown.
1. Gelatin not liquefied. Bact. brunificans, L. and N., page

a

292.
2. Gelatin liquefied. Bact. ferrugineum (Rullmann), L. and N.,
page 292.

II. COLONIES UPON THE NUTRIENT MEDIA ARE ROUNDISH AT THE
BEGINNING ONLY, IF AT ALL; LATER, THERE EXTEND MORE
oR LESS FROM WITHIN OUTWARD, RAY-, FORK-, BAND-, OR

( SAUSAGE-LIKE OUTGROWTHS.

_ In the Bact. vulgare, where these outgrowths may be absent, one

observes—best in 5%-6% gelatin—a swarming in the periphery of the

_ colonies in the plate. In the gelatin stab culture there sometimes

_ occurs the formation of branches. (Genus: Proteus Hauser. )

(a) With spontaneous motion and peritrichous flagella.

1. Gelatin not liquefied. Branching very beautifully developed.
Causes putrid decomposition. Bact.- Zopfii (Kurth.), L.

A and N., page 293.
2. Gelatin usually liquefied. No branching. Causes intensely

* eS Bu 8. See

1 For transition forms between these varieties, consult the detailed
descriptions.

a

200 IMPORTANT VARIETIES OF FISSION-FUNGI.

putrid decomposition. Bact. vulgare (Hauser), L. and N., —

page 295.

(b) Without spontaneous motion and flagella. Gelatin slowly
liquefied.

1. Gelatin colony resembles a bone corpuscle; delicate center,
with a series of irregular outgrowths. In gelatin stab:
nodules, prickly balls, and branches. Bact. erysipelatos
suum (Loffler, Schiitz), Migula, page 302.

2. Gelatin plate similar to the above, or (usually) with very
delicate, almost invisible colonies. Branches in the stab cul-
ture are very delicate and regular. Bact. murisepticum
(Fliigge), Migula, page 300.

Bacterium nitrosomonas (Winogradsky), Lehm. and
Neum.!

Nitrosomonas europzea (Winogradsky). A. P. ty,
v ; and Arch. des sciences biolog. de Petersbourg, 1, 1892.
The morphology is very briefly described. Elliptical and
short spindle-shaped, quiet cells, often united in short
chains (about 14 broad, and 1.1-1.8y long). Upon
silicic acid nutrient media the organisms form compact,
sharply contoured, brown colonies, from which, after
about two weeks, motile swarms wander out (appearing
as a pale halo). In fluids there is first a slight sediment ;
then after about eight days diffuse cloudiness due to the
motile form, which in one or two days again settles quietly
to the bottom.

The organisms thrive only upon inorganic nutrient
media : gelatinous silica or water to which is added, in a
liter, about 1.0 gm. potassium phosphate, 0.5 gm. mag-
nesium sulphate, a trace. of chlorid of calcium, 2.5 gm.
ammonium sulphate, and some solid magnesium car-
bonate. They form nitrite, but no nitrate, from salts of
ammonia.

Growth of the pure culture is difficult, and so far but
rarely accomplished.

Bacterium nitrobacter (Winogradsky). L. and N.

Literature.-—Winogradsky (C. B. L. 11, 415); Winogradsky and
Omeliansky (C. B. L. Vv, 329). The statements of Burri and Stutzer,

1 We select this name because it has many advantages over the un-
meaning one of Bact. europzeum.

BACTERIUM NITROBACTER. 201

as also those of Stutzer and Hartleb, regarding a polymorphous salt-
_ peter fungus are incorrect. Compare Frankel (C. B. L. Iv, 8, 62)
_ and Gartner (C. B. L. Iv, 1, 52, 109).

Microscopic Appearance.—Short rods, 1 » long, 0.3-
— O0.4y thick. Stain poorly. Whenstained with warm gen-
_tian-violet solution and washed with a 10% solution of
chlorid of sodium, a stained capsule surrounds the bacilli,
which are unstained. With carbol-fuchsin the rods are
gradually stained, the pointed ends escaping. Alkaline
methylene-blue first stains the ends, then the central
portion.
Motility is never observed. No growth occurs upon
_ the ordinary nutrient media, rich in organic substances
(bouillon, agar, gelatin), but it grows upon the following :
_ Nitrite-agar, which contains pure sodium nitrite 2 gm.,
sodium bicarbonate 1 gm., potassium phosphate and agar
15 gm., water 1 liter; or nitrite solution, which contains:
_ sodium nitrate 1.0, potassium phosphate 0.5, magnesium
sulphate 0.3, sodium bicarbonate 0.5-1.0, sodium chlorid
_ 0.5, a little iron sulphate, distilled water (distilled twice
over permanganate) 1000. If soda is used instead of
sodium bicarbonate, then also free CO, must be present.
The addition of more than 0.4% peptone, or of small
quantities of sugar, prevents the growth and the produc-
tion of nitrate.

Nitrite-agar Colonies.—Deep: granular, dense, small,
sharply outlined, strongly refracting, appearing only after
weeks. On the surface delicate, cloud-like, homogeneous,
scarcely at all granular droplets develop equally slowly.

Nitrite-agar Stab Culture.—Somewhat more luxuri-
ant, dirty white, greasy.

Isolation from Soil.—Numerous plates are prepared
from nitrite-agar with larger and smaller quantities of soil
suspended in it. After standing for three or four weeks at
about 20°, test the plates to determine whether nitrate has
been formed. Inoculate from a number of the smallest
colonies into nitrite solution, and after about three weeks
prepare new plates of nitrite-agar from the tubes which
contain no nitrite, but nitrate. The pure culture should
behave as follows : (1) A scarcely perceptible precipitate
should appear, which rises as a column on shaking; (2)

Srey

“leg

202 IMPORTANT VARIETIES OF FISSION-FUNGI. —

upon gelatin and agar plates no colonies develop ; (3) in
tubes with nitrite solution the reaction for nitrite should
disappear after about eight days.

Bacterium influenzz (R. Pfeiffer). Lehm. and
Neum.
Literature.—R. Pfeiffer (Z. f. H. x11, 357, 1893), with 7 plates ;

Delius and Kolle (Z. H. xxiv, 327) (immunity, production of
toxins); Grassberger (C. B. XXIII, 25).

Microscopic Appearance.—Very small, short rods,
about 0.4 » broad, 1.2 » long, often in pairs, often in
sputum within the cells, more rarely united in short
threads. (68, v). Grassberger observed typical cultures
with a marked tendency to form thin and thicker appa-
rent threads,+ which in. part were swollen into spindle-
form, and at times branching could be seen. This must
be studied further.

Spontaneous motion is absent.

Staining Properties.—Somewhat poorly with the ordi-
nary aqueous solutions of anilin dyes, better with alka-
line methylene-blue, and best by the application of a very
dilute carbol-fuchsin solution for five minutes. With
faint staining, the ends are somewhat more deeply stained.
Not stained by Gram’s method.

Relation to Oxygen.—Obligate aerobe.

Requirements as Regards Nutrient Media and
Temperature.—Grows only upon agar smeared with
blood (or hemoglobin) or blood-bouillon. Optimum,
37°. Upper limit, 43° ; lower, 26°-27°. ;

Agar Streak.—(Surface smeared with blood.) Clear,
like glass, small, hardly confluent, almost structureless
colonies.

Bouillon with Addition of Blood.—lIf the nutrient
medium is placed in a thin layer, the Bact. influenze
develops as delicate, white flocculi.

Special Nutrient Media. —According to Grassberger,
a mixture of agar and defibrinated blood, heated for one

1The pseudo-influenza bacilli described by R. Pfeiffer (7. ec.) grow
as large thick rods and false threads, but are identical with the L. B.,
according to Grassberger.

a ee SS ee ee ee 4

LF -_— soe

hi ero ae

.

BACTERIUM INFLUENZA. 203

hour to 50°-60°, is a specially favorable nutrient medium.

According to Grassberger, the influenza bacterium grew
with very much greater luxuriance upon unheated blood-
media in proximity to colonies of the Micr. pyogenes. It
may be supposed that heat and the growth of the Micr.
pyogenes alter the blood-medium in a similar manner

_ (Z. H. xxv, 453).

Vitality and Duration of Life.—In water, even in the

dark, they die in from twenty-eight to thirty-two hours ;

Py were) o!* 26

in agar and bouillon cultures, after two or three weeks.
In fresh sputum they are preserved about the same length
of time. Rapid drying kills in two hours ; slower drying,
in from eight to twenty-four hours.

Distribution.—(a) Outside the body: Not found.

(6) In influenza in man: Very abundant in the charac-
teristic, clear, yellowish-green, lumpy, tenacious sputum.
They are found purest in the secretion of the finer bronchi ;
at first free in clumps, later especially within the. pus
cells. Also, extensive colonization occurs in the lung tis-

sue, leading to lobular and pseudo-lobular influenza pneu-

monia. They are often abundant in the nasal secretion in

_ eases of influenza. R. Pfeiffer found them rarely in the

blood, and never cultivated them from the blood. In the

‘organs, especially the brain, they are demonstrable rel-

atively seldom (Nauwerck, C. B. xvu1, 395; Pfuhl, Z.
H. xxvi, 112). E. Frinkel traced a suppurative menin-
gitis to the I. B. alone (Z. H. xxvu, 315).

Animal Experiments.—Influenza can be transferred to
the monkey only, among all the numerous available exper-
imental animals. Devitalized cultures in large quantities
are intensely toxic (dyspnea, paralysis) for animals, espe-
cially rabbits. 2

Immunity and Serum Reaction.—Animals which
are treated for a long time with I.-toxins do not yield a
serum with antitoxic or bactericidal properties, but suc-
cumb to infection with a larger quantity of culture
(Delius and Kolle).

Special Culture Methods. — The bronchial mucus
washed in sterile water is triturated somewhat superficially
with a little sterile water ; and of this, small quantities are
smeared over slanted agar and slanted agar smeared with

204 IMPORTANT VARIETIES OF FISSION-FUNGI.

blood. If the first remain sterile, while delicate, drop-
like colonies develop upon the second, it speaks in favor —
of influenza. Bouillon and agar mixed with sterile pigeon
blood are highly recommended.

Related Varieties.—The ‘‘ bacillus of pneumonia in rabbits,” eulti-
vated by Beck, R. Pfeiffer’s assistant, from rabbits dying sponta-
neously, is closely related (Beck, Z. H. xv, 363, 1893). Small, —
fine, non-motile bacilli, twice as long and thick as the influenza
bacillus, obligate aerobe, not stained by Gram’s method. Does not —
grow on potato. Upon gelatin it resembles the streptococcus. Upon —
agar, grayish-yellow, with granular, sharp border, of tough, mucoid —
consistency. Guinea-pigs, rabbits, and mice are susceptible. Prin- —
cipal changes upon section are pulmonary hyperemia and atelectasis,
and fibrinous deposit upon the pleura.

Bacterium xgyptiacum (L. and N.).
Ordinary Name.—Koch-Weeks’ bacillus.

Entire literature by Kamen a B. xxv, 449), with beautiful |
photographs.

Microscopically, very Sah thin rods (1-2 » long); in
recent cases are often exceedingly numerous in the secretion

Fig. 16.—Bact. egyptiacum Fig. 17.—Bact. duplex (L.
(L. and N.). and N.).

from the eye; sometimes they form short chains. Non-
motile, do not stain by Gram’s method. The cultures
resemble in every way those of the influenza bacillus ; |
their growth is always poor, best upon nutrient media
smeared with blood. Optimum at 37°. They live only
a short time—about four days. Scarcely at all pathogenic

»
+-

&

:

_ BACTERIUM TUSSIS CONVULSIV 4. 205

_ for animals. Often associated with organisms of the
xerosis group.

A differential diagnosis from the Bact. influenze ap-
pears at present scarcely possible.
It produces in Europe, especially in summer, epidemic

conjunctivitis. The disease develops gradually during

_ two or three days ; after three or four days the inflamma-

- tion is more severe, and may be accompanied by abundant

_ purulent secretion. The affection continues severely for a
_ week, and more lightly for two or three weeks.

Frequent in Egypt (Koch), but also observed in Eng-

- land, Paris, Hamburg, Czernowitz, as the cause of epi-

demics. Never has been observed in Wirzburg.

Bacterium tussis convulsive (Czaplewski and
Hensel), L. and N.

Literature.—Czaplewski and Hensel (Deut. med. Woch., 1897, 586,

and C. B. xxur, 641); Koplik (C. B. xxi, 222) and Czaplewski

(C. B. Xxtv, 865); Zusch (Miinch. med. Woch., 1898, 712, and C. B.

_ XXIV, 721 and 769); Vincenzi (C. B. xxIv, 850). (See also Koplik,

Johns Hopkins Hospital Bulletin, 1x, 79, 1898.—ED. )
' Microscopic Appearance.—In smears of the expec-

_ torated mucus small short bacilli, often only oval forms,

of 0.75 to 1.5 » in length. Sometimes united in very
short chains (68, 1). Koplik describes individuals in old
cultures with slightly clubbed ends. Im glycerin and
sugar-agar there are sometimes longer forms, reminding

_ one of the coryne-bacteria.

Spontaneous motion absent (according to Koplik,
present).

Staining Properties. — Tendency to polar staining
when dilute staining solutions are employed. Strong
staining solutions give the organisms a plumper appear-

ance.

Relation to Oxygen.—Facultative anaerobe.
Intensity of Growth.—Usually very modest ; often an

inoculation from a culture one day old upon the original

plate is without result. Growth on agar, poor ; better on
glycerin-agar ; best upon Léffler’s serum.

Temperature,—Not below 25°; grows well only in the
incubator.

206 IMPORTANT VARIETIES OF FISSION-FUNGI.

Spores are absent.

Serum-agar Plate.—Young colonies exceedingly deli-
cate, like dewdrops ; single colonies may be as large as
2 mm., grayish, somewhat lobulated. Rarely are they.
elevated like a pinhead. Magnified from sixty to a hun-
dred times they are finely granular.

Agar Plate.—Smaller, poorer, and usually drier than
upon serum. On the contrary, Vincenzi observed better —
growth on agar than on serum. He describes, in the ©
agar stab, faint growth in the stab canal and no surface
growth.

Bouillon Culture.—Scanty growth, little turbidity.

Milk and potato growths are so far unknown.

Resistance to drying is minimal.

Distribution.—According to the authors previously
mentioned, the organism is regularly present in the glairy,
transparent sputum of typical cases of whooping-cough,
and from it it has often been cultivated. We have also
made, with Dr. Hirai, a series of successful cultures. We
always found the cultures to remain alive for an extraordi-
narily short time.

Animal experiments have failed with all animals and
in the hands of all investigators.

Special Cultures and Methods of Recognition.—
The sputum is obtained with as little contamination as
possible (when possible, after washing out the mouth),
and typical, glairy, tenacious balls are allowed to stand
in abundant sterile water about one hour. The water is
then poured off, and the clumps washed in several changes
of distilled water, and finally from them smears on cover-
glasses and streak cultures upon ascites-agar are prepared.

Bacterium duplex (L. and N.).

Ordinary Name.—Diplobacillus Morax.
seers —Morax (A. P. x, 337); Axenfeld (C. B. xx, 1), with
pia

Microscopic.—Rather large, plump rods, often arranged
in pairs or short chains, about 1 » thick and 2 to 3 long,
non-motile, not stained by Gram’s method, without any
capsule of importance.

BACTERIUM ULCERIS CANCROSI. 207

_ The organisms are very particular as to cultivation.
They grow best upon ascites-agar as small transparent
droplets ; upon ordinary agar, a growth is rarely obtained.
Pure, solidified blood-serum is slowly liquefied on the sur-
A ee. Cultures have little durability. It causes a con-
junctivitis, usually insidious in onset and running a
chronic course with slight catarrhal symptoms, abundant
ecretion, and redness of the conjunctiva, especially upon
the edges of the lids and inner angle of the eye. The
organism is found abundantly in the secretion (Fig. 17).
a he disease may be transferred by means of pure cultures
fo healthy individuals. It has been found infrequently
ir various places as the cause of epidemics ; also, on one

_ occasion, in Wiirzburg.

Bacterium ulceris cancrosi (Ducrey-Kruse), L. and N.
4 Synonyms.—Streptobacillus of soft-chancre Ducrey, Bacillus ulceris

Canecro si
Literature. —Ducrey (C. B. xvii, 290), Petersen (C. B. x11, 743),
x ina (C. B. xvut, 234), Kruse (Fliigge-Kruse, Bd. 11, 456).

_ It is now universally acknowledged that Ducrey rightly
‘recognized a small, thin bacterium (0.5 » broad, 1.5 u
a arranged in long chains, which can be demon-
strated, with no great difficulty, in sections of soft chancre
as the cause of the process. By successive inoculation of
‘chancre secretion from one place on the-skin to others, in
ach resulting ulcer a purer condition is found. Staining
of the sections with Léffler’s methylene-blue is not espe-
sially difficult, if the alcohol is allowed to act very briefly.
. ~The bacteria are not stained by Gram’s method. They
a e also found in the chancre secretion, but only rarely in
‘the contents of buboes. Cultures are rarely successful ;
Petersen obtained non-characteristic, faintly growing col-
‘onies deep in serum-agar.

208 IMPORTANT VARIETIES OF FISSION-FUNGLI.

Bacterium septicemiz hamorrhagice.! Hiippe.
(Plate 12.)

Literature.—Complete by Voges (Z. H. xxii, 261; Xxvill, 33); —
Karlinski (Z. H. xxvitl, 407); Th. Smith (C. B. xxv, 241); Voges —
and Proskauer (Z. H. XXVIII, 20); Preisz (C. B. xx1mI, 666).

Microscopic Appearance. —Short rods, from the ©
animal scarcely ever more than twice as long as thick, very
small (0.3-1 » long). Very often (always typically) in —
the short rods with somewhat smaller ends only the poles —
stain (plasmolysis) (12, rx and schematic, 12, x), so that
pictures resembling diplococci result. Heim once observed —
typical capsules. In cultures, likewise, there are mostly —
short rods (12, 1x), rarely short threads. :

Spontaneous motility and flagella are absent.

Staining Properties.—Not by Gram’s method.

Dependence upon Temperature and Nutrient —
Media.—About like the Bact. coli. Facultative anaerobe.

Growth upon Agar and Gelatin.—As shown in Plate
12, differing but little from the Bact. colli. 7

Milk Culture.—Behave differently. Our Berlin chicken ~
cholera presents the typical properties; it renders milk
alkaline and leaves it fluid; similar effects are produced
by a culture of Léffler’s swine plague from Berlin and one
of Honl. On the contrary, one obtained from C. Frankel
coagulated milk with formation of acid.

Potato Culture.—Often no growth, especially when
cultivated freshly from the animal, or only a very scanty

1 Our description is based upon a culture of ‘‘ chicken cholera,’”’ —
obtained from the Hygienic Institute in Berlin, whose properties agree
excellently with those described in the literature. Two cultures,
of ‘‘ chicken cholera ’’ and ‘‘ rabbit septicemia,’’ which have been culti-
vated in our institute for about six years, and which originally came
from a trustworthy but now unknown source, behave like typical
Bact. coli in the sense of the definition in our key. Unfortunately,
the connection between these can not be explained ; a contamination
seems to be excluded, a transformation is improbable. One might see
a proof in this observation of the identity of the Bact. sept. haemor-
rhag. with the motile organism of hog-cholera, so long maintained
especially by Voges. It does not seem possible to draw more far-
reaching conclusions from the observation, especially as Voges and
Proskauer now again maintain sharp differences between the causes of
the diseases from the biologic characteristics (fermentation of sugar,

BACTERIUM SEPTICEMLE HEMORRHAGICH. 209

one. Old laboratory cultures grow as faintly yellowish-
_ white ; after alkalinization the growth is more abundant.
Production of Gas and Acid from Carbohydrates.
—Often much acid is formed, both from grape- and milk-
‘sugar, but no gas. }

Indol and H,S.—Both abundantly formed (according
to Karlinski, not). According to Hoffa, methylguanidin is
to be looked upon as the poisonous principle of the organ-
ism.

Resistance.—Against drying, slight. Heating to 45°-
46° destroys the virulence in half an hour. On the con-
trary, cultures remain viable and virulent for months.
Cold and mixture with putrefactive bacteria do not reduce
the virulence. .

Distribution.—(a) Outside the body: Demonstrated by
Gafiky in water of the Panke. Inoculation of the same
into rabbits produced a fatal infectious disease. Also found
in water and soil ; undoubtedly widely distributed.

(b) In the body: Never in man. On the contrary, they
were found by Gamaleia in the feces of normal pigeons,
but with little virulence, and by Karlinski in the nasal
mucus of swine. Have been demonstrated to be the cause
of a series of destructive diseases in animals, in various
biologic races, and designated by various names.

Voges was unable to produce, in any way, a true, last-
ing immunity against any of those diseases.

We will describe only four of these varieties somewhat
~ more extensively.

1. Bacterium suicida Migula (Bacillus suisepticus
Kruse), cause of the so-called German (Lé6ffler’s)
_**Schweineseuche.’’ Compare Léffler and Schulz (A. G.
_ A. I, 55 and 376). It is a wide-spread and destructive
_ disease of swine, which usually kills in from one-half to

NT PO

ee a RS ee a he We Coen ar ie nae NEN FP) 2

ai.

etc.) recently in use. We remain, therefore, for the present with a
_ preponderating majority of authors who occupy the standpoint of a
_ duality. A new culture, obtained from Honl, in Prague, of Bact.
suicida Mig. corresponds with the scheme.

1 This statement in the literature, which Th. Smith recently again

_ verified, corresponds to our cultures of chicken cholera and new

** Schweineseuche ’’; on the contrary, both of the old motile cultures of

“‘Schweineseuche’’ produce gas from grape-sugar. According to

Karlinski, sometimes there is formation of gas, and sometimes none.
14

ee ee ee ee

210 IMPORTANT VARIETIES OF FISSION-FUNGL

two days. Usually a lobular, multiple, necrotic pneu-
monia is most prominent. Many cases pass as croupous
pneumonia ; other forms, with bacteria of less virulence,

lead, during a chronic course, to the formation of multiple «

caseous areas, which are often confused with.tuberculous
areas. Compare Ascher and Hirsemann (Z. H. xxyt,
148). Also diseases of the intestine (gastro-enteritis) occur
when a complication or secondary infection by the Bact.
cholerze suum is not present. Swine are very susceptible,
and, of the experimental animals, guinea-pigs ; birds are
very slightly affected.

For an exhaustive differential diagnosis from American
‘‘Schweineseuche,’’ see page 239. - |

2. Bacterium multocidum ! (Kitt), L. and N. (Bact.
bipolare multocidum Kitt, Bac. bovisepticus Kruse),
cause of the ‘‘ Wild-’’ and ‘‘ Rinderseuche”’ (Bollinger,
Kitt), which, while not very frequent, still has raged very
destructively among deer and cattle.? Hogs are rarely
affected.

There are found hemorrhagic enteritis, with either
pleuropneumonia and pericarditis, or very acute edema

of the head and neck, with hemorrhages in the mucous.

membranes of the head.

3. Bacterium of Barbone in buffalo disease in Italy
and Hungary (Oreste and Armanni, 1886; von Ratz,.C. B.
xx, 289; Sanfelice, Loi and Malato, C. B. xxm, 32).
Buffalo die in from twelve to twenty-four hours ; there is
severe hemorrhagic edema of the subcutaneous connective
tissue, especially about the larynx, trachea, etc., with the
small intestine reddened and hemorrhagic. It is patho-
genic for guinea-pigs.

4. Bacterium avicidum Kitt, cuniculicida (Gaffky)
Fltigge (Bacillus cholere gallinarum Kruse). It is the
cause of extensive epidemics in chickens (chicken cholera,
Perconcito, Pasteur), isolated by Gaffky from canal-water
(Mitt. Gesundheitsamt 1, 80) and described as the cause

1 Closely related : ‘‘ New Infectious Disease of Cattle,’’ of Basso
(C. B. x x11, 537). Stained by Gram’s method, non-motile, ferments
glucose.

2 According to Gmelin, the causes of many cases of infectious in-
flammation of the navel also belong here (C. B. XXIII, 295).

j BACTERIUM SEPTICEMIH HEMORRHAGICH. 211

_of rabbit septicemia (Davaine’s septicemia). It is differ-

entiated from Nos. 1-3 by a more abundant growth on
potato, and in milk sufficient acid is formed to produce

- coagulation.

For chicken cholera the following are susceptible :
Chickens, turkeys, ducks, geese, pigeons, in general all
domestic fowls, sparrows, finches, rabbits, and white mice.
Usually guinea-pigs are slightly susceptible. Compare, on

the contrary, Tjaden (C. B. xxv, 224). Every method of
- inoculation (also with only very small quantities) as well

the eee, gees
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as feeding are successful, death occurring in birds usually
after from twelve to forty-eight hours, rarely after from
seven to twelve days. Superficial inoculation by cuts into
the pectoral muscles with a lancet is most generally em-
ployed.

Postmortem Findings.—In pigeons, at the place of inocu-
lation in the muscle, there is a whitish-yellow, thick,
nodular swelling and discoloration of the muscle; in

_hens often more of a cloudy, edematous infiltration—an

appearance of diagnostic value. Dead animals have large
ecchymoses in the serous membranes (especially in the
pericardium), besides serous or fibrinous pericarditis,
hemorrhagic enteritis, and serous lobular pneumonia
(Kitt). (Dogs and cats devour dead birds without
injury.) During life the birds present suddenly develop-
ing choleraic symptoms, together with loss of appetite,
weakness, giddiness, ruffled plumage, thirst. Rabbits and
mice die quickly, without local manifestations, or an

_ abscess forms at the point of inoculation, which for weeks
_ contains the characteristic bacteria. 3

Special Methods of Demonstration.—lInoculation
of a pigeon by very shallow cutaneous incisions in the
breast, 2-3 cm. long. Characteristic organisms abun-
dantly present in the blood of the inoculated animal
(bipolar staining); change at the point of inoculation
(necrosis ).

The bacillus gallinarum E. Klein is a variety (C. B. v, 689; v1,
257; and XVIII, 105).

Closely related are : The disease of ring-doves of Leclainche (A. P.,
1894, 490) and the duck cholera of Cornil and Toupet (C. B. rv,
333); hens are immune to both these latter. Similar also are the

anti. ap ea

212 IMPORTANT VARIETIES OF FISSION-FUNGI.

parrot cholera (Nocard), Fiorentini’s septicemia of swans (C. B. x1x,

932), and a series of diseases in animals, which have usually been ob- ;

served but once.

Bacterium haemorrhagicum (Kolb), Lehm. and Neum.
(Plate 20, VII, VIII.)

Literature by Babés (C. B. 1x, '719); Kolb (A. G. vit, 60); Afanasieff
(C. B. x11, 402); Finkelstein (C. B. xvuit, 64).

Very closely related to, indeed, only biologically differ-
ent from, the Bact. septic. hemorrhag. is an organism
closely studied by Babés, Tizzoni,; and Giovannini, but
especially by Kolb (illustration, literature), which causes
purpura—Morbus maculosus Werlhofii—in man and ex-
perimental animals, which usually terminates fatally.
There occur hemorrhages into the skin, serous mem-
branes, lungs, kidneys, etc., and albuminuria.

Microscopic Appearance.—Short, oval bacteria, 0.8—
1.5 » long, 0.4-0.8 » thick, usually in pairs (20, viz) with
a small capsule in the animal body; in cultures, short
rods and threads. Non-motile. By Gram’s method they
stain poorly or not at all. - Facultative anaerobe.

Gelatin Culture.—Grow rather slowly; delicate, thin,
whitish, spreading but little, never liquefying. Agar cul-
ture: Uncharacteristic, white to whitish-yellow, spread-
ing somewhat flatly. Upon potato, whitish, moistly glis-
tening, not spreading much, not tenacious. Regarding
the relation to sugar solution nothing is stated ; since in
anaerobic cultures, which bear the addition of sugar well,
nothing is said by Kolb of gas-formation, it does not
appear to cause fermentation. The varieties isolated by
the three above-mentioned authors were different in their
pathogenic effects upon experimental animals. Kolb ob-
tained the greatest effects upon mice, less in guinea-pigs
and dogs; the organism of Tizzoni and Giovannini, on the
contrary, was not pathogenic for mice, but very pathogenic
for dogs and guinea-pigs. The animals often present
marked hemorrhages, with the same localization as in
man.

BACTERIUM PESTIS. 213

Bacterium pseudotuberculosis rodentium. Preiss.
(L. and N.)

_ Synonym.—Bacillus pseudotuberculosis A. Pfeiffer.
Entire Literatwre.—Delbanco ( Ziegler’s Beitrage xx, 477).
Microscopic.—Plump, short rods, motility absent or
oubtful, flagella not found, often arranged i in short chains
in cultures, stain best with alkaline methylene-blue, not by
Gram’s method.

Cultures.—Somewhat like Bact. coli, grow readily and
luxuriantly upon most nutrient media (only upon potato
poorly), forming yellowish-white to salmon-colored and
yellowish-brown growths. Bouillon is first diffusely cloudy,
then presents a thick sediment, but no pellicle. Abundant
formation of crystals in the cultures from the formation
of alkali (basic phosphate). No fermentation of sugar
‘with formation of gas. Milk is not coagulated. No

indol.
Distribution.—Frequently found as the cause of tuber-
-culous-like, caseous, granulation swellings (especially in
the abdomen) of rodents (rabbits, guinea-pigs). Appears
widely distributed; may also cause epidemics.
Detection.—The organism may be easily found in
‘stained smears from the swellings. Growth occurs read-
‘ily, and thus it is differentiated from tuberculosis.

Bacterium pestis.1 (Kitasato, Yersin.) L. and N.
(Plate 13.)

Literature.—Yersin (A. P. vitt, 662); Aoyama (C. B. xrx, 481);
Ogata contra Kitasato (C. B. xxi, 77 1). Much newer literature is
cited in the text. Especially important is: Gaffky, R. Pfeiffer, Dieu-
‘donné, Sticker. Report of the German Pest Commission (A: G. A.
/XvI, 1899).

Microscopic Appearance.—Short rods with rounded

ends, two to three times as long as thick, here and there
‘united in pairs (13, x a). In smear preparations from ex-

_. 1 We have had at our disposal for study and illustrating, through

the great kindness of Dr. Dieudonné, besides the living cultures, also

* series of preserved original Indian cultures and original prepara-
ions.

214 IMPORTANT VARIETIES OF FISSION-FUNGI.

udates or fresh portions of the body, the organisms show
the usual polar staining with anilin dyes, as in those of
septiczemia hzemorrhag. (13, 1x). In bouillon there occur
streptococcus-like chains (13, x 6). The bacteria are pro-
vided with a capsule, but it is not easily rendered visible
(Report of the German Pest Commission). In connection
with bacteria from a pure culture, we have not often seen
them, yet they may at times be demonstrated by using
dilute staining solutions.

Spontaneous Motility.—Absolutely non-motile, no
flagella. It must be noted that Kitasato observed very
sluggish motion, and likewise Kasanski saw movement of
the bacteria (C. B. xx, 25). Gordon stained, according
to the method of van Ermengen, flagella, which are usually
single and polar, rarely in pairs and at the sides (C. B.
xxu, 170); compare also N. Schultz (C. B. xxim, 594).
According to the statement of the German Pest Commis-
sion, what was supposed to be motility was only molecular
motion, and the flagella observed may be supposed to be
simply precipitated staining materials.

Staining Properties.—Stain with all anilin dyes. In
preparations from pure cultures, the polar staining is not
clearly observed. Not stained by Gram’s method. In

Per
ee ee Ee

opposition to Kitasato, the statement is made that the —

bacteria in the blood stain by Gram’s method. According
to Kasanski, the polar staining succeeds especially in blood
and old pus.

Relation to Oxygen.— The bacteria are obligate
aerobes. Growth is stopped by the exclusion of oxygen.

Dependence upon Temperature. — Optimum oF ies
but it also grows very well at 22°.

Intensity of Growth.—On all nutrient media, toler-
ably rapid. After two or three days a luxuriant deposit
is observed. In bouillon diluted with three times its
quantity of water, the growth is very much slower. In
dilutions of 1:10 it is almost entirely absent (Report of
the German Pest Commission).

Liquefaction.—Absent.

Spores.—None formed. The vegetative cells die com-
pletely at 55°-60°.

Involution Forms.—Very characteristic and ane

ae

BACTERIUM PESTIS. - 215

able involution forms are produced, the like of which are
said to occur in no other variety. The cell bodies are
swollen in the center, similar to yeast cells, or they become
rounded, like spherical forms. Very often there appear
cells many times larger than normal cells. The staining
properties in these forms are lessened (18, vim). Accord-
ing to the statement of the German Pest Commission,
upon Hankin’s 8% chlorid of sodium-agar involution
forms are produced almost exclusively (C. B. xx, 488).

Gelatin Plate.—(a) Natural size: Small, crumbly,
gray, transparent colonies, which are directly elevated
above the surface. After a longer time, they spread out
but little (13, v d).

(b) Magnified sixty times: Answering to the elevation
which occurs, there is observed a marked reflection from
the surface. The colonies are roundish, smooth-bordered
to lobulated, sharply outlined, with a yellowish to a green-
ish shimmer. Very often the superficial colony is sur-
rounded by a very delicate, transparent lobulated zone,
which occurs also in somewhat altered form upon other
nutrient media. The structure varies from homogeneous
to faintly granular. The deep-lying colonies are similar,
but never present this delicate zone (13, vt).

Gelatin Stab.—In stab-canal, a faint, homogeneous,
whitish, thread-like growth. On the surface the growth is
like the colony in the gelatin plate.

Agar Plate.—The description refers to cultures which
have been cultivated a long time. Cultures recently ob-
tained from pest cadavers behave somewhat differently.
(See under agar streak. )

(a) Natural size: After forty-eight hours the colonies
are plainly visible macroscopically, have wavy, smooth ©
borders, are slightly elevated, and cannot be differentiated
from those of the colon bacterium. They are gray to
grayish-white, and have an oily or moist luster (13, v a).

(6) Magnified sixty times : Colonies roundish, transparent
at the periphery, in the interior yellowish to yellowish-
gray. They are universally very crumbly. Sometimes
one is reminded of a very granular colony of diphtheria
or of a delicate sarcina colony (13, vira). The better the
nutrient medium, the more luxuriant the growth. Thus

216 IMPORTANT VARIETIES OF FISSION-FUNGL 1

the, colonies on glycerin-agar (13, vir 5) and ascites-agar
(13, vu c) are less transparent and darker in color.

The delicate, tiny, drop-like colonies which occur when
cultures are made direct from pest material (13, 1), when
magnified sixty times, appear less crumbly, sometimes
homogeneous, almost smooth-bordered, and elevated like
the head of a pin. Here, as in the colonies in gelatin, the
older colonies present a very delicate, transparent, finely
punctated peripheral zone, upon which the colony proper
appears built up as a hemisphere (13, v1). Young colo-
nies are gray; old ones, grayish-yellow to brownish-
gray.

Agar Stab.—Difficult to differentiate from the colon
bacterium. The surface growth is somewhat whiter than
with the colon. Stab is uncharacteristic and thread-like.

Agar Streak.—The growth upon the surface from fresh
pest material consists of tiny, delicate, transparent, drop-
like colonies, which lie close to each other and appear as a
delicate surface-layer. The minute colonies are not conflu-
ent and do not become much larger. The entire layer ap-
pears grayish-yellow (13, 1). The growth of cultures
which have. been long under cultivation cannot be differ-
entiated from that of a well-developed colon culture, but
is perhaps a little whiter.

Bouillon Culture.—At first is slightly turbid ; in course
of time a pellicle forms, which at first is delicate, and later
becomes denser. Very old cultures are often clear, with
an abundant, crumbly sediment. In sugar bouillon the
sediment is more marked, also the pellicle is more luxu-
riant.

Milk Culture.—Growth in milk is slight; the milk
is not coagulated.

Potato Culture.—Grows slowly. Deposit is whitish
to whitish-yellow, faintly shining, somewhat elevated,
erumbly. It is sharply outlined from the potato.

Special Nutrient Media.—Upon boiled rice, at 30°-
37°, there is an abundant growth in the form of a gray
film (Report of the German Pest Commission).

Chemical Activities.—(a) Chromogenesis, production
of odoriferous and gustable substances, liquefaction of
gelatin, and H.S are absent.

re

ee oe ee

BACTERIUM PESTIS. 217

(6) Indol reaction: After a long time; without the
addition of nitrite, slight ; with the addition of nitrite,
pronounced.

_ (ce) Toxins: Fluid cultures, devitalized by heat, never
contain soluble toxin. By extraction from cultures eight
to twelve weeks old which are killed with formalin, a fluid
is obtained which is very rich in toxin, and from this, with
‘ammonium sulphate or alcohol, a solid toxin may be ob-
ained, of which +545, of the body-weight is fatal for mice.
Still, in the serum of animals treated with large doses of
toxin antitoxins are absent (Wernicke, C. B. xxiv, 859).
“Markl obtained similar results. He obtained the largest
‘quantities of toxin in shallow bouillon cultures quite
rapidly (in a few days); he obtained sera with limited
‘antitoxic action, but without any effect against infection
with living bacteria (C. B. xxrv, 641). Roux, who pro-
‘duced strongly active sera, found them strongly antitoxic,
but not bactericidal.

_ Resistance and Viability.—The pest bacillus is not
very different from other fission-fungi. It withstands
drying from three to seven days ; in water it dies in from
three to eight days, according to the composition. In
‘buried bodies the duration of life is from twenty-two to
thirty-eight days. Kasansky proved that they stand the
‘Russian winter for months (C. B. xxv, 122). For par-
ficulars consult Toptschieff (C. B. xxi, 730), Gladin
(C. B. xxv, 588), Hankin (C. B. xxtv, 587), Wladimiroff
(C. B. xxiv, 424).

_ Distribution.—(a) Outside the body: In India, Hankin
and Yersin have many times cultivated non-virulent varie-
ties of bacilli, resembling very much the pest bacillus,
from the environs of men in houses infected with pest.

_ (6) In healthy body: Never.

_ (ce) In diseased human body: Is widely distributed.
Most abundant in the buboes, primary cutaneous pus-
fules, and the sputum of pest pneumonia. Rarely found
in the blood and organs (compare below).

_(d) In animals: Pest occurs spontaneously in rats.
Epidemics of pest in rats often precede those in man. It
appears as if certain tropical soil bacilli first become accli-
mated to the rat’s body, and then are transferred to man.

218 IMPORTANT VARIETIES OF FISSION-FUNGI

Pathogenic Properties for Man.—Cause of the true
oriental bubonic or glandular pest ; also of the pest pneu-
monia. Mortality 50%-80%. The gates of entrance are :
(1) Skin. The bacteria usually first become localized
and grow in the nearest lymph-glands (gland-pest), but
often there develops at the place where the bacterium
enters a pest pustule, which may be of the character of
a boil or carbuncle, and may contain very many bacteria.
Death may occur ‘without. the pest bacteria extending
beyond the local area, but usually it follows a dissemina-
tion of the bacteria throughout the entire body (pest
sepsis). Rarely pest bacteria also occur in great numbers”
in the internal organs ; at times in the urine. (2) Lungs;
pest pneumonia. In the sputum there are very numerous
pest bacteria, often also in the blood. Complication with
streptococci is frequent. (3) Digestive canal ; uncertain.
In animals it has been demonstrated. _

Experimental Investigations Regarding Patho-
genic Effects.—Almost all animals are susceptible to pest.
Pigeons are immune ; dogs and cows but slightly suscepti-
ble (Gosio, H. R., 1897, 855); more susceptible are swine, ©
horses, cats; yet more, monkeys and rabbits; and most
of all, guinea-pigs, mice, and rats. Compare Nuttall (C.
B. xxu, 87). The pest bacillus may also become ac-
climated to frogs (Devell, C. B. xx, p. 382).

Guinea-pigs inoculated intraperitoneally die in two days”
of an acute septicemia with few bacteria in the tissues.
After infection with small quantities of pest bacilli, death
occurs on the sixth day, when the mesenteric glands are
swollen and there are hemorrhages in the liver and lungs,
together with submiliary abscesses and nodular thickenings
of the omentum. The spleen contains whole swarms of
bacteria, which are united in a zooglear mass. These
zooglese are formed by very much swollen capsules. Honl
(C. B. xxu, 100).

Guinea-pigs are easily infected through the digestive
tract, in which case there is a special tendency to chronic
forms. (Nodules in various organs, including lungs.)
Bandi and Stagnitta-Balistreri (Z. H. xxvii, 261).

Flies transport pest bacilli; bugs and fleas remove pest’

Pt
«

_ bacilli together with the blood from animals with pest, yet

+

BACTERIUM PESTIS. 219

_ a transfer to healthy animals appears rare.

Immunity and immunization (consult the report of
the German Pest Commission and Dieudonné, Miinch.

: med. Wochenschr., 1898, 166).

Passive immunity may be obtained in animals, and to a

_ certain degree also in man, by the subcutaneous injection
_ of serum from horses which have previously been treated
many times with intravenous injections of devitalized cul-

tures; curative power is also possessed by such serum

_ over sick men and animals, yet only in a modest measure
and in very large doses. According to Roux, the action
_ of the serum is only antitoxic, not bactericidal. Accord-
_ ing to Haffkine, active immunity is obtained more easily,
_ more cheaply, and also without any danger, by injecting

subcutaneously 24-3 ¢.c. of a well-grown bouillon culture
after it has been heated to 70° for one hour. The symp-
toms (fever, pain) are usually moderate, and the injection

| is best repeated after ten days. If the protection is not

absolute, and some of those injected die later of pest
(1.6% instead of 24.6%), yet the majority are entirely
protected or are only affected very mildly.

Special Methods for Demonstration and Culture.—
1. Not to incise non-fluctuating glandular swellings or
boils of the skin for diagnostic purposes is a professional
failure. In the pus from discharging ulcers, and especially
in the sputum of cases of pest pneumonia, the micro-
organisms are found in abundance. Here a _ probable

- diagnosis is easily made microscopically from the bipolar

staining.
2. To certainly demonstrate the pest bacteria in a drop
of blood (more readily done in spleen- or liver-juice) after

_ Staining alone, is often impossible. It is more easily done
_ in cultures upon gelatin at 22°, by observing the small, ele-

vated colonies with delicate, transparent borders. Absence

_ of spontaneous motion.

3. It is important to observe the involution forms upon
38% sodium-chlorid agar after twenty-four hours’ growth.

4. The serum from cases of plague agglutinates pest
eeria.

220 IMPORTANT VARIETIES OF FISSION-FUNGI.

Bacterium acidi lactici. Hiippe.
(Plate 14.)

Literature.—Huppe, Mitteil, aus dem Gesundheitsamt 0, 309. More
recent literature up to 1891 is given byScholl ; Die Milch (Wiesbaden,
1891). Compare also Kayser (A. P., 1894, p. 737), where there are
described 15 organisms which produce lactic acid.

Microscopic Appearance.—Short, somewhat oval rods

(0.6—2 » long, 0.4-0.6 » broad), usually in pairs, rarely in

longer chains (14, 1x).

Motility and flagella are absent.

Staining Properties.—Stain by Gram’s method, but
not very well.

Requirements as to Nutrient Media and Temper-
ature.—Grows abundantly at room and incubator temper-
ature upon the various nutrient media. It grows better
aerobically, and not at all deep in shake cultures prepared
from non-saccharine media. With the addition of sugar, it
grows well anaerobically.

Growth upon Gelatin and Agar.—Not essentially
different from Bact. coli, very abundant, especially upon

agar, and moist and slimy. Upon gelatin, delicate. In ~

thin plate, the colonies may become 5—10 mm. in diameter
(14, v).

Bouillon Culture.—Diffuse cloudiness, abundant sedi-
ment.

Potato Culture.—Somewhat widely spreading, wavy,
smooth-edged growth, somewhat elevated, at first grayish-
to yellowish-white, later sometimes brownish-yellow. After
longer standing, bubbles arise which often are strongly
refractive, and later may burst (14, x).

Milk Culture.—Compact coagulation, with expression
of clear serum; a few little gas bubbles are always
present.

Chemical Activities.—It forms from grape- and milk- —

sugar a mixture of lactic and acetic acids, and sometimes
traces of alcohol, together with an abundance of gas. The
lactic acid may be optically inactive fermentation lactic
acid, but so far special investigations are lacking. As first
observed by Hiippe, the powers of producing lactic acid

eee ee a ee

BACTERIUM AEROGENES. 931

_and of coagulating milk are gradually lost after long culti-
_yation upon gelatin or agar.
_ Upon nutrient media free of sugar, there is a slight pro-
duction of indol, but none of H,S8.
_ Distribution. "Constantly cultivated from sour milk
by Hiippe in Berlin, and by his pupils with slight modi-
fications (consult Scholl). In Wiirzburg, since 1888
(compare Dissertation by Joh. Claus, Bakteriologische
Untersuchung der Milch im Winter 1888-89 in Wiirz-
burg), we have never failed to find the organism in milk
which had soured spontaneously and naturally, and until
recently we had no doubt that it was the most important
producer of lactic acid in milk, as Htippe assumed. Milk
which has soured spontaneously contains, in Wtrzburg,
considerable quantities of volatile acid. As soon as possi-
ble the question as to the most important cause of lactic
acid fermentation will be restudied in Wtrzburg. Com-
_ pare page 224.
Demonstration and Differential Diagnosis.—As dif-
fering from Bact. Giintheri, the Bact. acidi lactici grows
well upon the ordinary nutrient media, and produces gas
vigorously. As regards the staining by Gram’s method,
variations occur. In order to bring the findings into a
scheme we call the forms not stained by Gram’s method
Bact. lactis aérogenes (see below), and leave the question
open as regards the kind of relationship existing between
_ these ‘‘species.’’

Bacterium aerogenes.! (Kruse.) L. and N.

___Synonyms.?—Bacterium lactis aérogenes Escherich, Ba-
-cillus aérogenes Kruse.

_ __ Literature.—Escherich, Die Darmbakterien des Sauglings, 1886,
page 57.

— 4A Bact. lactis aérogenes obtained from Kral presented from 1 to 3
irregularly arranged, long flagella, and was thus, according to our
_ ideas, a typical Bact. coli. It also produced indol very vigorously.

__ #We cannot understand how Kruse designates the Bact. acidi lac-
tici as a variety of the Bact. aérogenes, which was described many
‘years later. If one name is to be eliminated, according to priority, it
Must unquestionably be that of Bact. aérogenes.

222 IMPORTANT VARIETIES OF FISSION-FUNGLI.

This variety, first isolated from the milk-stools of in- |
fants by Escherich, is, according to our investigations, and
according to Escherich’s own statements, to be differenti-
ated from the Bact. acidi lactici merely by the absence of
staining by Gram’s method 1—a characteristic upon which
no great value can be placed according to recent experiences.

A further difference, which Escherich understands from
Hiippe’s description, that Hiippe’s organism was an obli- —
gate aerobe, we cannot recognize according to our investi-
gations as present, for as often as we isolated the Bact.
acidi lactici from sour milk in Wiirzburg, it always pro-—
duced fermentation anaerobically. We cannot place any —
great value upon the luxuriant, sometimes hemispheric, —
slimy growth upon the surface in the gelatin stab, which —
he likens to the growth of the Bact. pneumoniz. Escherich —
has even seen exceptions. |

Metabolic Products.—Alcohol, acetic acid, active lac-
tic acid, succinic acid, and, according to Nencki (C. B. x, —
82), also CO, and H. According to Smith, about 30%-
40% CO,, 60%-70% H. Indol is not produced. |

For us Bact. lactis aérogenes is the name for a form —
without flagella, parallel to the typical peritrichous Bact. —
coli, or for a Bact. acidi lactici which is not stained by —
Gram’s method. Transition forms certainly may exist,— —
compare remark 1,—but one proved to be well founded is
not certainly known to us. Very closely related is the
Bact. diatrypeticum casei Baumann (C. B. xiv, 494), —
which is widely distributed in milk, water, and soil, and
causes the cavities in cheese, or perhaps aids in their for-
mation. Composition of gas: 63% CO,, 37% H,. It is ©
provided with a capsule.

We can see no final proof in the investigations of
Scheffer (A. H., 1897, xxx, 291) by which he attempts
to make a distinction between the two varieties dependent —
upon immunization and agglutination experiments, for we —
remember that the different varieties of the streptococcus —
furnish no reciprocal immunity, and that each form of the —
Bact. coli furnishes a serum which strongly agglutinates —
only the form concerned.

1 Wiirtz and Lendet find both varieties identical.

BACTERIUM GUNTHERI. 223

Here belong the following non-motile ea which
lerment grape- and milk-sugar :

_ Bacterium cavicida Brieger. Zeit. f. phys. Ch., 8.

_ Bacterium neapolitanum Emmerich. Cultivated from a series of
‘cholera cadavers in Naples and once from the blood of a cholera
patient. It is not the cause of cholera. According to Buchner, the
- moderate vibratory motion is not purely molecular. Flagella are not
known. If it possessed flagella, then it would be considered as Bact.
-eoli. Compare Weisser (Z. H. 1, 315).

__ Bacterium of septicemia of cats Lehm. and Neum. Cultivated
From a cat which died spontaneously. Killed cats with typhoid
3 ptoms. A more detailed description is still lacking.

| - Bacterium of dermatitis epidemica exfoliativa Russel (C. B. xv,

et) Unknown to us.

: Bacterium caucasicum. (Kern.) L. and N.

. Synonyms.—Dispora caucasica Kern. Bacillus caucasicus v. Freu-
| Benreich.

__ Literature.—v. Freudenreich (C. B. L. m1, 47, 87, 135).

__ Microscopic : Rods, about 5-6 long, 1 u broad, which often present
ll, clear, globular swellings at the ends (are ‘not spores!). Very
ightly motile.

_ Fresh cultures grow poorly or not at all upon gelatin or milk-sugar

tin ; on the contrary, old cultures grow well. Upon milk-agar

re develop whitish-gray, flat colonies with a somewhat jagged

border due to outward projection of individual bacteria. Milk is not

eater] Little gas-formation in milk ; grows well in milk-sugar
pouillon. Growth at 22° is feeble ; 37° is ‘the optimum.

_ According to Kern, it is the cause of kephyr fermentation. v.

-Freudenreich obtained kephyr in sterile milk most often (not always)

‘if he mixed together four varieties: (1) The kephyr yeast ; (2 and

3) two streptococci isolated from kephyr ; (4) the Bact. caucasicum ;
t also with the yeast and the two streptococci there resulted a
‘tolerable production of kephyr.

Bacterium Giintheri. Lehm. and Neum. Giinther
_ and Thierfelder (A. H. xxv, 164).

Literature.—Giunther and Thierfelder (A. H. xxv, 164). Leich-
(C. B. Xvi, 826). Consult especially Leichmann (C. B. L. v,

WT deca gay ge

we

_ Nomenclature.—Giinther and Thierfelder have not
‘named their organism. In our first edition, published in
y, 1896, we gave it the name Bacterium Giintheri L.
and N. This name must stand, for also Leichmann, who

ad received the organism from Giinther and Thierfelder,

224 IMPORTANT VARIETIES OF FISSION-FUNGL.

but did not especially study it, designated it by the name
Bacterium lactis acidi for the first time, so far as we can
see, in December, 1896 (C. B. L. u, 777). Aside from
the question of priority, it is very impractical to introduce
a Bacterium lactis acidi, together with a Bacterium acidi
lactici. Besides, Leichmann has also called a longer, |
slender, thermophilic, non-sporulating, acid-producing
variety Bacillus lactis acidi. Later than our name is also
Bacillus lacticus Kruse. Lately Kozai has introduced |
Bacillus acidi paralactici (Z. H. xxx1, 337).

Microscopic.—Short rods, 1 long, 0.5-0.6 p» thick, a
pairs or short chains; at the ends somewhat pointed ;
stains by Gram’s method ; non-motile, facultative aerobe. —
Upon the gelatin plate: Punctiform colonies, never more
than 0.5 mm. in diameter upon medium which does not
contain sugar; when sugar is added, they are a little
larger, but always very delicate, and never liquefying. In
the stab culture there is often scarcely anything but a deep |
growth. Upon the agar plate: Delicate transparent growth, —
like the finest dewdrops. Jn bouillon: Slight cloudiness
when no sugar is present, marked turbidity when sugar or
milk are added. Milk: Coagulated ; reaction strongly
acid. From grape- and milk-sugar pure dextrorotatory
lactic acid (no other acid) is produced, but no gas.
Upon potato there is a limited growth.

Distribution.—According to Leichmann, Giinther, >
Thierfelder, and Kozai, it is found in abundance in all
spontaneously coagulated milk, and is either the general
producer of lactic acid or, at least, the most important for
certain places and times. Yet the single fact that spon-
taneously soured milk contains preponderantly the long-
known inactive fermentation lactic acid, shows that other
varieties besides the Bact. Giintheri are concerned in the
process. Compare Leichmann (C. B. L. 1, 777).

Kozai (Z. H. xxx1, 337) has demonstrated for Halle
that, especially at higher temperature, two varieties, which
produce lactic acid, work together. They are given the
names Bacillus acidi levolactici and Micrococcus
acidi paralactici liquefaciens Halensis. By this last
name the necessity of the binomial nomenclature migh
be strikingly pointed out. Why not Micrococcus

|
! BACTERIUM PNEUMONIZ. 225

halensis ? The Bac. acidi levolactici resembles, morpho-
logically and biologically, the Bae. acidi lactici Hiippe,

_ but at room temperature only coagulates milk slowly (often
milk becomes a thick fluid only after twelve days), while
in the incubator it coagulates milk rapidly. The acid
formed is levorotatory lactic acid. The coccus is pro-
vided with a thick capsule, liquefies gelatin, and forms
dextrorotatory lactic acid. During the final reading of
our proof-sheets, Leichmann, in a partial work, claims

to find in sour milk, besides his Bact. lactis acidi, also
the Bact. acidi lactici—in the layer of cream, often even
in preponderating number (C. B. L. v, 344).

Special Culture Methods.—Ordinary gelatin or agar
plates do not give good results because of the minuteness
of the colonies. The best medium to employ is a chalk
medium (see Technical Appendix) which contains grape-
_or milk-sugar. Upon this the colonies are surrounded by
-aclear halo. Also good results are obtained with milk-
_ peptone gelatin. One pays attention to the small colonies.

Bacterium pneumoniz. Friedlander.1
(Plate 15.)

Literature.—Friedlinder (Fortschr. d. Med., Bd. 1, '715, ete.).

_ Synonyms.—Pneumonia bacillus of Friedlander, cap-
_ sule bacillus of pneumonia; also compare pages 227 and
228.
Microscopic Appearance.—Short rods (0.6-3.2 »
long, 0.5-0.8 » broad), with rounded ends. When from
_ the animal body, they present a thick gelatinous capsule,
which is developed only in milk among the nutrient
media. |
Spontaneous motility is absent.
Staining Properties.—Stains by the usual methods

. 1 The Bact. tholeeideum Gessner is only differentiated by its effect
- upon mice (A. H. rx, 129). Also the Bact. butyri colloideum Lafar
(C. B. xm, 807), constantly present in butter, according to Lafar,
appears also related, although not yet sufficiently described biolog-
ically. =

226 IMPORTANT VARIETIES OF FISSION-FUNGI.

even in the cold, but not by Gram’s method. The cap-

sules, which are colorless after the usual stain, may, how-

ever, be stained. (See Technical Appendix. )
Requirements as Regards Nutrient Media and
Oxygen.—Grows luxuriantly upon all the nutrient media
employed, both with and without oxygen.
Gelatin Plate.—(a) Natural size. Superficial: Round

or roundish, moist, white colonies, with even border,

usually much elevated, rarely flat, witha slimy-fatty luster.
Deep : Roundish to whetstone- -shaped, yellowish-white (15,

V).

(b) Magnified fifty times. Sunerfoial Round colonies
with smooth border, reddish to yellowish-brown, trans-
parent only at the periphery. Sometimes there extend
outward from the center rays which appear as dark brown
thorns and points upon the lighter underlying part (15,
vir). Usually a structure can scarcely be distinguished.
Deep: Roundish to whetstone-shaped, smooth border,
brown, opaque (15, vr).

Gelatin Stab.—Stab : Well developed, yellowish-white,
like a string of pearls. Surface growth: Elevated, like the
head of a nail. The gelatin is sometimes a little brownish
about the puncture, but never liquefied(15, 1).

Agar Plate and Stab.—Similar to the growth in gela-
tin, only the colonies are perhaps still more luxuriant and
moister.

Sometimes we observed in plates, instead of the roundish deep col-
onies, single deep veil-like spreading colonies, some of which are
reproduced in Plate 15, VIII.

Agar Streak.—Growth spreading moderately, whitish- |

yellow to gray, with a moist luster, much elevated, espe-

cially in the middle. The border is smooth, wavy, and —
the periphery transparent. Water of condensation is —

cloudy, with a slimy deposit (15, 1).

Bouillon Culture.—Very cloudy, with a slimy deposit —
at the bottom, which upon shaking becomes homogeneous. —

Bouillon becomes somewhat thickened.

Milk Culture.—Not coagulated after twenty days. Abel —

never found milk coagulated by true Bact. pneumonie,

but the opposite was observed by others; for example, —

|

BACTERIUM PNEUMONI. 227

a te (A. P., 1894, 292). Compare the observa-
ons of Denys and Martin, page 229.
- Potato Culture. —Thick, moist, highly shining growth,
w with smooth but scalloped border, bright yellow to grayish-
‘brown. It is gradually separated into padded, connected
‘sections, especially at the border.
| Chemical Activities.—From grape- and milk-sugar the
bacterium produces abundant acid, together with CO, and
/H, (40% Co,, 58% H,, Th. Smith). P. Frankland demon-
‘strated as fermentation products: ethy! alcohol, acetic acid,
‘a little formic and succinic acids. It is surprising that
‘lactic acid is not mentioned. Indol and H,S are scanty.
_ Distribution.—(a) Outside the body: Cultivated by Em-
‘merich from the foul floor of a prison.
(6) In healthy organism: Sometimes in saliva.
_ (©) In diseased human organism: As the cause of a few
cases of pneumonia and bronchitis, then occasionally, but
not very often, as the cause of inflammatory and suppura-
tive processes in almost all the organs of the body; rarely
as the cause of pyemia and septicemia. Often also found
im the blood. Rarely it causes cystitis (Montt-Saavedro,
fm, B. xx, 171).
_ (ad) In animals: The cause of pneumonia in horses, dis-
‘covered by Schtitz, is morphologically almost identical
| (Arch. Tierheil., XIII). Nail-head cultures usually are
king and the growth upon gelatin is flatter. The organ-
isms are abundant in the lungs and pleura, i. ¢., especially
‘in the necrotic parts, but sparingly in the blood. Fiedeler
si bstantiated the findings in all points (C. B. x, 310).
_ Immunity and Serum Diagnosis.—Active immuniza-
tion is possible ; the serum causes agglutination, although
the B -P. is non-motile. Landsteiner (Wien. klin. Wochen-
‘schr., 1897, 439).
fs Results of Experiments upon Animals.—Mice be-
“come sick after subcutaneous, more certainly after intrapul-
monary injection, also after inhalation, and soon die, with
ne appearances of septicemia. Also guinea-pigs and dogs
“are susceptible, but rabbits are not.
_ Of the numerous closely related varieties! we must

a Also the species in the following list (capsule bacilli of authors)
“taust be considered as forms which are identical with or closely related

a

228 IMPORTANT VARIETIES OF FISSION-FUNGI.

mention two somewhat more extensively, because they ard
found in typical infectious diseases of man, even though
they differ morphologically from the other forms only in
the insufficient characteristics already mentioned in the
key to the recognition.

Bacterium ozzenz (Abel). Lehm. and Neum.

Bacillus mucosus ozeenee (Abel, Z. H. xxi, 89); Lowenberg (A. P.,
1894, 292). Paulsen: Bacterium of atrophic thinitis (C. B. xiv, 249).

Rods of very variable length, capsule in the body often double tl a
width of the bacillus on each side, sometimes capsules oceur in milk eul+
tures. The cultures are not different from those of Bact. pneumoniz,
only they are somewhat more fluid. The formation of gas upon potato
or coagulation of milk was never observed. Sometimes marked, some-
times slight fermentation of grape-sugar. Old cultures sometimes
become a little brownish, but without a brown color of the nutrient
medium being produced.

Nt ee ee

to the Bact. pneumoniz, because, after all we know to-day, we cannot
recognize, as true characteristics of species, slight differences of adapta-_
tion to a certain variety of animal, the luxuriance of growth, the
imperfection of Gram/’s stain, or greater or less ability to produce fer.
mentation:

sre pneumonize Friedlander (Fortschritte der Medizin,
I, 715 a
Bacillus pseudopneumonicus Passet (Aetiol. der eitr. hig crags:
Berlin, 1885).

Proteus hominis capsulatus Bordoni-Uffreduzzi (Z. H., Bd. 1, 188%

. 333).

: Capsule bacillus from canal-water von Mori (Z. H., Bd. Iv, 1888, .
p. 47). 7

Capsule bacillus of R. Pfeiffer (Z. H., Bd. VI, 1889, p. 145). {

Capsule bacillus of Mandry (Fort. d. Med., Bd. yvir1, 1890, 205
C. B. vu, 570).

Capsule bacillus of Kockel (Fort. d. Med., Bd. 1x, 1891, 331).

Bacillus capsulatus mucosus Fasching (C. B. x1, 304).

Capsule bacillus of v. Dungeren (C. B. xtv, 541).

Capsule bacillus of Marchand (C. B. Xv, p. 428).

Capsule bacillus of Nicolaier (C. B. xvi, p. 601).

Capsule bacillus in keratomalacia of Loeb (C. B. x, 369; mu
literature).

Bacillus sputigenus Pansini (C. B. rx, 566). Somewhat more p
nounced differentiation.

Bacillus sputigenus crassus Kreibohm (C. B. Vil, 312). (Stained b
Gram’s !)

Bacillus aérogenes sputigenus capsulatus Herla Ne B. XXv, 359).

Bacillus capsulatus chinensis A. Hamilton (C, B Bz IV, 230). (AE i
ways forms capsules ; literature résumé.)

TOO AD

a BACTERIUM RHINOSCLEROMATIS. 229

_ The organism occurs regularly in ozena (foul), but also in pure
atrophic rhinitis without odor. The significance of. the organism in
t ae production of the ozena is therefore very questionable, Just as is
ee cance of the pseudodiphtheria bacillus, which is often simul-
taneously found. Jurasz and Hecht go so far as to question the sig-
nif ficance of bacteria in ozena, and speak of a trophic neurosis of the
nose with a putrid secretion. Compare Hecht (Minch. med. Woch-
~ chr., 1898, No. 7, 198).
_ Mice die in from one to four days after subcutaneous inoculation ;
‘| Tats and guinea-pigs are more difficult to infect, and rabbits are
- Immune.

Bacterium rhinoscleromatis v. Frisch.

| Literature.—Paltauf (C. B. 1, 236); Bender (C. B. 1, 563); Dittrich
(Cc. B. 1, 89, 483); Babés (C. B. 1, 617); Dittrich (C. B. v, 145);
| > g i (C. B. vi, 450). It behaves in all essential properties like the

. pneumoniz, yet many authors (Dittrich, Zagari) find it stains
y Gram’s method, but others do not. The growth in the gelatin stab
vs the nail-head form, is more of a transparent gray, and not quite
E 0 Se hite as in the Bact. pneumoniz. Further differences can not be
und even by the vigorous advocates of a difference between the Bact.
Be cocioromstis and Bact. pneumoniz. According to Paltauf, milk
is coagulated ; according to Abel, it is not. It is found in all cases of
491 ical rhinoscleroma (infrequent, hard, round-celled tumors of the
, partly subcutaneous, partly submucous ; more rarely in throat
; nd larynx) and claimed to be the cause of the same. In animal and
Fs lm aman experiments a reproduction of rhinoscleroma has never suc-
_ ceeded. De Simoni doubted that the organism is different from the
above members of the group of Bact. pneumonize, and, above all, that
it is the cause of rhinoscleroma (C. B. xxv, 625). The constancy of
_ the occurrence of the organism in all cases of rhinoscleroma examined
1 Be iologically remains as an incontestable, significant fact. Dittrich
found the organism generally to be scarcely at all pathogenic ; others
“observed mice to be about as susceptible to it.as to the Bact. pneu-
_ moni, and guinea-pigs less so.

“Critical Remarks Regarding Bact. acidi lactici, aéro-
genes, pneumonia, rhinoscleromatis, and ozene.

_ These varieties are, as appears from the description, at
least closely related, and only to be differentiated by
biologic characteristics which are known to be variable.
' Besides, Denys and Martin (La Cellule, rx, 1893, p. 261;
. B. xvi, 127), by repeated cultivation of pure "cultures
: ir milk, have brought the Bact. pneumonie, from three
“different sources, to a condition where it coagulates milk
J with the greatest, energy, and also produces gas from milk-
Sugar. Inversely, after being grown for eleven months
_ upon gelatin the power of breaking up grape- and milk-

ae

-

a.

:
5

sugar with liberation of gas was lost, the cultures then
growing thin and delicate upon potato, but still coagu-
lating milk. They thus resembled the Bact. Giintheri,
but it is stained by Gram’s method.

For us, consequently, all the above-mentioned forms are
botanically only biologically characteristic adaptation forms
of the same organism, which must come under the oldest
name of Bacterium pneumonie Friedlander. For practi-—
cal purposes we will, as formerly, differentiate the ‘‘ varie- —
ties,’’ but we must be conscious of their close relationship —
and of the possibility, in part proved, of their being con-_
verted into one another. These conclusions agree essentially —
with the statements of Kruse and Wilde (Fliigge-Kruse’s
Lehrbuch, 11. Aufl., p. 886, and Wilde, Diss., Bonn, 1896),
founded upon special studies. They have made observa-—
tions regarding the variability of flagella, especially in this
group, which correspond exactly with what occurs in ~
other groups, as we know from our own observations or ~
from what is found in literature, so that the relationship
with the colon group stands out yet more strongly. We
have failed to distinguish, like Kruse, a Bact. coli immo- —
bile, yet the less moist forms of Bact. aérogenes, according
to our own and Kruse’s judgment, can not be distin-
guished from the Bact. coli except by a lack of motility.

230 IMPORTANT VARIETIES OF FISSION-FUNGI.

Bacterium lactis viscosum. (Adametz, C. B. ix, 698.)
Lehm. and Neum.

Resembles the Bact. pneumoniz both macroscopically and micro-
scopically. Upon the gelatin plate it often appears as elevated
droplets. Non-motile, with capsules, staining by Gram’s method.
The surface growth in the gelatin stab is wide-spread but not very
luxuriant; upon agar and potato abundant, white, tenacious. Neither
grape- nor milk-sugar is fermented; little indol and no H,S are formed.
Milk and bouillon gradually become viscous, slimy, and may be
drawn out in long threads. The milk is not coagulated, and is feebly —
alkaline; bouillon becomes very cloudy. The slime is a carbohydrate,
which originates from the capsules of the bacteria. In our culture,
obtained from Kral, nothing was to be seen of the spore-formation ~
which Zimmermann claims to have seen. The organism was discovered —
by Adametz as an important enemy of the butter industry, the cream —
becoming slimy and the butter obtained therefrom spoiling and ~
becoming soft and pale. Found by Zimmermann in water. Leich- —
mann’s bacillus, which is somewhat thermophilic, does not form —
spores, and ferments sugars, appears different (C. B. XVI, 122).

1

Fre OY agit PEs

a

BACTERIUM PHOSPHORESCENS. 231

The Bacterium Hessii Guillebeau (C. B. x1, 439) is different. It
is actively motile, liquefies gelatin, and forms no capsule. It like-
wise makes milk tenacious and no spores are described. In the same
place may be found some further statements regarding varieties which
render milk tenacious. Compare also Micr. Freudenreichii Guil.

(p. 174).

: Bacterium Pfliigeri! (Lassar) Ludwig. Bacterium

phosphorescens. Bernh. Fischer (Z. H. ii, 92).

Literature.—Ludwig (C. B. 1, 372); K. B. Lehmann (C. B. v,
785); Beijerinck (C. B. vir, 616, 651); Katz (C. B. rx, 157).

Microscopically, short, plump rods, single or in pairs. Also spher-
ieal and short oval forms occur. Striking involution forms appear in

_ old cultures. There are neither spontaneous motion nor flagella.

Beijerinck claims to have observed spontaneous motion in sea-water.
Facultative anaerobe, but does not emit light when air is excluded.
The addition of 3% of sea-salt is favorable. Optimum at 20°, maxi-

- mum at about 39°, minimum at 0°. Upon gelatin and agar it is

indistinguishable from the Bact. acidi lactici ; once we obtained upon
gelatin plates colonies exactly like those in Plate 19, I, with most
peculiar outgrowths. Older gelatin and agar cultures exhibit a
tendency to become yellowish and yellowish-brown. Gelatin is never
liquefied. Potato cultures are yellowish, moist, sometimes with gas
bubbles. Grape- and milk-sugar and maltose are converted into acid,
accompanied by abundant formation of gas. Milk is coagulated.

The emission of whitish, greenish light is intense if oxygen is
admitted as long as the cultures are frequently transferred to fresh
nutrient media containing salt ; but if this is omitted, the emission
of light is soon lost. For a time the photogenic function may be
regenerated by transplantation upon salt (herrings) gelatin, but it is
permanently lost in time if the bacteria are cultivated upon ordinary
media with infrequent transfer. Concerning the photogenesis, com-
pare page 57. A few drops of phosphorescent bouillon culture may

_ give a milky luster to a liter of sea-water.

Neither the bacterium nor its metabolic products in small amounts

are harmful. It lives in the northern seas, causes occasionally phos-

phorescent sea, more often phosphorescence of fish, meat, ete.

The Bacterium of Giard (C. B. vi, 645; vit1, 177), which is
pathogenic for crawfish, and makes the living, inoculated animal
phosphorescent, appears, from the incomplete description, to be simi-
lar. Phosphorescent gnats (mycetophila), observed as rarities in
Germany, must owe this property to bacteria. Henneberg (C. B.
XxXvy, 649). The phosphorescent bacillus described as Photobacte-
tium javanicum Eykmann (C. B. Ix, 656) is plump and motile.
Regarding a second group of photogenic micro-organisms, see under
Vibrio albensis Lehm. and Neum.

1 Beijerinck distinguished B. phosphorescens from B. Pfliigeri by

_ biologic characteristics.

232 IMPORTANT VARIETIES OF FISSION-FUNGI.

Bacterium typhi. Eberth, Gaffky.
(Plates 16 and 17.)

- ] _
f- q ;
f
y

’ @

fe

}

q

Ordinary Names.—Typhoid bacillus, Bacillus typho- —

sus Kruse-Fltgge.

Literature.—Exhaustive list of literature (689 in number) by Lése-
ner (A. G. A XI, 207).

Microscopic Appearance.—In organs usually short,
rather plump rods (1.0-3.2 # long and 0.6-0.8 broad);
much less often found in short chains. In cultures all
forms, from short rods to long threads occur, threads being
especially well developed upon potatoes of acid reaction.
The shining polar bodies are not spores (see below). Ac-
cording to Leo Miller, however, the abundance and regu-
larity of the occurrence of these bodies upon feebly acid
nutrient media distinguishes the Bact. typhi from the B.
coli (A. K., 1. Band, Heft 1, 1894) (17, vu).

Spontaneous Motion and Flagella.—<Active motion
of the short rods; in threads a very beautiful snake-like
motion is seen. The flagella are long and tortuous and
are located all about the surface of the bacteria in num-
bers of 8 to 14 (17, rx, x).

Staining Properties.—Not by Gram’s method. -

Requirements as Regards Nutrient Media, Tem-
perature, and Oxygen.—Grows best as aerobe, also as
anaerobe, and fairly well in CO,. Grows well upon all
nutrient media employed, and bears acid well. Optimum
about 37°. Upon non-albuminous nutrient media, such
as Uschinsky’s and similar combinations, it grows but
scantily. According to Proskauer and Capaldi, it, in
contrast to the Bact. coli, does not require the amido and
ammonium nitrogen of the body for its growth.

Gelatin Plate.—

(a) Natural size. Superficial: At first small, yellow-
ish, punctiform colonies, becoming, in a short time,
roundish, irregularly notched or delicately lobulated,
and shining. The periphery is clear, transparent gray,
the center whitish, opaque, grayish-yellow, sometimes
slightly elevated. Deep: Punctiform, later roundish or
usually whetstone-shaped, yellowish (17, 1).

in 2 vila th Ua

phe vale gle

BACTERIUM TYPHI. 233

7
7
: (6) Magnified fifty times. Superficial: Up to forty-eight

_ hours the colony is entirely colorless and transparent. The
border is smooth and lobed. The surface presents wavy
- elevations, and numerous branching, strongly reflecting,
_| whitish, winding lines, which look like incisions or

_ scratches. The colony then appears fairly homogeneous,
‘| grayish-yellow, with white, curved lines and bands extend-
- ing from the center, between which are to be seen concen-

_ trically arranged lines. These lines are the expression of

_ folds in the colony (17,1). When more highly magnified

(xX 150) (17, 1), indistinct parallel curved lines may be
| seen. Not infrequently the superficial colony is thicker,

and then there occurs, instead of the windings, etc., in

_ the yellowish almost opaque appearing colony, only an

indistinct development of figures resembling hen-tracks.

_ (Compare 19, vi.) All the forms reproduced in Plates 18

_ and 19 of the Bact. coli may also occur; also spirals and

tail-like appendages, as in Plate 19, 1. Deep: Round to
roundish colonies, bright yellow, homogeneous, delicate,
shaded with gray, with even borders (16, v1; compare also

19, v).

Gelatin Stab.—Stab: Thread-like, slightly granular,

_ whitish-gray (16, m1). Surface growth: Thin, white, gray-

ish-green, iridescent, extremely transparent, roundish,

_ notched, with a dry luster, not elevated, extending to the
_ glass (16, Iv).

Gelatin Streak.—Somewhat spreading, white and thin
growth, as on the surface of the puncture.

Agar Plate.—

(a) Natural size. Superficial: Irregular, roundish, gray-
ish-white, shining, slightly elevated colonies. Deep: Punc-
tiform, gray (17, Iv).
: (b) "Magnified ‘sixty times. Superficial colonies: Round or
_ roundish, smooth border, bright yellowish, becoming darker
toward the middle, finely to coarsely punctated and trans-
_ parent at the edge; from the center usually dark yellow,
_ winding or jagged lines extend outward; morule are rare
(17, v1). Deep: Roundish to whetstone- -shaped, border
smooth or rough, brownish-yellow, opaque, without inter-
nal markings or finely granular (17, v).

Agar Stab.—Stab: Thread- like, sometimes a little

“ee ee ee Fe Ue

234 IMPORTANT VARIETIES OF FISSION-FUNGI.

granular, gray (16,1). Surface growth: Irregularly round-

ish, with an almost even border, whitish-gray, with an

oily luster, soon extending to the wall of the tube. Later —

it becomes yellowish-gray (16, 11).

Agar Streak.—Moderately spreading growth, wavy,
with a smooth edge, whitish-gray, shining, sometimes
appearing to be transparent in many places because of
porosity. The water of condensation is clear with slight
precipitate (16, v).

Bouillon Culture.—Cloudy with abundant sediment
which is homogeneously distributed upon shaking.

Milk Culture.—Milk is not coagulated even after stand-
ing for weeks. In spite of active multiplication, only very
little acid is formed.

Potato Culture.—From the line of inoculation the
growth spreads quite widely as an extremely delicate,
moist, often almost entirely invisible layer (17, v1),
which, when touched with a platinum needle, may some-
times be drawn out into delicate slimy threads. This was

first described by Gaffky (Mitt. a. d. G. A. m, 872) as.

characteristic, and was long held to be an important
specific characteristic, but it is not present in many cultures.
Other cultures, which grow typically upon acid potatoes,
present, at least upon alkaline varieties or alkalinized
portions, an atypical, luxuriant, grayish, white, or brown-
ish-yellow growth, which sometimes is moister, sometimes
drier, often not spreading much, and resembling the B.
coli. (Compare 18, Ix.)

No Spore-formation.—The formations which were formerly held
to be spores, and which appear especially upon faintly acid potatoes,
are of two varieties, as first pointed out by H. Buchner (C. B. Iv,
353). In unstained bacteria refractive polar bodies may be mistaken
for spores, but they stain especially easily with anilin dyes (quicker
than the bacteria) and do not impart any increased resistance to the
bacteria. In heated and stained preparations there occur vacuoles
which: have a resemblance to spores because of their form and size,
and from not being stained by the ordinary methods, but such a
vacuole is never stained by the special stain for spores. According to
H. Buchner, these roundish vacuoles are located especially in the ends
of the rods ; according to Leo Miiller, especially in the middle, while
the stained masses occupy the poles. The Bact. coli shows much more
irregular and very inconstant vacuole formation.

he ko

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fre vo De tebe

ee Lone per ge

he tly

De a 7 i
mee ey iH Wms 4

te oh a el a

BACTERIUM TYPHI. 235

Resistance.—
(a) Against drying: They tolerate preservation in the
dry condition for months ; according to Uffelmann, in soil

and clothing even, for one or two months. They do not

withstand such a complete drying as is necessary in order
to reduce to dust. Germano (Z. H. xxiv, 403). Compare

’ also Ficker (Z. H. xxrx, 1).

(b) Cold and heat: Janowski (C. B. vin, 167, 417, 449).

| _ They withstand cold well.

(c) In manure and feces: Over a week (Gartner).

(d) In water: From a few hours to many days. Com-
pare page 40.

(e) Chemical disinfecting agents: Compare Kohler (C. B.
xiv, 89).

The duration of life in the human body may be very
considerable. They have been demonstrated by Sahli in
pleural exudate fifty days after the beginning of the
disease, and by Hintze in the pus of periostitis ten months
after a case of typhoid fever.

Chemical Activities.—There is no production of pig-
ment nor odoriferous substances. It reduces solutions of
litmus, converts nitrate into nitrite, and gradually leads
to a disappearance of nitrite. They form levorotatory
lactic acid from grape-sugar (feebly from milk-sugar) and
no visible gas bubbles from any carbohydrate (verified by
Buchner, A. H. 111, 425, 1885). They form H,S strongly.
Indol is not produced. The cultures are rich in toxins ;
the germ-free filtrates are actively pathogenic.

Distribution.—

(a) Outside the body: So far, in a few cases in water and
soil which have come in contact with typhoid dejecta.
Recently demonstrated by Loésener in five instances in
Specimens of soil, portions of cadavers, and stools, where
there was no suspicion of the presence of typhoid bacteria.
Similar results were obtained by Remlinger and Schneider
(H. R., 1896, 743).

(6) In healthy body: So far, never.

(c) Indiseased human body : In cases of typhoid fever, as
the cause of the disease. Cultures are obtained with ereat-
est certainty from the spleen and lymph-glands, in which
the bacilli are always distributed insmall clumps. Often,

236 IMPORTANT VARIETIES OF FISSION-FUNGI.

also, it may be successfully demonstrated in the~ blood
(blood from the heart, veins, rose-spots); but, to be sure,
it is so often not found that the importance of the blood
examination for substantiating the clinical diagnosis is not
great. Kiihnau found the typhoid bacillus in the blood
from the veins of the arm in 10 out of 41 cases (Z. H.
xxv, 492). Neufeld obtained very good results in the
blood from rose-spots by making use especially of fluid
nutrient media (Z. H. xxx, 498). It has also been very
frequently demonstrated in the kidney, liver, and bile
(Chiari), and especially in the urine (H. Neumann, C.
B. viu, 80). Regarding the occasional presence of enor-
mous numbers of typhoid bacilli in urine, see Petruschky
(C. B. xxi, 577). Statements regarding successful cul-
tivation from typhoid stools are comparatively rare (com-
pare p. 237). The typhoid bacterium can itself cause the ~
various complications in the clinical picture of typhoid.
It has been demonstrated with certainty as the only cause
of cases of serous and suppurative inflammations of the
spinal cord, of the brain and its membranes, of the lungs
and kidneys, and in the erysipelatous, phlegmonous, and
suppurative processes of typhoids (in bones, skin, testicle,
lymph-glands, parotid, thyroid, spleen, etc.). The pyo-
genic function of the B. typhi is no more contested, and
has also been demonstrated by experiments upon rabbits.
However, in many (the majority ?) cases mixed infection
with Micrococcus pyogenes, Streptococcus pyogenes or lan-
ceolatus, etc.,must be held responsible for the complica-
tions.

Results of Experiments Regarding Pathogenic Ac-
tion in Animals.—<According to the fairly universal view
in Germany at present, the production of an infectious dis-
ease, analogous to typhoid fever in man, has never been
successfully accomplished in any animal by any mode of
infection. As a rule, bacteria introduced subcutaneously
rapidly die, at least, do not multiply, and the injuries re-
sulting may be produced in a similar manner by filtered
cultures. Thus, the results are due to intoxication and
not to infection. (Sirotinin, Z. H. 1, 465.) Also Pe-
truschky favors the idea of an intoxication rather than an
infection (Z. H. xu, 261).

[3
;
4
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BACTERIUM TYPHI. 237

Chantemesse and Vidal (A. P., 1892, 755) and Sana-

-relli (A. P., 1892, 721) were able, on the contrary, to so
increase the virulence by all sorts of artificial means that
‘they obtained varieties which are truly pathogenic for
animals. Chantemesse (H. R., 1897, 1103) was even able

to produce sickness in rabbits and monkeys by highly
virulent cultures introduced into the stomach, and the
animals died with typhoid symptoms (clinical and ana-
tomical). Thus the Bact. typhi becomes acclimated to
the animal body..

Special Methods for the Demonstration of the Bact.

Aran

typhi.

It is usually easy to cultivate them from the spleen and
lymph-glands of a fresh typhoid cadaver ; still, not infre-

— quently more colonies of the Bact. coli are obtained than

aay an

typhoid. The case is different when the bacteria are to be
sought for in water, feces, etc. The fact that the demon-
stration of the Bacterium typhi when in mixtures with
other bacteria appears to be very difficult for all investi-
gators! has led to numerous suggestions to replace the
simple gelatin plate method by better ones. A great dis-
trust is aroused against all of these suggestions, since every

‘new author criticizes the suggestions of his predecessor and

usually discards them.

The two principal methods which have been employed
are :

1. Preliminary Culture.—The suspicious water is placed
in nutrient media which contains an antiseptic, and kept
twenty-four to forty-eight hours in the incubator. Water
bacteria, especially a number of liquefying varieties, die,

_ while the Bact. typhi and coli, which are more resistant

to disinfectant agents, multiply in the incubator. Unfor-
tunately, the rapidly growing forms of Bact. coli, besides
Bact. vulgare, streptococci, and oidium, multiply more in-
tensely than the Bact. typhi, and when plates are prepared

1 An idea of the difficulty is given by the fact that many authors
were not able at all to isolate typhoid bacteria from typhoid stools,
and that Nicolle, Grimbert, and Chantemesse declare it to be impos-

sible to recover typhoid bacteria from water, containing abundant
Bact. coli, to which they had been added.

238 IMPORTANT VARIETIES OF FISSION- FUNGI.

from the preliminary culture, almost with absolute cer-
tainty many coli forms are obtained, but also, according
to most of the critical writers, much fewer typhoid bac-
teria than were in the original fluid (Lésener).

2. The direct preparation of plates from gelatin which con-
tains materials interfering with growth: phenol, hydrochloric
acid, methyl violet, potato juice, etc. Lésener, who has
tested all these methods, recommends the following as the
only useful one : Plates are prepared directly upon gelatin
containing 0.03 to 0.05% phenol. The plates are best pre-
pared, according to Kruse, by inoculation upon the surface
(Tech. Appendix). Upon this carbol-gelatin the colonies
of Bact. typhi and coli grow in the usual manner; many
others, especially liquefying varieties, are, on the contrary,
greatly retarded. From all colonies resembling typhoid
inoculations are made into liquefied 2% grape-sugar agar
(about a dozen tubes) and the shake cultures thus pre-
pared are placed in the incubator. The tubes in which
there is no fermentation are studied further, as indicated
on page 239.

Almost simultaneously with Lésener, Elsner studied, in
Koch’s Institute, the methods for the ready demonstration
of typhoid bacteria by means of special nutrient media, and
instead of the potato-gelatin of Holz,1 which had given
unsatisfactory results in the hands of many writers, he
recommended a new feebly acid potato-gelatin containing
1% iodid of potassium. (See Tech. Appendix.) (Z. H.
XxI, 25.)

According to Elsner, scarcely any bacteria except Bact.
typhi and coli grow upon his nutrient medium, the lique-

fying varieties not at all. Bact. coli grows very well, and.

after twenty-four hours presents already perfectly developed
colonies.

In contrast to this, the Bact. typhi grows very slowly;
after twenty-four hours the colonies are scarcely visible
with low magnification, and after forty-eight hours they
appear as small, clearly shining colonies, like water drop-

1 According to Holz, if carbolic acid is added to potato-gelatin, even
the typhoid bacteria eTow in a non-characteristic manner; if the addi-
tion is omitted, then very many liquefying germs are not at all nine
turbed in their growth.

"cipher cance

Sn

BACTERIUM TYPHI. 239

lets or exceedingly finely granular, contrasting with the
large, markedly granular, brown-colored colonies of the
2 Bs ct. coli.
_ The method is said to give very good results, and usu-
uly allows of the isolation of the typhoid bacterium from
’ stools, and the results are said to be most perfectly in har-
_mony ‘with Pfeiffer’s typhoid reaction (see below). Com-
“pare also Jemma (Miinch. med. Woch., 1897, No. 33)
t ed Sterling (C. B. xxm, 334).

_ Special Differential Diagnosis of the Bact. typhi, Es-
pecially from the Bact. coli.

_ The following peculiarities must all be demonstrated:
1. Rods, short to thread forms; active motility; abun-
di nt, long, peritrichous flagella; ‘not stained by Gram’s
“method.
_ 2. White film upon gelatin which is not liquefied.
_ 98. No formation of gas from grape- or milk-sugar in a
shake culture.
4. Uniform cloudiness of sugar bouillon in fermentation
tubes without formation of gas. No formation of acid
from milk-sugar, abundant from grape-sugar.

9. No coagulation of milk.

_ 6. Indol not produced in peptone solution.

_ 7. Finally, Losener places value upon the demonstration
‘by means of culturesin Petruschky’s litmus whey (at 87°)
eu Baat the questionable typhoid bacterium in about forty-
ght hours does not produce more than 3.0 ¢.c. of deci-
ae formal acid from 10 c.c. of milk, while the coli bacteria
form more than 8 c.c.!

as Marked agglutination by specific serum (see below).

_ 9. Of less value in the diagnosis are: (1) The microscopic appear-
‘ance of the gelatin plate, as it may be almost identical with the Bact.

a _ * Upon all these points a very satisfactory uniformity has been
Teached. To be sure, the uniformity depends in part upon an agree-

= ent, which is, that all those bacteria which do not present these pecu-

le rities of the typical typhoid culture are simply declared to be differ-
® i from typhoid, under the assumption that the typhoid bacterium
esnot vary. How little probability this assumption possesses in the
oe the enormous variability of the closely related Bact. coli, re-
—q Bs $s no discussion.

240 IMPORTANT VARIETIES OF FISSION-FUNGI.

coli. (2) The delicate growth upon potato, since there are typhoid —

bacteria which grow as luxuriantly as Bact. coli. In order that a — ;

potato culture may be of diagnostic value, two pieces from the same
potato must be placed in a dish and inoculated, one with the culture
in question, the other with a certain typhoid culture (Germano and
Maurea). According to these authors, with whom Losener agrees, a
deviation from the growth of true typhoid bacteria upon the same
potato is sufficient to exclude a diagnosis of typhoid. (3) Growth upon
nutrient media to which are added antiseptic substances (phenol, form-
aldehyd, acids, etc.). The Bact. coli always tolerates these me ecse
better than the typhoid bacterium.

The Diagnosis of Bacterium typhi is excluded :

If one of the following properties is demonstrated:

1. Absence of motility, flagella absent or located at the
pole, typical spores, staining by Gram’s method.

2. Absence of growth at body temperature.

3. Coagulation of milk. Formation of gas in grape-
sugar agar or fermentation tubes.

4. Liquefaction of gelatin.

A beautiful example of a thorough differential diagnosis

between mud and typhoid bacteria is given by Houston —

(C. B. xxv, 518).
Serum Diagnosis of Typhoid.1

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tt tt te i te het ebm

-

ole Ree ay

In doubtful cases the typhoid diagnosis may very often |

be verified by the serum test. Since we have been

acquainted with the Gruber-Durham agglutination reaction —

in vitro, almost always this is employed instead of R.

Pfeiffer’s more detailed reaction in the abdominal cavity —
of the guinea-pig. Cultures upon slanted agar, eighteen —
to twenty-four hours old at 37°, are used for the test, and —

1 Tf one has no immune serum, still, according to Laschtschenko,

cal ia we

he may differentiate the Bact. typhi from the Bact. coli in the follow-

ing manner (H. R., 1899, No. 3): Several test-tubes, each containing —

2 c.c. of fresh defibrinated rabbit’s blood, obtained by venesection, are —

provided. To these are added two drops of a dilute suspension of the
culture in question. The suspension is prepared by mixing 1 loop-
ful of an agar culture with 10 c.c. of bouillon, and then diluting 0.5
c.c. of this with 9.5 c.c. of bouillon. In the case of Bact. coli which
have not been cultivated too long, the bacteria are never dead in
six to seven hours, and usually are much more numerous, while
the Bact. typhi (ten cultures!) were always much less in number,
no matter whether the culture had been isolated for a short or long
time.

BACTERIUM TYPHI. 241

of such a culture 2 mg. are finely divided in 0.5 c.c. of
| bouillon (p. 105).
2 i Testing Doubtful Cultures by Means of Known Typhoid
Serum.—<Active serum is prepared, according to R. Pfeiffer,
as follows (compare also Fodor and Rigler, C. B. xxm1,

: A rabbit weighing from 14 to 2 kilos is injected subcu-
‘taneously with a suspension of the bacteria from three
lanted agar cultures of Bact. typhi, twenty-four hours old,
jhe suspension having been kept in a water-bath for one
hour at 65° before injection. Ten days after the injection
the animal is bled into a tall narrow glass cylinder, and
iter the blood has stood twenty-four hours in an ice-box,
slear serum is obtained.! (See p. 105 concerning this and
the dilution of the serum.) Such a serum agglutinates

ig. 18.—Smaller and larger clumps of agglutinated Bacterium typhi.

true typhoid bacteria in dilutions of from 1 to 30, to 1 to
100 or more. By means of a series of tests the limits of
‘the value of the reaction are determined. The organisms
to be diagnosed must also be agglutinated by a similar
dilution. If it should be affected much more feebly (2. e.,
only with a higher concentration: for example, 1 to 20)
E than the standard culture, this may depend upon the
sreater virulence of the first, because the greater the viru-
ce, the more easily the effect is produced. If, in spite
of lower virulence, the effect is much less than in the
standard culture, then the bacteria in question are not
_ typhoid bacteria.

2. Testing the Serum of Patients Who Are Suspected of Hav-
__ +Immune serum with the addition of 0.5% thymol is very stable,
out also serum may be absorbed by filter-paper in certain quantity

“and each time a test is to be made a piece of the paper may be added
te to diluted typhoid bouillon (Richardson, C. B. xx1, 445)
| 6

242 IMPORTANT VARIETIES OF FISSION-FUNGI.

ing Typhoid with Known Typhoid Bacteria.—The test con
sists in the demonstration that the serum, properly co
lected and diluted, according to the instructions on pages
104 to 110, agglutinates typhoid bacteria from a twenty-four
hours’ agar culture inside of an hour at 37°, when the
dilution is 50 times; or even, if possible, when dilutec 1
100, 500, or 1000 times. |
Following exactly the instructions on page 105, different
tests are made with varying strengths of serum and the
limit of the activity of the serum determined. If the
serum does not cause agglutination in dilutions highe
than 1 to 50, then no decision is possible, for serum:
which are active when thus diluted occur without typhoic
fever being present or having previously occurred (25%
of healthy persons furnish serum which is active in dilu-
tion of 1 to 10). On the other hand, the absence of ¢
glutination does not absolutely prove that typhoid foveal a
does not exist, especially at the commencement of the dis-
ease, for before the third week the reaction is not so very
rarely absent; after the third week, it is lacking in only
1% of the cases (compare Bieberstein, Z. H. xxvut, 347).
The diagnosis of typhoid is most certain when, during the
course of the disease, the reaction having been previously
absent, it develops with j increasing vigor (vy. Leube). For”
many details and the entire literature, consult Kasel (Ver-_
handlungen der phys.-med. Gesellschaft in Wiirzburg,
xxxt, No. 6, 1899); in abstract, Kasel and Mann (Miinch. -
med. Wochenschr., 1899, No. 18, 581).
Not infrequently coli. bacteria are influenced more
strongly than typhoid bacteria. by serum from typhoid
patients, and this is explained upon the entrance of coli
bacteria through the intestinal ulcers during the typhoid, ~

1 The proposal to employ blood instead of serum in the diagnosis
of typhoid is often made and has been found practical. With a
pipet 0.1 c.c. of blood is placed in a small graduated cylinder and
diluted to 2¢.c. This blood mixture is tested and corresponds in its
effects to a serum diluted 40 times. ° Compare Babucke (C. B. XXII, ~
1092). If one possesses a Thoma-Zeiss counting apparatus for leuko-_
cytes, 0.5 ¢.c. of blood may be drawn into the melangeur and then
10 c.c. of water. Of the mixture, a drop is mixed with a drop of sus-
pension of bacteria. Thus the dilution becomes 1 to 40. (Rostoski,
Miinch. med. Wochenschr., 1899, 209. )

BACTERIUM COLI. 243

ete., thus producing a mixed infection (Stern, Berl. klin.
“Wochenschr., 1897, 225). Thus the serum of patients
(in distinction to animal serum) can be employed as a
test for typhoid bacteria only when it has been previously
‘found entirely or almost without effect upon known Bact.

TC

3 -eoli

Regarding the relation of the Bact. coli to the Bact. typhi there has

much work done and still more written. Objectively considered,

the case is that numerous things speak for the probability that the

- Bact. typhi may originate from the Bact. coli bya loss of certain zymo-

_ genic and the acquisition of pathogenic properties, but the possibility

! of such a change is not proved by any experimental investigation.

We may believe what we will, but for the present the Bact. coli

and Bact. typhi are two different organisms. Nothing pertinent to

"the question is afforded by the demonstration, coming from various

i, that the Bact. coli may lose its ability ‘to produce indol and

te milk. Also, the circumstance that Peckham (Jour. f. ex.

Med, 1 1897, 11, 549; C. B. XXIII, 986) was able to stimulate typhoid

bacteria to vigorous production of indol, and that cultures absolutely

like typhoid are found which present no agglutination with typhoid

serum, do not certainly prove the transformation of one kind into the
_ other, but only point to an extraordinarily close relationship.

Bacterium coli (Escherich). L. and N.
(Plates 18 and 19. )

Synonym.—Bact. colicommune Esch. (Darmbacterien
des Siuglings, Stuttgart, 1886). Compare page 250 and
forward.

baa

___ Literature.—Kiessling, Sammelreferat (Hygien. Rundschau, 1893,
m1); Lésener (A. G. A. xI, 207); Germano and Maurea (Ziegler’s
_ Beitriige, x11, 494; C. B. xv, 60) ; Tavel and Lanz (C. B. xrv, 705);
- Von Stécklin (C. B. xvI, 130); Gordon (Jour. of Path. and "Bact.,
"ty, 438).

& Common Names.—Colon bacillus, Coli bacillus,
_ **Coli Escherich.’’

: Microscopic Appearance.—According to the nutrient
-medium and the age of the culture, the B. coli occurs as
almost isodiametric, oval forms, or (and as a rule) as
- short rods, 2 to 4 » long and 0. 4 to 0.6 » broad; more
rarely i in the form of shorter or longer threads. The ends
"are rounded; not infrequently two rods lie together in

its also chains of bacilli occur. In unfavorable con-

tions (old potato cultures, soda bouillon) there occur

4

244 IMPORTANT VARIETIES OF FISSION-FUNGI.

readily staining polar bodies at the ends of the rods, while
the middle remains unstained. Young rods are always
actively motile. Concerning non-motile forms, see under
Bact. aérogenes (19, vill, Ix).

Staining Properties.—Easily stained, even with cold
solutions, by ordinary methods. It is not stained by
Gram’s method. ?

Form and Arrangement of the Flagella.?—Most
authors, like ourselves, have found the flagella similar to
those of the Bact. typhi, but a little less numerous—~. ¢.,_
4 to 8 peritrichous, long, slightly wavy flagella. Stécklin
has found very great variation in individual ‘‘ yarieties "7
of Bact. coli in this respect; some correspond to the
description given above, a oreater number possess 1, 3, ont
5 flagella, a few generally only a single flagellum at ¢

os

end. In the very painstaking work of Remy and Sugg
the flagella of the B. coli are described as somewhat
shorter than in the Bact. typhi, and very fine (C. B. xtv,
70). We cannot entirely confirm this, for among the
different varieties (about twelve) which were stained by
us there were some with very long flagella. Sometimes

en Piggahs mn

the flagella extend outward from a colorless capsule.
Relation to Oxygen.—Grows best as aerobe, espe- —
cially upon nutrient media containing sugar; anaerobically
it does not grow quite so well, and still more poorly when
sugar is absent. It grows also in CO,, but not quite so”
well. ‘
Requirements as Regards Temperature and Nutri-_
ent Media.—It grows rapidly at room temperature and
very well at 37°, is satisfied with the various nutrient
media, tolerates quite strong acid reaction, but in nutrient
media containing sugar it not infrequently produces more

1The statement of Alexander Schmidt (C. B. x11, 761) that by
cultivating upon nutrient media containing fat the Bact. coli acquires
the property of staining by Gram’s method, we could not verify, and _
no more could Jacobsthal (H. R., 1897, 849).

2 We group provisionally the forms which have only one or a few
polar flagella, instead of a large number of peritrichous flagella,
as special ‘‘forma polaris’? Lehm. and Neum. (compare p. 252). The
non-motile forms we place under Bact. aérogenes.

a
ee 4

BACTERIUM COLI. 245

cid than it can tolerate, and so dies out. It grows well
n nutrient media which contain no albumin.

Remarks Regarding the Macroscopic Growth of
the Bact. coli.—All closely observing recent authors
(Dunbar, Ferrati, Lésener, etc.) state that the Bact. typhi
anc Bact. coli cannot be differentiated with certainty by
means of their cultures, but only that generally the coli
bacteria grow more luxuriantly upon the various nutrient
ai The color is white, often, in thick growths, be-
soming gray or yellowish- white.

_ Gelatin Plate.—(a) Natural size: Like the Bact. typhi,
; except that here the moist, opaque forms (not uncom-
monly somewhat raised above the medium in the form of
d ops) are more frequent than the thin, delicate, iridescent

_ growths, which are the rule in the Bact. typhi (19, 11).
F-(b) Magnified seventy times: Not to be differentiated with
sinty from Bact. typhi (compare 17,1), but on account

. the greater thickness of the film, the beautiful striking
system of furrows is rarely well developed in Bact. coli
= (19, li, tv, and y1). We have often observed the deep

? olonies, which, as a rule, are roundish or whetstone-
a paped (compare Lis ¥; and 15, v1; both types are very
frequent), present wonderful, drawn- out, lobulated, tailed

Btorms, which remind one of the zooglese ‘of Bact. vulgare,
_ and for which we can make only high temperature (soft-
ness of the gelatin) responsible (19, 1). Similar forma-
tions have since been described by W.. Rosenthal (Deut.
_ Arch. klin. Med., tv, 513). Compare also the complete
_ representation of Klie, who observed and represented
= such forms of colonies of typhi and coli bacteria, espe-
ec Belly upon soft nutrient media, containing little gelatin
mec. B. xx, 49).

3 “Gelatin Stab and Streak.—Like Bact. typhi, only
‘somewhat thicker, more opaque, and more rapidly grow-
ing. Never liquefying (18, 1, 11).

_ Agar Plate.—Colonies exactly like Bact. typhi, only

usually somewhat thicker and moister. Magnified seventy
- nes, the deep colonies often appear somewhat rough and
: knobby (18, v1), the superficial usually roundish, finely
- punetated, almost structureless, and opaque; at other times
_ they are finely lobulated, with markings like a mulberry.

+
-

246 IMPORTANT VARIETIES OF FISSION-FUNGL

Agar Streak and Stab.—Like Bact. typhi, often more>
luxuriant (18, m1, Iv, v).

Bouillon Culture. —Cloudy, with a moderate, slimy
precipitate, which upon shaking rises up and becomes
homogeneously distributed. Sometimes there is a distinct
pellicle formed on the surface of the bouillon.

Milk Culture.—Milk is usually rapidly coagulated;
more rarely, slowly. In connection with the ability to
break up milk-sugar, coagulation of milk is never absent.
Regarding non-coagulating forms, see under Bact. cholerze
suum.

Potato Culture.—Growth with a wavy outline, at first”
yellowish-white to grayish-yellow, later pea-yellow to yel-
lowish-brown and grayish-brown, partly flat, partly much
elevated, usually with a moist luster, less often dss and
dull. The potato in the region of the growth is usually —
discolored (18, 1x). Rarely the Bact. coli produces a-
delicate, almost invisible potato growth resembling that of
Bact. typhi.

Resistance to various injuries is about like that of the
Bact. typhi. It is even more resistant to acids, formalin, —
and other chemicals. According to Walliczeck, it bears
drying poorly (C. B. xv, 947).

Chemical Activities.—

(a) Chromogenesis: Only upon potato and always mod-
erate (yellowish-brown).
(6) Odoriferous and gustable substances : Uncharacteristic,
ill-smelling substances are developed upon agar and gelatin, —

but especially upon potato cultures.

(c) Gas and acid production from carbohydrates: Grape-—
and milk-sugar are fermented, with the production of a~
mixture of acetic, formic, and ‘lactic acids. According to —
Oppenheimer, there are formed 7 0% volatile and 30% non-
volatile acids, and some iodoform-forming substance (alco- —
hol). Many cultures ferment cane-sugar also. With this
fermentation there occur abundant CO, and H, in varying ©
proportion; we found about one-fourth CO,, the rest being —
H and some N, but no marsh-gas. According to Péré (A.
P., 1893, 737), three different Bact. coli formed levorota-
tory lactic acid from nutrient media with grape-sugar,
which contained peptone as a source for nitrogen, just as

ae

ee ae ee aes

w etn wae

BACTERIUM COLI. 247

done by Bact. typhi. But if ammonia was the source
' the nitrogen, then only the Bact. typhi and one Bact.
sli isolated from man produced levorotatory lactic acid
a remarkable manner; both the other coli cultures (from
: se and animal feces) produced dextrorotatory lactic

(d) Vigorous production of H,S from peptone; usu-
lly abundant indol. We have never failed to find traces
' indol.

a - Karplus found, in the urine of a patient, an organism resembling
he typhoid bacterium, which produced H,S and methylmercaptan
jundantly from the substances containing sulphur in the urine (C.
3. XVI, 701).

ee _(e) Decomposition of urea occurs with many cultures,
bt by no means in all (Barlow, Mann). Compare page
0. Hallé and Dissard, and recently Mann, have demon-
_ strated very minutely the decomposition of urea. Kashida
‘found it so constant that he described the production of
“ammonia in a lactoso-urea nutrient medium as a charac-
| teristic peculiarity as opposed to Bact. typhi (C. B. xx1,
802), while Melchior (Cystitis und Urininfektion, Berlin,
1807 considers the Bact. coli to be the most common
ause of cystitis (after previous injury to the bladder),
but denies that it can produce ammonia by breaking up
ua irea. Similar negative results were previously obtained
y Schnitzler and Krogius.
5 eatribution —
(a) Outside the body: In canal-water, impure water,
but also in springs which can scarcely "be suspected of
pollution, there occur very often organisms which corre-
: spond to Bact. coli (v. Freudenreich, Lehmann and
eumann). We never failed to find them in water sus-
v« sted of containing typhoid bacteria.

_The narrower the definition is made, so much the more is the
nur aber reduced. Thus, for example, Schardinger (C. B. xvi, 853)
= eclares water organisms, resembling Bact. coli, which ferment grape-
Sad grow in the incubator, to be frequently present, but in
Spite of it that the Bact. coli is rare. Most of the producers of fer-
1% Besos are easily differentiated from the Bact. coli by the milk-
ite, slimy, tenacious growth upon plates (see below).

3 "Regarding the occurrence in dough, compare page 255.

Gordan found it constantly in decomposing Hae (C. B.
L. Iv, 247). |

(b) In the healthy body: In intestinal canal even in the
first milk-stool. It is never absent in any normal human
or animal intestine. In the bodies of 82 healthy persons, ”
which were examined from twenty-four to thirty-six hours
after death, the B. coli was present 16 times, especially in”
the liver and kidneys, doubtless having wandered out
from the intestine. Wurtz and Hermann (C. B. xu,
388).

(c) In diseased human body (the motile and obo aera
forms are not often separated): As the cause of numerous |
diseases, especially of the abdominal organs : peritonitis, —
cystitis i (partly alone, especially when the urine is acid, —
sometimes associated with the Bact. vulgare ; see under the
latter), urethritis, pyelonephritis, suppurative nephritis,
perinephritis. It occurs remarkably often in suppurative —
strumitis. A number of intestinal affections appear to be —
associated with virulent forms of the colon group; at ~
all events, according to Dreyfuss (C. B. xvi, 581), the ©
forms isolated from the diseased intestine are much ~
more virulent for rabbits than those isolated from the —
healthy intestine. Regarding its relation to dysentery, —
see page 251. Many authors ascribe also certain cases ©
of cholera nostras to it. (Vaughan and Perkins found an ~
organism related to the colon group to be the producer —
of poison in confectioner’s ice. C. B. L. m, 799.) Most
cases of ‘‘typhoid’’ or choleriform disease from the eating —
of diseased meat depend upon it (see below). Axel —
Host traced the Norwegian disease from the eating of ©
‘“knetkise’’ to infection with the colon bacterium (C. B. —
xx, 160). More rarely the Bact. coli is the cause of ©
pneumonia (Klein, C. B. v, 625), leptomeningitis of —
infants, icterus gravis, Winckel’s disease (Lubarsch,
Virch. Arch., cxxi), melena neonatorum, puerperal
fever, panophthalmia, infection of wounds (wound-diph-
theria). Thoinot and Masselin hold it to be the cause of

248 IMPORTANT VARIETIES OF FISSION-FUNGI.

1 The cystitis microbes, which do not liquefy gelatin, described by
different authors (Rebland, Clado, Hallé, Albarran, etc.) under the
most various names, appear to be almost always Bact. coli ; compare
page 247.

ee TS ee

4

BACTERIUM COLI. 249

many cases of myelitis, as they can produce such ex-

_ perimentally i in rabbits (C. B. xvi, 919).

oa

-.
*

a

(d) In animals: In septic infections (puerperal fever,
- septic inflammation of the umbilical cord, etc.) of cattle.
_ Compare hog cholera, page 252.

Experimental Observations Regarding Pathogenic
Action.—(a) Jn animals: Just like the Micr. pyogenes,

the Bact. coli possessed most variable degrees of virulence;

the various morphologically and biologically variable

_ characters are entirely useless for determining anything

regarding the virulence. According to Valagussa, the

virulence of the colon bacteria from the intestine of ex-
_ perimental animals is greater the sicker the animal.
_ In eats vegetable diet produces considerable increase of

virulence of the colon bacteria, milk diet a marked at-

_ tenuation. Subcutaneously the Bact. coli sometimes
- causes only suppuration, sometimes septicemia ; intra-
_ peritoneal injection of 1 c.c. of bouillon culture, according

to Gabritschewsky, is always fatal for guinea-pigs in
about fifty hours. Fifty separately isolated Bact. coli
cultures behaved exactly alike in this; bacteria were
always present in the heart’s blood (C. B. xvu, 833).
According to Vallet, cultivation in filtered, sterilized
urinal refuse increased the virulence very much (C. B.
XIv, 325).

Immunity and Serum Diagnosis.—Active immun-
ization in the usual way is possible. The serum aggluti-
nates coli bacteria. According to many writers (for exam-
ple, Pfaundler, C. B. xxi, 9, 71, 131), the agglutinating
action of the serum is much greater against the coli
culture employed in the immunization than against other
cultures, and it is even absent against many other cultures.

_ The new form of serum reaction observed by Pfaundler

was only observed in the action of serum upon the culture
employed to produce the immunity. It consists in the

_ absence of agglutination and the formation of balls of

long threads in twenty-four hours.

(b) In man: Pathologic etiologic observations, which
have the significance of experiments, have been made in
man with B. enteritidis Gartner and B. morbificans bovis
Besenau, which are to be considered as examples of the

250 IMPORTANT VARIETIES OF FISSION-FUNGI ’

colon bacterium. When ingested in meat, they make
men sick. Similar observations have been communicated
by Gaffky and Paak regarding meat (sausage), and by
Gaffky regarding milk.

Also heated cultures are injurious. Repeated, subcu-
taneous injections of small quantities, according to
Sanarelli, produce an immunity against virulent Bact.
coli cultures (not against typhoid). Introduced into the
stomach, boiled cultures are less injurious. The gastro-
intestinal canal soon becomes accustomed to large quanti-
ties of poison, without the occurrence, on this account, of
an immunity against the subcutaneous injection of devital-
ized or living cultures (A. P., 1894, 353).

Special Methods of Demonstration and Culture.—
If coli bacteria are abundantly present (stools), the agar
plate at 37° is employed for their isolation. After twenty-.
four hours shake cultures in liquefied 2% grape-sugar agar
are prepared from numerous colonies. After sixteen to
twenty-four hours all colon bacteria present abundant gas
production, which leads to a breaking up of the nutrient
medium. (Fig. 11, p. 89.) The varieties which cause
fermentation of grape-sugar agar are examined micro-
scopically (to determine whether they are short rods
without spores, and whether they are motile) and are
transferred to lactose agar, milk, potato, ordinary and
grape-sugar bouillon, and peptone water (indol). If few
coli bacteria are present (water), then the water concerned
has 2% grape-sugar and 1% peptone added and is allowed
to stand for twenty-four hours in the incubator, and then
plates are prepared. It has also been recommended to add
to preliminary cultures 1% to 2% carboliec acid, 0.75%
anhydrous soda, and 1% hydrochloric acid, but we have
found no advantage from it.

Forms of the Bact. coli described under separate
names.
In the scheme for the peritrichous Bact. coli, as we have just

described and represented it, there are included very many subvarie-
ties, described as separate species. +

1Some investigators—for example, von Stécklin—undertake to
characterize separate forms of coli in relation to the number, length,

CS cS Ean al al el i eee Te

jell aa

oe

a a ee on er

eae Ait MA Beane, a

BACTERIUM COLI. 251

_ We ean find no sharp separation between these subvarieties in spite
of every effort todo so. Many descriptions are drawn up without any
reference as to how the variety being described is related to those next

te it, or the differential diagnosis is built upon one or another charac-

eristic whose inconstancy has long since been established either for the
colon group itself or even for other exhaustively studied groups (Micro-
S pyogenes, Streptococcus pyogenes, Streptococcus lanceolatus,

Ss Recciom coli, var. dysentericum Celli.—Maggiora traced an
extensive epidemic of dysentery in northern Italy to the Bact. coli.
_Arnaud candidly declared the Bact. coli to be the cause of dysentery
_ in hot countries. Celli (C. B. xvi, 309, and xxv, 481) found, as the
cause of dysentery in Italy, a form of the Bact. coli which he called
Bact. coli dysentericum, and which differs from the Bact. coli only in its
z, ee i properties and not in other peculiarities. It grows delicate-
ly, more like the Bact. typhi, and ferments grape-sugar and coagulates
milk slowly. With this the Bacillus dysenteriz Shiga (C. B. xxXII,
_ 599, and XxIv, 818) may be considered identical. Both organisms,
~ in distinction to other coli forms, were agglutinated by the serum from
eases of dysentery or from an imals immunized against this form of Bact.
coli. Shiga gives an extensive review of the literature, with illustra-
_ tions; also a criticism of the works which advocate amebee as the cause
of dysentery. There is still a decided possibility that the clinical pic-
+ ture of dysentery, as especially Kruse and Pasquale suggest, is caused
_ by entirely different agents in separate epidemics. Literature relating
_ to the ameba question is given by Kruse and Pasquale (Z. H. xvi, 1)
4 and Fajardo (C. B. xtx, 753), who consider amebe to be the cause of
Pe

oe

tropical dysentery. Ciechanowski and Novak cannot convince them-
selves of the importance either of amebz or of forms of the Bact. coli;
_ for many cases certain streptococci appear to them to be primarily
_ responsible (C. B. xxii, 445). Regarding the questions connected
_ with dysentery, consult also the critical review of the literature by
_ Janowski (C. B. x x1, 234).
Bacillus enteritidis Girtner. —Morphologically identical, flagella
_ unknown. According to Lubarsch, milk is coagulated, but it was
_ not observed by Giinther and Th. Smith. Cause of poisoning by meat;
even the broth prepared from the meat was also poisonous. (Kor-
S Bacitiue, des arztlichen Vereins fiir oe 1888, No. 9. age
illus of Ferret Plague of Eberth.
3 Corresponds, according to our investigations, in “all respects to the
oe . coli. It has four or five long, peritrichous flagella (C. B. v, 454,
and vi, 87).
Bacterium brassice acide of Lehmann and Conrad.—Found by
_ Conrad in many samples of sour-crout, and the cause of the fermenta-
tion of sour-crout. Has 4 to 10 very long, thin flagella. Often stains
slightly by Gram’s method. It is differentiated by its production of
_™arsh-gas upon cabbage broth. Besides about 80% CO,, there is

<

_ and staining properties of the flagella. We should be glad to separate
_ the atrichous (Bact. aérogenes), peritrichous, and mono- and lopho-
_trichous Bact. coli, if it had not been impossible to carry it out.

q

252 IMPORTANT VARIETIES OF FISSION-FUNGLI

formed 18% H,, and 2% marsh-gas. It ferments milk-sugar and co-
agulates milk (A. H. xxIx, 56).

Bacillus of the Marseilles Swine Plague Jobert and Rietsch.
(C. B. rv, 270.)

Bacillus of Spontaneous Rabbit Septicemia Eberth and Man-

+ ’

dry (Fortsch. der Med., vir, 1890, 547).—Milk is coagulated. We do —

not know regarding the arrangement of the flagella.

Bacillus aphthosus Kruse (Bacillus of mouth and foot disease, —
according to Siegel; Deut. med. Wochenschr., 1891, No. 49, 1328, and —

1894, Nos. 18, 400, and 19, 426; C. B. x1x, 728).—There is no cer-
tainty that it has anything to do with mouth and foot disease. Accord-
ing to Kruse, who found the cultures to be motile, it is a typical Bact.
coli.

Bacillus indigogenes Alvarez.—In the maceration and boiling of
the leaves of the indigo plant, it brings about the formation of a blue
pellicle from the pre-existing ‘‘glycosid, indican.’’ The bacterium
is motile, but otherwise, macroscopically, microscopically, and cul-
turally (capsule, fermentation of sugar, etc.), is very much like the

Bact. pneumoniz Friedlander. The latter is also able to break upin- —
dican. The indigo bacillus is also pathogenic (C. B. 11, 441). Ac- —

cording to recent authors, indigo is formed without aid from bacteria,
but by only the combined action of diastatic and oxidizing ferments.
(Compare also Bréaudat, C. B. L., Bd. v, 167.)

Bacterium coli § polaris. Lehm. and Neum.
(Plate 18, x11.)
Not distinguishable from the Bact. coli morphologically or biolog-

ically except that the flagella are always only at one or both poles. —

Cultivated by us from cheese (‘‘ Emmenthaler ’’) and from the organs
of a dead deer ; by Stécklin (C. B. xvi, 130) from feces ; cultivated
by F. Gartner from the organs of a dead guinea-pig, and closely
studied and found pathogenic for guinea-pigs (C. B. xv, 1).

Lucksch has photographed a similar form as Bact. coli (C. B. x1,

428), only it appears remarkable to us that he comes to the conclusion —

that the Bact. coli always have 1 to 3 flagella. We, like Stocklin,
have found, among many isolated ‘‘coli forms,’’ only a few with a
single flagellum, which, so far as we now know, possess this as a con-

stant property. We have not been able to enter into special investi- —

gations regarding this.

Bacterium cholere suum. (Migula.) Lehm. and
Neum. (Bacillus suipestifer Kruse.)

Synonyms.—Cause of hog cholera (Salmon), of Svin-
pest (Bang and Selander, C. B. 11, 360; x1, 339; XII,
203), of the Danish swine plague (‘‘Schweineseuche”’ ),
swine plague (Billings), swine fever (Klein, C. B. xv1u,

BACTERIUM CHOLERA SUUM. 253

105). Bacillus cholere suum Migula. Recently the
" disease is often spoken of in Germany as ‘‘Schweinepest ”’
or ‘‘ American Schweineseuche.’’ ?

Principal Literature.—Raceuglia (C. B. vitt, 289); Th. Smith (C. B.

1X, 253; Xvi, 231); Silberschmidt (A. P. Ix, 65) ; Voges (Z. H.
| xxmit, 149) ; Karlinski (Z. H. xxvitt, 373).

_ This organism is not different morphologically from the

| Bact. coli. Macroscopically and microscopically (multi-

ple, long, peritrichous flagella), it furnishes a typical form
of the Bact. coli.

- The following biologic peculiarities, which are confirmed

by our study of a culture from Rubner’s Institute, serve

to differentiate the organims:

1. From milk-sugar it forms neither acid nor gas, and
inoculated milk is not coagulated, and does not become

acid, but alkaline.

_ 2. The gas produced from grape-sugar is one-third CO,,
_ two-thirds H,. (The Bact. coli yielded us similar propor-
tions.) According to Smith, one-half CO, and one-half
= 4..

8. Does not produce either indol or phenol.

_ The cultures studied by us were always motile.* Ferrier (Lyon

_ Médical, 1894, No. 40) found the hog cholera, after being cultivated

_ for five months upon agar, to present short, very actively motile rods,

_ with multiple flagella, 35 “ to 55 wlong. The micro-organisms 1 / long

_ had the appearance of spindles. After passage through an animal

_ several times, the rods were longer, the cilia fewer and shorter.

_ Pathogenic Significance.—The organism causes de-
_ structive swine plague in northern countries, such as
_ America; recently also in England, and for about five
_ years in Germany (Graffunder, Deupser, C. B. xvu, 49);

_ 1 Voges and Proskauer, in their latest publication (Z. H. XXvIIt,
_ 20), designate a form as ‘‘schweinepest’’ which ferments all varieties
_ Of sugar, and so corresponds to the type of Bact. coli. However, with
_ the addition of caustic potash to fermenting sugar bouillon, in twenty-
_ four hours, with the admission of air, a red, fluorescent, eosin-like
_ color appears. This color occurs with none of the cultures of Amer-
ican hog cholera, and Voges then also states that in Germany he has
_ so far seen only swine plague (‘‘Schweineseuche’’), and no hog
_ cholera. We have found nothing concerning this motile, special
_ “Schweinepest ’’ bacterium in other authors.

_ ?Th. Smith has described a non-motile form (without flagella)
(C. B. xxv, 241).

254 IMPORTANT VARIETIES OF FISSION-FUNGI.

recently also in Hungary, Bosnia, etc. Th. Smith (C. B.

1x, 253) describes the following forms :

Acute Form.—Hemorrhagic septicemia; hemorrhages especially
observed in the lungs, kidneys, and serous membranes (stomach,
intestine). Marked splenic tumor. Death in a few days.

Chronic Form.—Animal emaciated, gait tottering. Larger and
smaller necrotic areas (ulcers) on lips, palate, tongue.
of stomach very red, in places showing ecchymoses.
(sometimes dry, nodular infiltration, sometimes broken-down ulcers)
are sometimes seen in the small intestine and rectum, and more often
in the cecum and colon. The lungs are not much changed, but there
may be some atelectasis or bronchopneumonia. The kidneys are

Mucous lining ~
Necrotie areas —

almost always diseased, albumin and casts appearing in the urine.

There is a splenic tumor, usually necrosis in the liver.

to four weeks.

Animal Experiments.—Guinea-pigs, rabbits, and pi-

geons are susceptible.

Differential Diagnosis.—

AMERICAN SWINE-PLAGUE.
(Swine-pest = Hog cholera. )
Bacterium cholerze suum. L. and

Very actively motile.
Ferments grape-sugar.
Luxuriant growth on potato.
-Rather luxuriant growth on agar,
very friable.
No changes at the point of infec-
tion.
Multiple areas of coagulation
necrosis in the liver.
Few bacteria in blood.

Very few bacteria at the site of

inoculation.

Pigeons very susceptible, guinea-
pigs less so.

GERMAN SCHWEINESEUCHE.

Death in two — .

a aie, ae

(Léffler and Schiitz. See p. 209.)

Bacterium suicida Migula.

Non-motile.

Does not ferment grape-sugar.
Little or no growth on potato.

Slow growth, on agar, coherent

(Karlinski).
Marked changes at the point of
infection.

Liver often the seat of fatty de- — .

generation.

Abundant bacteria in blood of

the heart and large vessels.
Abundant bacteria in the inflam-

matory edema at the point of ©

inoculation.
Guinea-pigs very susceptible,
pigeons less so.

The following are closely related to the Bacterium cholere

suum, and differ somewhat more from the Bact. coli on

account of the absence of some biologic (not morphologic)
peculiarity.

Bacillus of Intestinal Diphtheria Ribbert (Deut. med. Woch-
enschr., 1887, No. 8, 141).—This peritrichous organism is indis-
tinguishable morphologically from the Bact. coli, yet the culture in
our institute (cultivated for eight years upon non-saccharine nutrient

‘.
q
.
ied
fa
.

BACTERIUM CHOLERZ SUUM. 255

media) decomposes grape- and milk-sugar, with intense production
of acid, but without gas.

Bacillus diphtheriz columbarum Léffler.—A culture obtained
from Kral, which we studied carefully, corresponded exactly, morpho-
logically and biologically, with Bact. cholerzee suum: bouillon very
cloudy, suggestion of pellicle, milk unaltered, potato at first yellowish
then yellowish-gray, finally brown, almost the same as glanders.

Bacterium leyans Wolffin (A. H. xxi, 268).—Cause of fer-
mentation in leaven. Many long flagella, milk not coagulated, indol
formation overlooked by Wolffin, still it is present after prolonged
standing. It also brings about the most varying true coli fermenta-
tion of dough (acetic acid, lactic acid ; 75% CO,, 25% H,) in steril-
ized flour. More recently we have regularly isolated from sour
dough and fermenting bread-dough absolutely typical Bact. coli
which at least possess toxic action. Dissertation of Felix Frankel,
Wurzburg, 1896.

Bacterium morbificans bovis Basenau (A. H. xx, 241).1—Not
distinguishable morphologically and biologically from Bact. cholerz
suum. It ferments grape-sugar feebly, never coagulates milk, and
thus appears not to affect milk-sugar.

Cultivated many times from cattle suffering from a septic disease
in which the spleen is enlarged and there are necrotic, whitish-yellow
areas in the spleen and liver. The organism is found in the blood,
internal organs, and muscles of the diseased animal. Mice, white
rats, and guinea-pigs are killed by feeding. Rabbits and the other
animals die after infection of the subcutaneous tissue, the peritoneum,
or the interior of the puerperal uterus. The organisms escape in the
milk. Compare the Bacterium of Nouvelle septicémie des veaux of

_ Thomassen (C. B. xxiv, 800).

Compare, further, Bact enteritidis Gartner, page 251, which, as
it coagulates milk, is related to the Bact. coli. To one of these two
forms appears to belong Gaffky’s organism, which, if taken in fresh
milk by man, causes severe disease (C. B. XII, 389).

The Swedish Gaustadt bacillus of Holst is closely related. Eighty-

_ one persons in the institution for the insane at-Gaustadt became sick

in 1891, of which four died (C. B. xvi, 717). The disease depended
upon the eating of meat. Often there was an initial chill, many
times severe backache, sometimes herpes and erythema. The principal

_ Symptoms were : fever, vomiting, diarrhea. The organism does not

change the reaction of milk. It is motile, having 6 flagella.

_ Varieties of which nothing is written regarding motility, so

far as we know, but which still appear to belong to the
Bact. coli (or Bact. lactis aérogenes) :

Bacillus aérogenes vesicze Schow (C. B. xt, 745).
Bacillus of a pigeon plague of Sanfelice (Z. H. xx, 23). Causes
sero-purulent peritonitis. Perhaps belongs to Bact. septic. hemor-

; rhagice.

1See there, also, Basenau’s investigations, undertaken to establish
the difference between his organism and other similar ones.

256 IMPORTANT VARIETIES OF FISSION-FUNGI.

Bacterium Guillebeau, a and 6, v. Freudenreich. (See C. B.
Xvu, 487.) The organisms, described in the Annal. de micrographie,
which is inaccessible to us, produce simultaneously fermentation of
milk (inflation of cheese) and inflammation of the udder.

Bacterium of white or yellow calf dysentery. There is not much
to be gained by reference to the works of Piana, Mazzanti e Vigerzi,
Monti e Veratti (C. B. XVIII, 653).

Bacterium icteroides. (Sanarelli.) Heim.

Synonyms.—Bacillus icteroides Sanarelli, Bacillus of
yellow fever (febris icteroides; Spanish, febre amarilla).

Literature.—Sanarelli (A. P., 1897; C. B. xx1r, 181 and 668).
The more recent volumes of the C. B. contain many confirmations of
Sanarelli’s findings by American clinicians and some European authors.
Sternberg (C. B. xx1I, 145; xxi, 829; xxXIv, 376; XXv, 655).

Regarding the contention whether Sternberg’s Bacillus
X is the same as the Bacterium icteroides, as Sternberg
maintains, we will only say that Sternberg’s Bac. X is a
typical Bact. coli; the Bact. icteroides resembles more the
Bact. typhi.

Sanarelli accepts as the cause of yellow fever a short
bacillus, characterized by no special diagnostic peculiarity.
It is extraordinarily like the Bact. typhi and many cul-
tures of Bact. coli.

Microscopic.—Short, motile rods with gli a
flagella, not staining by Gram’s method.

In the description of the gelatin 1! and agar calfan, noth-
ing is found different from a delicately growing Bact. coli
or Bact. typhi. Single peculiarities accentuated by Sana-
relli are scarcely constant (compare Agramonte); thus, for
example, in agar cultures at room temperature there is
shown an elevated ring about a thinner growth (seal eul-
ture). The potato cultures resemble typhoid, being
delicate, colorless, but, according to Agramonte, they be-
come partly brownish later. Milk is not coagulated. In
milk-sugar bouillon, no or very little gas is produced.
On the contrary, in grape-sugar bouillon it is produced
abundantly, but none is formed in cane-sugar bouillon.
It is a facultative anaerobe.

1 Cultures which we obtained from Kral present moderate liquefac-

tion of gelatin and no spontaneous motion, but otherwise corresponded
very well to the descriptions given in the text.

BACTERIUM ICTEROIDES. 257

-Sanarelli rests the principal evidence as to the signifi-
es nce of the organism upon the following:

1. He found it in 58% of cases (the dead bodies).

_ 2. Germ-tfree filtrate of cultures is claimed to produce
in man the entire typical complex of a case of yellow fever.
_ 8. Serum from cases of yellow fever causes agglutination
‘of the Bact. icteroides. Serum from animals which are
immunized against the Bact. icteroides is said to operate
prophylactically and therapeutically against yellow fever.
_ 4. The B. icteroides is pathogenic for mice, guinea-pigs,
‘rabbits, goats, and sheep. Intravenous injections are
4 ollowed by vomiting, and bloody enteritis, scanty albu-
minous urine, and once (!) marked icterus.

5. The pathologic changes in the inoculated animal
correspond to those of yellow fever. Often extreme fatty
degeneration of the liver occurs. The most convincing
' preparations are obtained from the dog.

g Also Foa (C. B. xxiv, 890) finds grave specific changes

in the bone-marrow of ‘animals: fibrinous thrombosis of
the peripheral vessels, necrobiotic areas, etc.
_. We cannot yet look upon these proofs as completely
sufficient. While no objection to its pathogenic signifi-
- cance is to be found in the fact that especially sharp pecu-
liarities are not possessed by the Bact. icteroides,—one
‘only has to remember the characterization of the Vibrio
cholerse as compared with water vibrios, or the similarity
between the Bact. typhi and Bact. coli, "still it is to be
‘admitted that certain varieties of the~Bact. coli are also
‘found in many cadavers and may produce similar disease
symptoms in animals. It appears also objectionable that
yellow fever is a typical disease of the warm zone, and
ceases in places and at times with lower temperature,
while the B. icteroides possessed about the same resist-
ance as the B. coli to lower temperature. Yet this can
also be understood, since the cold may operate upon the
“intermediate host, carrier, etc.

4 Bacterium alcaligenes. (Petruschky.) L. and N.
Bacillus fecalis alcaligenes Petruschky. (C. B. x1x,

87.)

17

258 IMPORTANT VARIETIES OF FISSION-FUNGI.

Morphologically very similar to the Bact. coli; luxu- —

riant growth on potato with brown discoloration of the
nutrient medium.
No decomposition of any sugar with the liberation of

gas; milk is alkaline and not coagulated. Also upon —
litmus milk alkali is formed. The organism corresponds —

to the Bact. coli, which has lost its power of decomposing
sugar. Differentiation from the Bact. typhi usually not
very difficult. Found in the intestine, also in spoiled
beer. Compare also Pollak (H. R., 1897, vm, p. 22).

Bacterium Stutzeri. Lehm. and Neum.

ae a

Bacillus denitrificans u, Burri and Stutzer (C. B. L. 1,

257).

Mention is here deserved by the first completely described bacillus,
which, without the aid of synergetic organisms, was able to break

up saltpeter, with the liberation of nitrogen. It is a short, motile

bacillus (2-4 « long, #4 thick), without spores, and with tapering ends.
It grows upon gelatin plates as small, dry, tough, white disks, which
are traversed by characteristic radiating ribs, which become united by
arches at the edge. The surface growth in the gelatin stab culture is
similar ; in the stab it grows as a whitish streak. No liquefaction.
Agar growth not very characteristic. Upon feebly alkalinized potato, a
padded, rib-shaped, thick growth, from a pale flesh-color to peach-red.
In bouillon a pellicle forms. _In 0.3% nitrate of potassium bouillon
there is energetic development of nitrogen. It grows both at room
and incubator temperature, equally well without and with oxygen;
yet with an abundant supply of air, fermentation of saltpeter is inter-
fered with. The relation to carbohydrates is unknown. Isolated
from straw. Found by Kinnemann in straw and horse-manure
(C. B. L. rv, 906).

Bacterium typhi murium (Loffler). (C. B. xi,
129.) L. and N.

According to Loéffler himself, it is very similar in every
way, morphologically and biologically, to the Bact. of hog
cholera (grape-sugar is converted into acid with accom-
panying gas-formation). The culture studied by us, like
the Bact. typhi, produces acid vigorously from grape-
sugar, but no gas; and neither acid nor gas from milk-
sugar. }

1 According to Loéffler, feeble acidity is produced in milk, but not
sufficient to cause coagulation, Mereshkowski’s bactertum from the

BACTERIUM TYPHI MURIUM. 259

Milk remains fluid exactly as with Bact. typhi and is
rendered alkaline. Our culture is thus difficult to differ-
entiate from true typhoid, especially since its flagella
correspond in number and length with the best flagellated
typhoid cultures. On the contrary, the potato growth is
remarkably luxuriant.

_ Upon feeding, the bacterium is pathogenic only for
"mice; house mice (Mus musculus) and field mice (Arvi-
cola arvalis), but not for Mus agrarius and the various
domestic animals. It has been successfully employed to
‘combat the plague of field mice (compare, for example,
Zupnik, C. B. xxi, 458, and Appel, C. B. xxv, 373),
since the animals, after eating bread soaked in cultures of
the bacterium, die; and then, when eaten by their com-
panions, spread the disease still further. The ingestion of
_ 200 germs is certainly, and of 20 almost certainly, fatal
_ (Appel).

_ Bacillus of mouse plague of Laser (C. B. x1, 184).
_ Almost identical; not studied by us. However, according
to Laser, it is stained by Gram’s method.

Motile Varieties, Partly Incompletely Described, Re-
lated to the Bact. coli or Bact. cholerze suum.

_ (Statements are lacking regarding the arrangement of flagella or
the fermentation of carbohydrates. )

_ Bacillus of grouse disease of Klein (C. B. vi, 36, 592; vit, 82).
Epidemic of the Scotch grouse (Lagopus scoticus).

_ Bacillus loxiacida Tartakowsky. Cause of crossbill plague. In
g deg spre a little more Bact. typhi. No coagulation of milk,
no indol.

_ New gas-producing, aerobic Bacillus of Laser (C. B. x11, 217).
Cause of an epidemic among calves.

__ Bacterium of an epidemic of young pheasants. Klein (Jour.
of Pathol. and Bact., 11, 1893, 214).

__ Bacterium in melzna neonatorum Girtner (C. B. xv, 865).
Typical peritrichous flagella; relation to milk-sugar unknown.

__ Bacillus pyogenes fcetidus Passet. Untersuchungen iiber eitrige
Phiegmone, Berlin, 1885. Compare also Rabe, Bact. coli as cause of
disease in animals (C. B. xxI, 282).

‘Spe mophilus gattatus, a variety of ground squirrel (C. B. xv1, 612,
and Xx, 176), appears similar to our culture.

260 IMPORTANT VARIETIES OF FISSION-FUNGI.

Bacterium caniculz. (Galli-Valerio.) L. and N.

Cause of an epidemic in dogs.
After earlier investigations had given either negative or
uncertain results (Microc. pyogenes as cause), recently two — i
authors! (Galli-Valerio, C. B. xvm, 677 ; xix, 694; and —
Jess, C. B. xxv, 541) claim to have discovered the cause

of an important disease of dogs in short, small, motile —
bacteria (1 to 2 » long, 0.3 to 0.6 or even 0.9 y» thick).
The disease can be successfully transferred to young dogs —
and cats; according to Jess, also to rabbits and guinea-~
pigs, but not to mice, the symptoms then corresponding _
to those of the natural, multiform disease, in which the —
picture is dominated by fever, ocular and nasal catarrh,
protrusio bulbi, and bloody diarrhea. |
The statements of both ‘authors do not deviate in any
important way. .
According to the description of Jess, who needlessly
places his findings in contrast with those of Galli-Valerio, —
the organisms, which are readily cultivated at room tem-—
perature, appear to be related to the colon group. They
are motile, having a single polar flagellum; gelatin is not
liquefied; upon agar there is a gray growth; upon potato —
a white, velvety growth. In gelatin there occur a few gas _
bubbles (grape-sugar?). There is a tendency to polar
staining by Gram’s method. From the description of
Galli-Valerio, who places great value upon the form being —
sometimes shorter and sometimes longer, it appears that
old gelatin cultures present funnel-shaped depressions”
without liquefaction, and that the growth in milk is not”
accompanied by coagulation or fermentation of milk-_
sugar. No indol is formed.

ee tal

rl, ey ws Nt ar ag pe

Supplement to the Non-liquefying, White, Non-
motile, Short Bacteria.

The Bacteria of Acetic Acid Fermentation.

A small group of very closely related varieties form.
acetic acid from dilute alcohol (for example, beer-wort to

1 More extensive reference to the literature is given by these authors, ;

|

ee a re

yared

™

moniz, lactici and coli.

ACETIC ACID BACTERIA.

which is added 0.5% of alcohol).
selves studied these varieties, which have so far been
studied upon solid media with little thoroughness.
scopically the cultures resemble those of the Bact. pneu-
Thus far, three ‘‘ species ”’
_ been distinguished, Bacterium aceti Hansen, Bacte-
rium Pasteurianum Hansen, and Bacterium Kiitzin-
_gianum Hansen, and they are characterized as follows:

261

We have not our-

Macro-

have

Bacterium aceti
Hansen.

Bact. Pasteurian-

um Hansen.

Bact. Kiitzingian-
um Hansen.

‘Pellicle on sterile ale,
at 34° after 24 hours:

Slimy, smooth,
moist, shining,
showin g a ten-
dency to veining
like marble.

Dry surface, early
beginning to
wrinkle, some-
what elevated
above thesurface.

Similar to Pasteuri-
anum, but the
membrane climbs
up even on the

wall of the tube.

_ When flasks in which
growth has taken
ase at 34° are
rought into room
temperature:

3

Fluid
clear.

remains

Fluid
clear.

remains

Fluid becomes
cloudy and gradu-
ally, under sedi-
mentation, it be-
comes again clear.

Microscopic character
of the cells of the

Short rods with
hour-glass-like

Like Bact. aceti.

Short rods, usually
single; at most in

young membrane: constrictions in pairs; no chains.
chains, Long rods
and thread forms
uncommon.
_ Staining with iodin | Not at all. Blue. In older | Blue.
of the mucilaginous membranes the
material holding the blue staining of
bacilli together in the mucus is only
. young membranes : presented in
places; and in
still older, dead
cultures, it is en-
tirely absent,
_ Staining of the bacte- | Yellow. Yellow. Yellow.
rial cells with iodin:

_ We gave above a general presentation of the statements
_ of Hansen, the most successful investigator of this group,
in tabulated form. Literature: Lafar (C. B. L. 1, p. 129);
most important is Hansen, Recherches sur les bactéries
- acétifiantes (Travaux de Carlsberg, 1, 182, and C. B. L.
mt, dl).

All three forms of acetic acid bacteria possess a wide
_ range of forms, depending especially upon the temper-

262 IMPORTANT VARIETIES OF FISSION-FUNGI.

ature. This is especially marked in the Bacterium Pas- .

teurianum, according to Hansen.

At temperatures below the optimum of 34°, beautiful |
chains of short rods are formed; at higher temperatures —

the short links grow out into long undivided threads. The

latter, when again brought to a temperature of 34° or less, —
in part break up into new short rods, and in part present —
characteristic bulging. Also the bulging structures are —

gradually changed, at least partly, under elongation, into
short rods, yet the widest parts nevertheless disintegrate.
According to Lafar, moreover, the swollen forms are partly
dependent upon the action of acid.

During recent years an entire series of new varieties with
names have been added to these three old species, which
appear to be very difficult to differentiate. (Compare Hen-
neberg, C. B. L. m1, 223; 1v, 14, 67, 138, and 933.) Also
Beijerinck (C. B. L. tv, 209) and his pupil Hoyer (C. B. L.
1v, 867) have made surprising new communications
regarding acetic acid fungi. Unfortunately, according to
the material accessible to us, the description of the mor-
phologic peculiarities leaves much to be desired.

Beijerinck calls Hansen’s Bact. aceti, Bacterium rancens
Beijerinck, and regards the Bact. Pasteurianum (including
Kiitzingianum) as a variety. Bacterium rancens is the
bacterium of sour beer. Here also belong Bact. acetosum
Henneberg and Bact. oxydans Henneberg. The true
rapidly forming acetic acid bacterium, which Pasteur first
isolated, and which Beijerinck now calls Bacterium aceti
Pasteur, is entirely different. Hansen, on the contrary,
did not know that the. Thermobacterium aceti Zeitler
belonged here. Finally, Beijerinck distinguished a Bac-
terium xylinum.

Their recognition may perhaps be accomplished by
means of the following key:

(A) Vigorous growth forming a covering in a mixture of tap-water
100, alcohol 3, ammonium phosphate 0.05, potassium chlorid 0.01.
Upon beer forms very delicate coverings. Very slight growth upon
beer-gelatin, but very luxuriant, slimy growth upon beer gelatin
which contains 10% of cane-sugar. Bacterium aceti Pasteur-
Beijerinck.

(B) No growth on the above media. Upon beer a vigorous growth
forming a covering.

ese

ns Ch aa

ocean be EEN: |

ie gn

Re)

a ee ee ee ee ee en ee vee iy ee

BACTERIUM DISCIFORMANS. 263

(a) Upon gelatin a soft white growth, uninfluenced by cane-sugar.
(a) The mucilaginous material secreted remains unstained with
iodin. Bacterium rancens!? Beijerinck.
we tee secreted slime is turned blue by iodin. Bacterium
asteurianum Hansen.
(b) Upon gelatin, dry, tough, leathery mass ; upon beer, there is

at first a slimy and then a thick, leathery seum, which gives the reac-

tion for cellulose. Cane-sugar influences the luxuriance of the growth.
Bacterium xylinum Brown.

How this classification will stand the test of more exact
morphologic investigation remains undecided. In the
mean time the Bact. xylinum should be considered as
a leukonostoc—i. e., it then belongs to the streptococci.
Of the other varieties, which are represented as non-

- motile rods, usually motile forms are also observed. The
work of Hoyer is very rich in biologic detail regarding

the nutrition of those which produce acetic acid, ete.

Bacterium disciformans. Zopf.

Synonyms.—Bacillus disciformans Zimm. (1, p. 48),
Bacillus azureus Zimm. (1, p. 24).

Short rods (0.3-1.4 u long, 0.3-0.5 thick), non-motile, not stained
by Gram’s method, grows also as an anaerobe. Upon the gelatin plate :
Very young deep colonies, rather coarsely punctate, round, trans-
parent ; superficial colonies, partly like typical young typhoid cultures
(especially in the B. azureus), partly somewhat more compact, re-
sembling more the colon type. The superficialcolonies liquefy after
the second day, when there is frequently seen a narrow hair-like
zone. ‘The mass lying on the bottom of the plate is a little denser in
the disciformans than in the azureus, and with both there are open
spaces. The deep colonies later present little tubercles, and, when
they come to the surface, liquefaction and a hair-like rim. In gelatin
stab: Funnel- to tube-shaped liquefaction, developing rather rapidly.
Bouillon: very cloudy, abundant H.S and a little indol produced.
Upon agar: Dirty white, slimy, luxuriant growth. The agar is col-
ered brownish to rosy red. Upon potato: Grayish-yellow to reddish-
brown, moderately elevated, moist growth. Grape-sugar is fermented,
with abundant formation of gas. Milk is first coagulated, then again

_ rendered fluid.

1 We found the Bact. rancens not growing very rapidly and some-
what elevated upon solid nutrient media with an oily gloss. No
liquefaction of gelatin, no anaerobic growth. Grape-sugar is fer-
mented, with formation of gas and acid.

264 IMPORTANT VARIETIES OF FISSION-FUNGI.

This variety corresponds, except in the liquefaction of gelatin, toa + —

Bact. lactis aérogenes.

We obtained this variety twice from Zimmermann, once marked
Bae. disciformans, the second time Bac. azureus. The varieties corre-
sponded in no way with the description which Zimmermann gave
them, but, on the contrary, the two were identical even in the smallest
details.

Bacterium punctatum. (Zimm.) Lehm. and
Neum.!

Synonym.—Bacillus punctatus Zimm. (1, p. 38).

Short rods (0.8 long, 0.5 « thick), often also forming long threads.
Actively motile from one polar flagellum. Not stained by Gram’s
method. Superficial colonies are first roundish, smooth-bordered,
transparent, punctate disks; gradually the border becomes finely
notched and finally presents beautiful hairy border (somewhat like
41, v). Simultaneously liquefaction begins as a shallow saucer, in
which the remnant of the colony can be seen at the center. The
border of the saucer is surrounded with a delicate grayish-white zone,
sometimes presenting sinuous decorations. The gelatin stab culture
at first resembles cholera, but liquefaction rapidly becomes complete.
Upon agar and potato the growth is not characteristic, resembling
that of the Bact. coli. Milk is coagulated and then the coagulum is
liquefied. Grape-sugar is actively fermented, with production of gas.
Abundant production of H,S and little of indol. According to
Kruse, this organism, for which he unfortunately suggested the super-
fluous name Bac. aquatilis communis, is one of the most common
water bacteria. It corresponds to a Bact. fluorescens without the
production of pigment. We have also obtained this organism very
frequently from water (if we leave out of consideration the fermenta-
tion of sugar, which we rarely tested), and also have often observed
forms which were colorless at first for a long time and then became
feebly fluorescent.

We found the Bacillus annulatus Zimmermann (11, p. 30) very
similar in all morphologic and biologic peculiarities; nevertheless it is
very well distinguished by habitually producing liquefaction in gelatin
in the form of holes. The marked, white accumulation of bacteria
which is present beneath the undermined edges of the colonies in the
plate culture, the colonies looking as if cut out by a punch, gives a
very striking picture.

Bacterium vitulinum. (Weissenberg.) L. and N.

Short rods, motile, not staining by Gram’s method, re-
sembling the Bact. coli. Facultative anaerobe. Young

1An organism, similar in every way, but not forming gas from
sugar, was isolated by us from gastric contents.

BACTERIUM AGILE. 265

gelatin colonies resemble those of B. coli; then there soon
occurs a liquefaction of the surrounding medium and the
_ growth breaks up into a crumbly mass. In the gelatin
- stab there is marked liquefaction, at first funnel-shaped,
_ then cylindric. The content of the funnel is very cloudy,
-and there is a delicate pellicle on top. Rather marked
little hairs, directed downward, which grow out into the
' solid gelatin about the funnel of liquefaction are quite

~ remarkable.

ee or ee Te vee ve y

ep ee eee ey

eae oe

Bouillon very cloudy, with a delicate pellicle. Abundant

: H,S, and'little indol are produced. Upon agar and potato
_ the growth is nearly the same as that of the B. coli; the

otato culture has a very putrid odor. Grape-sugar is
ermented, with abundant gas-formation, and milk is not

- coagulated.

Determined to be the cause of a dysentery in calves in

_ Silesia and given to us by Weissenberg.

The four following are closely related to, perhaps identical with,
the described ‘‘liquefying varieties of B. coli,’ and are known to us
through the descriptions only:

Bacterium foetidum liquefaciens. (Tavel.) L. and N.

From one to three short flagella extending out from an unstained
capsule (von Stécklin, Recherches sur la groupe des Coli-Bacillus,
1894).
Gelatin in stab is liquefied, and has a strong fecal odor. Sugar is
fermented, with liberation of a vast amount of gas. Milk is not coagu-
lated. Bouillon becomes turbid and a pellicle develops upon the sur-
face.

Bacterium cloace. (E.0. Jordan.) Lehm.and Neum.

(Compare Th. Smith, ‘‘ The Fermentation Tube,’’ 1893, 215.) Sur-
face growth in gelatin is thin, with a somewhat irregular outline.
Abundant, uncharacteristic, yellowish-white growth on potato. Ac-
tively motile. Very abundant and rapid formation of gas from dex-
trose and saccharose, the closed end of the fermentation tube contain-
ing from 50% to 95% (about one-third H and two-thirds CO,). Gas
is produced more slowly from lactose. Milk is coagulated in eight

days.

Bacterium agile (Schou, Fliigge). Lehm. and Neum.

_ Synonym.—Bacillus pneumonicus agilis Fliigge.

266 IMPORTANT VARIETIES OF FISSION-FUNGL

Cause of the aspiration pneumonia following division of the vagus.
Fligge: Mikro-organismen, tI. Aufl., page 287, and G. Neumann
(C. B. 11, 755).

Bacterium pseudomelanosis. P. Ernst (V.A., Bd. 152,
: p- 418).

In this relationship belongs the interesting organism

which Ernst isolated from a case of pseudomelanosis, and
recognized as the cause of the same. About the bunches
of bacteria there lay in the tissue dark green deposits of
sulphid of iron. The organism produces H,S very actively,
forms gas from sugar, liquefies gelatin, has many flagella
and no spores, and is not stained by Gram’s method.

Bacterium salmonicida. (Emmerich and Weibel.)
Lehm. and Neum.

Bacillus of a trout epidemic of Emmerich and Weibel (A. H. xx).

Non-motile, short rods, more rarely longer rods and threads, not
stained by Gram’s method. Facultative anaerobe. Plate cultures in
gelatin - Very young cultures resemble those of the streptococcus; then
they sink deep into the gelatin, without real liquefaction, the border
of the colony becoming irregular and notched. Gelatin stab cultures at
first also resemble those of the Streptococcus pyogenes ; later (after
five to seven days) there occurs a funnel-shaped, steep-walled, deep
cavity about the inoculation line, on the sides and at the bottom of
which are delicate, whitish bacterial masses. Agar stab cultures
present flat, moistly shining, irregularly outlined growths of a grayish-
yellow color, which after many weeks become brown in the center,
and simultaneously the upper part of the agar is discolored brown.
Bouillon remains clear, only near the top a delicate cloud is formed
upon the glass wall, which, upon gentle shaking, sinks very slowly to
the bottom as cloudy flakes. A plentiful, whitish sediment gradually
collects at the bottom. No growth upon potato. No growth at 37°;
optimum 10°-15°. We are not acquainted with it.

The organism was cultivated by its discoverers from trout which died
in an epidemic in upper Bavaria. Healthy trout were killed by inocu-
lation as well as by adding the organism to water. The principal
symptoms of the disease were: at places, where at first there are lentil-
sized defects in the scales, furuncle-like swellings gradually develop;
then, secondarily, hemorrhagic, suppurating areas form. The organism
was abundant in the dead fish, especially in the blood of the heart.
The following organism is very similar:

-

BACTERIUM CREMOIDES. 267

Bacillus devorans Zimmermann (i, p. 48).

Found in well-water. It possesses very active locomotion, but
nothing is known of its pathogenic properties.

_ Bacterium turcosum. (Zimm. ii, p. 32.) Lehm.
and Neum.

Very small rods, 0.2-0.3 4 thick and 0.3-1.5 u long, with slug-
_ gish movement, which is due to a polar flagellum.

_ Upon gelatin plates: small, intense turquoise-yellow, transparent
- colonies, which gradually sink into the gelatin; microscopically, struc-
tureless and more or less transparent. The growth in the gelatin
stab culture develops slowly, is smooth, roundish, of an intense yellow
passing into a greenish color, and sinks in very slowly, without lique-

faction. The agar cultures are similar. Upon potato: scanty, green-

- ish-yellow dry or slightly shining growth. In bouillon there is a little

_ turbidity, without formation of H,S or indol worth mentioning.

_ Grape-sugar is not perceptibly affected. Milk is not coagulated.

_ Isolated by Zimmermann from water. In examinations of prepu-
tial secretion we have twice obtained cultures which correspond to
Zimmermann’s original one.

Bacterium cremoides nobis ad interim.!

| Short rods, 0.5-0.8 thick, 0.8-1.6 4 long, non-motile, staining by

Gram’s method. Gelatin plate: natural size, gray to grayish-yellow
_ disks; magnified 60 times they are finely granular, later opaque and non-
liquefying. The gelatin stab is not characteristic ; the surface growth
_ gradually becomes thick, whitish, reddish, or cream colored and has
an oily luster. In the agar stab the growth has a moist luster and is

_ cream colored. The water of condensation is clear with a pellicle and

little sediment. Bouillon is similar. Little indol and H,S are
_ formed, and no gas from sugar. Milk is not coagulated.
Obtained from the tap-water of Wurzburg.

1 Bacterium synxanthum (Ehrenberg) L. and N. Ordinary name:
Bacillus of yellow milk. According to J. Schroter, it is characterized
_ as follows: Actively motile, short, thin rods, producing a yellow
_ pigment, which is readily soluble in water, but not at all in ether and
alcohol. It is decolorized by acids, but the yellow color returns upon
treating with alkalis. Milk is colored a bright yellow, the casein is
_ dissolved, and the milk becomes alkaline. The culture which we
_ obtained from Krdél possessed no motility, clouded the bouillon, and
produced a prominent pellicle; coagulated milk, with formation of

_ acid; formed gas from grape-sugar; furnished very luxuriant, moist,

_ yellowish-gray growths, resembling those of the B. coli upon agar and
gelatin, without liquefaction, and upon potato developed as a light
_ yellow, much elevated growth with a fatty luster. Stains by Gram’s
_ method.

268 IMPORTANT VARIETIES OF FISSION-FUNGI.

Bacterium erythrogenes. (Grotenfelt.) Lehm. and
Neum.

Bacillus lactis erythrogenes Grotenfelt. Bacillus of red milk.
Literature: Grotenfelt (Fortschritte der Mediz., 1889, vit, 41) and A.
Baginsky (C. B. vi, 137).

Non-motile, short rods, 0.8-3.0 4 long and 0.5-1.0 4 thick. Stains
by Gram’s method. Upon the gelatin plate, grayish-yellow, roundish
disks, which gradually sink into the gelatin and liquefy it. When
magnified 60 times, at first both the superficial and deep colonies
resemble very much those of the Bact. coli; later, when liquefaction
begins, the border of the colony, now having become opaque, is
beset with fine hairs, and later appears irregularly eaten out and
coarsely granular. The intensity of liquefaction is decidedly variable
in different colonies. Upon the gelatin stab there develops a sulphur-
yellow, thick, slowly sinking growth; later the liquefaction is cylin-
dric. The surface growth upon agar is yellow and moist. Agar and
gelatin (especially in the dark) become colored intensely rose-red to
garnet. Our cultures also did so in diffuse daylight. According to
Grotenfelt, the pigment presents two lines between D and E, and one
in the blue portion of the spectrum. Potato culture is sulphur-yellow,
elevated, partly dull and partly moist. The cream separates from
milk (cream, yellow), the casein forms a flocculent precipitate (with
alkaline reaction), the clear serum becoming rose-red. No gas is
formed from grape-sugar. From bouillon there is formed abundant
Aarts but little H,S. Our description is from a culture obtained from
Kral.

Bacterium helyolum. (Zimm. i, p. 52.) Lehm. and
Neum.

Plump, rather thick, short rods (1.0-3.6 4 long, 0.8-1.2 u thick),
non-motile, staining by Gram’s method. Gelatin plate : Colonies are
roundish, lively lemon yellow, flatly elevated, and later they sink in
the gelatin. When magnified 60 times: homogeneous, hardly at all
transparent in the middle, clearer at the edges, border smooth, and
with beginning liquefaction the sharp border becomes slightly
crumbly.

Upon the gelatin stab culture a luxuriant, shining, intense lemon-
yellow growth, which slowly sinks into the medium. Agar culture:
yellowish-gray, moist. Potato culture: dull, broad, greenish-yellow.
Bouillon becomes cloudy, with a delicate pellicle. Abundant forma-
tion of H,S, but none of indol. No gas is formed from grape-sugar.
Milk is coagulated.

We obtained an organism from air which corresponded exactly with
Zimmermann’s description. Bacillus luteus Fliigge appears identical,
except that liquefaction is absent. Also the following appear very
closely related: the non-liquefying Bac. constrictus Zimmermann
(I, p. 42) and the Bac, subflavus Zimmermann (I, p. 62).

"er.

a a er rae Tron igex

BACTERIUM NUBILUM. 269

Bacterium lactis saponacei. (Weigm. and Zirn.)
Lehm. and Neum.

As the Bacillus lactis saponacei, Weigmann and Zirn

_(C. B. xv, 463) have described a short rod, which in gela-
tin plates forms white colonies with yellow centers, which

later become yellow throughout, but without special

markings. Gradually liquefaction takes place. In the
_ gelatin stab a funnel forms, at the bottom of which lie
yellow flocculi. In the agar stab the luxuriant growth is

yellow in the center only at first, then throughout the
whole growth. Upon potato a waxy-yellow, slimy growth.
Milk is not coagulated, but becomes slimy and slightly
tenacious. The culture has an odor like soap or lye.
Optimum at 10°. Regarding soapy milk, the first com-
munication was by Herz, Ch. Zeit. Rep., 1892, page
34.

Bacterium nubilum. (P.and C. Frankland. Z. H. vi,
p. 386.) Lehm. and Neum.

Non-motile short rods, 1-2 long, 0.3-0.5 4 thick, staining by
Gram’s method. The colonies on the gelatin plate present beautiful,
polymorphous forms. In the younger stage they are yellowish, of ir-
regular forms, and provided with many thick and thin lateral out-
growths, similar to mites in shape. The more compact nucleus at the

center gradually disappears, while the projections become arranged

more in the form of a star. Now liquefaction of the gelatin begins.
The periphery of the colony slowly dissolves into delicate little frag-
ments, and in the fluid contents of the saucer of liquefaction there
remains a framework of radiating threads, which later become ar-
ranged like the spokes of a wheel. Finally the entire colony breaks up
into irregular fragments. Macroscopically the colony does not appear
unlike that of the Bac. subtilis. In the gelatin stab the growth sinks
in, with the form of a saucer, and then cylindric liquefaction occurs.
The liquefied zone is slightly cloudy. The growth upon agar is jagged,
undulating, fairly luxuriant; in the center, pale rose color; at the
edges, yellowish-brown, with a fatty luster. The water of condensa-
tion is clear with a yellowish-brown sediment. The growth upon po-
tato is at first entirely reddish-white, faintly shining to dry; later it
becomes intensely brownish-yellow. Milk is not coagulated, and is
alkaline in reaction. No gas is formed from grape-sugar. It forms
but little indol. Bouillon becomes cloudy. Isolated by Zimmermann
from water (I, p. 28). Our description is from one of Zimmermann’s
cultures,

270 IMPORTANT VARIETIES OF FISSION-FUNGLI.

Bacterium ochraceum. (Zimmermann, i, p. 60.)
Lehm. and Neum. ~

Short rods, 0.5-0.8 1 thick, 1.2-3.6 u long, actively motile from
polar flagella, staining by Gram’s method. Gelatin plates at first pre-
sent forms like those of the Bact. coli and typhi; later the borders are
fringed, while the gelatin becomes liquefied. Pellicles varying in color
from gray to grayish-yellow float upon the liquefied medium, and they
may be of a tougher or more delicate character. The more delicate
pellicles often appear as a net with irregular meshes. The gela-
tin stab culture presents a yellowish-gray surface growth, but it sinks
in at once. Later there is a cylindric, turbid liquefaction, with
grayish-yellow sediment. The agar growth is dirty, light grayish-
yellow, then spreads out. The water of condensation is clear, with
moderate precipitate. Bouillon becomes lightly cloudy, with moder-
ate sediment and slight pellicle. Indol and H,S are formed in abun-
dance. Milk is not coagulated, and becomes somewhat slimy. No gas
is formed from grape-sugar. The growth on potato is yellowish.

This organism, isolated by us from gastric contents, corresponds in
all the main points with Zimmermann’s description. We isolated a
very similar but non-motile bacillus from Secale cornutum. From
this we cannot distinguish a Bacillus plicatus Zimm. (I, p. 54), which
we obtained from Zimmermann, but it did not form folds any more.
Also Bacterium carnosum (Tils, Zimmermann, I, p. 4) is very
closely related. We were unable to find the spores, seen by Tils ;
also, the color of the culture obtained from Zimmermann could not be
distinguished from that of the Bact. ochraceum.

Bacterium fulvum. (Zimmermann.) L. and N.

Rods, 0.3-0.5 » thick, with a length varying from 1.0 #
to long threads. Non-motile, without flagella, staining
by Gram’s method, sometimes liquefying, sometimes not.

Gelatin plates: Shining, orange-yellow colonies, sometimes
more drop-like, sometimes more spreading, with moderate
or no liquefaction. The non-liquefying, superficial colo-
nies, when magnified 60 times, are at first very much like
those of Bact. coli; they are irregularly roundish to leaf-
shaped, somewhat transparent, grayish-yellow, homo-
geneous, often having furrows and markings resembling
the Bact. coli. The liquefying colonies present an essen-
tially different appearance: The yellow, superficial disks
have a threaded border resembling subtilis (compare 40,
11); later the colonies break up into a crumbly mass
which lies at the bottom of the liquid.

BACTERIUM FULVUM. 271

In the gelatin stab there is no striking growth. The sur-
" face growth is leather-brown to orange and reddish-orange.

- When liquefaction occurs, there is formed a funnel, filled
_ with turbid fluid ; later the liquefaction became cylindric
"and sometimes there isa pellicle.

_ Agar stab: Succulent orange-yellow to yellowish brown-
_ish-red. (Compare, for example, 5, v.) Potato growth is
the same.

_ Mik: It is not coagulated, but both of our liquefying
4 forms changed it into a yellowish turbid fluid, with an
- orange sediment, upon which the yellowish cream floated.

mA non-liquefying form coagulated milk (original culture
- of Bact. tremelloides Schottelius). No gas is formed from
‘sugar. Littleindoland noH,Sare produced. Found by
us in water and milk.

_ We consider that the following varieties, which we have
_ ourselves investigated, belong here : Bacterium bruneum
_ Schroéter, which we obtained from A. Fischer ; Bacterium
_tremelloides Schottelius, obtained from the discoverer
himself. The description of Zimmermann’s Bacillus
_ fuscus Fliigge corresponds completely. ?

_ The Bacterium mycoides roseum Scholl appears
_ very closely related, although deviating somewhat in color
(Fort. d. Med., vn, 46).

_ What we obtained from Hauser as Bacillus arbores-
_ cens Frankland is also the same, and neither corresponds
with Frankland’s original description (Z. H. vt, 3879) nor
with that of Zimmermann. The deviation from Frank-
land consists in the loss of liquefaction (absence of bun-
‘dles); in Zimmermann’s description it is said not to stain
by Gram’s method. We have never been certainly con-
_ vinced regarding motility, and so far have been unable to
stain flagella.

* The description given by Schréter himself of his Bact. bruneum
_ corresponds very poorly, as does the description of Fliigge of his Bacil-
lus fuscus. Therefore we select the oldest of the newer names, which
‘ischaracteristic, and the description of which corresponds well with
- our cultures,

272 IMPORTANT VARIETIES OF FISSION-FUNGLI.

Bacterium chrysogloea. Zopf.!

According to Zimmermann’s description (1, p. 12), it is
only distinguished from the preceding by active motility.
We found in gastric contents an exactly corresponding
form with peritrichous flagella and active motility, which
stained by Gram’s method. Chrysoglcea and fulvuam may
be related, as Forma mobilis and immobilis. Proof is still
wanting.

Bacterium latericium. (Adametz.) Lehm. and
Neum.
(Plate 20, I-VI.)

Short rods, somewhat pointed at both ends (0.8-1.6 ~ long, 0.4-0.6
# thick), non-motile, and stained by Gram’s method. Upon the gel-
atin plate the deep colonies appear as roundish, reddish-brown, opaque —
disks with smooth edges. The deep ones are jagged, sinuous, trans-
parent at the edge, very crumbly, and reddish (20, m1). In the gel-
atin stab no liquefaction occurs, the surface growth is from vermilion
to reddish-brown (20, 11). The growth upon the agar streak is the
same (20, 1). The growth upon the agar plate is not especially char-
acteristic; round disks, coarsely crumbly, border granular, and in the
deep ones smooth (20, v). Upon potato the bacterium grows very
slowly only and very scantily (20, Iv). Bouillon remains clear. Milk
is not coagulated. Neither gas nor acid is formed from sugar. No
H,S, and only traces of indol are formed. Isolated by us from the air;
corresponds, so far as can be judged from Eisenberg, with the descrip-
tion of Adametz. The organism does not belong here, according to its
natural relationship, but more properly with the Bact. acidi lactici.

Catiano has described two other bacilli, which are motile, beauti-
fully provided with flagella, produce red pigment, and do not possess
spores: Bac. rubiginosus and coccineus (Cohn’s Beitr., Bd. vu,
1896, H. 111, 537). We could not study these.

Bacterium prodigiosum. (Ehrenberg.) Lehm. and
Neum.

(Plates 21 and 22.2)

Synonyms.—Monas prodigiosa Ehrenberg, Micrococ-
cus prodigiosus Cohn, Bacillus prodigiosus Fliigge.

1 Migula places Bact. chrysoglceea with the non-motile varieties,
and designates Bact. aureum Frankland, Bact. aurescens Frank-
land, and Bact. egregium Zopf as closely related.

2 The plate drawn for Bact. kiliense has forms which also occur

ATER

BACTERIUM PRODIGIOSUM. 273

_ Most Important Literature.—Schottelius (C. B. 11, 439); Wasserzug
(A. P., 1888); Kiibler (C. B. v, 383); Scheurlen (A. H. xxvI, 1).

q _Microscopic Appearance.—From solid nutrient me-
lia, very short bacilli, often looking like cocci. The ends
are somewhat pointed or rounded. The greatest diameter
is 1 » (21, xr; 22, 1x). In bouillon, especially if it is
faintly acid, there occur longer forms, distinct rods, and
shorter and longer threads.
_ Motility.—In young bouillon cultures there is active
“motion, produced by from 6 to 8 long, peritrichous flagella
(21, xr; 22, x1). On the contrary, older agar and
bp otato cultures appear non-motile, and in them the bacil-
tus produces abundant slimy material, which limits mo-
‘tion. Scheurlen attributes the mucous formation to the
abundant production of alkali.
_ Staining Properties. — Easily stained, but not by
Gram’s method.
_ Relation to Oxygen. — Facultative anaerobe; grows
better as anaerobe. Alsoas an anaerobe it liquefies gelatin
(also with the addition of 2 per cent. sugar), but forms
“no pigment.
_ Requirements as Regards Temperature and Com-
position of Nutrient Media.—Optimum at 22°-25° ; in
the incubator, especially at 38°-39°, the formation of sae
ment is suspended. A more prolonged cultivation at a
higher temperature permanently lessens the formation of
‘pigment.! It grows also, with production of pigment,
upon non-albuminous nutrient media.
Gelatin Plate.—(a) Natural size: At first the super-
ficial colony is a grayish-white point, and the gelatin is
Piquefied at once. The area of liquefaction is shaped like
a plate. The peripheral zone is lighter than the central
“zone. Original colonies are often colored reddish, but often

, ere) the Bact. prodigiosum, since both are identical (compare p.
276

_ 14Itmay be here remarked that, without known cause, chromo-
genesis by the Bact. prodigiosum is often much reduced. As is often
seen, of 20 cultures made at the same time and from the same origi-
nals upon the same nutrient media, many form pigment abundantly
and others very feebly. Also, upon plates fainter and more deeply
- colored ere always occur side by side.
8

274 IMPORTANT VARIETIES OF FISSION-FUNGI.

they remain white and disappear with the increasing size -
of the area of liquefaction. Thus the paler zone disap-
pears, and the entire liquefied area becomes colored uni-
formly gray (21, v1; 22, mr). :

(6) Magnified seventy times: Superficial colonies, at first
delicate, granular, roundish, with a smooth border ; later
the central zone is colored rosy red, is delicately crumbly,
and sometimes has a faint suggestion of streaking. The
peripheral zone consists of continuous little tufts of hairs,
which terminate externally in very fine points (21, vir;
22, Iv). Besides this form, there are often atypical ones
with a brownish center, the separate zones being lost, and
the whole colony appearing covered with extremely deli-
cate hairs. One form passes into the other. The deep
colonies are uncharacteristic, yellowish-brown, granular,
whetstone-shaped.

Gelatin Stab.—After six hours liquefaction begins
at the surface of the gelatin in a saucer shape. The
liquefaction extends along the stab canal, forms a tube-
or cone-shaped funnel, and continues to possess a funnel
form in the advanced stage. Only after a very long
time does the liquefaction become cylindric. The funnel
of liquefaction is filled with whitish or rose-red floceuli,
among which more deeply stained clumps are swimming.
When liquefaction has advanced very far, a cloudy, red-
dish to deep red precipitate is at the bottom and the
supernatant fluid remains red. When the culture grows
atypically, no red color is seen. The form of the funnel
of liquefaction is most variable (21, 1; 22, 1).

Agar Plate.—(a) Natural size: The colonies appear
as minute red points even after thirty-six hours. Those
lying upon the surface increase in size perceptibly and
become colored from rose-red to dark red. Also, uncolored
colonies occur together with these. They are irregularly
roundish, sometimes lobed, often with alternating paler
and darker zones and distinct cloudy center (21, v;
22, v1).

(b) Magnified seventy times : Both the deep and superficial
colonies at first are roundish, of irregular form, pale yel-
low with smooth border. Later the deep colonies take on -
-a brownish color with a reddish luster, the border remain-

BACTERIUM PRODIGIOSUM. 275

ing smooth and the structure coarsely granular. On the
contrary, the superficial colonies are transparent, pale
rose-red to red, very finely punctated, with borders almost
_or entirely smooth (21, vI; 22, vir).

Agar Stab.—Stab: Thread- like, without nodules, white
to reddish. After keeping longer, a whitish cloudy zone
forms about the stab canal (21, 11). Surface growth: Al-
ready after forty-eight hours completely covered with a
smooth, shining growth, the color of which varies from
atypical white to typical purple (21, Iv). . Often it is
_whitish-gray, shaded with red. The agar, especially be-

_ neath the surface growth, after a Jonger time becomes

colored a garnet-red.

Agar Streak.—The growth remains limited to the
streak; compare agar stab. The water of condensation
presents a reddish cloud with a red sediment (21, 0;
22, 1).

- Bouillon Culture.—Diffuse, marked turbidity, with a
_ more or less red-colored, delicate pellicle upon the surface.
_ The bouillon becomes of a gelatinous or oily consistency.
_ Milk Culture.—After twenty-four hours it is firmly
coagulated; later the coagulum is dissolved and a yellow-

_ ish color produced.

Potato Culture.—At first a rosy red, moist, flat growth,

_ limited to the inoculation streak. Later it becomes darker

in color, is elevated, with a wavy, smooth border, and after
_ five or six days has attained its dark purple color (21, 1x;
_ 22, x). Sometimes the surface then exhibits a greenish-
golden reflex, similar to dry fuchsin. Also the potato
_ culture develops atypically at times, as does that upon
_ agar, and becomes only whitish-gray, orange, or rose-red,
instead of dark red (21, x).
Chemical Activities.—
_ (a) Thered pigment (prodigiosin): Develops best upon
_ agar and potato, is insoluble in water, and only externally
in color and golden luster is it like fuchsin ; according to
Scheurlen, it is apparently also free from nitrogen besides
containing no sulphur nor phosphorus. The pigment is
readily soluble in alcohol and ether, is turned orange-yellow
_ by alkalis, and from carmine to violet-red by acids. With
zine and hydrochloric acid the pigment, notwithstanding

276 IMPORTANT VARIETIES OF FISSION-FUNGL

contrary statements, is decolorized, as are all red pigments
of this group. In light it fades rapidly as well when
dry as when in solution. The pigment, spectroscopically,
is sharply characterized; more detailed communications
thereon will soon follow from this laboratory.

(b) Olfactory and gustable materials: Especially upon
potato it forms methylamin and ammonia. According to
Schottelius, the odor is proportional to the pigment pro-
duction, but we found also colorless cultures with a marked
odor, as of herring.

(c) Production of gas and acid from grape-sugar: Fairly
active, according to Schottelius and other authors; on the
contrary, our prodigiosum culture formed acid without
gas (but our kiliense formed gas). A prodigiosum iso- —
lated by Cramer from the tap-water of Heidelberg also
formed no gas. Scheurlen demonstrated the production
of formic and succinic acids.

(d) Urea is converted into carbonate of ammonia, but
not by all cultures.

(e) Traces of indol, no H,S.

Distribution.—Upon cooked potatoes, moist bread,
paste, especially upon starchy substances, occurring epi-
demically often, especially in the late summer and autumn.
(Compare Scheurlen. ) Cause of the ‘‘ bleeding host. ag
Sometimes found in water-pipes.

Pathogenic Significance.—If injected alone, is not
pathogenic, but may be when combined with other bac-
teria. The proteins of the prodigiosum have been studied
many times and found to be poisonous.

Varieties Identical with or Closely Related to the Bact.

prodigiosum.
Bacterium kiliense. (Fischer and Breunig.) L.
and N.
(Plate 22.)

Compare Kieler Wasserbacillus, Breunig, Dissertation, Kiel, 1888.
Laurent (A. P., 1890, 465; C. B. 1x, 105).

The culture which we used for preparing illustrations (Plate 22)
is distinguished from the Bact. prodigiosum (Plate 21) by more of a

497

BACTERIUM VIOLACEUM. 277

_brick-red or orange-red color. This, however, according to our more

recent observations, is not constant; prodigiosum may grow with
- orange, and kiliense with bluish-red color. The formation of alkali
_is most important as to the color: with abundant production of alkali

it is yellowish-red; in other cases, bluish-red. We also found to be

absolutely identical Bacterium miniaceum ! (Zimmermann, L. and

'N.) and the Bacterium indicum (Koch, L. and N.), isolated by Koch

from an Indian monkey, of which we obtained beautiful red cultures

from Kral and carefully studied them.
It is very probable that these are also identical:
Bacterium of red pus Ferchmin (C. B. x11, 103), which differs in

_ being non-motile and staining by Gram’s method.

Red water-bacillus Lustig (C. B. vim, 33).
Bacterium plymuthicum Fischer. (L. and N.) Compare Voges

»(C. B. xtv, 301).

Bacillus fuchsinus Boekhout and Otto de Vries (C. B. L. Iv,

4},
The following is, at any rate, closely related.

Bacterium piscatorum. Lehm. and Neum.

Microbe rouge de la sardine of the French. Causes, in combination

with an anaerobic bacillus, panaritium in fishermen, apparently

originating in spoiled bait. In boxes of sardines it causes a red color
(Du Bois Saint Severin, A. P., 1894, 152). The pigment is soluble in
water (?), usually poorly developed upon agar, and is produced at 37°-
39°. More extensive studies are required to establish the constancy of
these characteristics.

Bacterium violaceum. (J. Schréter.) L. and N.?
(Plate 23.)

Synonym.—Compare page 279. Bact. janthinum Zopf.

- Schriter’s name is older.

Microscopic Appearance.—Thin rods, 1.6-5 4 long,

_ 0.5-0.8 » thick, with rounded ends; the smallest are often
_ oval; sometimes threads form. In the interior unstained
- areas sometimes remind one of chicken cholera.

1 What we obtained from Kraél as Bac. rosaceus metalloides
Dowdeswell is entirely different. We have called this Bact. rosaceum,
and found it to be a fine, small, motile rod, which something grows

_ like the Bact. coli on ordinary media, but with a brick-red color.
_ The pigment is not prodigiosin. Milk and bouillon present brick-red

pellicles. No gas is formed from grape-sugar. Milk is not coagulated.

_ Not stained by Gram’s method.

* Twice in cultures, according to Migula’s method, upon quince-

_ juice we have seen pictures which may have been spores.

278 IMPORTANT VARIETIES OF FISSION-FUNGI. 7

Motility.—Active, serpentine motion. We found the
flagella to be sometimes peritrichous (3-4, long, tortuous),
sometimes polar (1-2) (23, x1 and xm).

Staining Properties.—Stains by Gram’s method.
Growth is moderately rapid, and best at ordinary tem-
perature. ;
Gelatin Plate.—WNatural size: At first, small, yellow
points, later violet. If the liquefaction is rapid, then there —

is a gray saucer-shaped depression with violet, alternating
concentric rings (23, vit). Where colonies do not liquefy, —
or do so late, they appear as lobulated, fringed, shining,
yellowish to violet growths (compare 23, vill). Magnified
siaty times : In both weakly and actively liquefying colonies,
they almost always at first resemble those of the typhoid.
When sunken in, the colonies become crumbly, have a —
streaked peripheral zone consisting of little hairs, and
finally disintegrate into crumbly masses (23, vimt). Colo-
nies which liquefy very late are internally of a darker, ~
yellow, finally bluish color and opaque, with a crumbly
structure.

- Gelatin Stab.—In freshly isolated varieties the lique-
faction after two or three days is funnel-shaped; and along
the stab canal, tube-shaped. The contents of the funnel
are grayish-violet with colored fragments (23, 1). After
longer cultivation (as in our culture, after two years)
liquefaction is almost entirely lost. The surface growth
now is shining, lobulated, dirty yellow to violet. Only
after two to three months is there a very shallow saucer-
shaped depression.

Agar Culture.—Moist, shining, somewhat elevated, of
the same color as the colonies upon gelatin. In the plate,
when slightly magnified, the colonies resemble those of
the Bact. coli, and are yellowish-gray and faintly granular
(23, v).

Potato Culture.—Wavy, somewhat elevated growth,
moist, shining, violet to violet-black. We have also
observed, in numerous potato cultures, dirty yellow to
brownish- -green growths, resembling those of Bact. coli .
and fluorescens (23, x).

Bouillon.—Faintly or strongly turbid, sometimes pro-
vided with a thick, sometimes with a delicate pellicle. In

BACTERIUM VIOLACEUM. 279

favorable cases the pellicle may assume a pale violet
color.
Milk.—In some cases it is coagulated, but it usually re-
mains fluid and is violet in color, at least forms a violet
cream layer. |
Chemical Activities.—In grape-sugar bouillon there
is formed little acid and no gas. It produces abundant
HS and a moderate amount of indol.
Regarding the pigment (janthin), see page 67.
From the one just described we are unable to dis-
_ tinguish, by any peculiarities worth mentioning, the
_ Bacterium janthinum Zopf (Sweden and America),
obtained from Zimmermann, and a similarly named _ bac-
-terium from Kral, and a bacterium isolated during the
summer of 1894 from the well of the local fort.
_ A beautiful chromogenic culture obtained in 1898 from
-Hohnl (Prague) corresponds entirely with the description
except that the liquefaction was prominently punched-out
in appearance and it did not stain by Gram’s method.
Also, it seems to us, from a study of the literature, that it
is scarcely possible to differentiate a Bacillus violaceus
_ Laurentius (Lustig, p. 103), cultivated from the water of a
7 filter basin of Lawrence, a Bacillus violaceus Macé (Ann.
: d’ hygiéne, 1887), and the Bacillus violaceus (Lustig, p.
j

-—

75), from tap-water of Berlin and London. The latter,
_ according to Voges, is identical with the Bacillus lividus
_ of Plagge and Proskauer (Z. H. 1, 463), except that the
_ latter is differentiated from the violaceum by growing less
well upon potato and by rapid liquefaction. All these
_ characteristics, as follows from what has already been said,
_ are not sufficient for determining a separation of species.
_ Also closely related is the Bacillus membranaceus
amethystinus (Eisenberg, 1891, 421), cultivated by
_ Jolles from well-water. It produces large violet pel-
licles upon gelatin and is non-motile. Germano likewise
_ cultivated a membrane-forming organism (C. B. x11, 516),
_ which he named the Bacillus membranaceus amethyst-
-inus mobilis. It agrees with the preceding except in
- being motile. Also here it is probable that two identical
_ varieties are found, the one motile, the other non-motile.
_ This is in accord with Ward’s discovery of an organism

280 IMPORTANT VARIETIES OF FISSION-FUNGL.

4

.

=

which belongs here, and which sometimes was motile and —

sometimes not (C. B. L. tv, 902).

Bacterium indigonaceum. (Claessen, Schneider.)
L. and N. -

Obtained from Kral, from Prague. Rods, 1.6-3y long, —

0.8-0.9» thick, somewhat thicker than violaceum, some- —
times curved. Upon the gelatin plate, which is not lique-_

fied, there appear, macroscopically, small, blue, drop-like

growths. When slightly magnified, they are sharply —

rounded, yellowish disks, slightly granular and _ later

becoming indigo-blue from the center outward. Upon the ©

agar plate they are similar. The surface growth in the q

gelatin stab is sky-blue, moist, sometimes also remaining —

white. The growth upon potato is deep indigo-blue,

somewhat granular; later it presents a coppery-red,

metallic luster, very similar to solid indigo. It renders
bouillon cloudy and forms a pellicle upon it. Milk is not

coagulated, but is colored bluish-green. The bacterium is ©

not motile. We have not examined it for flagella. Re-
garding the pigment, see page 67.

The original description of Claessen (C. B. viz, 18) and —

the description of Voges of the Bacillus indigoferus,
which was obtained from tap-water in Kiel, differ only in
the statement that the latter organism is actively motile,
and that this depends upon a polar flagellum. We ex-
amined a culture from Kral and verified all the statements
of Voges, so that here also are two varieties which differ
only as regards flagella, and which really belong together.

Bacterium ceruleum. Voges. (L. and N.)

Literature.—Voges (C. B. xtv, 301). Our description is from a —

culture from Kral.

Microscopically, longer and shorter motile bacilli, resem- :

bling the Bact. coli. Do not stain by Gram’s method.
They grow well also anaerobically.
Gelatin stab : surface growth thin, with a dull luster ;
deep blue, slowly becoming depressed. Stab, thread-like,
‘with little nodules. The surface growth in agar has a

(de eae ab em), 2

ey et a er ee

BACTERIUM PYOCYANEUM. 281
poist luster, is scarcely at all elevated, with a gray zone at
the periphery, and a sky-blue one at the center, and with
‘some diffusion of the pigment intothe agar. Upon bouil-
lon there is a thick, tough, somewhat wrinkled, deep blue
pellicle, the bouillon becoming moderately turbid. Milk
is unchanged, the surface light blue. Upon potatoa layer
‘is formed, which is light blue at first, and later becomes
dark blue to dark blackish-green. Potato becomes gray-
_ish-green throughout. No gas is formed from grape-sugar.
We found a trace of the pigment soluble in glacial acetic
_ acid, but it is entirely insoluble in all ordinary solvents.

Bacterium pyocyaneum. (Gessard, Fliigge.)
L. and N.

(Plate 24.1)

_ Synonyms.?—Bacillus pyocyaneus Fliigge, Pseudo-
_monas pyocyanea Migula, Bacillus of greenish-blue pus,
_ “green or blue pus.”’

Literature to 1893 by Jakowski (Z. H. xvi, 475).

_ Microscopic Appearance.—Slender rods, often grow-
ing into threads. Thickness, 0.4; length, 1.4 to 6 uz.
_ Other authors have also observed transition forms, from
_ slender rods to short, plump, even almost round forms
» (24, rx).
_ Motility.—Actively motile by means of a polar flagel-
_ lum (24, x).
Stains with anilin dyes and by Gram’s method.
Requirements as Regards Nutrient Media, Tem-
_ perature, and Oxygen.—Usually is a strict aerobe, but is
also cultivated from closed abscess cavities. Jakowski
_ (Z. H. xv, 474) has cultivated from an intestinal fistula a
_ form growing anaerobically and in carbonic acid. It is
_ not very particular as to nutrient media and grows rapidly
_ at room and incubator temperature.

1 Our plate is painted from a culture which was not entirely typical,
_ as it only forms a little pyocyanin. The color may be much more
bluish-green.

2 See page 285, et seqg., for related forms.

282 IMPORTANT VARIETIES OF FISSION-FUNGI. {

Gelatin Plate.—(a) Natural size. Deep: Roundish to
whetstone-shaped, yellowish-white to greenish-yellow.
Sometimes also there is a roundish, spreading, transparent,
greenish-yellow extension with the original colony in the
center. Superficial: At first roundish, uneven, delicately —
spreading, but immediately saucer-shaped liquefaction —
occurs. There is often a lighter peripheral zone. The —
liquefied material is cloudy, and gray to greenish-gray. —
The original colony appears as a crumbly mass at the —
center (24, v). There is intense fluorescence about the
colony.

(b) Magnified fifty times: Both the superficial and deep
colonies are yellowish, roundish, with smooth border, and —
delicately punctate at first. After twelve to twenty-four
hours the superficial colonies have a transparent, ragged
border (like Bact. coli), and are also sometimes beset with
little hairs or fringes. Then immediately begins the de-
pression of the colony (24, m1). The color becomes brown- —
ish, the irregular form and ring of hairs are partly lost,
the contents of the liquefied area are uniformly crumbly.
The periphery and the structure of the colony appear with
the greatest variations, sometimes ragged, sometimes gran- —
ular, sometimes punctate, sometimes lighter, sometimes
darker, until the colony falls entirely apart. The middle
portion of the colony usually survives and is darker in
color (24, Iv). (Compare also 25, v and x.)

Gelatin Stab.—Liquefaction begins very early, is at
first cup-shaped, later cylindric, and more rarely is shaped
like a pointed funnel. The liquefied material is slightly
cloudy, with a greenish-yellow to bluish-green fluorescence.
There is gradual liquefaction along the stab canal, the con-
tents being yellowish and crumbly (24, 1).

Agar Plate.—(a) Natural size. Deep: TRoundish to ©
whetstone-shaped, non-characteristic, yellowish. Super-
ficial: Roundish, smooth-bordered, with a moist luster, —
greenish-white to yellowish. There is intense greenish-yel- _
low fluorescence of the surrounding medium (24, vt). i

(b) Magnified fifty times. Deep: Roundish to whetstone-
shaped, with a border partly smooth, partly delicately
wavy, delicately punctate or granular (like Bact. coli),
light yellow to greenish-yellow. Superficial: Usually

BACTERIUM PYOCYANEUM. 283

round disks, with border almost smooth, more or less
strongly granular, very often also moruloid, light yellow to
sreenish-yellow. Except for the color, it is not distin-
“guishable from Bact. fluorescens, putidum, and coli (24,
‘ym). (Compare also 25, v1; 26, vu. )
| Agar Stab.—Stab: Non-characteristic, thread-like, and
a little nodular. Surface growth : Whitish- -gray to greenish,
dull to moistly shining. In forty-eight hours it is uni-
formly spread over the entire surface. The agar has a yel-
_lowish-green to bluish-green fluorescence.
_ Agar Streak.—Somewhat spreading growth, with a
“moist luster, wavy, smooth border, yellowish-green in
color. The agar shows marked blue to yellowish-green
BE cacence. The water of condensation is almost clear;
‘there is a white precipitate and a whitish pellicle on the
surface (24, 11).
_ Bouillon Culture.—Marked yellowish-green fluores-
cence. Very turbid. Moderate quantity of sediment,
which is broken up with difficulty upon shaking. Pellicle
“upon the surface.

_ Milk Culture.—Milk is coagulated, and later again
liquefied. The liquefied portion presents yellowish-green
fluorescence. Reaction is always alkaline.
_ Potato Culture.—At first a yellowish growth, with
a moist luster, wavy irregular border, and but slightly
elevated; later, brownish-yellow to brown or reddish-
‘brown. Often there is a fluorescent zone about the growth
(24, vit). According to the character of the potato,
there is very great variation in the luxuriance, fluores-
cence, and color, and so the growth cannot be distin-
guished at any time with certainty from that of other
fluorescent varieties. (See also 25, rx.)

Sensitiveness to Injurious Agencies.—Drying kills
rapidly. The action of the sun’s rays for four hours does
not entirely suspend chromogenesis.

Chemical Activities.—

(a) Chromogenesis: In its typical cultures the Bact. pyocyaneum
; forms two pigments: a green-yellow, fluorescent bacteriofluorescein,

‘Soluble in water, and the beautiful blue, crystalline pyocyanin, soluble
in chloroform (see p. 68). There are cultures, however, “like the one
represented in our plate,—which produce scarcely any pyocyanin, only
much bacteriofluorescein. We have often seen cultures which form

284 IMPORTANT VARIETIES OF FISSION-FUNGI. I

upon wafers abundant pyocyanin, which can be easily extracted with -
chloroform from nutrient media containing water. There are also.
cultures which, at least on certain nutrient media (it is recommended

to employ 1% peptone, 1.5% agar boiled in water, and, finally, 5%

gelatin added), produce only pyocyanin, and, finally, there are thom
which produce no pigment. The brown color of old cultures comes

from a changing of pyocyanin into a reddish-brown pigment. Pyo-

cyanin is easily changed into yellow pyoxanthose. Regarding pyocy- :
anin, see also Borland, C. B. xxv, 897.

Regarding interference with the formation of pigment brought
about by other bacteria (for example, Micr. pyogenes, Bac. anthracis),

see Miihsam and Schimmelbusch (C. B. xv, 430).

(b) Other products: Upon all nutrient media there is
present at first a delicate aromatic odor (compared to
linden blossoms). We have also often perceived this
odor in other cases; for example, in Sarcina lutea, Micro-
coccus luteus. Old cultures smell disagreeably ‘of am-
monia. It forms neither indol nor H,S, and from grape-
sugar little acid and no gas are produced. Even the
boiled bouillon cultures are strongly poisonous. They
contain, besides proteins, toxic metabolic products. Nitro-
gen is liberated from nitrates and nitrites (Lehm. and
Neum.). Weissenberg has, in our institute, demonstrated
this property in all the. four cultures of B. pyocyaneumt
examined (A. H. xxx, 274).

Experimental Observations with Animals.—It is
usually weakly pathogenic for animals; when injected, it
causes suppuration. Schtirmayer found in mice, after
subcutaneous injection, clear edema and serous exudate
into the body cavities. Virulent cultures kill guinea-pigs
when injected subcutaneously and intraperitoneally.

Immunity.—The very interesting studies of Wasser-
mann (Z. H. xxi, 263) are mentioned on page 110. For
more details the original must be consulted.

Distribution.—

(a) Outside the body: So far, has not been certainly
found. :
(b) In healthy body : Sometimes in the mouth and intes-_
tine and upon the skin of healthy persons.

(c) In diseased body: Not infrequently (espociallall
formerly) in pus from open wounds, also in the dressings -
from wounds, sometimes in epidemics i in the rooms of the :
sick. Usually the organism appears only in association —

aS on er

BACTERIUM FLUORESCENS. 285

with the suppurative process in combination with the
rell-known causes of suppuration. Through its pigment
it colors the pus blue, bluish-green, or green. In a series
~f cases the organism has occurred alone in connection
with disease processes (otitis media, pericarditis, bursitis
reepatellaris), so that it may very properly be looked
ipon as pathogenic for man, especially for children
(Kossel). General septic infections are but rarely caused
by this organism alone. Krannhals has collected some
such cases (C. B. xv, 481); recently Escherich has de-
scribed a small pyocyaneum epidemic among infants (C.
B. xxv, 117). Its relation to diseases of children, where it
is only found in the stools, remains doubtful (Baginsky).
_ Related Varieties.—According to our conviction, it is
impossible to sharply separate this organism from the
bacterium fluorescens. Closely related also is a disagree-
ably smelling organism, cultivated by Galtier from a pig
dead of a septic disease, and pathogenic for rabbits (C.
a. IV, 109).
_ Schiirmayer observed, as descendants of original cultures, forms
which scarcely liquefy any more, representing short rods, forming tough
coherent gelatin growths and a firm covering upon the liquefied gel-
atin. Many colonies in gelatin plates present marked, radiating stri-
ation (observed by us in Bact. fluorescens).

Bacterium fluorescens. ! (Fliigge.) Lehm. and Neum.
a (Plate 25.) ;

Bacillus fluorescens liquefaciens. Fliigge.

Literature.—Ruzicka (A. H. xxxtv, 148). Kurt Wolf: Die fluo-
Tescierenden Bacterien des Dresdner Ell- und Leitungswassers, Zeit. f.
sewasserkunde, 1898. Not accessible to us and only known to us
through an abstract.

After the detailed description of the Bact. pyocyaneum
itis unnecessary to also describe the Bact. fluorescens in
detail, since we found it identical in all essential prop-
erties. :

1 A transitional form to the following variety occurs in an organism
Which we obtained from A. Fischer as ‘‘ termoiahnlichen Bacillus.’”’ At

irst the gelatin remains solid, and liquefies very slowly after eight to
‘ourteen days.

286 IMPORTANT VARIETIES OF FISSION-FUNGI.

5

At first sight, absence of production of pyocyanin and of
denitrifying action (very many cultures were investigated
in vain in these respects by Weissenberg) appear suffi-
cient to separate the organism from the Bact. pyocyaneum;
but this is not the case, for the following reasons:

1. Ruzicka has’ also obtained Bact. fluorescens with
formation of pyocyanin.

2. We and other writers have had cultures of Bact.
pyocyaneum which no longer produce any trace of pyocy-
anin, and Ruzicka has observed, in aerated cultures of
Bact. pyocyaneum, a marked reduction in the formation of
pyocyanin (possibly transformation into pyoxanthose?).

3. Not only have Stutzer and Burri found a non-lique-
fying, fluorescent, denitrifying organism, but Kiinnemann”
claims to have cultivated from the soil, besides a denitri-
fying Bact. pyocyaneum, also a denitrifying Bact. fluores-
cens (C. B. L. 1v, 906). Most recently Kurt Wolf has—
found the Bact. fluorescens to be frequently denitrifying
(H. R., 1899, rx, 538).

4. The more restricted growth of the Bact. fluorescens
in the stab canal as compared to Bact. pyocyaneum may
be explained by acclimatization to higher temperatures;
thereby also the pigment produced by the fluorescens”
takes on a bluer tone (Ruzicka).

5. Also the difference that the Bact. pyocyaneum,
when introduced into the animal body, remains alive
there very well, while the Bact. fluorescens after three
days at the latest is dead, is not conclusive. |

In short, the methodic investigations of Ruzicka agree
absolutely with the impression which we obtained from our
most careful comparison of the cultures, and which we
advanced in the first edition. ;

We have studied most minutely four different cultures.
of the Bact. fluorescens isolated from water and soil.

Microscopically we found rods which were partly plump, ~
and partly slender, with polar flagella. Threads were rareg
ly wanting. In Plate 25, vill, a plump form is reproduced. —

It stains poorly or not at all by Gram’s method. Upon

;

1 We are not acquainted with the non-motile Bact. butyri fluores-_ :
cens, Lafar (A. H. x11, 1), constantly present in Munich in butter. .
It does not change the color of agar,

PW ton cs

BACTERIUM PUTIDUM. 287

the nutrient media, we are unable, either microscopically
‘or macroscopically, to see any difference between it and
the Bact. pyocyaneum, except that milk is never coagu-
lated, but rather clears up gradually, with a yellowish-
green coloration. The yellowish-green ring about the
owth on potato we have rarely seen. Usually a slight
Bi rmation of indol is observed, but no H,S. We have not
‘conducted any experiments upon animals.
The organism, with different variations of chromogenesis
_ and fluorescence’(yellowish-green, bluish-green, abundant,
; slight), is one of the most common inhabitants of water
_ and soil, also it is very often found in milk, gastric contents,
‘ete. The literature contains descriptions of a number of
varieties claimed to be specific. We have not been able to
study them, but are very skeptical regarding them because
_ of the great variability of the Bact. fluorescens. E. Klein
has cultivated from lupin tubercles a form which belongs
here (Jour. of Path. and Bact., m, 1893, 205). (See p.
83.) Also Bact. viridans Symmers, from the vesicles
_ of herpes (C. B. xu, 165), is entirely identical, in spite
_ of its ability to grow also anaerobically.

_ Bacterium ranicida. (P. Ernst.) Lehm. and Neum.

‘a
Bacillus ranicida Ernst. (Ziegl. Beitraige, vi11, 203.) Bac. hydro-
_ philus fuscus Sanarelli (C. B. rx, 193). (See also F. H. Russeil, Jour.
of Amer. Med. Assoc., June 18, "1898. —ED.)

Judging from the description and illustration of this organism, it
t appears to belong here. It is pathogenic for cold-blooded animals
(frogs, fish), but, according to Sanarelli, also for warm-blooded
animals. The rods are actively motile, and on many nutrient media
_ grow into long threads. The cultures upon agar and gelatin exhibit
_@ bluish fluorescence. Potato cultures are brown. They liquefy
_ gelatin and ferment sugar, which was not done by any of the eleven
fluorescent forms studied by us. The arrangement of the flagella
; may perhaps give further light upon their relationship.

{ Bacterium putidum. (Fliigge.) Lehm. and Neum.
$ (Plate 26. )

| _ Synonyms.—Bacillus fluorescens putidus Fliigge, Bac.
fluorescens non liquefaciens Autorum. Compare also
; remarks on page 285.

’

288 IMPORTANT VARIETIES OF FISSION-FUNGLI.

Microscopic Appearance.—Small, slender rods, often —
growing into exceedingly long threads. Thickness, 0.4——
0.8 #; length, 1.6-5 » (26, vi, rx). i

Motility.—Actively motile, dependent upon one, rarely —
two polar flagella. .

Staining Properties.—Not by Gram’s method.

Requirements as to Temperature, Oxygen, and —
Nutrient Media.—Strict aerobe, not particular as to
media, grows fairly rapidly and best at 25°-30°.

Gelatin Plate.—(a) Natural size. Deep: Roundish
to whetstone-shaped, yellowish. Superficial: At first like -
the deep ; after forty-eight hours, 2 or 3 mm. wide, trans- —
parent, lobulated, ragged, shining, yellowish-green. The ~
gelatin shows yellowish-green fluorescence (26, tv). It
gradually enlarges until its size is 1 sq. cm. Fi

(b) Magnified fifty times. Deep: Roundish, smooth- —
bordered, light yellow, homogeneously shaded, usually —
with a somewhat darker concentric ring (26, 11). Swper-—

.

ae

oi

ficial: Both in the early and later stages it is indistin- —
guishable from the colonies of Bact. typhi and coli except —
from the fluorescence (26, 11). There are here also mani-
fold variations. ;

Gelatin Stab.—Stab: Not characteristic, thread-like. —
Surface growth : Lobulated, jagged, transparent, dull or with —
a fatty luster, whitish-gray to yellowish-green. The ©
gelatin shows yellowish-green fluorescence (26, I).

Upon agar, potato, milk, and bouillon it is indistin- —
guishable from Bact. fluorescens.

Remarks.—Aside from the liquefaction of gelatin, the Bact. putidum
and Bact. fluorescens are scarcely different, and it appears entirely
justifiable to place them together under a Bact. fluorescens, with —
forms a liquefaciens and § non liquefaciens. We have also reached
the conclusion that the Bacillus fluorescens albus Zimmermann
and fluorescens longus Zimmermann, which we received directly
from Zimmermann and studied carefully, do not deserve to be desig-
nated as varieties. Both forms were identical with one isolated by us
from soil ; another, obtained from water, which we have cultivated for
years in our institute, now forms very long threads almost exclusively,
which we do not remember it to have done previously. A third form,
isolated by us from soil, corresponds somewhat with the Bacillus
fluorescens aureus Zimmermann, and is distinguished by a dirty
yellow growth upon agar and gelatin, but this characteristic is not

I iy SPS Pa al ig NN ay A AS lI oN

BACTERIUM SYNCYANEUM. 289

constant. Compare also Lesage (C. B. 111, 8, and Iv, 135) regarding
the Bacterium of green diarrheas.

The same experience occurred to us with the Spirillum fluorescens
of Kral. It corresponded exactly upon all nutrient media with the
Bact. putidum ; microscopically it presented rods with a single flagel-
lum, 0.4-0.6 « thick and 0.8-3 “ long. We may here add that it may
pmetimes be very difficult to reach a certain decision as to whether
we have to deal with a vibrio with a single flagellum, or a member of
_ the fluorescent group with single flagella, since there occur almost
“Straight vibrios as well as bent rods. At any rate, the fluorescent
‘group forms the transition to the vibrios. The following appears to
_ belong in this connection :

Bacterium denitrificans. Stutzer and Burri.
(L. and N.)
Bacillus denitrificans I. Stutzer and Burri. Liberates gaseous ni-
trogen from nitrite, and from nitrate only when reducing bacteria

(Bact. coli and others) are present. For details regarding this inter-
‘ ig organism, see C. B. L. I, 257, and Weissenberg (A. H. xxx,

Bacterium syncyaneum. (Ehrenb.) Lehm.
and Neum.

(Plates 27 and 28.)

Literature.—Hiippe (Mitt. a. d. Gesundheitsamt 0, 355), Heim

(A G. A. v, 518), Thum (A. K. 1, 291).

_ Synonyms.—Bacillus cyanogenes Fltigge, Pseudomo-
nas syneyanea Migula. Bacillus of blue milk.

Microscopic Appearance.—Small rods, with blunt or

pointed ends, 0.5 thick, 1.2-3 » long. Threads could not

be seen (27, vit).

_ Motility.—Active motion dependent upon from 1 to 5

flagella at one pole, rarely (before division) upon bipolar

flagella (27, vim).

_ Staining Properties.— With anilin dyes and by Gram’s

“method. In staining plasmolysis sometimes occurs, so

_ that the bacteria have stripes like a zebra.

Requirements as to Temperature, Nutrient Media,
_and Oxygen.—Obligate aerobe, grows best at room tem-
perature, perceptibly less at 30°, and at 40° it soon dies.
Tt grows with moderate rapidity.

Gelatin Plate.—(a) Natural size. Deep: Roundish

19

4
i
:
&

a

—. eee’ =

290 IMPORTANT VARIETIES OF FISSION-FUNGI. 7

to whetstone-shaped and yellowish. Superficial (after —
three days): Irregularly jagged lobulation, with a moist
luster, a little elevated, sharply outlined from the surround-
ing medium, yellowish to grayish-white (28, v1). Later
they become grayish to brownish-lavender. The gelatine
is variously colored. (See also 28, vu.)

(6) Magnified fifty times. Deep : Round or roundish, ;
yellowish, delicately granular (28, vu i). Superficial :
In the youngest stages are not distinguishable from those
of Bact. typhi and coli. Also later they are still very —
similar to them, only the colonies appear much more
delicately granular. Often the original colony appears at_
the middle as a yellowish-brown nucleus. Every possible
variation of form, structure, and color is observed. The
color usually is yellowish and the form irregularly lobu-—
lated (28, vim e).

Gelatin Stab.—Stab: Not characteristic, thread-like. | ©
Surface growth: From whitish and bluish-gray to greenish-_
yellow, with a moist luster, slimy. The color of the gela-—
tin varies exceedingly. A culture obtained from Berlin |
in the summer of 1895 usually furnished light to dark
blue growths, while a culture of our own, which had been
cultivated in the institute for about six years, exhibited,
upon the same nutrient medium, brownish-green, dark
brown, and light yellowish-green growths with more or less —
fluorescence. A Bact. syncyaneum # cyaneofluorescens
Zangemeister (C. B. xvi, 321) behaved very similarly. —
A year later also the Berlin culture produced no blue color
upon either acid or alkaline nutrient media, but only dirty
colors, from light or dark brown to light yellowish-green —
and deep brownish-green (27, I, J, I). (Compare also”
27, Iv.) <A culture freshly isolated from milk in Wiirz-—
burg gave beautiful blue pigment with little fluorescence. —

Agar Plate.—(a) Natural size: Like those on the gelag
tin plate.

(6) Magnified fifty times. Deep: Roundish to whetstone-_ j
shaped, yellow, gray, or brownish, with even border,
homogeneous, shaded. Superficial: Round to roundish, —
smooth edge, light yellowish to grayish-brown, homogen- _ i
eously shaded or finely granular. It is similar to the colony
of Bact. fluorescens (28, v).

4

BACTERIUM SYNCYANEUM. 291

_ Agar Stab.—Just like that in gelatin. Usually the
surface growth is a little more luxuriant (27, Iv). Com-
yare 27, I-11.

Agar Streak.—Moist, usually grayish-white growth,
ith a smooth, wavy border. Water of condensation
sloudy, with a grayish-white sediment. The agar presents
most variable colors. Sometimes cultures can not be dis-
tinguished from those of the Bact. putidum.

- Bouillon Culture.—Moderately cloudy, at first gray-
ish-green, later with many cultures, becoming blue to
bluish-green. Precipitate moderate, ‘whitish- -gray, feebly
coherent. Pellicle formation is observed in some Cases,

and not in others (27, v).

_ Milk Culture.—Bluish-green color; otherwise unaltered.

Reaction alkaline (27, v1). With the addition of hydro-
chloric acid the color becomes blue, if a culture is employed
which forms syneyanin. Upon unsterilized milk the color
s deep to sky blue on account of the acid produced by the
Bact. acid. lactici.

_ We obtained beautiful blue milk by adding 1% grape-
sugar to sterilized milk or, better, whey; grape-sugar,
particularly, is converted into acid by Bact. syncyaneum.
_ Potato Culture.—<According to the variety of potato,
owths resulting from inoculation with the same culture
lay vary widely. The growth is greenish or brownish-blue,

blackish-blue, dark brown, yellowish-brown, or gray,
always elistening, sometimes a little elevated. The potato
is discolored greenish, brown, gray, blue, etc. (28, 1-11).

In many cases it can not be distinguished from Bact.

fluorescens, especially if the formation of blue pigment is
‘slight or absent.

Other Nutrient Media.—Grows and produces pigment
upon non-albuminous nutrient media. As pointed out by
‘Hippe, tartrate of ammonium serves as the source of car-
bon and nitrogen.

Resistant Properties.—Against drying, five to seven
months (Heim). Spores are certainly not present (Heim).
_ Chemical Activities.—Chromogenesis: Most cultures
form two pigments, the fluorescent yellowish-green bac-
feriofluorescein, and also the blue syncyanin. We have
ossessed cultures which no longer produced any trace of

292 IMPORTANT VARIETIES OF FISSION-FUNGLI.

blue pigment, but only bacteriofluorescein, and Hiippe
and Thumm (A. K. 1, 291) have observed those which
produced a pure blue pigment. Finally, all chromogen-
esis may be lost (Heim, /. c.; Behr, C. B. vim, 485).
Regarding syncyanin, we still know little; we have been
unable to find a good solvent for it; spectroscopically it
furnishes a powerful absorption band. Weak acids do not
alter the color, strong hydrochloric and sulphuric acids
change it to violet, acetic acid produces a dirty color.
Sodium hydroxid produces a rosy to yellowish red; the
change in color occurs as the acid reaction disappears; upon
standing, the color passes into brownish-red, thus explain-
ing the color of old cultures.

Acid is not formed from milk-sugar, but is from grape-
sugar without liberation of gas. In peptone bouillon
no H,S is formed, but traces of indol are produced. The
bouillon smells disagreeably aromatic, and abundant am-
monia is formed. .

Distribution.—(a) Outside the body: Often found in
blue milk, sometimes occurring epidemically. Such milk
is not harmful. (6) Jn the body the organism has not
been found.

Bacterium brunificans. Lehm. and Neum.

Actively motile, does not liquefy gelatin, isolated from foul pus by
Scheibenzuber (C. B. v1, 441). Instab cultures upon various nutrient
media it discolors the medium dark brown in the form of a sack—#. e.,
above for a short and below for a greater distance from the stab canal.
Upon the potato there develops a brown growth, about which is a ring
of dark brown discoloration.

Bacterium ferrugineum. (Rullmann.) L. and N.

According to the description, it is closely related to the
preceding. It is actively motile. Cultures yellowish or
reddish, but usually dark brown, with marked rusty
brown discoloration of the nutrient medium. Pigment is
soluble in water, alcohol, and acetone. Upon meat infu-
sion-glycerin-agar there is, at 37°, a greenish fluorescence.
Gelatin is feebly liquefied. Found by Rullmann in canal-
water (C. B. xxv, 465).

BACTERIUM ZOPFII. 293

Bacterium Zopfii. Kurth. (Botan. Zeit., 1883.)
(Plates 29 and 30.)

Microscopic Appearance. — There occur all forms
from long threads to short rods. Often the threads break
up into nearly spherical members (80, 11).

_ Motility.— Very active, dependent upon numerous

i eh

; peritrichous flagella (80, rx).
_ Staining Properties.—Stains well by Gram’s method.
_ Requirements as to Nutrient Media, Temperature,
and Oxygen.—Facultative anaerobe, satisfied with the
_ greatest variety of nutrient materials; grows at room and
- incubator temperatures.
_ Gelatin Plate.—(a) Natural size: Delicate, whitish-
_ gray colonies, resembling spider’s web or mold mycelium
_ (29, v1). Later there appear little branches upon the
_ threads, which are more distinct the more superficially
_ they are located. The colonies (29, v) then resemble
those of the Bacillus mycoides (87, vi, Ix).
(6) Magnified 50 to 100 times: Very characteristic. The
original colony serves as a central part, from which radiat-
- ing threads pass outward on all sides, which are more or
_ less branched and matted together. Between these lie
zooglez of the most variable forms: resembling loops of
_ hair, corkscrews, whipcords, and sausage with marked re-
flection (29, vir). When magnified 90 times, the indi-
vidual threads appear as wavy strings with wide lumina,
arranged in a most irregular manner (29, vit). The
_ ribbon-like zoogleic forms, when magnified 90 times,
appear composed of highly refractive, often granular
threads (30, 1). The elongated, sausage-like forms appear
decidedly irregular. They consist of oval, overlapping,
yellowish-gray, homogeneously shaded clumps. At the
end of such a chain there are usually bough-like branch-
ings. Between these lie younger zooglee, bounded by two
notched lines (30, viz).

Gelatin Stab.—The stab is provided with very delicate,
fine, parallel outgrowths, which are longest near the sur-
face and become shorter as they become deeper in the
tube. Surface growth is delicate, transparent, gray,

:
4

294 IMPORTANT VARIETIES OF FISSION-FUNGI.

shining, sometimes presenting very beautifully a combina-—
tion of small boughs (29, 1). .
Agar Plate.—(a) Natural size: After twenty-four hours
the colonies are 2 to 4mm. wide, grayish-white, with deli-—
cately fringed border, and are surrounded by a thin trans-—
parent zone (30, 1v). Inashort time the whole plate is _
covered with a gray veil.
(6) Magnified fifty times: After twelve hours the colonies —
are like exceedingly delicate balls of hairs, and visible
only with a very narrow opening in the diaphragm (80, —
v). Later the colonies take on a more intense yellowish —
color, the branching increases, and the extension takes place —
rapidly but irregularly. The colony can not be distin-—
guished from a deep subtilis colony (30, mr). Afterafew —
days the colony has become yellowish-brown, and is |
exceedingly matted and tangled. When magnified 90
times, it is seen that the delicate veils about the superficial —
colonies consist of thin layers of bacteria (80, vr).
Agar Stab.—Similar to that in gelatin (29, m1). .
Agar Streak.— Exceedingly delicate, grayish-white, —
transparent, shining. In the middle there sometimes isa
paler streak. In a short time the entire surface is over- ~
grown ; hairs are not usually distinguishable ; the water —
of condensation remains clear, with a whitish precipitate.
Bouillon Culture.—Clear or slightly turbid, with a
little sediment.
Milk.—Not coagulated, amphoteric reaction.
Potato Culture.—Slight, yellowish-gray growth.
Chemical Activities.—Produces typical putrefaction
with pronounced foul odor upon nutrient media rich in
albumin. It is remarkable that Kuhn (A. H. xm, 40)
could never demonstrate indol production in our institute, —
while we now find some indol. .
Distribution.—(a) Outside the body: Isolated by Kurt —
from hen dejecta ; by Kuhn, repeatedly from putrefying —
mixtures. (b) Inside the body it has never been found. |
Related Varieties.—The Bact. vulgare, forma Zenkeri,
which has so far been but little studied, is most closely
related. The principal difference lies in the beautiful little —
hairs, bristles, and threads sent out from the stab culture. —
According to Hauser’s description, it appears that also the

BACTERIUM VULGARE. 295

elongated, sausage-shaped zooglez are lacking in his Pro-

| teus Zenkeri. In Kuhn a confusion of our organism with

re) er . Ce Ee eee ji" as

_ the Prot. Zenkeri is met with ; in his work it is every-
_ where called Bact. Zopfii instead of Proteus Zenkeri (A. H.
xi, 40).

| Bacterium vulgare. (Hauser.) Lehm. and Neum.

(Plates 31 and 32.)

Synonyms.—Proteus vulgaris Hauser, Bacillus vulgaris

| Macé, Migula. Proteus Hauseri Autor., Bacillus albus cada-

veris Strecker and Strassmann (C. B. 1v, 67), Urobacillus
liquefaciens septicus Krogius, Bac. fotidus ozene Hajek,

_ Bacillus Proteus vulgaris Kruse.

Ordinary Name.—Proteus.
Literature. — Hauser, ‘‘Ueber Faulnisbakterien,’’ Leipzig, 1885.

_ Meyerhof (C. B. xxIv, 18), extensive review of literature (152 num-

bers).

Microscopic Appearance.—Slender, thin rods, aver-
aging 1.6—4 » in length, and 0.4—0.5 » in thickness. It is
often found as long threads, but it also occurs in isodia-
metric forms and as spiral winding threads. The mul-
tiplicity of the microscopic growth-forms has led to naming
the organism as Proteus. Upon acid nutrient media, very
short rods are especially produced (81, vit and 1x).

Motility.—Very active, due to very abundant, long,
peritrichous flagella. At present our cultures exhibit
active motion only when examined while very young, in
spite of a good development of flagella (31, rx).

Staining Properties.—Stains well by Gram’s method.
When treated by Gram’s method, it was found by Meyer-
hof to be easily decolorized (C. B. xxiv, 27), and by Sil-
berschmidt to be unstained. We have demonstrated
anew with many cultures that it stains well.

Requirements as Regards Oxygen and Nutrient
Media.—It grows equally well aerobically and anaerob-
ically, also in CO,. The most variable nutrient media
(also non-albuminous) are suited to it. It grows very
rapidly. Hauser found that, in the absence of oxygen
and in carbonic acid, growth was poor, also upon non-

5)

296 IMPORTANT VARIETIES OF FISSION-FUNGI. 4

albuminous nutrient media. It grows at quite low tem-
peratures (ice-box) and in the incubator. According to
Levy and Meyerhof, gas production is most abundant

when oxygen is freely admitted, as when the bacteria are

grown in shallow dishes.

Gelatin Plate.'—(a) Natural size: Gray, delicate, trans- ;

parent growths which present a shallow depression even
after twelve to twenty hours. After three days the cups
of liquefaction are already 0.5 to 1 cm. wide, with turbid
contents. The deep colonies are punctiform and not char-
acteristic. Before liquefaction there is usually seen about
the superficial colonies an irregularly jagged zone, consist-
ing of highly refractive zooglee (31, v).

(6) Magnified sixty times: Upon very young plates two |

kinds of colonies are seen—the one, roundish, grayish-
yellow, sharply outlined, with even borders, homogeneous
or finely granular, which lies deep in the gelatin; the
other, transparent, colorless, delicate, with wavy lobu-
lations, difficult to distinguish from those of Bact. typhi,
which lie upon the surface. The latter spread out more
and more, and then there appears in the center of the
colony a lively, interesting motion of the bacterial masses.
After a longer time the motion ceases, while liquefaction
extends at the periphery. The colony then is of an irreg-
ular form, and when the entire colony is almost completely
liquefied, the thin shining peripheral portion remains.
The deep colonies often present hairs, which later are
mostly arranged about the periphery. ?

Gelatin Stab.—Stab: At first is thread-like and not

1 Sugar gelatin is not liquefied, the growth upon this medium being
entirely similar to that of the Bact. Zopfii, but in the stab the out-
growths are absent (Kuhn).

2'The representation given is taken from a culture which has been
long cultivated and often studied. Not rarely, especially in freshly
isolated cultures, one observes on gelatin plate cultures sausage-shaped,
spiral zoogleze exactly identical with those which we have described
and illustrated so minutely in the Bacterium Zopfii, and which Hauser
has so beautifully photographed. Schedtler(C. B. m1, 437) appears to
have submitted cultures similar to those represented by us. We have
sometimes noticed the swarming islands at the periphery of the colo-
nies, which, according to Hauser, are especially observed upon 5%
gelatin.

hei

Str

BACTERIUM VULGARE. 297

sharacteristic ; later, there is tube-shaped liquefaction.
The surface growth at once forms a saucer-shaped lique-
faction, and later the liquefaction becomes cylindric. The
iquefied material is turbid or cloudy (31, 1). (The picture
is a little too violet. )
_ Agar Plate.—(a) Natural size: Entirely non- -character-
istic. The surface colonies are delicate grayish-white; the
deeper, yellowish-white (381, 1).
(6) Magnified sixty times. Deep colonies: Roundish, very
¢ mbly, later often moruloid (33, Iv below). Super-
ficial: Delicately transparent, exceedingly finely granular,
at t the center yellowish and becoming colorless toward the
periphery. The periphery assumes all possible irregular
forms, from the wandering outward of the bacteria (32,
“yit). At first it is always roundish (31, Iv). Typical,
} este sausage forms, etc., such as occur on gelatin,

Agar Stab.—Stab: Not characteristic, thread-like.
Surface growth: Gray, slimy, moist, transparent.
__Agar Streak.—Veil-like, thin, transparent, moistly
‘glistening growth, which already after twelve hours has
“overgrown the entire surface. Water of condensation very
cloudy, whitish-yellow.
it Culture.—Very cloudy, abundant precipi-
tate
_ Milk Culture.—Firmly coagulated after two to three
days, and later again liquefied. Still later, milk becomes
yellowish and feebly acid.
Potato Culture.—Very scanty growth; whitish-yellow,
usually limited to the inoculation streak, somewhat
rumbly, dull or with a fatty luster, somewhat elevated.
Chemical Activities.—
_ (a) Odoriferous substances: Albuminous bodies under-
go putrefactive decomposition with very abundant foul
odor, and a strong alkaline reaction.
(b) Formation of gas and acid from carbohydrates: It
forms abundant gas from grape-sugar; according to Th.
Smith, still more from cane-sugar, and none from milk-
sugar. According to Smith, the gas consists of one-third
CO, and of two-thirds H,. Upon sugar media no foul
odor i is present (Kuhn).

298 IMPORTANT VARIETIES OF FISSION-FUNGI.

'(c) HS and indol are produced abundantly. :

(d) Urea is vigorously transformed into ammonium
carbonate. (Compare p. 70.) i

(e) Toxins: Hauser observed the production of exceed-
ingly powerful toxic metabolic products, which could be
obtained free of bacteria by passage through clay filters.
Tito Carbone has isolated cholin, ethylendiamin, gadinin,
and trimethylamin from meat cultures (C. B. vin, No.
768).

The sepsin of Schmiedeberg, from putrid yeast (Mediz.
Centralblatt, 1868, No. 32), acts just like the metabolic
products of the Bact. vulgare (Levy), and appears to be a
product of that organism.

Resistance.—Considerable against chemical and therd
mal injuries, but is killed by 60° in one-quarter to one-’
half minute (Meyerhof). .

Distribution.—

(a) Outside the body: Very common in putrid meat and
other putrid objects. Cause of foul-smelling decomposi-
tion ; occurs in water from contamination with putrid ma-—
terials. It never occurs in gelatin plates from air, but is
easily obtained if sterile- or sterilized meat is allowed to.
stand uncovered ; thus it is present in air. ;

(b) In healthy body : Throughout the entire alimentary |
tract.

(c) In diseased human organism: Often alone it produces
severe catarrh of the bladder with ammoniacal urine;
often, also, in association with Bact. coli (Schnitzler, C
B. xIv, 218), It is also the cause of other diseases of the
urinary organs. The urobacillus liquefaciens septicus of
writers is at least in part identical with the Bact. vulgare. —

While Bact. vulgare occurs rather frequently together -
with other causes of disease (in foul phlegmons, abscesses,
pulmonary gangrene, decubitus, foul carcinomas, ete. ), it
has relatively seldom been demonstrated to certainly be
_the cause of diseases in man, as in a few cases of abscess,
inflammations of serous membranes, ete.

Booker found varieties of the proteus in 18 cases of
cholera infantum (C. B. x, 284).

Levy has demonstrated the Bact. vulgare to be the
cause of meat poisoning: Eighteen persons were taken

a
4

BACTERIUM VULGARE. 299

‘sick with severe vomiting and diarrhea (vomiting of
blood), of whom one died. Compare also the epidemic
‘described by Wesenberg (Z. H. xxvut, 484).
In an instance where several soldiers became very sick
‘with Weil’s disease (infectious, febrile icterus) after
bathing in impure water, Jiger (Z. H. xu, 525) found
‘the Bact. vulgare in a feebly fluorescent form; in two
cases postmortem, in great numbers in some of the organs;
= four out of six lighter cases examined, in the urine.
It was also possible to demonstrate that the same or-
_ ganism was present in the bath water, which, besides,
caused a sickness in fowls. Jager pointed out the ereat
variability of his organism, and believes that sometimes
_ the Bact. vulgare may be actively pathogenic. Also a
series of other cases of infectious icterus are to be referred
_ to proteus infection—whether all, is questionable.
Experimental Observations Regarding Pathogenic
Properties.—Hauser did not obtain true infection; his
animal experiments are all intoxications with the meta-
_ bolic products (dyspnea). Meyerhof has produced, with
large quantities of slightly virulent proteus cultures, fatal
_ disease in mice, rabbits, and dogs, accompanied by an
_ increase of the bacteria introduced, it thus being a true
_ infection. The filtrate of the cultures was very weak,
_ devitalized (chloroform) cultures having but little effect.
___ Virulent forms of proteus, when injected subcutaneously
in an animal (rabbit), produce putrid abscess. This
_ occurs much more easily if other organisms (for example,
; streptococci) are simultaneously introduced into the body.
: Slightly virulent, pathogenic varieties (staphylococci,
;

= a TS le

streptococci) increase in virulence if they are injected
_ simultaneously with dead or living proteus cultures.

_ QO. Wyss has convincingly demonstrated Bact. vulgare
_ to be the cause of a disease in fish (Z. H. xxvu, 148).
__ Immunity and Serum Reaction.—According to Car-
bone, it is possible to immunize animals against the living
bacteria by means of their metabolic products. Accord-
_ ing to Pfaundler, the serum of animals which pass through
- an afebrile proteus infection agglutinates the proteus indi-
_ viduals of the same stock. If, however, the sickness is
accompanied by fever, then no agglutination occurs, but

300 IMPORTANT VARIETIES OF FISSION-FUNGI.

in such a serum the organism grows out into long threads.
Similar results—the serum being active only or especially
against the particular culture employed to produce the
sickness or immunity—were obtained also by others.
Compare 8. Wolf (C. B. xxv, 317).

Related Varieties.—Hauser (J. c.) has designated —

eu pbe + eae

with the name Proteus mirabilis a form of the Bact. vul-

gare characterized by somewhat more feeble liquefaction,
and producing especially striking involution forms. An-
other he has designated as Proteus Zenkeri, which does
not liquefy gelatin and does not any longer cause vigorous
putrefaction. These forms were later recognized by
Hauser as transformable into each other (C. B. xm, 629).

Here also belongs Gerdes’ eclampsia bacillus. On the ~

at we ye eter, e

contrary, a Proteus hominis Bordoni-Uffreduzzi (Z. H. m1, —
333) certainly belongs among those related to the Bact.

pneumonie; it does not exhibit motility, formation of —

zooglee, nor the production of putrefaction. (Compare —

p. 228.) The case may be considered as similar to Banti’s ©

four ‘‘ varieties of proteus’’ (C. B. v, 207).

Bacterium murisepticum. (Fliigge.) Migula.
(Plate 33. )

Synonyms.—Bacillus murisepticus Fligge. Bacillus —

of mouse septicemia Koch.

Literature.-—Koch, R., Wundinfektionskrankheiten, p. 40; Preisz
(C. B. x1, 110); Liffler (C. B. x1, 129).

Microscopic Appearance.—In the culture, beautiful,
slender rods, 2-4 » long, 0.4—6 » thick, straight or curved,

often arranged i in threads (33, vut). In preparations of
smeared blood the organisms are only about 1 » long and ~

0.2-0.3 » thick (33, Ix).

Motility is absent.

Staining Properties.—Stain well by Gram’s method.

Relation to Oxygen.—Facultative anaerobe. Liborius
found it an obligate aerobe. Many cultures grow de-
cidedly better when air is excluded.

Intensity of Growth.—Grows rather slowly.

Gelatin Plate.—(a) Natural size: After three or four

i
- BACTERIUM MURISEPTICUM. 301

days there is a very shallow depression, in which the
-— colony rests as an exceedingly delicate veil, only distin-
_guishable from the surrounding medium with difficulty
_ (88, v). The plate represents the colonies as too distinct.
_ (6) Magnified fifty times: The colony is visible only
-_ with a very narrow opening in the diaphragm. It is not
without difficulty that one observes the exceedingly slight,
| delicate, gray growth of homogeneous or finely granular
character and differentiated from the surroundings with
“little sharpness (33, v1). When magnified 90 times,
_ there is seen what suggests a tangle of threads. Other
__writers—for example, B. Preisz—describe the colonies as
~ somewhat denser; from a homogeneous or matted nucleus
_ there radiate outward ramifying and interlacing threads,
_ which sometimes wind like a corkscrew.
_ Gelatin Stab.—After a few days the stab canal repre-
_ sents the structure of an exceedingly delicate fir-tree, with
branches of equal length throughout the entire length

(33, 111), which after a longer time become in part more
and more confluent and remain as delicate, transparent
clouds in the gelatin. Upon the surface there gradually
forms a slight, pointed depression. The atypical anthrax
culture (34, v) presents a similar, but very much coarser
picture.

Agar Plate.—(a) Natural size: Small, very insignifi-
cant, whitish-gray points, which are barely visible only
when examined upon a dark background.

(6) Magnified fifty times. Superficial: At first gray,
delicate, veil-like; later, more brownish or yellowish.
The homogeneous structure becomes finely or moderately
coarsely granular and sometimes looks not unlike the
granulation of a finely granular sarcina. Deep: Round-
ish to whetstone-shaped, yellowish, homogeneous (38,
vil), with a smooth or granular border.

Agar Stab.—Similar to that in gelatin, a little less
luxuriant. The branches may be entirely absent. Sur-
face growth exceedingly delicate, transparent, spreading
but little, colorless. Sometimes the growth is indicated
only by a little luster (33, Iv).

‘Sem Streak.—Delicate, exceedingly thin growth (33,
II),

Bouillon Culture.—No pellicle, little turbidity, very
little sediment, which is very slightly coherent. |

Milk Culture.—No coagulation, ampHees or feebly
alkaline reaction.

Potato Culture.—No perceptible growth.

Chemical Activities.—No formation of pigment or
odoriferous substances. Our culture produces no HS or ~
indol. Petri and Maassen observed vigorous production
of H,S. A little acid is formed from grape-sugar and —
gelatin i is very slowly liquefied.

Distribution.—

(a) Outside the body: Isolated repeatedly by Koch and —
others from canal-water and putrid mixtures (putrid meat, —
yeast). ;
(6) In the body: Not found in man, for whom the or- —
ganism is not pathogenic. It causes mouse septicemia, —
an artificial infectious disease, discovered by Koch, which —
also occurred spontaneously in one instance in Greifswald. —

Special Culture Methods.—Inoculation of a white —
mouse with the suspected material. Stained smear prep- —
arations, plates, and stab cultures are made from the
blood and spleen, where the bacteria are present in abun-
dance.

Pathogenic Effects upon Animals. — Pathogenic
(death in two or three days) for house mice (not for field
mice). Symptoms: Eyelids stuck together, head drawn in,
a tendency to sleep. Also pigeons die in two and one-half
to three and one-half days (Th. Smith). Rabbits and
guinea-pigs withstand large quantities of bouillon culture.
In swine it produces only. transitory indisposition.

3802 IMPORTANT VARIETIES OF FISSION-FUNGL

Bacterium erysipelates suum. (Loffler.) Migula.

(Swine Erysipelas pro parte, Bacillus of Erysipelas of Swine.)
Bacillus rhusiopathice suis Kitt.
Literature.—Léoffler (A. G. A. I, 46). Preisz (C. B. x1, 110).

This organism is very closely related to the one causing
mouse septicemia, being identical microscopically, and the
stab culture is extremely similar, only the branches are a
little more sturdy and bristly (383, 1). When the inocu-

BACTERIUM ERYSIPELATOS SUUM. 303

: tion is made in gelatin from the blood, according to
Lorenz, the branches are sometimes almost entirely absent
rom the primary cultures, and, instead, only nodules and
y Gules are seen in the stab. The principal difference lies
in the gelatin plate colonies, which were described by
Loffler as minute, distinctly visible growths, with a few
sular radiations (like bone corpuscles), and which
ere found by us to be constantly somewhat between 31,
e, and 31, vil, on an average. Compare Lésener, A.
A: XI, 448, There is intense production of H,§, ‘and
| Mitle of indol. Some acid is formed from grape-sugar.
_ Regarding a rather considerable resistance to pickling
and smoking, consult Petri (A. G. A. v1, 266).
It causes an important disease of swine, young animals
of the choicer varieties being especially affected. Older
| eas, and also younger ones of ordinary breeds,
re more or less immune. Upon section the animals
Tshow, besides a patchy or diffuse redness of the skin,
which is often very marked, also subcutaneous edema,
"rec ness of the pharynx and ‘of the gastric and intestinal
eucosa, swelling of the mesenteric glands and spleen,
“parenchymatous nephritis, hemorrhages in the kidneys,
and red spots in the lungs. Consult Graffunder, Berl.
- tieriirztl. Wochenschrift, 1896, No. 2.
_ The organism is not pathogenic for man, also the flesh
of swine affected with erysipelas is harmless.
_ Mice sicken and die after feeding, and more rapidly
after inoculation; also rabbits usually succumb to inocu-
lation.
_ The differential diagnosis from other diseases of swine
is easy, from the characteristic form of the individuals
| and from the cultures of the organism. It must not be
q orgotten that redness of the skin in patches occurs in
many diseases of swine, as in Léffler-Schutz’s swine plague
(see p. 254).
Protective Inoculation.—Animals may be actively
“immunized with attenuated bacteria (Pasteur), with de-
_yitalized bacteria, with body juices (Emmerich) and blood-
serum (Lorenz, C. B. xrx, 168).
_ According to Voges and Schiitz (Z. H. xxvin, 38),
‘no method has stood practical tests. The serum of ac-

304 IMPORTANT VARIETIES OF FISSION-FUNGI.

tively immunized animals possesses active bactericidal su mf
stances. In the place last referred to is also found ¢
extensive review of the literature.

Bacterium of Brick-pock (Backsteinblattern).
Lorenz (C. B. xi, 672).

A disease of swine,—evidently to be considered as a form of swine —
erysipelas,—which almost always runs a favorable course, has been —
described by Lorenz as brick-pock, and he has ascribed it to an organ
ism which, upon subcutaneous inoculation, stands midway, as to viru-
lence for swine, between mouse septicemia and swine erysipelas.
After inoculation with it, swine become immune to swine erysipelas
Rabbits, on the contrary, in a very interesting manner are much more
susceptible to brick-pock than to swine erysipelas. They always s
cumb to brick-pock infection, but may be immunized with swine
erysipelas against brick-pock. Lorenz likewise holds that swine ery-
sipelas, mouse septicemia, and brick-pock are produced by forms of one
organism, even if the transformation of one form into the other has
not been entirely successful.

2. Bacillus F. Cohn, emend. Hiippe.

Straight rods, often growing into threads, often of con-
siderable thickness, rarely less than 0.6, usually more
than 0.8 ». They form endospores.

Key to the Recognition of the More Important
Varieties of the Genus ! Bacillus.?

I. AEROBIC VARIETIES, thriving only scantily anaerobically. The
pathogenic ones never form spores in the animal body, but only in”
cultures with oxygen admitted (compare also p. 310). Almost all in
cultures grow into long threads with central spores.

1 Regarding our insufficient knowledge of this genus, consult the
statements upon page 306 and in the discussion of the anaerobes. —
Numerous new aerobic varieties are described by Burchard (A. K. ©
ay,c 4).

2The genus Tyrothrix Duclaux is included in the bacilli. It
designates primarily varieties originating from milk and cheese, which
form spores and grow into long threads. Two species are described
below: Bac. tenuis and Bac. geniculatus. The most remarkable
statements of W. Winkler regarding extraordinary biologic and mor-—
phologic variability, especially in the Bact. tenuis (C. B. L. 1, 657), —
could not be confirmed either by ourselves (see first edition) or by —

KEY TO GENUS BACILLUS. 305

= (4) Stab culture in gelatin with projecting branches :
. Branches distinct, usually only in the upper part of the stab.
r plate colonies, when magnified 60 times, have beautiful regular
os s. Agar streak culture without branches, wide, white with “‘sil-
very vesicles.’’ Never motile. Pathogenic for animals. Bac, an-
hracis Cohn and Koch (p. 307).
2. Branches delicate, extending along the entire length of the stab.
Vher magnified 60 times, the colonies in the agar plate exhibit irreg-
lar outgrowths in the form of roots or the mycelium of molds. Agar
st trez k culture has long, delicate, parallel transverse branches. Slug-
gishly aaa Not pathogenic for animals. Bac. mycoides Fliigge
— (p. 316
_ (B) Stab culture in gelatin without projecting branches ; motility de-
endent upon peritrichous flagella :
7 A. Potato growth at first moist and flat, later (about eight days)
with a distinctly mealy sprinkling. Bac. subtilis Cohn (p. 317).
3 2. Potato growth moderately elevated, not characteristic, resem-
bli Bact. coli. Bac. oxalaticus Zopf, butyricus Hiippe, mega-
therium De Bary (pp. 321, 322, and 323).

3. Potato culture luxuriant, moist, intensely yellow. Agar moist,
mustard yellow. Later resembles vulgatus. Bac. luteus L. and N.!
: 2 Potato is not characteristic during the first days; later, there

s a distinct, wrinkled elevation.
re The folds are padded, like coils of intestine. Bac. vulgatus
( Fli ) Migula (p. 323).
_ (0) The folds are low, reticulated. Growth yellowish. Bac,
mesentericus (Fliigge) Lehm. and Neum. (p. 326).
: — (e) Growth moist, wrinkled, besides the potato is deep black. Bac.
4 terrimus Lehm. and Neum. (p. 328).
(d) Growth rose-colored, a little wrinkled, gelatin smoky brown.
Compare also Bac. mesentericus ruber. Bac. gangrene pulpz
Arkovy (L. and N.) (p. 328).
z 5. The potato growth is delicate, syrupy, clear. Bac. liodermos
_ (Fliigge) Lehm. and Neum. (p. 328).
II. ANAEROBIC VARIETIES (of which, certainly, partially aero-
_ bic forms exist). Only exceptionally form ‘long threads. Staining by
Gram’ s method rarely well developed (the Bac. tetani stains well).
Spontaneous motion dependent upon peritrichous flagella is rarely
lacking. The spore is usually located at the end (paraplectrum form)
_ or at the middle, usually with some bulging (clostridium form). In
“most varieties both forms of sporulation occur. The recognition of
the individual varieties, which is often very difficult, and even impos-
sible, may be rendered somewhat easier by means of the following
_ scheme:
(A) Pathogenic Varieties.—

a.
ws
1
id

: Wittlin (Cc. B L. 1, 475) ; and since then, so far as we know, it has
_ not been upheld.

1 For more details regarding this organism, compare Bacillus luteus
'sporogenes Wood Smith and Baker (B. C. L. Iv, p. 788).
A 20

3806 IMPORTANT VARIETIES OF FISSION-FUNGI. 1
‘

2

1. In a deep cutaneous pocket in animals no local symptoms “7
produced, but only or preponderantly nervous symptoms. !

(a) Produces tetanus. Bac. tetani Nicolaier (p. 332).

(8) Causes symptoms of botulism: disturbances of the innerva-
tion of the pupils and accommodation, aphonia, paresis in the
region of the tongue and pharynx, disturbances of the salivary
a mucous secretions, etc. Bac. botulinus y. Ermengem (Pe
337

2. When introduced into a deep cutaneous pocket in animals, i
causes local bloody, often emphysematous edema. The organi
spreads in the body, especially in the edema. Guinea-pigs are es
cially susceptible. ?

(a) Motile.

(a) In the edema growing into long, jointed threads, usually not
present in the bile. Very pathogenic for rabbits. Brain
nutrient media darkened. Not stained by Gram’s method.
Bac, cedematis maligni (Koch) Fligge (p. 341).

(8) No long threads in edema; usually only pairs. Usually
slightly pathogenic for rabbits and mice. Always found in the
bile. Brain nutrient media not darkened. Usually stained by -
Gram’s method. Bac. Chauveei of French authors (p. 339). :

(b) Not motile. Disease picture similar to symptomatic anthrax,
but there is a tendency to grow into long threads. Bac. phiegoaociall :
emphysematose E. Frankel (p. 344).

3. Only known as injurious to bees. Bac. alvei Chesire and
Cheyne (p. 345).

(B) Zymogenic Varieties.— ,

A large group which has not yet been sufficiently cleared up. Com-_

pare page 345, etc.; also especially page 348. Here belong many
forms producing butyric acid.

Introductory Remarks to the Special Description of
the Aerobic Varieties Here Given. !

(Common Characteristics. )

All of the varieties described in what follows,—Bace. —
anthracis, mycoides, subtilis, megatherium, butyricus, —
vulgatus, mesentericus, aterrimus, liodermos,—which are
very closely related to each other, have the following
biologic properties in common, which may be given here
for all of them: |

1 Non-pathogenic cultures cannot always be diagnosed with cer-
tainty from the morphologic and biologic properties.

2Compare also the Bac. sporogenes (Klein) L. and N. (p. 346),
which occupies a place midway between malignant edema and symp-
tomatic anthrax; also the pseudoedema bacilli (p. 343).

BACILLUS ANTHRACIS. 307

1. Gelatin is liquefied.
2. Milk is alkaline or very feebly acid in reaction, is
‘coagulated, and later the coagulum is dissolved.

_ 3. All form little acid from grape-sugar (see Table I, at
end of the book, for quantitative statements) and no gas.
From milk- -sugar there is formed little or no acid. ;
_ 4. No indol is formed. The production of H,S is

_ variable, never abundant.
_ 5. All are stained by Gram’s method.
The equipment with flagella appears also in this group
_ to be valuable for the diagnosis of species only with great
precautions. When present, they are peritrichous.

= 2) are HR.

Bacillus anthracis. F. Cohn and Koch.
(Plates 34, 35, and 36.)

Ordinary Names.—Anthrax bacillus, Bactéridie du
_ charbon.

Microscopic Appearance.—In the animal body it
occurs as large vigorous rods, 3-10 » long, 1-1.2 » thick,
which are often arranged in longer or shorter chains (36,
1). The ends in fresh specimens are a little projecting
(rounded ); after drying and staining, they appear square-
eut or slightly concave. To demonstrate the capsules,—

_ which are always well developed in the animal body, fluid
_blood-serum, and brain-agar mixture,—the directions
_ given in the technical appendix are to be followed. Ac-
cording to Kern, capsules may be demonstrated in old
_ cultures upon most variable nutrient media. !
In artificial nutrient media the bacilli grow into long
_ threads, placed parallel or somewhat twisted and en-
_ tangled (36, 11), which either produce spores (see below)
or perish in the formation of bizarre involution forms
(36, v). The threads, even when unstained, give indi-
cations of their being composed of separate bacilli (36,
vi). This is especially distinct after staining.

: 1 Noetzel has also demonstrated capsules in undoubted ‘ cadaver
bacilli,’’ and this points out how unsafe it is to allow the diagnosis of
_ anthrax to rest upon the demonstration of capsules, which is often
_ very much overestimated by veterinarians. (C. B. x1x, 498.)

308 IMPORTANT VARIETIES OF FISSION-FUNGI.

Motility.—Always is absent. To this no exception is

known.

Staining Properties.—Stains with all anilin dyes and —

by Gram’s method.

Relation to Oxygen.—Grows best wige oxygen is”

admitted. When oxygen is excluded, it grows poorly
and without liquefaction. There is no growth i in CO,.
Intensity of Growth.—Grows rapidly, especially at
37°. Lower limit of growth at 14° (Kitasato).
Gelatin Plate.—(a) Natural size. Superficial colony:

Whitish, round; after three or four days, deeply sunken.
Also, upon longer standing the liquefaction only extends —

slowly. In the middle of the abrupt crater there lies a
white, crumbly, poorly defined mass, the remainder of the
contents of the liquefied area being rather clear, but the
outermost peripheral zone is somewhat turbid again (35,v).

(b) Magnified seventy times: The colonies when three
days old appear distinctly darker than on agar. Near the
center grayish-yellow, toward the edge more distinctly
transparent. At the periphery the formation of locks
is clearly seen, but toward the center they become very
dense and indistinct (36,v1). The liquefaction is recog-
nized as a grayish reflex. Later an irregularly outlined

-

H
:

ote ee.

So a eee nea areu he Ube Ardy 9 ees ee)

ball, devoid of distinct locks, floats in the liquefied —

medium.

Gelatin Stab.—Along the stab there forms a thick —
white thread, from which, asa rule, only in the upper part —
(34, 11), more rarely throughout the entire length, long ©

(34, 1) or short (384, m1), bristly, distinct outgrowths —
extend outward. Sometimes the growth of hairs fails.

entirely (34, Iv). Also the direction of the lateral out-
growths varies; many times they are tangled together
(34, v). After twelve to twenty hours there begins a
slowly progressing liquefaction, with limited depression of
the surface of the gelatin. The liquefaction at first is
cup-shaped, later cylindric. The content of the funnel
is sometimes diffusely cloudy with white crumby floceuli;
at other times the flocculi settle down, leaving a clear
liquid gelatin above. No pellicle is ever formed.

Agar Plate.—(a) Natural size. Superficial colonies:
Small, white, with a play of yellow, moistly shining, a

Powe. es ~~

Sl a

i BACILLUS ANTHRACIS. 309

i

- little elevated, roundish. Deep: Punctiform and perma-
- | nently small (35, 11). The structure is the same as given

_ for the agar streak.

_ (b) Magnified fifty times: Deep and superficial colonies
_| present great differences. The former are usually whet-
_ stone-shaped, roundish, greenish-gray, becoming yellowish
_ toward the center. The peripheral zone consists of coarse,

dark-colored, crumbly masses, which are continued as

shorter or longer outgrowths, composed of little hairs,
crumbs, and granules. If the colonies lie near the surface,
_ outgrowths are formed at the periphery which resemble
hairs or locks of hair (35, 1, 1). The same completely
surround the surface colonies (35, 1, e). The colony then
gives the impression of a ball of wool or tangled hair of a
_ yellowish-gray color.
. (c) Magnified 150 times. Superficial colony: The curly
_ hairs appear as exceedingly long threads, which at the
periphery lie singly, and toward the interior in parallel
collections. These are regularly arranged like locks of
hair (often intertwined like a whip-cord) (85, ur). Deep
colonies: The outgrowths of the deeply located colonies
present coarsely granular, very irregular clumps, which
are usually connected by means of nodular branches and
fine processes. The colony does not present any center
proper, but is very irregularly torn and exceedingly poly-
morphous.

Agar Stab.—From the stab canal, which remains
white, there extend outward longer or shorter hairs, which
become shorter as they are lower in the stab, and which
terminate at times in curls or small clumps (34, v1). The
surface growth is roundish, regularly spreading, with a
smooth border, a little elevated, with a fatty luster, and
gray, bluish, or yellowish-white in color. After a longer
time there is often observed a formation of concentric
zones (34, Ix), or also, in other cases, of distinct, radiating
folds passing outward from the center (34, vir).

Agar Streak.—The growth remains limited to the in-
oculation streak; smooth edge, usually wavy. The color
is grayish-white, somewhat trahsparent at the edge. The
entire growth impresses one as if there were innumerable
tiny, silvery air-bubbles lying beneath the surface. The

Fees »

310 IMPORTANT VARIETIES OF FISSION-FUNGL.

water of condensation is clear or slightly turbid, with a —

§ -

little cloudy sediment (34, vr).

Bouillon Culture.—Homogeneous precipitate; bouil-
lon clear, with most delicate, floating clouds. No pellicle
is formed.

Milk Culture.—See page 307.

Potato Culture.—Rather dull, grayish-white or white,
moderately elevated growth, limited to the inoculation

streak. The border is wavy, sometimes notched. The —

growth stands out distinctly from the potato only when

the latter is somewhat discolored. The appearance as of ©

‘* silvery vesicles ”’

streak (35, viz).

Conditions of Spore-formation.—At temperatures
above 12° there are formed in cultures which have the
necessary supply of oxygen, oval, highly refracting spores.
The higher the temperature (optimum 37°), the more
rapidly the sporulation occurs; at the optimum, sporula-
tion may be completed in eighteen to twenty hours.
Giinther gives the optimum at 28°; at higher tempera-
tures the sporulation is not so regular. Weil obtained the
most resisting spores at 37°.

Regarding the morphology of spore-formation, see page
26, etc. Plate 36, v1, shows the picture of fine bodies
(spore antecedents) at regular intervals, occurring after
four to eight hours at incubator temperature; Plate 36, 11,
represents mature unstained, and Plate 36, Iv, mature
stained spores. ?

Regarding the germination of spores, see page 27.

Spores are never formed in the living animal nor in un-
opened cadavers (poverty of oxygen); on the contrary,
they are formed upon anthrax meat after cutting it up, in
bloody dejecta, etc. Weil has also observed anaerobic

is also observed here, as in the agar

1 Chauveau and Phisalix (Comp. rend., 1895, 801) have described a
Forma claviformis, which sporulates like the Bact. tetani. Since we
are here dealing with an absolutely non-virulent form, cultivated only
in fluids, and not studied as to its morphologic properties upon solid
nutrient media, it appears to us that the possibility of a substitution
through a contamination is not’excluded, even though previous treat-
ment with this organism prolongs life a little in animals after the
introduction of virulent anthrax. The observation deserves much
attention.

—
.

er

ee Calta i rns

eee ee

BACILLUS ANTHRA CIS. 311

sporulation and a germination of spores without oxygen
upon pieces of potato, quince juice, etc. (A. H. xxxv, 355).
Upon fresh nutrient media spores germinate in a few

- Cultures which are not transferred for a long time often
lose spontaneously the ability to form spores. The bacil-
‘lus may be deprived of its ability to form spores by culti-
‘yation upon nutrient media containing ‘carbolic acid, or,
with more difficulty, upon media to which are added
__bichromate or hydrochloric acid. Different cultures vary
‘much as to the ease with which they become asporogen-
ous. All agencies which reduce the virulence also operate
unfavorably upon the sporogenous function, yet these
_ properties are not necessarily associated ; there are viru-
‘lent asporogenous and absolutely non-virulent sporo-
- genous varieties. Phisalix found in long cultivation at
42°, with frequent reinoculation, that the ability to form
spores at 42° was gradually lost, but later the bacilli
were also unable to produce spores at 30°. While at
_ first the sporogenous function was recovered by inocu-
lation of a mouse, after 14 reinoculations at 42° the
‘ sporogenous function was finally completely lost. The
remaining virulence, still present at that time, after the
5 twentieth generation at 42° was also lost (C. B. xm, 533).

i Viability and Resistant Properties of the Bacilli without Spores
_ (Compare Momont, A. P., 1892, 21).—(a) In cultures the B. anthracis
_ maintains itself (through spore-formation ! ) for many months.

In water: In an inhabited aquarium Hober found it dead in three
_ to four days.

__Insoil: Moist anthrax blood is rendered free of germs in twelve to
_ fourteen hours by sunlight.

(b) Drying : According to Koch, they remain alive, when dry, for

_ five weeks at most ; also in large dried pieces of meat they die ina
‘few weeks. Bacilli in dried blood endure 92° for one and one-half
_ hours, are killed by light in vacuum in eleven hours, and with admis-
sion of oxygen in nine hours.
: (e) Salting does not kill anthrax bacilli in ham in fourteen days,
__ but does in six weeks (Peuch).
: (3 ) Moist heat at 60° kills rapidly.
4 e) Cold: With an outside temperature of from —1° to —24°
__ (average of —10.4°) bacilli in agar cultures were dead in great part
in twelve days and almost completely in twenty-four days. The few
which remained alive produced cultures with lessened power of pro-
ducing disease and of liquefying gelatin.

lef) ls

—S
aie

312 IMPORTANT VARIETIES OF FISSION-FUNGI.

Vitality and Resistant Properties of the Spores.—Stored in adry
condition, the duration of life appears unlimited. Spores remained —
alive in different samples of water and earth (with different conditions _
as to moisture), in putrid spleen, and in sewer contents for one and —

one-quarter to two and three-quarter years (Sirena and Scagliosi,
B. xvul, 318).

Regarding the varying resistance to heat, see page 52; to chemicals, ~
see page 53; to light, see page 53. Mormont found the resistance to
light very great: spores in water die in sunlight after forty-four hours; _
when dry, they endure sunlight well for one hundred hours with ~
admission of air, and for one hundred and ten hours with exclusion
of air. The most resistant spores were obtained by Weil at 37°.

Chemical Activities.—Only those mentioned in the
introductory remarks (p. 307) are known, The acids
formed are acetic and caproic. There is slight formation —
of H,S, and none of indol. Specific toxins could not be
obtained from cultures by most writers. Compare the
most recent, entirely negative, critical and experimental
study of Conradi (Z. H. xxx, 286).

Distribution.— 4

(a) Outside the body: So far found only, and always in
the form of spores, in places or on objects which were
contaminated with anthrax blood, etc.; for example, barn
floors where anthrax cadavers had been skinned (G._
Frank), skins, wool, and hair of anthrax animals, brushes —
prepared from the same, etc. They have not been demon-—
strated in the water and soil of anthrax meadows. . 4

(6) In man connected with disease: As the cause of
cutaneous anthrax (malignant pustule), inhalation anthrax
(rag-pickers’ and wool-sorters’ disease, in the majority of
the cases), and intestinal anthrax. In the first form the
bacilli are only at the affected place and in the lymphatics
leading therefrom; in the other forms they are also found
in the blood.

(c) In animals: Common disease in cattle and sheep,
rare in horses (very rare in swine), which graze in anthrax
meadows. The infection takes place with preponderating
frequency through the intestines by means of spores.

Regarding the findings upon section see page 515.

Experimental Observations Regarding Pathogenic ~
Effects.—Especially susceptible are guinea-pigs and rab-
bits; somewhat less, sheep and cattle; much less, horses.
Rats, especially dark-colored ones, are often quite highly

on. ——

—— "" anieet

aod A

vst

BACILLUS ANTHRACIS. 313

| immune; white ones always succumb, at least to a manifold
infection. Swine, dogs, hens, and pigeons enjoy a very
considerable, and the adult animals not infrequently a
_ complete, immunity. (Regarding the variation of the
‘same, see p. 96.) Frogs are killed, when kept warm, by
ordinary anthrax; or, without warming, by anthrax
adapted to cold temperatures (Dieudonné) (p. 45). For
| the susceptible animals, every possible method of intro-
_ duction of anthrax bacilli and spores is successful. Feed-
i ing spore-free bacilli is especially uncertain (gastric juice
: kills); subcutaneous, intravenous, intraperitoneal, espe-
cially respiratory introductions of bacilli or spores are suc-
cessful. After subcutaneous inoculation the animals
show no symptoms for several hours. Frank and Lu-
_ barsch found that in the inoculation of guinea-pigs with
an anthrax culture, which killed in thirty-four hours
after subcutaneous infection of the animal, the bacilli
_ first appeared in the blood seventeen to twenty-two hours
after the infection. The section of the infected animal
usually presents the picture of a septicemia. Besides
hemorrhagic edema of the subcutaneous tissue (especially
in the region of the point of inoculation), effusion into
_ the body cavities, and splenic tumor, there are no espe-
_ cially characteristic changes. The bacilli are found in the
blood, local edema, and in all the organs, especially in the
spleen, but in variable numbers.
_ Variations in the virulence of the B. anthracis have
_ been especially carefully studied. The virulence in ordi-
: nary cultivation is not very easily nor very much reduced,
: yet it is very easily attenuated by heat, chemicals, etc.,
:

{alee eg

_ until no virulence remains. (See p. 94.) Tavel once
_ observed an anthrax bacillus (originating from smoked
ham) which killed mice only after many—as much as
_ thirty-two—days, and yet a man was killed from eating
this ham.
___ By inoculation of cows and sheep with cultures of little
_ virulence there is obtained a slight, and by subsequent
inoculation with more virulent cultures a "pronounced,
_ immunity (similar experiments fail in mice and guinea-
_ pigs, but sometimes succeed in rabbits), which, indeed,
_ does not protect against the deleterious effect of feeding
J

314 IMPORTANT VARIETIES OF FISSION-FUNGI.

large quantities of virulent spores (Koch), but has proved
very successful practically in anthrax regions (Pasteur).

oe
os Wel. oa yi +e oe YF

Hankin has tried to produce an immunity in animals —
with the metabolic products of anthrax without positive —

results; the serum from sheep in which a very high active ©

immunity has been produced possesses immunizing action
only for sheep and not for rabbits (Sobernheim). The
bactericidal power of the serum in vitro is not increased
over the serum from normal animals. ‘The serum does
not cause agglutination. Sobernheim -considers the in-
jection of a mixture of immune sheep serum (16 c.c.)
and attenuated bacilli (=, loopful) to be the most certain

method of conferring a more lasting vaccination protec-—

tion (Sobernheim, Z. H. xxv, 301; and xxx, 89).
Emmerich and Low have observed very noticeable cura-
tive and immunizing results in rabbits from pyocyanase

(p. 110).
Special Methods for Demonstration and Differen-

tial Diagnosis.—If the question, as is usual, is one of

diagnosis in an infected man or animal, very often a good
preparation of smeared blood stained by Gram’s method
gives valuable results. For the differential diagnosis ordi-
nary agar plates are especially to be prepared, which, after
seventeen to twenty-four hours in the incubator at 37°,

present spores within threads. Also observations as to
motility are useful, as also are sugar-agar shake cultures.

The Differential Diagnosis between the Varieties which most
Come in Question may be Represented as Follows :

Symptom-| Malig- Bact.
Anthrax. _ atic amar vulgare — Strep-
Anthrax, | Edema. |(Proteus).| °° tocaes.
Motility .. 0 + oP Se 3% 0
Gram’s stain | Very good. | Often good.| Usually Good. 0 Good.
negative.
Growth . . .| Aerobic. | Anaerobic. |! Anaerobic. Facultative anaerobic.
ee
Formation of
locks .. Good. 0 0 0 0 0
Formation of
threads .. Good. 0 Sometimes. + + 0
Fermentation
of sugar. . 0 + + + + Oand +
Spores ... aa a + 0 0 0

lis.

a so

Pre

ray

—

BACILLUS ANTHRACIS. 8315

j The result is always obtained with absolute certainty in
thirty-six hours.
For the differential diagnosis between symptomatic
ithrax and malignant edema, see page 314.
It may be difficult to differentiate an anthrax bacillus

: from soil from the closely related spore-forming varieties.

Tf a virulent form is in question, then the inoculation

"with a sample of earth into several guinea-pigs will often
decide the question. The dead animals will be examined
-as described above. Thus it is possible that one animal

dies of anthrax, others of malignant edema, tetanus, etc.,

_ the causes of which were all present as spores in the
sample of soil. Non-virulent forms of anthrax, isolated
F from soil, are only recognizable by comparison with known

anthrax, whereby the five species of the table (p. 314) are
“to be excluded.

The following are described as closely related to anthrax:

_ B. pseudanthracis Burri. According to Hartleb and

_ Stutzer, it is widely distributed in American meat powder.

_ The cultures isolated from different samples were not ex-
actly identical. The cultures are motile, especially when

grown in bouillon. Also it is important that in bouillon

_ there is first diffuse turbidity, then a clearing up, with the

_ formation of a precipitate and pellicle. All other charac-

teristics are deceptively like those of anthrax. Its viru-

_ lence for mice and guinea-pigs is slight. Compare C. B.

_ L. 1m, 81, where also is a description of cultures of B.

pseudanthracis II and III, which are still a little further
removed from anthrax.
B. anthracoides Htippe and Wood (from soil), which

_ we obtained from Kral and carefully studied. We found,

indeed, the agar cultures macroscopically very much

_ like anthrax, but microscopically they resemble B. sub-

tilis; also upon gelatin the similarity to Bac. subtilis

when magnified 60 times was much greater than to Bac.
anthracis. In young colonies loop-like projections extend
out, reminding one of Bact. vulgare. When magnified

1000 times, sluggish motion was unmistakable.

| B. anthraci similis McFarland (C. B. xxtv, 556). It

_ was once found upon a laboratory plate, and is entirely

non-pathogenic. Perhaps it was true anthrax.

b»
.

316 IMPORTANT VARIETIES OF FISSION-FUNGLI.

‘ ’
1 el

Bacillus mycoides. Fliigge.
(Plates 37 and 38, I-Iv.)

2 fpr wire
a

Synonym.—Root bacillus.

Microscopic Appearance.—Rather large rods, scarcel
at all rounded at the ends, 1.6—3.6 » long and 0.8 » thick.
Sometimes arranged in threads (38, m1). Forms oy
spores.

Motility.—We have seen no cultures which exhibit
active motility. Usually all the individuals are quiet ex
cept a few, which are only detected in motion after more
prolonged observation. Also in the stained preparatio
the impression is given of only a few being provided with
flagella. Formerly we believed we had observed entirely
non-motile cultures, but can not now demonstrate this.
Here belongs the non-motile Bacillus radicosus Zimm. »

Staining Properties.—Stains by Gram’s method.

Requirements as to Nutrient Media.—Slight; also.
grows without oxygen but scantily.

Gelatin Plate.—(a) Natural size: In earliest stage the
colonies consist of a scarcely perceptible circle of little
hairs (37, v1). After one or two days the gelatin is lique- ~
fied a little, while the colonies have become distinctly”
larger. The hairy circle ramifies more and more, ang
thicker branches are formed, especially at the center,
which, toward the periphery, are replaced by irregular, .
fine, root-like branches (37, Ix).

(b) Magnified fifty times: Colorless, more or less windingy
threads, interlacing in a most extraordinary manner. Tn :
the center the colony is sometimes felted and opaque, —
The branchings are only apparent, since always two closely |
lying threads diverge from each other at the point of |
apparent branching.

Gelatin Stab.—It is characterized by delicate little.
hairs of quite uniform length growing outward, parallel? —
to each other, along the stab canal (37, 1). The liquefac- |
tion of the gelatin begins in the form of a saucer and then

1In more advanced stages the hairs are often directed upward (37, —
11). The zone of liquefaction is usually clear or slightly cloudy.

F | BACILLUS MYCOIDES. 317

Bornes cylindric. Upon the surface of the liquefied

medium there is a thick white film, reminding one of a
overing of asbestos. If this falls to the bottom of the

liquid, a new one at once forms, so that cultures may be
ound having many such films.

| ae en Plate.—(a) Natural size: At first the colonies

very like those in the gelatin plate, but more sturdy
| (87, vu). The further growth is absolutely irregular,
there being found, as well, colonies with a dense center

‘and very distinct main branches, and also those with a
delicate central portion, and about this a growth in the

form of a circle (87, vui1).

(6) Magnified fifty times: Exactly like the colonies in
Bthe gelatin plate. Plate 38, 1, represents a colony with an
“open central part. Plate 38, Iv, represents a part of the
same magnified 150 times.

_ Agar Stab.—Stab: Parallel brush-shaped outgrowths,

“usually of unequal length, delicate gray, but a little
denser than in the gelatin stab (37, Iv). Surface growth:

_ Exactly like the colonies on the agar plate; light gray,
- moist, shining (37, v).

Agar Streak.—Grayish-white, moist, shining growth,

with root-like outgrowths, showing most extraordinary
abundant branchings. In a short time it covers the entire
surface (37, m1).
Potato Culture. —Extremely like the potato culture of
the Bac. subtilis. It is white, when older yellowish, a
little elevated, crummy, dull, provided with a delicate,
‘insignificant fringe at the periphery (38, Ir).

Chemical Activities.—See page 307. There is also no
formation of H,S.

Distribution. —Very common in soil.

i

5

. a ae al

Bacillus subtilis. F. Cohn. (Beitrage, Bd. i,
H. ii, 175.)
(Plates 39 and 40.)
Common Name.—Hay bacillus.

Microscopic Appearance.—Short (1.3-3 »), rather
_ thick (0.8-1.2 »), sturdy rods with rounded ends, often

318 IMPORTANT VARIETIES OF FISSION-FUNGI.

united in long strings, and not infrequently the separatior
into individual rods is indistinct, so that long threads
occur (40, v).

Spores.— When air is admitted, oval spores are readily
formed, which germinate at right angles to the long diam-
eter. (Compare p. 27.)

Motility.—The short forms are very actively motil
because of long, abundant, peritrichous flagella. The
chains of bacilli still show flagella, even when they are n¢
longer motile (40, vi, rx).

Staining Properties. —Stain by Grok s method.

Requirements as to Nutrient Media and Oxygen
—Grows upon the most various nutrient media at room
_and incubator temperatures, but when oxygen is ex
cluded it grows poorly and without sporulation. Growth
is rapid. :

Gelatin Plate.—(a) Natural size: In a short time the”
colonies sink into a saucer-shaped depression. The con-
tent of the liquefied zone is grayish-white. At the center
lies the whitish, ragged colony (40, m1). A later stage is”
shown in 40, Iv. |

(b) Magnified sixty times: At first the colonies are round- —
ish, even-edged,crumbly, yellowish, sometimes surrounded
by a delicate row of hairs (40, 1, 1). Later, especially in:
the case of the superficially located colonies, ‘the periphery
becomes wavy, and with advancing liquefaction of the
gelatin breaks up into innumerable tangled locks, which
surround the colony. The central part is still held firmly
together, being granular and yellowish to brownish until
after four to five days, when it also becomes completely
disintegrated (40, 1, e).

Gelatin Stab.—The surface growth is whitish-gray, ©
and, after thirty-six hours, sinks into the gelatin in the
form of a saucer. The gray liquefied content of the
saucer contains whitish bunches in suspension (39, 1).
The liquefaction progresses in a cylindric form, the con-
tents being grayish-white, and cloudy, especially below. —
Upon the surface is a thick white scum, firmly attached
to the wall of the tube (89, 11).

Agar Plate.—(a) Natural size: Small, irregular, shining, ~
grayish-white colonies (39, vuir). |
;

BACILLUS SUBTILIS. — 319

0) Magnified sixty times. Superficial: Decidedly ir-
regularly shaped colonies, rarely smooth-edged, usually
extraordinarily ragged and fringed. The peripheral part
consists of irregularly winding and interlacing threads,
which at times may be rolled together in an impenetrable
tangle. The center of the colony is yellowish, and finely
ranular. Deep: Similar to the superficial, but denser,
thicker, and more opaque. The branches are still more
oad and gnarled (39, vir).
Agar Stab.— Surface growth: Moistly glistening,
: roundish, even-bordered, soon extending to the wall of
the tube, a little elevated, dirty gray. Sometimes there
is formed a pellicle or radiating wrinkles (39, v).
( Bevom pare also 34, vi.) Stab: Thread-like or granular.
Agar Streak. —The growth upon the surface is like
‘that upon the agar stab. The water of condensation is
turbid, with a gray, cloudy precipitate (39, 111).
- Bouillon Culture.—Uniformly cloudy. There is
_ pellicle formation upon the wall of the glass, and some-
_ times also upon the surface of the bouillon. There is a
little whitish precipitate.
_ Potato Culture.—Dirty white to yellowish growth,
with an undulatory, scalloped border, somewhat elevated,
dull, never shining, spreading fairly widely, and upon
long standing it has a meal-dust appearance (40, I).
_ Chemical Activities.—See remarks upon page 307.
Charrin and de Nittis have obtained a pathogenic Bac.
“subtilis by cultivation upon nutrient~media containing
blood and by passage through animals. They always
_ employed 0.5-0.75 c.c. of the most pathogenic culture to
q produce death in guinea-pigs. The affection remained
- local and the picture as a whole was more that of an
ee cation. (Compt. rend. de la Soc. de Biol., 1897,
718.)
___ Distribution.—In hay, and widely distributed in soil.
_ Besides this, also other forms with spores are present in
hay, so that various kinds may be obtained by the method
: formerly recommended for acquiring the hay bacillus
- Gnoculation of a sterile nutrient solution with a small
_ quantity of fluid containing spores obtained by boiling
nay for a long time).

320 IMPORTANT VARIETIES OF FISSION-FUNGI.

It is not known to be of any practical importance. It |

cannot be transformed into anthrax, as was held for a long
time.
Closely Related Varieties.—Bacillus leptosporus

L. Klein, Bac. sessilis L. Klein (C. B. vi, 377), and—
Bacillus malarize Klebs (see Schiavuzzi in Cohn’s—
Beitriigen zur Biol. der Pflanzen, v, p. 245), which cer-_
tainly has nothing to do with malaria. Compare Golgi ©

(C. B. v, 516). The following are also very closely related:

Bacillus tenuis (Duclaux.) L. and N.

Tyrothrix tenuis Duclaux. Microscopically and upon gelatin —

plates, gelatin stab, agar streak, milk, bouillon, etc., it cannot be dif-

ferentiated from the Bacillus subtilis. There is no trace of gas-formation —

from dextrose. On the contrary, the growth upon potato resembles

that of Bac. vulgatus. The growth is pale red, much elevated, with —

a sinuous boundary, and traversed by voluminous rolls. The variety
stands midway between Bac. subtilis and vulgatus. It is worthy of

4|

:

§
3
7
‘

7
i
¢
=

notice that it does not stain by Gram’s method. We have not found ©

a form which ferments sugar.

Most closely related, if not identical, is the Bac.
implexus Zimmermann, which Zimmermann described
as non-motile, and which we ourselves always found non-
motile in frequently repeated examinations made for our

i a

ee

first edition (1895). The same cultures now exhibit —

lively motility, which is absolutely unmistakable. Con-
tamination is surely excluded. Compare Zierler (A. H.

xxxiv, 192) and Lehmann (J. c., 198). This observa-

tion is of the greatest interest.

Bacillus bernensis. L. and N.

Ordinary Name.—<Aroma-producing bacillus from Emmenthaler

cheese. Burri (C. B. L. m1, 609). In the same place is also found —

further literature regarding organisms with the smell of cheese.
Thick rods (1.5 4), facultative anaerobe. Spores are formed if
oxygen is admitted, which are twice as long as thick. Motion slug-
gish, rarely active. Gelatin plate cultures differ in appearance from
those of the hay bacillus type; gelatin stab and agar cultures are
somewhat like those of the hay bacillus. Potato cultures are moist
and smooth, lusterless, and without wrinkles. Bouillon becomes
cloudy, with pellicle formation. Milk is coagulated in about twenty-

four hours, the coagulum being later dissolved. After about forty-

BACILLUS MEGATHERIUM. 321

ht hours there is a strong, genuine odor of Emmenthaler cheese;
also this occurs upon sterilized casein, precipitated by rennet. There
never any gas formed from sugar.

acillus megatherium. (De Bary.) Vorles. iiber Bak.,
| ii. Aufl., 1887.

(Plate 41.)

= Microscopic Appearance.—Rods 1.6-5 u long, 0.6-
/0.8 4 thick, the ends not rounded, often united in long
“strings (41, x). These dimensions are a certain demon-
_ stration that the organism becomes smaller after prolonged
“cultivation (forma depauperata); we obtained this culture
rom the hygienic institute in Berlin in 1888. De Bary
Tepresents the thickness at about 3 4. (Compare Bac. oxa-
laticus, p. 323.)
_ Motility.—By means of many peritrichous flagella it is
rather slowly motile (41, xr).
_ Staining properties and requirements as to nu-
_trient media, etc., are the same as in the Bac. subtilis.
Gelatin Plate —(a) Natural size: Like Bac. subtilis
(41, 1).
4 (b) Magnified fifty times. Deep: Grayish, transparent,
“more opaque toward the center, finely to coarsely granu-
lar, as if beset over the entire surface with most minute
airs (41, tv). If the colony lies upon the surface, the
riphery supports a row of rather long, very fine, deli-
cate hairs, while the middle zone is a little lighter. The
. at the middle remains compact (41, v). Itresembles
ac. subtilis and Bac. mesentericus.
_ Gelatin Stab.—Tube- or sack-shaped liquefaction takes
place along the stab. The liquid is turbid; sometimes,
especially later, with cloudy flocculi. Later the lique-
7a becomes cylindric (41, 1).

Agar Plate.—(a) Natural size: White to grayish-white,
a little elevated, moistly shining disks (41, v1).

(b) Magnified jifty times: In the earliest stages the deep
colonies possess hairy or corkscrew-shaped outgrowths
(41, vu, i), while the superficial ones possess a delicate,
“extremely transparent zone (41, vu, e). The latter in
time becomes opaque, coarsely crummy, yellowish-brown,

21

nd.

322 IMPORTANT VARIETIES OF FISSION-FUNGI.

usually provided with winding, anastomosing lines. The
deep colonies later appear irregular in form, with smooth
edges, opaque, usually with outgrowths at the periphery
(41, vimt).

Agar stab and streak like those of the Bac. subti
41, 11).
Bouillon. — Moderately cloudy, often with pellicl
formation.

Potato Culture.—Very similar to that of the Bac.
subtilis. Thecolor is usually somewhat more yellow; th
mealy appearance also occurs (41, Ix).

Chemical Activities.—See remarks on page 307; n
indol, much HS.

Distribution.—Found by De Bary upon decomposing
cabbage leaves. The Bac. quercifolius Lehm. an
Detjen, from sausage, described from this institute by
Detjen (dissertation) in 1890, appears to be identical. .

Remarks.—The separation of this variety from the
subtilis is rather difficult ; it lacks the marked pellicle
formation upon the gelatin culture and the pronownegay
formation of curls upon the gelatin plate.

The following, described by Htippe, is very closely re-—
lated:

s
%

Bacillus butyricus. Hiippe. (Mit. G. A. ii.)
(Plate 38, v—-vir a.)

Judging from our studies of a culture which we have cultivated for —
six years, it seems to stand midway between the Bac. megatherium —
and mesentericus. The thinness of the rods appears to be a result of
prolonged cultivation. Slender rods, 1.2—4 u long, only 0.3-0.5 « thick —
(!), with ends moderately rounded. They are motile, because of
numerous peritrichous flagella, and stain by Gram’s method. Upon
the gelatin plates there occur, as in the case of Bac. vulgatus, typhoid
forms ; still, they are usually much scalloped, and often have a crater-
shaped depression at the center (38, vI). Later the crummy center
enlarges at the expense of the outer transparent zone (38, VII) until
finally the whole colony breaks up.

Upon the gelatin stab culture a pellicle is likewise to be found, only
the Bac. butyr. liquefies a little more slowly. The agar plate culture
is exactly like that of the Bac. mesentericus, perhaps a little more
delicate ; also the agar streak and stab, except that the brown color is
absent. The potato culture, on the contrary, presents no meshwork,
and is not distinguishable from the megatherium (38, v). Bouillon

BACILLUS VULGATUS. 323

mains clear, a pellicle forming upon the surface. Milk is coagulated,

but sometimes remains fluid. No gas or indol, but some HS is

‘formed. According to Hiippe, it elaborates butyric acid from salts of

lac’ acid ; also from milk-sugar when it is previously hydrated by
er bacteria.

Bacillus gastrophilus. L. and N.

_ This name is applied to a sporulating, motile, aerobic bacillus,
Which coagulates milk with the formation of lactic acid. Recently it
ias been repeatedly cultivated by Kaufmann and Strauss from the
iman carcinomatous stomach. The organism grows poorly upon the
wdinary nutrient media, best upon beer-wort agar and gelatin, upon
which it forms fine threads (according to the description, resembling
ax). Old cultures appear as if covered with fine dust (air

Ns ‘Details, together with literature, are given by Sternberg (Wien.
i . Wochenschr., 1898, 744), who cultivated the organism from an
ted hernia, and ‘naturally disputes its diagnostic significance

4. Bacillus oxalaticus. Zopf.

This organism possesses a greater interest because Migula (A. K., I.
: _~&Ba. ., p. 139) conducted valuable studies upon bacterial structure upon
“it. The culture which we obtained from Kral consisted of rods,
_ which, from their relatively small diameter (0.8-1.6 ), differ much

_ from the thick forms (2.5-4 ) which Migula observed. This is ap-

"parently due to cultivation. Motility and flagella were always
3 absent. Upon gelatin plates the colonies at first resemble those of

Bact. coli ; later they become crummy and settle into the gelatin with
fs wide zone of liquefaction. With longer growth they become like
the subtilis in appearance. In the gelatin stab the liquefaction is
_ funnel-shaped, later cylindric, the contents are turbid, and a scum
_ forms on top. The agar streak culture is not distinguishable from that
_ of anthrax. Upon potato is formed a pure white, dry, later moistly
shining, elevated growth. Bouillon remains almost clear. For
_ chemical activities, see page 307 ; no H.S nor indol.

z.

‘= Bacillus vulgatus. (Fliigge.) Migula.
; (Plate 38, VIII, Ix ; and Plate 42.)

: Synonym.—Bacillus mesentericus vulgatus Fliigge.
Ordinary Name.—Potato bacillus.

_ _ Literature.—Vignal: Le bacille mesentericus vulgatus, Paris, 1889.
q Not accessible to us. ,

_ Microscopic Appearance. — Slender rods, ends
pwreely at all rounded, 1.6-5.0 # long, 0.8 # thick, often

324 IMPORTANT VARIETIES OF FISSION-FUNGI.

united in threads. It readily forms roundish to oval
spores (42, x1). 7

Motility.—It propels itself by means of numerous peri-
trichous flagella (42, x1).

Staining Properties.—Stains by Gram’ s method.

Requirements as to Nutrient Media, Oxygen, etc.
—The same as Bac. subtilis; grows rapidly.

Gelatin Plate.—(a) Natural size: After one or two
days the colonies sink into the gelatin, grayish-white, deli-
cate, wrinkled films being formed, which, even later, after
liquefaction of the entire plate, do not disintegrate (42,
VII).

(6) Magnified fifty times: In the early stage the colonies,
especially at the periphery, resemble the smallest typhoid
colonies until they begin to sink into the gelatin. (Com:
pare also 43, x1.) Soon this transparent zone changes
into a crummy mass, which is coarsely granular internally
and possesses lobular markings, while the peripheral por=
tion presents lobules separating from each other. Finally
the entire colony acquires the appearance of a distinctl
moruloid, brown, loosely attached heap, resembling a
panther skin (42, vur and 1x). Besides the forms just
described, there are often forms furnishing a transition to
those of the Bac. mesentericus.

Gelatin Stab.—Upon the surface, a grayish-white, rag-
ged-edged growth with a fatty luster. Gradually from this
there is developed a dense film, which sinks into the gelatin ~
in the form of acup. The liquefied zone is cloudy with
a dirty grayish-white precipitate (42, 1).

Agar Plate.—(a) Natural size. Surface growth: White
to whitish-gray, moistly shining, even-bordered or slightly
granular, a little elevated. Deep: Roundish to whetstone-—
shaped, white. Sometimes in older cultures there occur
folded or rounded elevations (42, Iv).

(b) Magnified fifty times. Superficial: Roundish, gray,
homogeneous colonies, without markings, becoming
opaque toward the center, transparent at the periphery, |
and beset by long, often tortuous, plaited hairs (42, v1). .
Deep: Roundish to whetstone-shaped, gray, homogeneous, —
opaque, sometimes also with a limited development of
hairs (42, v).

BACILLUS VULGATUS. 325

a
at

rd _ Agar Streak.—Luxuriant growth, with a wavy scal-

oped border, grayish-white, with a fatty luster, especially
after a longer time becoming covered with numerous,
irregular, considerably elevated folds. Toward the edge
it is more transparent. The water of condensation is clear,
' and upon the surface of the same a firm film is formed
(42, 11). This description answers for the agar stab
: & ll).
Bouillon.—A little cloudy, upon the surface a firm,
yish-white film, which is not broken up by shaking.
_ Milk Culture. —Slimy coagulum formed ; strong alka-
line reaction. Sometimes no coagulation occurs.
__ Potato Culture. —Exceedingly variable. The typical
form at any rate presents abundant tortuous and confused
: “more or less padded elevations, rising and falling precipi-
tously, not unlike intestinal coils (42, x). The color is
partly whitish-gray, partly yellowish, yellow, or even rosy
| =e The coils may also be widely padded (38, 1x) or
appear as thick, moistly glistening elevations (like Bact.

coli) (38, vm).

_ Chemical Activities.—See remarks on page 306. No
_indol is produced and little H,S.

_ Djistribution.—Common in soil, thus a frequent con-
_ tamination of our potato cultures (potato bacillus!). Also
_ found in the intestine and in sausage (Detjen, Serafini).
Practical importance is slight. In incompletely unster-
ilized milk it, like the related varieties, occasionally
_ produces a gradual coagulation with strong alkaline
- reaction, and later a solution of the coagulum with pro-
- duction of bitter-tasting, injurious substances.

The ability of the bacillus to sometimes produce abun-
dant quantities of a slimy carbohydrate, especially in feebly
_ acid bread, through swelling of its membrane, at times

becomes troublesome. J. Vogel (Z. H. xxvi, 398; there

also the literature), who has carried out in Hamburg a

special study upon the bacilli of viscid bread, found

especially two varieties of bacilli concerned with it: Bacil-
lus mesentericus panis viscosi II Vogel, which essen-
tially completely corresponds with the bacillus mesen-

tericus L. and N. (see below), and B. m. p. viscosi I,

which is distinguished by lack of motility, and by the

qi

326 IMPORTANT VARIETIES OF FISSION-FUNGI.

flat, primarily non-characteristic, smeary growth o 1. .
potato, which later forms large, parallel folds. The resist-
ing spores withstand a short baking process. p

The Bac. gummosus Ritsert (C. B. x1, 730), obtained from gela-_
tinous infusion of digitalis, seems to be related. See also Happ
(C. B. xv, 175). According to both authors, this organism is only
able to elaborate this slimy material from cane-sugar and not from
grape- or milk-sugar. Besides, there originate mannite, grape-sugar.
lactic acid, butyric acid, and carbonic acid. The carcinoma bacillus
of Scheurlen (C. B. 111, 397) has also been shown to belong to this
group, but has nothing to do with carcinoma. —

Bacillus geniculatus (Duclaux). L. and N.

Tyrothrix geniculata Duclaux. The gelatin plates macroscopically
resemble Bac. vulgatus. When magnified 60 times, they present an
interesting exhibition. The colonies at first appear upon the gelatin
with delicate scalloping, like typhoid; with advancing liquefaction
. the scallops are replaced by curls, which in irregularity may compare
with those of anthrax; still later, the circle of locks falls away and —
the compact colony floats in the shallow liquefied area, surrounded by —
irregular disintegrating masses. Also in the growth upon potato and —
in other properties it resembles the Bac. vulgatus. We have —
nothing of the branches in gelatin as described by Winkler.

Bacilius mesentericus. (Fliigge.) Lehm. and .
Neum.

(Plate 43.)

Synonym.— Bacillus mesentericus fuscus Fligge.
(Fliigge, 3d Edit., p. 199.) j
Microscopic Appearance.—Slender rods with rounded —
ends, 0.8-2.4 » long, 0.7-0.9 thick, » having a tendency |

to form roundish spores.

Motility, staining properties, and conditions of life ©
are like those in the case of the Bac. vulgatus. :

Gelatin Plate.—

(a) Natural size: Minute, roundish,. grayish-white col-_
onies, which very soon sink into the gelatin. The zone of
liquefaction is flat, gray, cloudy. The colonies resemble ©
very much those of subtilis (48, x).

(b) Magnified fifty times. Superficial: In the earliest stages —
they resemble those of typhoid, as do those of the Bac. —
vulgatus (43, x1). (See also 16, vu.) With the onset —

Ie

BACILLUS MESENTERICUS. 327

of liquefaction the transparent peripheral zone becomes
delicately granular, at the periphery appears a row of
fine hairs, and the entire colony assumes the character of
a liquefying subtilis colony. The center is usually gray-
‘ish-brown and opaque (438, 1x). Deep: Grayish-yellow,
‘irregular; about the edge are curling, hairy outgrowths.
_ Gelatin Stab.—The colony after twelve to twenty-four
hours sinks into a saucer-shaped depression. The liquefac-
tion first assumes a funnel form and later progresses in a
eylindric manner. The contents of the funnel are cloudy,
_with a whitish-gray film upon the surface (48, 1).
Agar Plate |

_ (a) Natural size: Roundish, gray, thin, veil-like
_ growths, transparent, with the original whiter colony in
the center (43, v).
(6) Magnified fifty times: The original colony lying be-
neath ‘the superficial colony appears yellowish-brown,
moderately or very crummy, with an even border or with
curling outgrowth. When the colony reaches the surface,
it forms a delicately punctated, transparent, irregular
growth of a gray to yellowish color (43, v1).

Agar Streak.—Wavy, moistly shining, yellowish-
brown, in many places gray and transparent. Water of
condensation cloudy, with yellowish precipitate and a
pellicle upon the surface (48, 11).

Bouillon.—Moderately cloudy, pellicle on the surface.

Potato.—At first the growth is moderately elevated,
grayish-yellow, moistly shining, slimy (438, m1). Later it
is transformed into a meshwork of irregularly anasto-
mosing wrinkles, which are much elevated, of a yellow-
ish-gray color, and possess a dull luster (48, Iv).

Chemical Activities.—See preliminary remarks on
page 306. It forms little indol and abundant H.S.

Distribution, Practical Importance, Etc.—Like Bac.
vulgatus.

Bacillus mesentericus ruber. Globig (Z. H. iii, 322).

Slender bacilli, 1 to 3.2 4 long, 0.4 u thick, sometimes forming quite
‘long threads. Not motile, stain by Gram’s method, no spores. The
gelatin plate exhibits quite variable forms. At first all the colonies
present an appearance like typhoid; later some colonies retain the

328 IMPORTANT VARIETIES OF FISSION-FUNGI.

same, others form thick, moist, white growths, still others liquefy and
form pellicles, and yet others resemble subtilis colonies. Upon the

gelatin stab is formed a growth like typhoid, which still, after a
longer time, slowly sinks in, with the form of a funnel. Potato cul-

tures at first are like Bact. coli; later the cultures acquire a rose-color,
which finally is transformed into reddish-brown. Agar stab culture

is delicate, whitish-gray, transparent, moistly shining; later a netted

film forms upon the surface. Bouillon becomes faintly cloudy, with
a thin pellicle upon the surface. Milk is not coagulated, reaction
feebly alkaline. H,S and gas are not produced. -

Bacillus aterrimus. (Biel.) Lehm. and Neum.

A very striking, aerobic, motile, sporulating bacillus, possessing

all the peculiarities given on page 306, and producing black pig-
ment. The gelatin plate cultures appear to resemble Bac. subtilis and —
vulgatus. Gelatin stab cultures present funnel-shaped liquefaction ©

without coloration. Upon potatoes at first grayish-blue, then brown-

ish-black, wrinkled, moist pellicles are formed, the potato being black

throughout. Agar cultures become brown with yellowish-brown films.

The organism is not pathogenic. Compare Biel (C. B. L. 11, 137) and

Lunt (/. c., 572) regarding B. mesentericus niger. Gorini’s closel
related Bac. lactis niger (C. B. xx, 94) we obtained from 1
in 1895 and studied. It no more showed any chromogenesis, and
grew as a flat deposit upon potato, resembling the Bact. coli. Spon-
taneous motion could not be seen. It is questionable whether the
insufficiently described Bac. melanosporus Eidam of Schréter belongs
here.

Bacillus liodermos. (Fliigge.) Lehm. and Neum.
Bacillus mesentericus liodermos Fltigge.

This bacillus, described by Fliigge as short and very actively motile,
we have not certainly encountered during recent years. The gelatin
growths in plates and in the stab are like Bac. vulgatus. The potato
culture forms a smooth, shining, yellowish-white, syrupy growth,
which only after several days becomes a little wrinkled and cloudy.
The Bacillus mucosus Zimmermann (I, p. 8), from slimy water, ap-
pears to have some relationship.

Bacillus gangrenz pulpz. Arkovy.

Synonyms.—Bacillus fuscans Miller? Caries fungus of
Galippe and Vignal, Caries fungus of Jung.

Literature.—Arkoévy (C. B. xxii, 917).

According to the investigations of Dr. Zierler, made in
the Wiirzburg hygienic institute, which deviate somewhat

BACILLUS GANGRENA PULP. 329

“from those of Arkévy in some points,! the following are
_ the characteristics (compare Zierler, C. B. xxv1, 417):
. Microscopically.—Stout rods, 4 » long, about 0.8-1.0 Ut
“thick, often united in chains. Actively motile. Stain-
ing of flagella has not been successful. Upon all nutrient
__ media large, oval spores soon form, which are difficult to
r stain. Germination of the spores is equatorial, often
' oblique to the axis of the spore (Hirai). Grows luxu-
_ riantly on all nutrient media, best at 37°. Gelatin plate:
— Similar to subtilis, rapid liquefaction. Gelatin stab: After
- two or three days saucer-shaped liquefaction, which becomes
~ cylindric. Upon the surface a very tough, wrinkled
~ film, which usually remains connected by a fine string
‘ with the stab canal in the solid gelatin. The liquefied
_ mass gradually becomes colored smoky brown. After two
_ to four days fine, horizontal branches are seen throughout
_ the entire length of the stab canal. Anaerobic gelatin stab:
Without liquefaction and pellicle formation, the surface
gradually becomes depressed. The branches in the stab
_ are longer and more delicate. Agar plates: Thick plates
exhibit superficial colonies something like subtilis (389,
vi and vir); thin plates present dense, scalloped, concen-
trically striped, white growths, which, when magnified fifty
times, appear opaque, and from the rather sharp border
bunches of threads project outward. Deep colonies are
compact and often whetstone-shaped. Agar stab: The
growth rapidly spreads over the surface of the agar and is
whitish, dense, with a dull luster; soon it becomes a little
rough from the formation of small depressions and eleva-
_ tions. Potato: On first day, a delicate, membranous,
moist, spreading growth; after three to four days the same
_ becomes finely wrinkled and dirty red, and the neighbor-
hood of the growth is stained dirty violet. Bouillon is
very cloudy, with a dense, wrinkled surface pellicle. Ina
few days, with the formation of a rather crummy precipi-
tate and brownish discoloration, the culture becomes clear.
Neither H,S nor indol is formed. Milk is coagulated after
two or three days. In grape-sugar bouillon there is

1 Nothing was seen of the pleomorphism of Arkévy, and the “‘ cocci
forms’’ represented by Arkévy are often spores (C. B. xx11II; Plate
Xvi, 10

330 IMPORTANT VARIETIES OF FISSION-FUNGI.

abundant formation of gas (principally CO,) and moderate _
formation of acid. |

Arkévy always found the fungus in gangrenous teeth,
and frequently in saliva and in gangrenous wounds —
(decubitus). :

Zierler, like Arkévy, never failed to find the organism
in gangrene of the pulp (24 cases examined), those in ©
which the characteristic gangrenous odor was present.
Not infrequently pure cultures were obtained, and, what
appears especially remarkable, scarcely ever a culture of
any other sporulating bacillus.

Zierler can not bring forward direct proof of the signi-
ficance of the organism as the cause of gangrene. Arkévy
has inoculated sound, broached human teeth with the
bacillus and thus produced gangrene.

Further Sporulating Aerobic Varieties.

Here are included the thermophilic varieties which were —
spoken of from the biologic side in the general part. (See
p. 44.) For the characteristics of the individual varieties
we must refer to the original literature there cited, since
they have only been partly named and are without great
practical interest. They appear to be concerned in the
spontaneous heating of hay, manure, etc., also in the
hitherto puzzling bubbling fermentation. Laxa has
described an organism which belongs here (C. B. L. tv,
362), and Poupé (C. B. L. tv, 484) another thermophilic,
jelly-forming organism.

— Te

eo

Introduction to the Special Description 1 of the Bac.
tetani, Bac. Chauveei, and Bac. edematis maligni.

The three varieties have in common:

1. In pure cultures upon the usual nutrient media
(agar, gelatin, potato) they are more or less perfectly
anaerobic; on the contrary, they also grow very well

1 In the following, free use is made of v. Hibler’s critical ‘‘ Beitrage
zur Kenntnis der durch anaérobe Spaltpilze erzeugten Infektionser-
krankungen des Menschen, ete.’ Preliminary communication (C. B.
abs) 1899, 513, ete.). The detailed work has not yet appeared (June,
1899).

— = T ——s Ts ee

ANAEROBIC VARIETIES. 331

zt

aerobically upon boiled rabbit’s blood, which before the
‘inoculation is again briefly heated to 100° (especially true
of Bae. tetani). The latter then forms spores excellently
_ and its virulence increases (v. Hibler). It is also possible
_ to grow anaerobes upon nutrient media containing sulphid
_ of sodium, as stated on page 42. Regarding the behavior
of anaerobes in aerobic mixed cultures, see page 43, and
_ Kedrowski (Z. H. xx, 358).

_ 2. Gelatin is liquefied, and (similarly to Bact. vulgare)
fatty acids—from formic to caproic—and, besides, acids
_ with aromatic groups (phenylpropionic acid, hydropara-
_ cumaric acid, and skatol-acetic acid), are produced
_ (Nencki).

3. Also, without the sugar being present (!), according to

_ Nencki, there arise from albumin: carbonic acid, hydro-

- gen, H,S, mercaptan, marsh-gas, perhaps free nitrogen

_ (Bovet, C. B. vit, 174). The gases have a very foul

odor. Phosphoretted hydrogen, which smells like garlic
and darkens silver nitrate paper, but not lead paper, in
the latter differing from H,S, has been found by Marp-
mann.

4, With the presence of sugar there originates a mixture
of gases, with less of a putrid but with a sweet, repulsive
odor, and consisting mostly of H,S and CO,,

, Spontaneous motion produced by peritrichous fla-
gella.

6. Spores are partly in the middle and partly polar.
Attenuated as well as virulent cultures, when grown upon
saccharine nutrient media, usually form spores only in the
middle or incompletely in the pole, and of an oval form,
which may be much elongated. Upon blood and blood-
serum in all three varieties the spores are polar and round.
In general, sugar and glycerin make the nutrient medium
less favorable for spore-formation, and often it very soon
fails to occur. This influence is more marked in symptom-
atic anthrax and malignant edema than in tetanus.

7. Regarding their resistance to injurious influences,
consult the statements of Sanfelice. According to him,
they were not nearly so resistant as the aerobic soil spores,
being killed by 100° in live steam in fifteen minutes at
most, sometimes also by 80°-90° rather quickly. It re-

332 IMPORTANT VARIETIES OF FISSION-FUNGL

mains an open question whether the spores employed in
these investigations were of maximum resistance. Spores

stored dry in earth remain alive for months, and even —

years; also, if the spores are placed in water together
with soil, they live for months.

8. Upon rice nutrient media (rice covered over with a
solution of 1% peptone and 0.5% NaCl) the virulence of
all the varieties studied was soon lost (v. Hibler).

9. Most effective are intramuscular inoculations; less,
subcutaneous; and least, intraperitoneal. The effect is
more pronounced with extensive injury of tissue than
without.

10. Attenuated cultures of the varieties producing local

affections cause less edema and more cellular accumulation

than virulent ones. The less the virulence, the greater the — :

phagocytosis.

Bacillus tetani. (Nicolaier.) (Deutsch. med.
Wochenschr., 1884, 842.)
(Plate 44.)

Literature.—Kitasato (Z. H. v11, 225; x, 267), Kitt (C. B. v1, 297),
Knorr (Tetanusgift, Miinch. med. Wochensehr., 1898, 321 and 362).

Microscopic Appearance.—In AS rods, 1.2-3.6 —
» long, 0.5-0.8 » thick. In cultures (especially of little —

virulence) there are often very long threads,+ sometimes
also rods arranged in strings (44, 1x). Mature spores
are at the ends of the short rods, oval to round, 1.5-2.0 »
long and about 1.5 » thick (44, vi). Many times a
piece of a thread rests upon the end with the spore like a
scepter. Sometimes also the long threads sporulate (44,
x). Then in many places are seen short rods arising from
the thread containing very distinctly polar spores; in other
places one spore lies close to another, so that the entire
substance of the rod appears to be converted into spores.
Similar pictures appear to have been seen by Vincenzi
(C. B. xtv, 149).

Spontaneous motion is observed in anaerobic hanging
drops, dependent upon numerous, long, peritrichous fla-
gella; according to Votteler (Z. H. xxvu, 480), 50 to 100

1 Vincent and Kanthack claim to have observed branching.

BACILLUS TETANI. 333

a bacillus, which we have verified. According to
hwarz, there is only a single terminal flagellum!

_ Staining Properties.—Stains well by Gram’s method.

Requirements as Regards Oxygen.—When freshly

cultivated from the animal body (from wounds from which

tetanus has originated, from nails, etc., which have caused
tetanus), it is always an absolute anaerobe. After long

cultivation in the stab (deep culture) the organism often

is

adually becomes less susceptible to oxygen. Cultivation

_ is facilitated by the presence of certain saprophytes, which
_ grow when oxygen is admitted. Recently Carbone and

_ Perrero (C. B. xv, 193) have succeeded in cultivating

i virulent tetanus bacilli from the bronchial and tracheal

‘

_ mucus in a case of rheumatic tetanus, where absolutely no

injury was to be found anywhere. The bacilli thrived

- much better and more luxuriantly aerobically, but in pure
_ culture were no longer virulent. In the same place are also
_ found references to the literature regarding earlier obser-
_ vations of aerobic tetanus cultures (Belfanti). Kamen

eee er

OS eae Sq, os

(C. B. xvii, 513) and Ferran (C. B. xxtv, 28) made sim-
ilar observations.

Intensity of Growth aud Relations to Tempera-
ture.—Grows moderately rapidly, best at 36°-38°, and
at 14° growth no longer occurs.

According to vy. Hibler, the growths upon artificial nu-
trient media are the more luxuriant and sturdy, and
the liquefaction of gelatin the more vigorous, the less
pathogenic the organism is. Strongly-pathogenic cultures
often produce very slight growths. The findings of Tiz-
zoni and Cattani have been similar.

Gelatin Plate.—(a) Natural size: At first minute, white,
punctiform colonies, which, upon sinking into the medium,
become surrounded with a transparent gray zone of lique-
faction (44, Iv; see also 45, vr).

(6) Magnified sixty times: Usually the colonies have a
yellowish-brown, very crumbly center, from which there
extends first a rim of short hairs; later innumerable,
intertwining, interlacing, corkscrew-shaped threads. The
older the colony, the more developed, longer, and more
irregular these outgrowths become, and often they break
up in a crumbly manner (44, m1).

334 IMPORTANT VARIETIES OF FISSION-FUNGI.

Gelatin Stab.—Deep in the gelatin along the stab
there occurs first a cloudy growth, then cyst- or tube-
shaped excavations, which are filled with cloudy, granular
liquid (44, 11). |

Agar Plate.—(a) Natural size: Colonies whitish, _
roundish to ragged, usually surrounded by an exceedingly —
delicate veil (44, v).

(6) Magnified sixty times: The original colony appears
grayish-yellow, roundish, opaque, surrounded by a broad
zone made up of the finest interlacing hairs. Toward the
periphery it becomes transparent; toward the center,
grayish-yellow and opaque (44, vi).

Agar Stab.—In the stab prepared by a simple thrust
of the platinum loop there develops, deep in the agar, a —
scaly, ribbon-shaped growth (compare 45, m1). If one ~
rotates the loop in the agar, then the growth extends in a ~
wider zone and consists of a cone of cloudy layers (44,1), —
the surface of which, after a very long time, becomes
covered with points and fine serrations (45, m1).

Agar Streak.—No confluent growth, but only single —
discrete colonies (Votteler).

Blood-serum.—Sometimes liquefied, sometimes not.

Bouillon (anaerobic) moderately clouded.

Milk.—No (according to v. Hibler, very slow) coagu-
lation; reaction amphoteric.

Non-albuminous Nutrient Media. —Upon Uschin-
sky’s solution no distinct growth.

Resistant properties of the bacillus are without
practical interest, as it sporulates very readily.

Resistance of the Spores.—See page 331, and Tizzoni
and Cattani (C. B. 1x, 487).

Chemical Activities.—See page 331. The forms
studied by us form gas from sugar actively; a production
of acid could not be demonstrated (on account of simul-
taneous abundant formation of alkali). Brain nutrient
medium was darkened (v. Hibler). Extremely vigorous
production of H,S, little indol. Attenuated (slightly
virulent) forms, according to Tizzoni and Cattani, often
form more acid and grow more luxuriantly (C. B. x1, 150);
in general, the virulence is well preserved. Upon nutrient
media without sugar, we saw no gas production. The

F BACILLUS TETANTI. 339

chemical activities of malignant edema and symptomatic

anthrax are more vigorous. Regarding toxins, see page
74. The similarity of tetanus and strychnia poisoning,
‘according to G. Brunner, is only superficial (C. B. xxrv,
629); on the contrary, Lusini even claimed that tetanus

antitoxin had a favorable influence over strychnia poison-
ing (C. B. xxv, 325).

Distribution.—
(a) Outside the body: Wide-spread in garden soil, hay,

snd dust. Very often tetanus results in animals from
the inoculation with samples of soil and dirt-floors from

_ dwellings (Heinzelmann).

(6) In healthy body: In feces of horses and cattle, more

‘rarely of man.

*

(c) In man in cases of disease: Cause of traumatic tris-
mus and tetanus, puerperal tetanus, and tetanus neona-
torum through wound infection. The organism is found
only in the wound secretion, and usually in very small
numbers; never in the blood and internal organs. ‘‘ Rheu-
matic tetanus’’ appears (see above) to be due to tracheal
infection with aerobic forms of tetanus.

(d) In animals: Tetanus often occurs spontaneously in

horses; more rarely in sheep, goats, and other domestic

~ animals.

Experimental Observations Regarding Patho-
genic Effects.—

(a) In animals: The following are especially suscepti-
ble: Horses, guinea-pigs, goats, mice: Much less so:
Rabbits, sheep. Dogs, rats (v. Hibler saw rats usually

_ die), pigeons, and hens are almost immune, although the
_ toxin remains quite long in the body of the hen. More
details as to the immunity of the hen can be found given
_ by Asakawa (C. B. xxtv, 166).

About twelve hours after subcutaneous infection at the
root of the tail with virulent material, the mouse (simi-
larly guinea-pigs and rabbits), which is the most com-

monly employed, shows the first symptoms of tetanus in

a rigidity of the groups of muscles near the point of
infection (tail, hind leg), and it goes about after the
fashion of seals—i. ¢., with the extended hind leg dragging
upon the ground, Mild infection may cause unilateral.

336 IMPORTANT VARIETIES OF FISSION-FUNGLI.

tetanus and terminate in recovery. General exaltation of —
reflexes may be absent. In man and horses after subeu-_
taneous infection the first symptoms are not local, but
consist in a stiffness of special muscles—in man, the
muscles of mastication; in horses, the muscles of masti- —
cation, membrana nictans, and those elevating the tail.
Pure cultures cause no suppuration at the point of inocu-.
lation; the organisms remain limited to the site of inocu-
lation and do not spread throughout the body.
According to Vaillard and Rouget (A. P. vu, 755),
tetanus spores which are well washed, or freed from toxins
by long heating to 80°, are harmless: trauma, metabolic
products, admixture with other bacteria, protection of the
spores by coverings, are necessary, in order that tetanus
shall be produced. Other writers, as Roncali (C. B. xy,
439), dispute this; also, according ‘to Donitz, spores which
were heated to 65° for one hour in 10% solution of chlorid f
sodium retained their virulence (Deut. med. Wochenschr., —
1897, No. 27, 428). The introduction of sterile tetanus
toxin may kill animals with the symptoms of tetanus. A
high degree of active immunity against tetanus may be
produced by careful, repeated injections of sterile toxins, |
beginning with small doses and increasing gradually. By
means of the serum thus obtained, which is rich in anti-
toxin, other animals may readily be passively immunized, ©
and even small infected animals may be cured, but horses
only with difficulty. A female mouse immunized against —
tetanus transmits a high degree of immunity to her offspring —
(for two to three months), but the immunized male does —
not. The milk of an animal immunized against tetanus —
preserves or produces immunity in the sucking young of
herself or others.
(6) In man: Experimental infections of man with
tetanus are lacking. Curative results from injection of
tetanus antitoxin in cases of tetanus have often been
claimed. For the older literature, see Remesoff and
Fedoroff (C. B. xv, 115); for the latest condition of the
question, see, for example, Erdheim (Wien. klin. Woch- —
enschr., 1898, No. 19, 463), who, out of 22 new cases, re-
ports 11 failures. (Cc. B. xxIv, 634. ) Also in horses there —
are reported some favorable results and some failures,

—

BACILLUS BOTULINUS. 337

Special Demonstration and Culture Methods.—
he demonstration of the tetanus bacillus in the scanty
sretion of wounds, usually cemented over, in tetanus
patients may be difficult. In the first place, the wound
‘secretion, which is scraped out, is examined in microsco-
pic preparations for polar spores, whose demonstration
“under these circumstances may be looked upon as fairly

certain proof of tetanus. In the second place, and it is
never to be omitted, one inoculates a little of the secretion,
but especially fragments and splinters of any foreign
bodies found in the wound, into mice (p. 335), and
' finally an effort is made to cultivate the tetanus bacillus
‘by means of anaerobic sugar-agar plates. Kitasato has
recommended a preliminary heating for half an hour at 80°
‘to get rid of spore-free, disturbing organisms; yet the viru-
ence of tetanus spores is easily injured thereby. Heating
te 60°—65° for ten minutes is sufficient to kill all spore-

4 Related Varieties.—Tavel (C. B. xxi, 538) has
_ described as Bacillus pseudotetanus Tavel a bacillus
_ very similar to the tetanus bacillus, which is sluggishly
nd wi presenting only 8-16 flagella (tetanus, 50-100!),
_ and which is found in the human intestine. It is a strict
anaerobe and is not pathogenic for animals. Tavel is in-
_ clined, with Roux, to ascribe to the organism a significance
in the causation of appendicitis and peritonitis.
_ Zimmermann (1, 50) describes his Bac. gracilis Zim.
as a non-motile, facultative anaerobic bacillus with polar
“spores, which grows upon plates like the Bac. tetani.

Bacillus botulinus. van Ermengem. (Z. H. xxvi,1.)

Further Literature.—Brieger and Kempner (C. B. xx1r, 765), Mari-
- nesco, oa ge and Pollack (C. B. xxIv, 899), Kempner and Schepi-
* lewsky (Z. H. XXvil, 213).

_ Vigorous rods, 4-9 » long, 0.9-1.2 » thick, sluggishly

_ motile because of 4 to 9 flagella. Does not stain by Gram’s

_ method. Spores are usually polar. Sporulation is not

_ interfered with by the presence of sugar in the nutrient

~ medium.

In sugar-gelatin plates the colonies, according to y. Er-
22

=
=
=
.
=
=
~-
hal
-
7
é
4

338 IMPORTANT VARIETIES OF FISSION-FUNGI.

mengem, are at first characterized by a smooth edge, which
later develops a row of prickles, and by being composed
of rather coarse, refractive granules, which are in constant
motion. Later the border becomes much notched and
irregular. Gelatin is liquefied. Stab cultures are not
characteristic. In grape-sugar gelatin the growth is more
luxuriant and accompanied by more vigorous formation
of gas and liquefaction of gelatin; in ordinary gelatin it ig
not characteristic. We cannot see any essential difference,
upon plates, between malignant edema, symptomatic an-
thrax, tetanus, and Bac. botulinus.

The most important biologic properties are: while
grape-sugar is fermented very intensely, with accompany-
ing gas-formation, milk and cane-sugar are scarcely
affected. Milk is not coagulated. A marked foul odor
never occurs even upon nutrient media containing no
sugar, but only a sour, rancid odor. Sugar-bouillon be-~
comes uniformly turbid, with a strong odor of butyric
acid. ¥

Optimum temperature, below 35°; obligate anaerobe.
Growth is checked absolutely by more than6% of chlorid
of sodium. The spores are killed in half an hour by a
temperature of 80°.

Pathogenic Properties.—The organism, when intro-
duced by mouth or subcutaneously, produces the picture
of botulism, without multiplying in the body. Filtered
and devitalized cultures operate in the same way, since
toxins are formed in the cultures. Brieger and Kempner
have isolated the toxin, and have also obtained an anti-
toxin from the serum of animals which were poisoned for a
long time. Especially susceptible to the poisoning when
exhibited by mouth are guinea-pigs and mice; less, rabbits;
still less, rats and pigeons; and least, cats, dogs, hens. Cats
are also very susceptible to the subcutaneous introduc-
tion, but no local symptoms develop. The organism is the
cause of certain cases of meat poisoning in which the enteric —
symptoms give place to nervous ones—dilatation of pupil, —
disturbances of accommodation, aphonia, paresis in the —
region of the tongue and pharynx (paralysis of swallow-
ing), more rarely paresis of the extremities, and finally of
the muscles of respiration. Together with this are present

BACILLUS CHAUVGI. 339

alterations of the salivary, bronchial, and pharyngeal mu-
sous secretions (usually increased production), croupy
_ eough, and interference with the evacuation of urine, bile,
'-and feces. Consciousness is preserved. There is no fever.
Tn a limited number of cases of poisoning by meat in Elle-
zelles in Belgium, the organism was cultivated from spores
_ which were present in exceedingly poisonous ham, and also
' from the spleen of aman who had died after eating the
ham. It appears to be rare, and could not be found by
van Ermengem in the environs of men.

Bacillus Chauveei. Aut. gallic.
(Plate 45.)

— Synonyms.—Bacillus sarcemphysematis Kitt, Rausch-
brand bacillus, Bacille du charbon symptomatique, Bacil-
lus of symptomatic anthrax, Bac. anthracis sympto-
matici Kruse, Acetone or Forbicione of Italians.

_ __ Literatwre.—Kitasato (Z. H. vi, 105; vu, 55); Ellenberger and
_ Hofmeister, Path. der Haustiere (11, p. 458); Kitt (C. B. 1, 684, 716,
_ 71) (C. B. 111, 572, connected review ).

A detailed description of the cultures is unnecessary.
_ As is shown in Plate 45, any sharp differentiation between
_ this bacillus and the Bact. tetani by means of cultures is
not possible, unless one makes much of the somewhat
_ greater luxuriance of the cultures of symptomatic anthrax,
_ as v. Hibler has shown. According to Votteler, the form
_ of the outgrowths in the anaerobic culture upon slanted
_ agar is more in round and arborescent lobules, not in root-
like outgrowths, as in malignant edema.

‘The bacilli themselves exhibit active motion, dependent
_ upon peritrichous flagella (according to Votteler, 20 to 40,
with which our results correspond), show a tendency to
_ spindle forms upon nutrient media containing sugar, stain
_ well by Gram’s1 method, and often possess spores which
_ are a little nearer one end of the bacilli. Mature spores
__ are typically polar upon serum (see p. 331). Besides, the
bacilli contain easily staining, clear granules.

According to Kitasato, spore-formation occurs in the

1 According to Giinther, they do not stain by Gram’s method.

340 IMPORTANT VARIETIES OF FISSION-FUNGI.

animal only after death. The viability of the organisms _
with spores in the dried flesh of symptomatic anthrax
animals is very great. The chemical activities of the
organism have been fairly completely investigated, most
of the communications upon this being referred to upon —
page 331. Our cultures coagulate milk; v. Hibler found ©
gradual coagulation. As the cause ‘of symptomatic i
anthrax (‘‘ Rauschbrand’’) (a destructive disease of ©
cattle, localized in certain pastures, and formerly confused —
with anthrax) it is found in the bloody edema and mus-
cles, the intestinal contents, and, what is of diagnostic
value, always in the bile? of the affected animals. Cattle
usually die in one and one-half to three days, with the
development of a large crepitating swelling of the skin,
regional swelling of the lymph-glands, high fever, and
sopor. At the section there is found within the swell-—
ing a bloody, gelatinous edema, beset with gas bub-
bles, together with slight hemorrhagic exudate into the
serous cavities, and peritonitis. The spleen is normal.
The infection enters through injuries of the skin or .

+

mucous membranes. Of experimental animals, espe-
cially cows of one to three years (calves under six ©
months less), goats, and sheep,? and most especially —
guinea-pigs and mice (rats somewhat less), are sus- —
ceptible. Man is immune, also swine; dogs, cats, and —
rabbits are rarely killed by it. Im horses and kindred
animals the inoculation produces only a local reaction. —
Protective infections with attenuated cultures (or dried,
pulverized symptomatic anthrax flesh heated to 100° for
several hours) have proved very valuable. Immunization —
against symptomatic anthrax, according to Roux, protects
also against malignant edema; Kitasato found the oppo- —
site.

The so-called puerperal symptomatic anthrax, according
to Carl, is produced by the Bac. ced. maligni (C. B. xrx,
489). Schneidemitihl considers the Bacillus botulinus or

1Tt is always necessary to examine not only the subcutaneous
tissue, but also the bile.

2 The Norwegian sheep disease, Bradsot, is said to be symptomatic
anthrax (H. P. vii, 559).

BACILLUS G@DEMATIS MALIGNI. 841

- closely related organism to be the causative agent (C. B.
_ xxiv, 577).
?

Bacillus cedematis maligni. Koch.
(Plate 46.)

_ Synonyms.—Vibrion septique of the French. Bacillus
of malignant edema.
Literature.—Koch (Mitt. a. d. Gesundheitsamt I, 53); Kitasato (Z.

‘ HL vi, 111); Jensen and Sand (Deut. Zeit. f. Tiermed., XIII); Penzo
¢. B. X, 822) ; Horne (C. B. X1x, 77); Besson (A. P. IX, 179).

symptomatic anthrax, but with a ereater tendency (of

diagnostic value!), especially i in the cadaver, to grow into
_ long threads. Active motion is due to rather numerous
_ (20 to 40) peritrichous flagella, but it is only present in the
short forms, the long threads being usually scarcely at all
‘motile. In the short rods spores form at the middle some-
times, at the end sometimes, and they are oval or spheri-
eal. Our cultures were not stained by Gram’s method, and
most authors have obtained the same result. In cultures
we found the Bac. ceedematis maligni to be indistinguish-
able from the bacillus of symptomatic anthrax, as shown in
Plate 46; we have not placed more illustrations in this
plate because they would be only repetitions of those of
symptomatic anthrax. We found only that the growth was
_ somewhat more scanty than that of symptomatic anthrax,
_ and the vitality of the cultures of shorter duration. Of
_ importance is the universally demonstrated inability to
_ sour milk (to break up milk-sugar); the milk is coagulated,
with amphoteric reaction. Abundant formation of alkali
_ by the organism reveals itself by darkening of the brain
nutrient media.” The chemical products have been much
studied, and are essentially all enumerated on page 331.
_ Also, in addition, in media containing grape-sugar there
occur ethyl alcohol and inactive lactic acid (Kerry). A
mixed culture of the Micr. acidi paralactici Nencki and
the Bac. ced. maligni produces butyl alcohol abundantly,
5 4 neither variety alone can do it (Nencki, C. B. x1,
‘ ).

| Microscopically. — Vigorous rods, like tetanus and

ore

AH:

PES ie arr ay atien

ee Ss we

342 IMPORTANT VARIETIES OF FISSION-FUNGI.

Distribution.—Very widely distributed in soil, con-
taminated water, hay dust, etc. The inoculation of ani-
mals (best guinea-pigs) with samples of soil very readil
produces malignant edema (still more often than tetanus).
According to Horne, the most various septic diseases o
domestic animals are occasionally produced by this bacil-
lus. The section reveals, especially at the point of infee-
tion, a marked, bloody, gelatinous, often wide-spreading
edema, with enlargement of the spleen.

Animal inoculations are made in practice by the subcu-
taneous injection of anaerobic bouillon cultures, most con-
veniently with not too small quantities of the edematous
fluid from dead animals, or by the introduction of the
infectious material into a deep cutaneous pocket. Spores
without toxins infect with difficulty or not at all. The
administration of toxins or the negatively chemotactic
lactic acid increases the danger of infection very much
(Besson, A. P. rx, 179). Also, according to Penzo, as
tetanus, the toxins formed in vitro are of the oreatest i im-—
portance in the outcome of the animal experiment; small
doses of the pure culture he found to be without effect.
Very small quantities of very virulent cultures suffice. Of ©
the experimental animals, guinea-pigs and mice, and, in
distinction to symptomatic anthrax, also rabbits, are very —
susceptible, and, besides, cattle, sheep, goats, horses, and —
pigeons.

The symptoms of the experimental disease in guinea-
pigs correspond very beautifully with those in the sponta- —
neous disease. Also in the case of animals dying from —
other causes, especially in warm places, bacilli may be |

|

found in the blood (having wandered from the intestinal
canal) which are identical with or very similar to those of —
malignant edema; therefore care must be taken in the ©
consideration of cadavers which are not fresh!

In the blood of recently dead animals the bacilli are —
not usually found microscopically (but may usually be —
demonstrated by cultures without difficulty), and very —
soon after death they spread everywhere, especially in the —
form of long threads. In the mouse, which is especially _
susceptible, there is also marked multiplication in the
blood.

BACILLUS GDEMATIS MALIGNI. 343

: Infection occurs especially readily if the wound is con-
sed or, as is very often the case in natural infection,
other bacteria, hardly injurious of themselves, are simul-
iz néously inoculated: for example, Bact. vulgare or Bact.

# See the key, on page 306, for the differential diagnosis
_ between Bac. cedematis maligni and Bac. Chauveei. For
completing the diagnosis the following are to be carried
out:

1. Examination of a fresh preparation for motility.

2. Two smear preparations are made from the edema-
tous fluid or muscle juice and stained with fuchsin and by
_ Gram’s method.

3. Experiments upon guinea-pigs, and the examination
} of the phlegmon as to gaseous contents, and the bile for

bacilli contained in it.
@ 4. Experiments upon rabbits, which often give negative
_ results with the Bac. Chauveei.

eae RR m

& Related Varieties (Pseudo-edema Bacilli).

Bacilli have been described by numerous writers which
also kill experimental animals, with the production of
bloody and emphysematous edema, but which do not

_ correspond exactly with the bacilli of malignant edema
_ orsymptomatic anthrax. Recently v. Hibler has devoted
_ aspecial study to these varieties and found representatives
_ of most of the forms described which deviate somewhat
in individual properties.

All these forms differ from malignant edema in the
absence of thread-formation, growing as short rods, like
symptomatic anthrax, and in pairs, and no more distin-
guishable from one another than some of the water
vibriones. The following will give some idea:

Aside from the absence of thread-formation, the follow-
ing correspond very well with malignant edema: the
-pseudo-edema bacillus of Liborius (Z. H. 1, 163)
(according to Liborius, it has two spores in a single cell)
and the Bacillus emphysematis maligni of Wicklein
(Virchow’s Archiv, cxxv, 75), while Kerry’s new patho-

344 IMPORTANT VARIETIES OF FISSION-FUNGLI.

genic anaerobic bacillus (Oest. Z. f. Veter., v, H. 2 and
3) differs in the rapid fermentation of milk and absence —
of a dark color in brain nutrient media. Klein’s new —
bacillus of malignant edema (C. B. x, 186), and also —
Sanfelice’s Bacillus pseudocedematis maligni (Ann, —
Inst. d’Igiene di Roma, 1, 375), do not liquefy gelatin ~
nor coagulate milk, and darken brain nutrient media —
intensely. Compare also what is said under Bac. sporo- —
genes Klein. These findings naturally make the diagnosis

of symptomatic anthrax much more difficult. |

Bacillus phlegmonis emphysematose, E. Frankel.1

Synonym.—Bacillus capsulatus aérogenes Welch?

Literature.—E. Frankel (C. B. x11, 13; and monograph, Hamburg,
1893); P. Ernst ( Virchow’s Archiv, CX XXIII, 308); Welch and Nuttall
(Johns Hopkins Hosp. Bulletin, July and Aug., 1892; Journal of
Experimental Medicine, 1, 5, 45); Dunham (Johns Hopkins Hosp.
Bul., April, 1897).

We are very much in doubt whether the producers of
‘“oas phlegmons,’’ ‘‘foaming liver,’’? ‘‘development of
gas in the blood and internal organs,’’ described under these
names, are always the same organism, and in what way
they are related to other anaerobes. At any rate, con-
sidering the variability of bacteria, their identity is possi-
ble. All the organisms appear to have the following
properties in common: they are plump bacilli, which are
occasionally arranged in long pseudothreads, which present
no spontaneous motion, are stained well by Gram’s
method, and very rarely (best upon blood-serum) form
spores, which are observed either at the pole or middle of
the bacilli. Welch and Nuttall usually found a capsule,
the German writers say nothing about it. Grape-sugar is
very rapidly fermented.

In guinea-pigs emphysematous phlegmon is produced
(E. Frankel, Ernst), in which the tissues are often destroyed
like tinder. The effect upon mice is variously given.

1The name of Welch is a little older, but, in the first place, it is
not formed upon the binomial plan, and, in the second place, it is
liable to cause confusion, since Bacillus capsulatus has been used
repeatedly and there is a bacillus (or bacterium) generally known as
aerogenes.

be

w elch and Nuttall found, at least in their first cases, no
thogenic properties for animals, but observed marked
ention of gas in an animal which was killed soon after
the intravenous injection of 0.5 to 1 ¢.c. of the culture.
The organism has been rather infrequently isolated
from men with abscesses containing gas, etc. Here also
belongs the Bac. cadaveris butyricus Buday (C. B.
XxIv, 369).
Occasionally there also occur gaseous phlegmons and
similar diseases of internal organs, in which are found the
' Bact. coli alone or usually in combination with other
varieties, but without any anaerobes being present. See
Bunge (Fort. der Med., 1894, xm, 533).

BACILLUS ALVEI. 345

wide

Bacillus alvei. Chesire and Cheyne (Jour. Royal
Microsc, Soc., 1885).

Synonyms : Bacillus of the foul-brood of bees (French, ‘‘ Loque’’).
Microscopically: Straight, fairly sturdy rods (0.8 u thick and 2.5 to 5
long), sluggishly motile, large spores in spindle-shaped bulgings. The
Bepores show polar germination. Our culture did not sporulate. Gelatin
Colonies first round, then they become provided with peculiar
‘sturdy outgrowths, resembling wisps of straw or tendrils and very
tortuous. Similar appearances occur in the stab culture. Gelatin is
liquefied. In the stab there are often only single liquefying colonies,
which are surrounded by radially arranged liquefying outgrowths;
often the picture looks like an ink blot surrounded by fine outshoots.
_ Upon potato a yellowish, upon agar a white, growth. Milk is first
slowly coagulated; later the coagulum is dissolved and the reaction is
- faintly acid. The organism is only a facultative anaerobe ; our cul-
_ ture from Kral grows also aerobically. Aerobic potato cultures are
delicate. It produces neither indol nor H,S, and no gas upon nu-
_trient media containing sugar.
It is the cause of the characteristic disease of bees.

We are not familiar with the actively motile Bacillus piscidicus
_agilis N. Sieber, which was described as a facultative, anaerobic,
short bacillus with spores and which exhibited marked pathogenic
effect upon fish (C. B. xvii, 888). O. Wyss held it to be the Bact.
vulgare, which it certainly approaches very closely. N. Sieber, in his
reply to Wyss, makes no mention of spores. Z. H. xxvii, 143, and
XXVIII, 159.

The Anaerobic Producers of Butyric Acid.

While medical men have studied the anaerobic varieties
from the pathogenic standpoint, and only secondarily in-

346 IMPORTANT VARIETIES OF FISSION-FUNGI.

vestigated the zymogenic activities of the varieties isolated ‘
by them for purposes of differential diagnosis, whereby —
typical production of butyric acid was demonstrated in —
a number of varieties, also the ferment technicians have —
studied the anaerobic varieties as to their ability to form —
butyric acid, and have considered their pathogenic func- —
tions only secondarily or not at all.

We have not been able to study this group more closely,
and cannot refer to the extensive literature in the absence ~
of any comprehensive reviews,! but look forward to the ~
exhaustive publication by v. Hibler. A review was sup- i
plied by Baier in 1895 (C. B. L. 1, 17).

The following was found in Fliigge’s laboratory to be a —
vigorous anaerobic producer of butyric acid. ;

Bacillus butyricus. Botkin. (Z. H. xi, 421.)

Microscopically: Rods 1 to 3 » long, 0.5 u thick, often in chains in —
fluid nutrient media. They are motile and stain by Gram’s method. —
The spores are in the middle or at the end, about 1 » thick and are ©
formed only upon non-saccharine media. Obligate anaerobe. Opti- —
mum temperature is 37°. Upon sugar-agar the growth appears like
that of the other described anaerobes. Upon sugar-gelatin the col- —
onies possess a slightly undulating border, as if consisting of a mass —
of felted threads, without the formation of branches. Gelatin is
rapidly liquefied. After fifteen hours the casein of milk is precipi-
tated, and butyric acid is formed, with violent liberation of gas; later
the coagulum is soon dissolved. Upon nutrient media containing
starch the bacillus presents inclosures staining with iodin. Starch is
converted into sugar, and this directly into butyric acid. Also butyric
acid is produced from milk-sugar without an intermediate formation
of lactic acid. The organism is widely distributed and supposed to be
non-pathogenic. The following may perhaps appear as a pathogenic
form of this organism.

Bacillus sporogenes, (Klein.) L. and N.

Bacillus enteritidis sporogenes Klein (C. B. xvmt, 737;
xxu, 113, 577; xxv, 278).

This rather strictly anaerobic organism stands very
close to the Bacillus butyricus Botkin (see above), espe-
cially as regards the appearance, staining properties

1 Regarding Clostridium butyricum Prazmowski, see also vy. Hibler,
i. c. Regarding Clostridium butyricum M. Gruber I and II, see C. B.
I, 367.

BACILLUS SPOROGENES. 347

(Gram’s, blue with iodin), motility, and spore-formation.
| The flagella are located at the ends especially in little
| _ bunches; the motion is sluggish. Also, in the edema no

long threads are formed. The growths possess no mor-
_phologic value. According to v. Hibler, gelatin is slowly
liquefied. Brain nutrient media is very slowly darkened.
_ Milk is very rapidly decomposed, with vigorous formation
of butyric acid and gas. According to Klein, no spores
-are formed in milk in fourteen days, but they readily
develop upon serum. Spores withstand 100° for one hour.
_ Subcutaneous injection kills guinea-pigs in eighteen to

_ forty-eight hours, with the development of a wide-spread-
- ing, foul-smelling edema, containing abundant bacilli, and
gas. Sometimes there is injection of the intestine and
peritonitis. The spleen is not enlarged, and usually con-
_ tains only a few bacilli and no threads.
= In man the taking of milk which contains these bacilli

in abundance causes severe gastro-intestinal disturbances
- (enteritis). So far, such occurrences have been observed
only in England.

According to Klein, the bacillus is widely distributed in
milk, intestinal contents of children and in cases of diar-
rhea, in street dirt, sewage, horse manure, etc.

Diagnosis.—The differential diagnosis from sympto-
matic anthrax is decidedly more difficult than from ma-
lignant edema. Attention must be paid to the presence
of bacteria in the bile in symptomatic anthrax.

4 Other writers have described other producers of butyric
_ acid. We give here, according to Klecki (C. B. L. 1, 286),
- acomparison of three of them with the Bacillus butyricus
Botkin. The literature is found in the same place.

1 According to Klein, if spores are repeatedly transferred from
sugar-gelatin to sugar-gelatin, the organism loses the ability to form
gas abundantly in milk; the milk remains alkaline and has a foul
odor. The bacillus forms long, sporulating threads instead of the
short members of the typical form; the virulence is almost or entirely
lost (C. B. x x11, 581). If there has been no mistake here, and that
seems improbable, then these observations are of very great significance,
since they show how characteristics upon which two species were for-
merly based occur as two forms of one culture.

348

IMPORTANT VARIETIES OF FISSION-FUNGI.

| Bacillus ortho-

Bacillus sacciael

Bacillus amylo- Bacillus butyricus. . =
PERDRIX (1991), | BOTKEN (1892). Gurmeen (1508). von eee (1895),
0.5 w thick ; 2-3 | 0.5 «thick; 1-3 4 | 1.5 thick; 3-6
# long. long; in fluids} y long.
may be even 10
# long.
Gelatin unfavor- Round or _ oval
able nutrient} colonies with i
medium; not| slightly wavy borders and
liquefied. borders, which granular —
appear as if structure.
composed of a Gelatin not.
mass of matted liquefied. —
threads. Gela- Very slight
tin _ liquefied. effect upon the —
Casein is dis- casein
solved.
Motile. Slightly but dis-| Motile. Sluggishly mo-
tinctly motile. tile.
Anaerobe. Anaerobe. Anaerobe. Anaerobe.
Forms spores. | Sporesinthemid-|2-3 spores|Polar, oval
dle, butattimes| formed in one} spores.
in the ends.| rod.
Spores oval.

It causes fer-
mentation of
glucose, cane-
sugar, and
milk-sugar,
and from them
forms butyric
acid, acetic
acid, CO,, and
H,. It fer-
ments starch,
thus produc-
ing butyric
acid, amyl al-
cohol, some
ethyl alcohol,

It causes fermen-
tation of milk-
and grape-sugar
and starch, thus
producing buty-
ric acid, propi-
onic acid, acetic
acid, formic
acid, lactic acid,
succinic acid,
butyl alcohol,
some ethyl] alco-
hol,CO,,and H,.

It ferments gly-
cerin, mMan-
nite, glucose,
invert - sugar,
cane-sugar,
maltose, milk-
sugar, galac-
tose, arabin-
ose, starch,
dextrin, inul-
in, thus pro-
ducing butyl
alcohol, some
isobutyl alco-
hol, normal
butyric acid,
acetic acid,
formic acid,
CO,, and Hg.

It ferments ~
milk-sugar,
producing ©
butyric acid, —
formic acid,
CO,, and H,.
Besides buty- —
rie acid, there —
is also appar-
ently pro-
duced a higher
fatty acid (va-
lerianic acid).

CHEESE-RIPENING BACILLI. 349

_ Schattenfroh and Grassberger (C. B. L. v, 209) announce
the discovery in the Vienna Hygienic Institute of three
further types! of anaerobic butyric acid organisms, which —
were found while the Bacillus butyricus Botkin was being
sought for in market milk.

They could not certainly identify their organisms with
the numerous anaerobic varieties from milk which Fligge
has described very briefly and designated by numbers.

Special Sporulating Anaerobic Bacilli as Agents in
the Ripening of Cheese.

The interminably disputed and difficult question as to
the part played by bacteria in the ripening of cheese is
still not satisfactorily solved.

It is questionable whether non-living milk ferments

play a part, as is believed by Babcock and Russell
(C. B. L. mm, 615). It is certain that bacteria are con-
cerned in the ripening of cheese, and very evidently ya
different varieties in different cheeses.

Duclaux was certainly right when he said that sporu-
lating varieties play an important rdéle in the ripening of
certain soft cheeses (Cantal, Backstein, Limburger).

Weigmann described as Paraplectrum foetidum (C. B.
L. Iv, 820) an obligate anaerobe, very like the bacilli of
tetanus and symptomatic anthrax morphologically, but
not pathogenic, which produces a pronounced odor of
cheese. It is usually associated with the similar faculta-
tive anaerobe Clostridium licheniforme Weigmann,
which usually forms spores at the middle, with swelling
of the cell, and, perhaps, by exhausting the oxygen, it
_ prepares the soil for the Par. foetidum. Further details
are found in the original, which is accompanied by photo-
graphs. The work shows how much remains to be done
in the field of differential diagnosis of anaerobic varieties,
and also how insecure the genera paraplectrum and clos-
- tridium are. (See p. 125.)

1 All three form butyric acid, but no alcohol, accompanied by
abundant liberation of gas, from milk-sugar, starch, or grape-sugar.
On the contrary, they are without effect upon dextrorotatory lactic

acid. None of the three peptonized the precipitated casein; two are
motile, one is not.

350 IMPORTANT VARIETIES OF FISSION-FUNGL.

On the other hand, in the case of cheeses of the charac- c |
ter of Emmenthaler cheese especially, von Freudenreich —

(for the similar Cheddar cheese, Russel and Weinzierl,

C. B. L. m1, 456) has reached the conclusion that here —
the ‘ producers of lactic acid’’—~7. e., those related to the —
Bact. acid lactici, etc.—are essentially concerned in the —
ripening, and that aerobic and anaerobic varieties do not ~

necessarily participate, because they were found in ripe

Emmenthaler cheese in very limited numbers, together — :
with abundant generators of lactic acid. The addition of 4
various spores cultivated from milk scarcely influenced —

the ripening process (v. Freudenreich, C. B. L. 1, 168;
v, 241).

According to Orla J ensen, the normal cavities in —
Emmenthaler cheese are due to the activity of the lactic —
acid bacteria, and contain CO,, which is obtained from ~
albuminous bodies and does not originate from carbo- —
hydrates (C. B. L. tv, 217). According to v. Freuden- ~
reich, a number of acid-forming varieties gradually attack —

the casein if allowed to operate for a longer time, and
alter the character of the ripening.

Further, Gorini found that several organisms of the
subtilis group change sugar into lactic acid only at higher
temperatures, and regularly peptonize only when oxygen
is admitted (C. B. L. v, 44). Also Burri (see p. 320)
has described the Bacillus bernensis, a facultative aerobic,
sporulating organism from Emmenthaler cheese, which
has a pronounced cheesy odor.

The question is still being actively discussed. Those
interested will constantly find articles upon it in the
second part of the Centralblatt ftir Bacteriologie. (Com-
pare also, v. Klecki, C. B. L. m, 21.)

Olav Johann-Olsen says that in the Norwegian ‘‘ gam-
melost’’—a fine cheese with a flavor of ‘‘ apple, lemon,
and Camembert cheese’’—there must be a cooperation of
lactic acid bacteria, Chlamydomucor casei, and Penicillium
aromaticum casei; often also the process is aided by
Dematium and a tyrothrix (C. B. L. tv, 161).

NE, > ee

RETTING OF FLAX AND HEMP 351

ee) oy
‘|

Anaerobic Bacilli as Causes of Fermentation of
| Cellulose. |

_ While van Senus designated, as the cause of fermenta-
tion of cellulose, an anaerobic variety (Bacillus amylo-
-bacter v. Tieghem, according to van Senus) which operates
‘ . . E ° F : : ‘
“only in symbiosis with an aerobic variety, Omelianski
(C. B. L. 1, 358, and v, 433) isolated a thin, anaerobic
bacillus, which is not turned blue by iodin and forms
_ polar spores, and which alone in a nutrient saline solu-
tion with peptone very readily causes fermentation of
cellulose, with resulting formation of considerable quanti-
ties of volatile fatty acids (among them, normal butyric
acid), CO, and H, We might give a large number of
organisms causing decomposition of cellulose. The litera-
ture is given by Herfeldt (C. B. L. 1, 114).

As Amylobacter navicula Wehm., Wehmer has described a facul-
tative anaerobic bacillus, when sporulating assuming a clostridium
form, which is motile when young, is partially stained blue with
iodin, dissolves cellulose, and plays an important réle in the wet-rot
of potatoes. Wehmer has not carried out a sharp separation of this

_ variety from related ones (C. B. L. 1v,734). He here also describes a
second sporulating variety, but gives it no name.

_ Anaerobic Bacilli in the Retting of Flax and Hemp.

_ According to Winogradski and Fribes, the retting of
_ flax (isolation of the bast fibers by softening in water) de-
_ pends upon an anaerobic bacillus with terminal spores,
which breaks up the cementing material (calcium pec-
tate), with the production of butyric acid. Also the ret-
ting of hemp is brought about by an anaerobic bacillus,
but it presents central spores and a blue color after iodin.
Gerstner (A. K. 1, p. 152) has collected numerous anae-
_ robic, sporulating varieties in addition to these, and has
attempted—a perfectly thankless task—to arrange them
ina scheme according to the descriptions found in the
literature.

352 IMPORTANT VARIETIES OF FISSION-FUNGI.

Ill, FAMILY SPIRILLACEAE (MIGULA). SCREW
BACTERIA, 4

(For family and genus diagnosis, see p. 125.)

We have adopted the improved definition of genus as
originating from Léffler instead of those of Miiller, Cohn,
and Ehrenberg for vibrio and spirillum. 4

1. Spirals rigid:

(a) With one (rarely two or three) polar flagellum; very
rarely without flagella. Vibrio.

(6) With a polar bunch of flagella. Spirillum.

2. Spirals flexible: Spirocheete. .

Other writers have retained the somewhat older (1889)
classification of J. Schréter, which is as follows:

1. Cells, bent into more or less pronounced screw forms,
rigid, in the vegetative form actively motile, forming en- —
dogenous spores. Spirillum.

2. Vegetative cells slightly bent, rigid, usually with —
half a turn (comma form), actively motile, with arthro- 1
spores. Microspira. .

This seems to us to have no advantages, but, indeed, —
great disadvantages, since spores are entirely unknown in ©
most spirilla, arthrospores in microspira are denied by
most authors, and, besides, the name microspira has been
used by no one for ten years.

1Tt certainly does not appear possible to make a sharp separation of
the genera vibrio and spirillum according to whether they are pro-
vided with one or several polar flagella, and thus there is furnished a
new proof of the necessity of-great caution in establishing classifica-
tions upon the number and arrangement of flagella. According to
Giinther, his Vibrio terrigenus has a flagellum on each end, and often
bunches of flagella! Kutscher has found some bent forms which pre-
sent horny outgrowths, forkings, ete. Since Zettnow (Z. H. XxIv,
72) has photographed beautiful bunches of flagella upon the out-
growths, one cannot conclude that here involution forms are being
dealt with. Severin has made similar observations in the case of his
Vibrio denitrificans (C. B. L. m1, 504). Here, however, the formation ~
of branching forms is not under consideration, but triradiate forms
(resembling a uterus). Compare the remarks in connection with the
actinomyces.

- a ee
paeen

VIBRIO CHOLERZ. 303

1. Vibrio. (F. 0. Miller, emend. Loffler.)

Cells short, slightly bent, rigid, comma-shaped, some-
times united in screw-like forms, usually only one, excep-
tionally two, polar flagella. There are no endospores.
According to Hiippe, arthrospores are formed.

Key to the Recognition of the Most Important
Varieties. !

1. Motile without phosphorescence.

(a) Gelatin slowly liquefied. Nitroso-indol reaction. Young
gelatin plate colonies coarsely granular.

(a) Usually not pathogenic for pigeons. Vibrio cholerz (Koch)
Buchner, page 353.

(8) Very pathogenic for pigeons. Vibrio Metschnikovii Gama-
leia, page 366.

(6) Gelatin rapidly liquefied. No nitroso-indol reaction. Young
gelatin plate cultures finely granular, brownish-yellow. Vibrio Pro-
teus Buchner, page 367.

(ec) Gelatin not liquefied. Vibrio terrigenus Giinther and Vibrio
tonsillaris Stephens and Wood Smith (C. B. x1x, 929), page 371.

2. Motile with phosphorescence. Vibrio albensis Lehm. and
Neum., page 370.

3. Non-motile. (Spirosoma Migula). Vibrio nasalis Weibel, Vib-
tio lingualis Weibel, pages 375, 376.

Vibrio cholerz * (Koch). Buchner.
(Plates 47-51.)

Synonym.—Spirillum cholerze Koch.
Common Names.—Comma bacillus, cholera bacillus,
** Bacille virgule’’ of the French.

Literature.—Petri, der Cholerakurs, Berlin, 1893. It contains all
bacteriologic literature up to 1893. Voges has collected critically 139
more recent works (C. B. x1x, 466).

Microscopic Appearance.—Bent rods (about 2 »
long, 0.4 » thick), the ends not lying in the same plane.
The bending is often slight, scarcely perceptible; at other.
times pronounced (51, 1, m1), so that they are almost in

1 Because of the close relationship of the varieties, the brief state-
ments in the key can only point toward a diagnosis, and not furnish a
complete description. ,

2In the description illustrations of related varieties are és 3 re-
ferred to, when similar pictures occur exceptionally in cholera.

23

354 IMPORTANT VARIETIES OF FISSION-FUNGI.

the form of a semicircle. By the adhering together of

two vibriones there occur such forms as these: ( and

Under unfavorable conditions of growth (lack of oxygen,
lack of albumin, etc.) the vibriones grow into true screw

forms, which often cannot be recognized as composed of —

separate vibriones. According to Cramer, under espe-

cially favorable conditions (soda bouillon in a thin layer)

there occur especially short oval or cocci-like formations.
In old cultures there are manifold involution forms (51,
Iv). |
Motility.—Very distinct, rapid, turning motion, de-
pendent upon one, rarely two, long, terminal flagella
which are somewhat spiral in form (51, 11).

Staining Properties.—Stains with the ordinary anilin
dyes, but not especially easily; not by Gram’s method.
Usually carbol-fuchsin diluted ten times is employed for

staining, it being allowed to act for a few minutes when —

warm.

Relation to Oxygen.—Aerobically, and much more
slowly anaerobically, it forms powerful toxins.

Intensity of Growth.—Optimum at 37°, but also
very well at 22°. The lower limit of growth has been
found to be 10°-12°, sometimes 8°.

Gelatin Plate.—At first small, yellowish-white to yel-
low, roundish colonies, which as early as twenty-four to
thirty-six hours sink into the gelatin in holes, and later in
saucer-shaped areas of liquefaction.

(a) Natural size: The rapidly enlarging zone of lique-
faction at first remains clear (48, v1); later it becomes
cloudy, and usually gray, from the colonies disintegrating
more and more (48, vu). In many cases after a longer
time there are present in the liquefied zone concentric
rings (48, Ix), which increase from day to day (48, vm).

(6) Magnified sixty times: After sixteen to twenty-four
hours the colonies are visible as minute, pale-yellowish,
roundish, coarsely granular disks with more or less of a
crumbly character at the border (49, 1). Often at this
stage a beautiful, intensely red reflex appears at the per-
iphery of the colonies. The older the individual colonies
become, the more the granular character increases, and a

pitta t Atha hii

.

gan Ome. —

yori.

VIBRIO CHOLERZ:. 300

stage is soon reached where the colonies appear to con-
sist entirely of most minute, strongly reflecting fragments,

looking, according to Koch, as if covered with broken

glass (49, 1). This is the most characteristic stage. The

| liquefaction now rapidly advances. The peripheral parts
of the colonies disintegrate more and more (49, II, v),

the structure appears fragmented and very granular, and
sometimes a hairy border is formed at the periphery (54,
Vv) or a gray transparent zone (53, 1), until finally the
entire colony is broken up into single fragments and small
portions (49, vu). Sometimes also the colonies may per-
sist as compact masses in the areas of liquefaction (49,

Ix), when they are dark yellow to brown (50, Iv), and

there even occur forms which have absolutely no resem-
blanee to cholera (50, 1, 1, v). In general, the varia-

_ bility is extraordinarily great, as is sufficiently shown in

the illustrations (49, rv, vit; 50, m1; 53, v; 54, v, v1).
On one occasion in a gelatin plate of vibrio aquatilis

- irregularly formed secondary colonies, resembling those of
_ the Bact. coli, were observed, and similar ones of the
_ vibrio cholerse (53, viz) may also occur.

Gelatin Stab.—At first thread-like and not character-
istic (47, 1; 53, 1; 54,1). After a short time—twenty-four
to thirty-six hours—there occurs upon the surface of the
gelatin a very small perforating depression, which soon
extends further in the form of a large air-bubble (47, 1).
In the depth the liquefaction extends in the form of a
flattened funnel until the wall of the tube is reached (47,
mi, Iv). Later the liquefaction becomes cylindric. The
area of liquefaction is sometimes cloudy (47, 11), some-
times only filled with the finest fragments (47, Iv). In the
stab canal granular, yellowish-white masses are usually
implanted. It has been demonstrated by many observers
that freshly isolated cultures of cholera vibriones are able
to liquefy gelatin more vigorously than old laboratory
cultures; therefore one must guard against recognizing
rapid liquefaction of gelatin as evidence against the diag-
nosis of cholera. (See p. 61.) Such liquefactions as
shown in Plate 54, m, m1; Plate 53, 1, 1; Plate 52, 1, 1, are
very unusual, but do occur.

_ Agar Plate.—(a) Natural size: Roundish, light brown-

856 IMPORTANT VARIETIES OF FISSION-FUNGLI.

ish to white growths, with a moist luster, smooth borders,
a little elevated, transparent (47, vill, Ix), sometimes
resembling the colon colonies. (Compare also 18, vim.)

(b) Magnified sixty times. Deep colonies: Irregular

roundish and whetstone-shaped, with smooth or slightly

roughened borders, with delicate or medium-sized granules, |
and pale yellow (48, 1, um, 1, right). Only after standing a

very long time do they become darker colored (48, vy) or

present a brown central point with gray or greenish zones —

(48, 1v). Superficial colonies : Roundish, faintly yellowish,
transparent, at first extremely finely punctated (48, 1, 1),

later coarsely crumbly (48, m1). The picture after twenty —

days is shown in Plate 48, Iv.
Agar Stab.—Stab: Whitish-gray, not characteristic,
thread-like; later rough (47, v1). Surface growth: At

first light brownish-gray, with a moist luster, wavy, —
smooth border, a little elevated, and after a longer time —

becoming colored a yellowish-brown (47, vit). The agar
streak corresponds to this (47, v).

Serum Culture.—Solidified blood-serum at incubator
temperature is rapidly liquefied.

Bouillon.—At incubator temperature after ten to six-
teen hours there is a diffuse cloudiness, very often with the
formation of a distinct, more or less rigid or friable pelli-
cle. In cultures freshly isolated from the body, pellicle
formation may sometimes be entirely absent; when the
reaction is strongly alkaline, the pellicle becomes thicker
and firmer (Cramer). Sometimes we have met with very
compact, wrinkled pellicles, but in a subsequent culture
upon the same nutrient medium nothing striking was ob-
served.

Milk.—Koch described the vibrio cholere as having
no particular effect upon milk. More recently many
writers have isolated cholera vibriones from typical cases
of cholera which coagulate milk. The formation of acid
appears to most of the authors to be sufficient explanation
of the coagulation; a rennet ferment: has not been demon-
strated. For details, see Schoffer (A. G. A. x1, 262).

Potato Culture.—Upon faintly acid potato there is
either no growth or it occurs only at incubator tempera-
ture. According to Krannhals (C. B. xm, 338), there are

ne oe

VIBRIO CHOLERZ. | | 357

acid potatoes which become alkaline after standing and
then become a good nutrient medium. The acid reaction
may be gotten rid of by washing the sterile pieces of
potato in sterile 0.25% to 0.5% soda solution or 0.5% to
0.75% solution of sodium hydroxid until the fluid becomes
yellowish. If inoculation is made after washing off the
fluid, the cholera vibrio will surely grow; also 2%-3%
sodium chlorid solution performs the same service,
although the reaction of the potato remains acid. Upon
potatoes impregnated with sodium salts the cholera vibrio
grows at 20°, not only at 87°. (Voges, C. B. xin, 543.)
Upon ordinary potatoes not thus prepared the growth is as
follows: At first a dirty white to yellow growth, scarcely
- at all elevated, with a moist luster, not sharply outlined
from the surrounding medium (50, v1). After standing
longer, the yellow color is transformed into a brownish-
red, while the culture spreads over the whole potato
(50, vit).

Nutrient Media More Rarely Employed.—In sterile
eggs the cholera vibrio grows very well, and here many
varieties (also when every contamination is excluded)
form abundant H,S, while others form little, and still
others none. Thus the long contest regarding this is
settled. (See Abel and Drier, Z. H. xrx, 61.)

A solution of 1% peptone and 0.5% chlorid of sodium
in water (peptone-water) is much employed, especially
for the demonstration of the formation of pellicle and
indol. (See p. 371 regarding preliminary culture. )

The cholera vibrio grows very well upon Uschinsky’s
nutrient medium; according to Voges, with pellicle forma-
tion; but indol is never formed in it. |

Spore-formation.—The formation of arthrospores as
described by Hiippe (compare illustration on p. 25)
has been verified by most subsequent investigators at the
most in a botanical sense, and it appears to have no
practical significance as far as the resistance of the vibrio
isconcerned. Also, Friedrich could never observe germin-
ation of the ‘‘ arthrospores.’’

Viability.—

(a) In the sick: The vibrios have usually disappeared from the
intestinal contents of the sick after four to eight or ten days, rarely

358 IMPORTANT VARIETIES OF FISSION-PUNGT.

sixteen days; in rare cases living vibrios have been found after forty-
seven days (Rommelaire).

(6) In cholera stools the vibrios are usually alive after one or two
days; more rarely, twenty to thirty days; still more rarely, longer;
in one instance they were alive for one hundred and twenty days.
Very similar results obtain in the case of clothing which is kept moist.

(c) In cultures: The cholera vibrio belongs among the varieties
which die out easily. According to Gottschlich and Weigang, the
number of living individuals in agar streak cultures very rapidly
diminishes (Z. H. xX, 376).

Yet living individuals are usually found in cultures three months
old, still frequently in those six months old, and now and then in
those one year of age, if only too extreme drying i is avoided. Morpho-
logically such cultures consist almost entirely of involution forms.
(Compare 51, Iv.) According to Hiippe, also arthrospores. __

(d) In water: Very different results have been obtained by writers
as regards the viability of cholera vibrios when introduced into
unsterilized water, varying from one day to one year. Low tempera-
ture, exclusion of light, and the presence of salts favor preservation;
now and then, also, an increase is undoubtedly demonstrable. Most
often in well- and river-water death of the cholera vibrios is observed

in three to eight days. For more details see Ficker (Z. H. Xxr1x, 1).
Anaurding to Hankin, the water of many Indian rivers kills cholera
vibriones very promptly; these waters are said to contain ‘‘ certain
volatile, acid substances. ry

(e) Upon foods, usually a few days; coffee, one hour; beer, one or
two hours; red wine, ten minutes. For further details compare
Uffelmann (Berl. klin. Wochenschr., 1892, 1209) and Friedrich (A. G.
A. VIII, 87).

Resistance to:

(a) Desiccation: Some statements are found on page 41; the entire
literature is given by Ficker. Uffelmann upholds and William
contests the possibility that currents of wind occasionally may dis-
tribute living cholera vibriones in a partially dried state.

(b) Moist heat: Killed in ten minutes at 60°.

(c) The resistance to cold is given very differently by various
authors. All German investigators found them to withstand even
very low temperatures for a short time, but our winter cold (5°-10°)
was found sufficient to destroy them, often even in three, always in
eight, days (Renk, Uffelmann, etc.).

Others, especially Russian writers, found greater resistance. Thus,
Kasansky claims that neither a short exposure to a temperature of 30°,
nor the operation of four months of Russian winter and repeated
freezing and thawing, completely destroys the cholera vibrio. Similar
results were yielded by experiments with Vibrio Proteus, tyrogenes,
etc. (C. B. xv, 184).

(d) For the effects of disinfecting agents see Kasansky (C. B. XVII,
506). The resistance is slight; especially acids are poorly borne.
Todoform vapor injures the cholera vibrio more than the other vibrios
(Buchner, Bujwid).

VIBRIO CHOLERZ. 359

(e) According to investigations by Palermo, cholera vibrios in
bouillon are robbed of their virulence, but not killed in three to four
hours by sunlight, and in six to seven hours become non-motile.

Chemical Activities.—

(a) Chromogenesis: Slight upon potato only. For chol-
era-red reaction see below (54, Iv).

(6) Odoriferous and gustative substances: The disagree-
able odor of cholera bouillon cultures, which is difficult
to describe, was pointed out by Laser as of diagnostic
value, but it is not sufficiently specific.

(c) Formation of gas and acids from carbohydrates: Dex-
trorotatory lactic acid is formed in abundance from sugar
(grape-, cane-, and milk-sugar) without perceptible pro-
duction of gas (Kuprianow, A. H. x1x, 282). In 10c.c.
of litmus milk the cholera vibrio forms a blue pellicle on
top, the following layer is red, the deepest part is decolor-
ized (reduction); thus the formation of alkali is favored
by the entrance of oxygen, and the fermentation of sugar
and formation of acid by anaerobiosis (Hellin).

(d) Production of ferments: Besides bacteriotrypsin,
some inyertin; also, according to Sclayvo, rennet ferment.

(e) H,S: In peptone bouillon rather abundant. (See
ege culture, p. 357.)

(f) Phosphorescence: According to the statements of
Rumpel, two cholera cultures (‘‘ Oergel’’ and ‘‘ Elwers’’)
were photogenic. R. Pfeiffer assumes that there is here
a mistake, and denies that these photogenic cultures
belong to cholera, basing his conclusion upon his immun-
ity reaction described below (p. 373). It is also consid-
ered by most authors—for example, Dunbar—to be a
photogenic vibrio from water, etc., and not a cholera
vibrio at all. But recently Weleminsky, in Htippe’s
institute, has observed two cultures of cholera vibrios
become photogenic after passage through the body of
pigeons, which were not so previously (C. B. xvitt, 285).

(g) Indol: Usually abundant production of indol upon
nutrient media containing albumin or peptone. Accord-
ing to the number introduced, sufficient indol for demon-
stration is formed in peptone-chlorid of sodium solution
in three to six or nine to twelve hours. Since simultane-
ously, from the small amount of nitrate contained in the

860 IMPORTANT VARIETIES OF FISSION-FUNGI.

peptone and chlorid of sodium,! ete., some nitrate is
produced (Petri), indol can be demonstrated by the addi- —
tion of sulphuric acid alone: ‘‘ cholera reaction of Dun- —
ham and Bujwid,’’ nitroso-indol reaction of the authors.
After keeping the culture longer the intensity of the reaction
increases somewhat up to twenty-four or forty-eight hours; —
later the nitrite gradually decreases, and, in order to
demonstrate the quantity of indol, which increases for :
some days, some nitrite solution must be added (p. 78),
when a dark violet-red color is obtained. A large loopful
of an old agar culture will carry sufficient indol into 10 c.c.
of peptone water for demonstration. The indol reaction
rarely fails. (See p. 372.)

(h) Toxins: Manifold poisons have been produced from
cholera cultures, but all are much less poisonous than the
original material. According to R. Pfeiffer, these poisons
are to be conceived as secondary, altered ‘products from
the disturbing action of reagents. Much more powerful
but qualitatively similarly acting poisons are obtained
from the bodies of the vibrios by very careful killing of
the pure culture upon agar with chloroform or by brief
heating, but the filtrate of young cultures is not poison-
ous.2. Three times the quantity (about 0.5 mg. agar cul-
ture) of the minimum fatal dose of living bacteria, after
being killed, also kills a guinea-pig in sixteen to eighteen
hours. By longer heating the toxicity rapidly decreases.
The effects of all these poisons when injected intraperi-
toneally are exactly the same as those following the intro-
duction of living vibrios into the peritoneum : rapidly

11f the peptone and sodium chlorid are absolutely free of nitrate,
then a weak solution of nitrate must be added. According to Bleisch,
40 drops of a 0.08% solution of saltpeter to 100 of nutrient solution
was the proper quantity. If the nutrient medium contains too much
nitrate, too much nitrite is supplied and interferes with the nitroso-
indol reaction.

2 Metschnikoff, Roux, and Taurelli-Salimbeni have obtained by
means of all sorts of devices, fluid cultures of highly virulent cholera
organisms, the filtrates of which were very poisonous. With such
toxins also cholera antitoxins can be produced. While Pfeiffer’s anti-
bacterial serum protects animals very well from intraperitoneal infec-
tion, it is entirely without effect against infection through the sone
against which the antitoxic serum affords some protection (C. B

XX, 627).

VIBRIO CHOLERZ. 361

developing algid stage, muscular weakness, sleep, falling
of temperature to 30°, death in sixteen to eighteen hours.
Yet it must be emphasized that various proteins (from

Bact. prodigiosum, Bact. coli), when introduced into the

peritoneal cavity of guinea-pigs, produce the same symp-
toms (Hiippe, Klein, and others); also Voges obtained

similar results with papain. Regarding the theory of

Emmerich and Tsuboi (Mtinch. med. Wochenschr., 1893,
_ 473, 497), that cholera is a poisoning by nitrite origi-
nating in the intestine, see page 94.

Distribution.—

(a) Outside the body: Recently they have been found
not infrequently in water (wells, tap-water, rivers, har-
_ bors, canals), which had been contaminated with dejecta
from cholera cases, yet their presence is only valuable if
_ the differential diagnosis from the ‘‘ water bacteria resem-
bling cholera’’ is carried out with great caution. (Com-
_ pare p. 378, etc.)

(b) In the healthy body: Not infrequently, in times of
cholera, cholera vibriones have been found in healthy
persons without any pathologic symptoms (‘‘ Cholerage-
sunde’’). For example, Abel and Claussen, upon re-
peated examination, found cholera vibriones present at
~ some time in 14 out of 17 healthy persons who were mem-
bers of 7 families in which there were cases of cholera; in
_ many, for as long as fourteen days. Negative days inter-
_ yvened between the ones when positive results were ob-
tained. In Hamburg 28 such cases of ‘*cholera in health ”’
with absolutely normal feces were demonstrated.

(ce) In diseased human organism: Found only in cases
of cholera, and in no other disease. The principal location
is in the intestinal contents, especially in the mucous floc-
culi of the rice-water stool. There the cholera vibrio is often
in pure culture; usually at the height of the attack they
are present in large numbers, and generally decrease after
four tofourteen days. In fresh cholera cases the organism
is not usually found in the organs, except in the intestinal
glands, where sometimes the epithelial layer is broken
through. In exceptional cases, however, both in man and
experimental animals, the vibrios are also found in the
internal organs, as lungs, liver, kidney, spleen, and most

362 IMPORTANT VARIETIES OF FISSION-FUNGI.

rarely in the heart’s blood. The more virulent the
organisms, the more they spread into the organs.

(d) In animals: Spontaneous cholera in animals caused —
by cholera vibrios is unknown. (Compare Vibrio Met-—
schnikovii, p. 866.) Our domestic animals, etc., appear
to be immune to cholera infection, as it occurs in natural —
ways. (See below.)

Experimental Observations Regarding Pathogenic .
Effects.—(a) In anvmals: According to Sabolotny (C. B. —
xv, 150), the Spermophilus guttatus, a rodent of southern —
Russia, dies after being fed cholera vibrios with symptoms ~
and section findings resembling those of cholera. Positive —
results per os were also obtained by Metschnikoff in young —
rabbits, by Wiener in sucking kittens and young (five ©
days’ old) rabbits, and by Karlinski in young dogs. (See
Wiener, C. B. xx, 205, 595.) In adult guinea-pigs by the ~
natural channels, only an approximation to the picture of
a case of cholera can be produced. Usually, following ~
Koch’s method, 5 c.c. of a 5% solution of soda is first
introduced into the stomach, and shortly afterward 10 c.c.
of a cholera culture in bouillon; at the same time 1 c.c.
of tincture of opium to each 200 gm. of body-weight is
injected intraperitoneally to quiet the intestinal peristalsis.
Death occurs in twenty-four to forty-eight hours, preceded
by a falling of temperature and extreme prostration: The
intestine is reddened and contains abundant fluid, rich in
cholera vibrios. Other vibrios, Vibrio proteus, etc., pro-
duce similar but not so pronounced effects. It is easier
to kill animals (rabbits, guinea-pigs) by the introduction
of the organisms into the blood-vessels or serous cavities.
Death in peritoneal infection occurs in twelve to sixteen
hours, usually after a primary multiplication, from the
action of absorbed toxins originating from the dead
vibrios (R. Pfeiffer). In the peritoneum (and eventually
in the blood and organs) of the dead animal, living vibri-
ones are usually found only when the infection has been
produced with very large quantities. Many other bacteria
operate exactly the same. (See p. 360 regarding the poi-
sons of cholera.) If an animal withstands a single intra-
peritoneal infection with a small dose of living vibriones,
it becomes immune to larger doses, because the bacterici-

VIBRIO CHOLERZ. 363

dal power is heightened, but the animal is not really more
resistant to cholera toxin than it was originally. See below
- concerning R. Pfeiffer’s biologic cholera reaction. See
also R. Pfeiffer (Z. H. xv1, 258), M. Gruber, and Wiener
(A. H. xv, 241).
One principal difficulty in the animal investigation of
cholera is the variable, easily reduced virulence of the
cholera vibrio. Many methods are recommended to in-
- erease the virulence; for example, the anaerobic cultiva-
tion in hens’ eggs (Hiippe), which is contested by Wes-
brook (H. R., 1896, 241), also passage through pigeons
(Gamaleia, Salus, etc.). W. Rindfleisch, however, insists

_ that no example of the cholera vibrio can be cultivated

which is distinctly pathogenic for pigeons when injected
_ subcutaneously (Z. H. xxi, 247). ‘‘Young’’ cultures,
- upon which many writers place great value, are only
_ apparently more virulent, because they contain many
more living individuals than older ones (Gottschlich and
Weigang, Z. H. xx, 376).

According to Blachstein, the virulence of cholera vibriones is en-
tirely dependent upon the nutrient medium. It is said that a cholera
culture which is no longer virulent may be rendered virulent by culti-
vating it as follows:

1. Two days in a 2% peptone solution, which contains besides only
0.5% disodium phosphate and is cleared up with a little ammonium
citrate solution.

2. Nine days in a 2% peptone solution containing also 3% potas-
sium nitrate.

3. One day upon the solution given in 1, with the addition to each
100 c.c. of 1 ¢.c. of a cold saturated solution of ammonium-ferro-
sulphuric acid.

(6) In man: In a considerable number of cases, follow-
ing the example of v. Pettenkofer and Emmerich, previ-
ously healthy men, after swallowing small quantities of
pure cultures of the cholera vibrio, have developed the
symptoms of cholera of slight or medium severity. The
persons on whom the experiments were conducted usually
had previously taken some soda solution to counteract the
acidity of the stomach. Several severe and one fatal case
of ‘‘laboratory cholera’? have been known to occur in
men who were working with cholera vibrios. (See Reincke,
C. B. xvu, 202.) According to R. Pfeiffer, cholera in

364 IMPORTANT VARIETIES OF FISSION-FUNGI.

man arises, after destruction of the epithelial lining of the
intestinal canal, by the enormously multiplied vibrios and
the accompanying intoxication and absorption of poisons”
from the dead vibrios. We cannot here enter into a dis-
cussion of the teachings of Buchner, Nencki, and Metsch-
nikoff, that the immunity against cholera in many
localities is always or often dependent upon the absence of
a synergetic or upon the presence of an antagonistic
micro-organism in the intestine of the host.

Immunity and Immunization.— Recovery from
cholera or an artificial cholera infection is followed by a
certain immunity. In the peritoneal cavity of such an
immunized animal cholera vibrios become granular and ~
die (p. 374). The serum of the animal contains agglu- —
tinin (p. 374). With the cholera immune serum no con- —
siderable passive immunity in other creatures can be —
obtained, the conditions being very similar to those in —
pest. :
On the contrary, Haffkine has obtained very good results —
in India in the production of active immunity by means
of devitalized cultures. Kolle (Deut. med. Wochenschr.,
1897) has repeated the experiments in the institute for
infectious diseases, and found them confirmed in so far
~ that the serum of the experimental persons contained bac-
tericidal substances after about five days, which were most
abundant on the twentieth day, but could also be demon-
strated after a year. Various materials were injected; for
example, one-tenth of an agar culture suspended in bouillon
and heated for one hour to 56°. Virulent cultures operate
similarly to non-virulent ones. For two or three days
there is quite a painful infiltration at the point of injec-
tion. For the entire literature regarding cholera immu-
nity see Voges (C. B. xrx, 466).

Varieties and Variations of the Vibrio cholerz.

Since first D. Cunningham (C. B. x, 763, also xxi, 854) demon-
strated a considerable variation in cholera vibrios which he cultivated
from typical cases of cholera, many writers have described forms which
in part deviate very much. We can here only mention a few of these
experiences, and only those where it appears certain that vibrios from
true cases of cholera were in question.

A series of forms have been accurately described and photographed

|

_ by Friedrich (A. G. A. vitt, 87), yet they do not deviate very widely
from the typical organism.

VIBRIO CHOLERZ. 365

_ More interesting than the reports regarding varieties are
_ the observations regarding variability:

For example, the experiments made by Claussen in v. Esmarch’s
_ institute are very instructive. Vibrios freshly isolated from cholera
_ stools presented upon plates a tendency for the colony to disintegrate
- and exhibit a border asif eaten away. The nitroso-indol reaction was
absent. A guinea-pig did not die after the injection of 1c.c. ofa
bouillon culture. The stab cultures grew slowly and were not char-
acteristic. After repeated transfers in bouillon, a guinea-pig died after
_ the injection of 1 c.c. of the bouillon, and in the peritoneal exudate,

and even in the blood, cholera vibrios were found which possessed all
_ the characteristic peculiarities, including the nitroso-indol reaction
» (C. B. xvi, 325).

Vibrio romanus of Celli and Santori, isolated from numerous

typical cases of cholera in Rome in 1893, was cultivated from the

_ stool, was not pathogenic for animals, gave no indol reaction, did not
coagulate milk,and at 37° grew neither in bouillon nor on agar.
After being culvtiated for eight months it gave the indol reaction and
grew at 37°, but was still almost perfectly non-pathogenic (C. B. xv,
789).

Bordoni-Uffreduzzi and Abba cultivated from a typical case of
cholera a very rapidly liquefying, short vibrio, which grew atypically
upon gelatin and formed a yellow growth upon potato. After being
cultivated for nine months upon gelatin it was constantly like the
ea vibrio, both macroscopically and microscopically (C. B. XvI,
201).

The Varieties Most Closely Related to the Cholera

_ Vibrio.

When the cholera vibrio was discovered, its peculiarities
seemed so characteristic that its differentiation from other
bacteria was thought to be easy. Since then there have
been found in the environs of man first a few, then more,
and finally such an immeasurable series of vibrios that
for a long time they have no longer been designated by
separate names. The richest results have been yielded by
the methodic examination of certain rivers. Thus, Dun-
bar (Z. H. xxi, 295) has published an entire series of
Elbe water vibrios isolated from the water of Hamburg.
Abbot and Bergey have collected 110 cultures of vibrios
from the American Schuylkill River, on whose banks no
cholera has prevailed for a very long time. Part of these

366 IMPORTANT VARIETIES OF FISSION-FUNGI.

are very similar to the cholera vibrio, and correspond
most closely to the Vibrio Metschnikovii (Journal of
Experimental Medicine, m1, 535).

A detailed repetition of these descriptions would be
senseless +; the description of the individual forms which
are known by names is not even of much value, but in a_
measure serves to demonstrate the difficulty ‘of differ-
entiating ‘‘ varieties.’’ We give again a short description
of the varieties which were carefully studied for the first
edition, and, in connection with the same, refer continu-
ally to our illustrations.

Vibrio Metschnikovii. Gamaleia.

Principal Literature.—Gamaleia (A. P., 1888, 11, 482), R. Pfeiffer —
(Z. H. vit, 347).

It is the cause of a disease of fowls occurring in southern
Russia with symptoms resembling those of chicken cholera.
Since its original discovery it has been also found by
R. Pfeiffer in the north harbor of Berlin, and once by
Kutscher in the Lahn. (See also above.) In the affected
animals the vibrios are found in the intestine, and almost
always also in the blood (Vibrio septiczemia).

This exceedingly interesting micro-organism can not be
distinguished from the Vibrio choleree by any morphologic
peculiarities, therefore we have not made any illustrations
of it. The vibrios are often a little more sharply bent and
shorter than those of cholera (51, v). The liquefaction of
gelatin varies exactly as in the case of the Vibrio cholere.

It yields the nitroso-indol reaction without the addition
of nitrite, and, according to Kuprianow, forms levorota-
tory lactic acid from sugar (like the V. cholere).

The Vibrio Metschnikovii is remarkable for being highly
pathogenic for pigeons and young chickens. If a trace of
the culture is inoculated by a prick in the breast muscles,
it causes death with local and general symptoms like those
in chicken cholera (p. 210), only the intestinal findings
are more like those of cholera than in the latter, and the
spleen is rather shrunken than enlarged. The organisms

1The forms known before 1894 are found together: Dieudonné (C.
B. XVI, 363) and Brix (Hyg. Rundschau, 1894, Iv, 913).

+

he

1 VIBRIO PROTEUS. 367

are present in quantity in the blood and in the edema at
the necrotic point of moculation.

According to the statements of Gamaleia, cholera vibrios
behave similarly toward pigeons, but Pfeiffer could verify
this only by using very large quantities of cultures.

“Weibel (A. H. xxi, 22), Salus (A. H. xix, 342), Wlajeff
(C. B. xvu, 619), and others, on the contrary, obtain
inoculation results similar to those of Gamaleia with
cultures which are originally virulent or rendered so
artificially. The possibility of immunizing pigeons with
the Vibrio Met. against the Vibrio cholerz is advocated
from many- sources, and denied by R. Pfeiffer, who also
finds a reason for considering the Vibrio Met. a separate
organism in its refusal to give Pfeiffer’s serum reaction.

(See p. 373.)

Vibrio Proteus. (Finkler and Prior.) Buchner, A. H.
iii, 1885, 361.

(Plate 52.)

Vibrio ‘‘ Finkler and Prior ”’ of authors; ‘‘ Finkler.’’

Literature.—Finkler and Prior, Erganzungshefte z. Centralblatt f.
allg. Ges.-Pflege., Bd. 1, 279; Koch (Z. H. xiv, 329).

Microscopie Appearance.—More or less bent rods; on an average,

2.4 long and 0.4-0.6 4 thick, usually a little thicker than the Vibrio
cholerz (51, vr). .

Gelatin Plates.—With the unaided eye it only differs from the Vibrio

_cholerz in more rapid liquefaction and in the formation of larger

_ disks (52, 111). Magnified sixty times, the colonies are yellow, with
almost smooth borders, only slightly and finely granular (colonies of

the Vibrio cholerz are coarsely granular with finely pectinate or

crumbly borders). The surface colonies usually sink in rapidly and

: a a darker peripheral zone, sometimes with a row of hairs (52,

DLV).

. Gelatin Stab Culture.—Tube-shaped liquefaction along the stab,
without formation of any air space, and with marked turbidity of the
contents (52, I, It).

Agar Plate.—A little more luxuriant growth than in the Vibrio
cholerz (52,1x). When magnified sixty times, the colonies look like
those of Bact. coli (52, vit and vit). (See also 18, vi; 12, Iv.)

Chemical Activities.—Milk is coagulated, and later again liquefied;
faint acid formation; no gas formed from grape-sugar; indol reaction
faint and frequently absent; very little H,S developed.

| Distribution.—(a) Outside the body: Claimed to have been once found

- in surface water (Héricourt).

(6) In body: In the intestinal contents or dejecta of some healthy

368 IMPORTANT VARIETIES OF FISSION-FUNGI.

persons, of some cases of diarrhea, and of men suspected of hav
cholera. Since its discovery by Finkler, in 1884, in the evac
of persons said to be suffering from cholera nostras which had been.
kept a long time, this organism has been found but very rarely.

Pathogenic Significance in Man.—lIt is not the cause of the so-called
cholera nostras; at any rate, in the great majority of the cases. Sin
its discovery, although much sought for, it has searcely once been found
in cholera nostras. :

- In experimental animals it produces in general the same, nominally
somewhat milder, disease symptoms as the cholera vibrio.

B. Fischer found, in a case of suspected cholera, the Vibrio helco-
genes Fischer, which was pathogenic for animals and resembles the
Vibrio Proteus (C. B. XIv, 73).

According to Chantemesse, the Vibrio lissabonensis is identical
with or very closely related to the Vibrio Proteus. It was discovered
by Pestana and Bettencourt (C. B. xvi, 401, photographs) in the
spring of 1894 in numerous cases of an epidemic, widely distributed,
mild, choleriform disease in Lisbon, and was also found in the _ citys
water aqueducts. It is a slightly bent vibrio with polar flagella, giving
no nitroso-indol reaction, and without pellicle formation upon bouil-
lon. It produces liquefaction of gelatin in the upper part of the stab
culture in the form of a broad, flat funnel. In the gelatin plates there’
appear upon the surface colonies, which at first are round, smooth, and
only slightly granular; later they have a gray center surrounded by a
scarcely transparent, granular zone, which is limited externally by a
thick circle of fine radiating threads of considerable length. Because
of progressing liquefaction the characteristic appearance is lost by the
third day. Upon ordinary potato it grows very poorly, but upon
alkalinized potato very well as a shining gray growth. The organism
is slightly pathogenic for animals. It does not immunize against
cholera.

Vibrio tyrogenes. (Deneke.) Lehm. and Neum.

Synonyms. — Deneke’s cheese spirillum; Spirillum
tyrogenum Deneke (Deut. med. Wochen., 1885, 338).

Isolated by Deneke from an old cheese, but since then it has been
very rarely found. As regards intensity of liquefaction, it stands
midway between the Vibrio cholerze and Vibrio Proteus, and also in
other respects its peculiarities are usually so intermediate between
these two varieties that we have not illustrated them. The peculiari-
ties mentioned by Giinther (Bakteriologie, tv. Aufl., p. 361)—a
thick mold-like scum upon the gelatin stab culture and a marked
yellow color of the same—were not observed in our cultures, Our cul-
ture gives the nitroso-indol reaction like the Vibrio cholere. Accord-
ing to Kuprianow, it forms dextrorotatory, and the Vibrio cholere
levorotatory, lactic acid. Our old laboratory culture grows well at
37°,

fe . VIBRIO BEROLINENSIS. 369

Vibrio danubicus. Heider. (C. B. xiv, 341.)
(Plate 53, I, U1, IV.)

‘ Nothing peculiar microscopically (53, Iv). Gelatin is powerfully
liquefied. Stab cultures remind one of very actively liquefying
cholera cultures. In our cultures the form of liquefaction was always

_ more like a saucer than a flattened funnel. Upon very thick plates it

__ is very similar to the cholera vibrio; upon thinner plates, after twenty-

_ two hours at 22°, the surface colonies spread out exceedingly thin, are

irregular, and have a border which is wavy or provided with coarse

outgrowths. They are almost colorless and very delicately and uni-
formly marked with fine striations. Our illustration corresponds
with this in general (53, 011). Milk is coagulated; upon potato there
oceurs in the incubator a brownish, miserable growth. It gives the
indol reaction well. Pathogenic for guinea-pigs, less for pigeons. Cul-
tivated by Heider from the water of the Vienna canal of the Danube
_ at a time when no cholera was known to exist in Vienna; later
_ detached cases of cholera occurred.

¢ ger r

Vibrio aquatilis. Giinther. (Deut. med. Woch.,
1892, 1124.)

(Plate 53, 11, VII, VIII, 1X.)

Microscopically not specially different from the cholera vibrio (53,
vit). The colonies in gelatin plates, however, are easily distin-
_ guished from those of the cholera vibrio by the smooth or slightly
_ wavy border (never with granular irregularities) and very fine granules
_ (53, 1x). In Plate 53, vil, we have reproduced a quarter of a very
_ remarkable deep picture in a thinly sown gelatin plate. The sur-
_ rounding, numerous, secondary colonies are to be explained by soften-
ing of the gelatin (too high temperature). Older gelatin plate cul-
tures are similar to the cholera vibrio; the liquefaction is slow. There
is no nitroso-indol reaction, but a strong odor of sulphuretted hydro-
gen. Itis not pathogenic. Weibel found a similar vibrio in a well
which had been infected with cholera vibrios a long time before (C. B.
xi, 117).

Vibrio berolinensis. Rubner. (Neisser, A. H. xix, 194.)
(Plate 53, v, VI.)

Microscopically like the Vibrio cholerz (53, v1). We also found
the gelatin plate cultures very similar to those of cholera. There is
a tendency to the formation of coarser lobulations, and a finer granula-
tion of the colony is striking. Liquefaction of gelatin is minimal.
Strong nitroso-indol reaction. Considerably pathogenic for guinea-

pigs.
24

370 IMPORTANT VARIETIES OF FISSION-FUNGI.

Vibrio albensis. Lehm. and Neum.
(Plate 54.)

Synonyms.—Phosphorescent Elbe vibrio of Kutclem
Dunbar.

A detailed description is unnecessary in the face of the fact that the -
best judges of the photogenic vibrios do not presume to differentiate
them morphologically from those of cholera. Our cultures show very .
constantly—as they are usually described—luxuriant growth, vigorous
liquefaction in the stab eanal, pellicle formation on bouillon, and vig-
orous indol reaction. The gelatin plate colonies we were unable to
certainly distinguish from cholera (54, v1). We often observed in
old superficial gelatin plate colonies a pretty circle of hairs, as is pre-
sented by many vigorously liquefying varieties, but which we have

>

never met with in the cholera vibrio. In the six cultures of photo-—
-
'

genic Elbe vibrios obtained, the phosphorescence was vigorous, but
often, through insufficiently frequent transfer to fresh nutrient media,

it was completely lest, and in some experiments it could not be re-—

gained by employing herring nutrient medium. Marpmann refers the ~

phosphorescence to the formation of phosphoretted hydrogen.

7

Judging from the descriptions, a number of photogenic. |

inhabitants of the sea, described as bacilli or photobac-
teria, appear very closely related to the Vibrio albensis.
We may place them here, naturally without expressing
ourselves as to how far they are different ‘‘ species.”’

Vibrio indicus (Beij.) Lehm. and Neum. Bacillus phosphores-
cens Fischer (non Bacterium phosphorescens Fischer, which is found
on page 231). Photobacterium indicum Beijerinck (non Bacillus
indicus Koch, which is found on page 274). West Indian photogenic
bacillus. The gelatin plate and stab cultures are described as like
cholera throughout; the liquefaction is intense. Microscopically:
small rods, two or ‘three times as long as thick, very often in pairs,
more rarely threads. In chlorid of sodium milk, screw forms occur.
Active serpentine motion. The light is bluish-white and intense.
Minimum, 15°; optimum, 30°-35°; maximum not much higher.
According to Beijerinck, it is also able to emit light upon non-sacchar-
ine nutrient media, but also does so with the addition of a little
su

Katz considers the Bac. cyaneophosphorescens Katz, obtained
from Australian seas, to be closely related (C. B. rx, 156 od Pree
cording to Katz, however, this organism occurs as straight motile rods
and curved non-motile threads.

1 In the same place Katz has also described completely four other
‘¢ varieties’’: Bacillus argenteophosphorescens I, II, III, and arg.-
phosphorescens liquefaciens. They appear in part to be also vibri-
ones.

DEMONSTRATION OF CHOLERA VIBRIO. 371

a? hae

Vibrio luminosus (Beij.) L. and N. (Photobacterium luminosum
Beijer.), obtained from the North Sea. It is very closely related to
_ the Vibrio indicus, according to Beijerinck. It liquefies vigorously
and presents vibrios and spirilla. According to Beijerinck, it also is
: photogenic without the addition of sugar. Slight addition of sugar
favors photogenesis; a little more (1% or more of dextrose) inhibits it.
__-Vibrio balticus (Beij.) L. and N. (Phot. balticum Beijer., C. B.
yur, 616). ‘‘ Native phosphorescent bacillus’’ Fischer (C. B. “Ht,
105), from the Baltic Sea. Described by Fischer as very similar to the
Vibrie indicus. Light, bluish-white. In the description of the
- microscopic character and the appearance of the cultures, Fischer him-
self often compares it to the Vibrio cholere. Minimum, below 5°.
_ It produces light, according to Beijerinck, only upon nutrient media
_ which contain sugar. It bears very well a large proportion of sugar
(3%-5% of cane-sugar). The freshly isolated cultures liquefied very
little. Beijerinck finally obtained very vigorously liquefying cultures
_ by longer cultivation on gelatin. It does not ferment sugar.

Vibrio Fischeri (Beij.) L. and N. (Photob. Fischeri, Beijerinck;
_C. B. vit, 616). According to Fischer, it stands very close to the
_ Vibrio balticus. When freshly isolated, it liquefied very vigorously,
and gradually almost completely lost this property. Traces of cane-
_ sugar favor the photogenesis ; 0.5% or more lessens it. It does not

ferment sugar.

yo a 7

et 29

Vibrio terrigenus. Giinther (C. B. xvi, 746).

Does not liquefy gelatin at all, forms a delicate pellicle upon gela-
tin. It is interesting, from the standpoint of classification, that it
_ possesses either a single flagellum or a bunch of flagella at each end.
_ Gelatin colonies are smooth-edged and structureless; the superficial -
_ ones form little heaps. Older deep colonies are brownish and studded.
__ It produces a good yellowish-white growth upon potato. Sugar is not
_ fermented, milk not coagulated. It is not pathogenic for animals,
_ and is an obligate aerobe. Obtained from Berlin soil. The Vibrio
ie a, 8, y Weibel appear to be similar (C. B. Iv, 225, 257,
289).

_ Special Methods for the Demonstration of the Cholera
Vibrio.
The examination should usually be completed in twenty-four to
thirty-six hours.
A. In the evacuations of cases of cholera or suspected cholera.!
1. Microscopic preparation (usually from a flake of mucus !): The
presence of abundant vibrios (especially if arranged parallelly like

1 The demonstration is conducted in the same manner in the case of
milk and other foods, soiled linen, old dried laboratory cultures, etc.
Here often the direct microscopic observation can be omitted.

372 IMPORTANT VARIETIES OF FISSION-FUNGI.

a school of fish, according to Koch) speaks strongly in favor of
cholera, for vibrios resembling those of cholera, if present in the
stools at all, are usually only scanty. If the stool is of nearly nor-

mal consistency, the direct microscopic examination may be omitted. —

One should avoid mistaking the thin spirilla (Sp. hachaizze) for vibrios. —
2. Testing of a fresh, minimal specimen of the stool which con-
tains living vibrios in great number with serum, as on page 373.

3. Infection of an alkaline peptone chlorid of sodium solution? —
(about 50 ¢.c.) with a flake of mucus or with 1 to 5 ¢.c. of the stool.
This is to be kept at incubator temperature. (Preliminary cholera —

culture.)

(a) Observation of the pellicle formation. After three hours indica-
tion of pellicle formation may be present. After about sixteen to
twenty-four hours the pellicle does not become more distinct. (Many
micro-organisms form pellicles ! )

(6) Microscopic demonstration of vibrios in pellicles. Here the

i

1
i

occurrence of vibrios demonstrates much less the presence of true ~
cholera vibrios than does a large number in the stool. Also vibrios —

resembling cholera may develop into pellicles.

(ec) Agar plates from the pellicle (37°) after eighteen hours must

not be phosphorescent.

(d) Gelatin plates from the pellicle (22°). After sixteen to twenty-

four hours, when magnified sixty times, the characteristic shining and
coarsely granular colonies are found. The form of the growth in
elatin is one of the principal characteristics. The suspicious colonies
if not numerous, all are considered ) are inoculated as soon as practic-.
able into gelatin (flattened funnel-shaped liquefaction) and tubes of
peptone chlorid of sodium solution (indol reaction).

(e) Indol reaction (without nitrite being added) with part of the
tubes after three hours. The indol reaction is usually certainly present
in cholera after eighteen hours. By rapid transformation: of the
nitrite into ammonia, various water bacteria can frustrate the direct
cholera reaction. See page 359 regarding the failure of the indol
reaction in pure cultures of certain cholera.

(f) Potato cultures from the pellicle. Chlorid of sodium potato
(p. 357) at 37°. Yellowish-brown to brownish-red color is in favor of
cholera.

4, Gelatin plates prepared directly from the stool (3 dilutions).
Abundant colonies of vibrios with a form like those of cholera speak
very strongly for cholera even if the liquefaction appears too vigor-
ous.

5. Agar plates smeared over very thinly with very much diluted
stool and kept at 37°. Photogenic colonies are not looked upon as
cholera.

6. All vibrios isolated in these ways must be examined with the

1 For the preliminary cholera culture, in order to produce energetic
alkalinization, there is always added to 100 c.c. of nutrient medium,
neutralized with phenolphthalein, 2 c.c. of normal sodium hydroxid
or 1% crystalline or 0.3% anhydrous soda, in which way many water
bacteria are eliminated.

an, aT

DEMONSTRATION OF CHOLERA VIBRIO. 373

Gruber-Durham test, which is to be looked upon as the most certain
reaction which we possess up to this time (see below).
With a negative result in these examinations cholera may still be

“present, for in very rare cases the occasional absence of vibrios from
‘the stools of undoubted cases of cholera has been proved. Thus, for

example, Rumpel failed to demonstrate the vibrios in the first 50 c.c.
of rice-water stool from a fresh typical case of cholera.

_ B. In suspected water.

(4a ate

The water in question is placed in half-filled flasks in quantities of
500 c.c. to 1 liter, together with so much of a strong peptone chlorid
of sodium solution (20% peptone, 10% NaCl) that the water contains

1% of peptone; and to this is added also alkali in excess (26 c.c.
normal sodium hydroxid, 1% crystalline or 0.3% anhydrous soda).
_ The further examination is carried out exactly asin A, 2-6. Great

ek

art

skepticism is demanded in water examinations.

As especially shown by the detailed work of Dunbar, we may
from the first exclude a great number of vibrios resembling cholera in
the diagnosis of cholera by means of gelatin plates, potato cultures,
photogenesis, etc.; but there were a considerable number of cultures,

_ in which all morphologic and biologic means of separation were lack-
_ ing, which were pointed out by the serum reaction, exactly analo-
-gously to the typhoid-coli diagnosis.

This was carried out according to Pfeiffer’s method (Z. H. xrx,
75; XX, 198), since at the time of the last active interest in cholera

_ the Gruber-Durham reaction was still undiscovered. Here, unfor-

tunately, all the cultures were excluded which proved to be non-patho-
genic for experimental guinea-pigs, and which could not be rendered

_ viulrent by means of the introduction of definite, large doses into

animals. (Compare p. 95.)

The cholera serum which is used for these investigations is obtained
as follows: A rabbit weighing 1.5 to 2 kilos is injected subcutaneously
with the culture substance from three slanted agar cultures (twenty-
four hours, 37°), together with about 5 to 6.¢.c. of bouillon. The
animal becomes somewhat feverish, and on the sixth day is bled, and
yields, following the directions on page 105, an active serum, which
keeps for months in a dark ice-box if 0.5% phenol is added.

Pfeiffer indicates the working strength of serum as follows: He
designates as a titer of serum the smallest quantity of serum which
certainly suffices to cause solution of 2 mg. of living normal culture
inside of an hour, if it is mixed with 1c.c. of bouillon and injected
into the abdominal cavity of a young guinea-pig weighing 200 gm.
The most active guinea-pig serum had 0.5 mg. to the titer. (Serum
from i convalescent cholera cases in man had 2.5 to 20 mg. to the
titer.

Of this serum, now, about 10 to 30 mg. (ten times the minimum
efficient dose), together with 1 c.c. of bouillon and a loopful of viru-
lent cholera vibrios, are introduced into the peritoneal cavity of a

1 Tf cholera serum is generally introduced as a diagnostic aid, then
reliable firms or State institutes must undertake its preparation.

_

374. IMPORTANT VARIETIES OF FISSION-FUNGL

young guinea-pig (200 to 300 gm.). This is accomplished by
a slight cut into the corium with scissors and gently forcing a bl
Koch’s syringe through the abdominal muscles. After twenty mi
utes one removes little drops through the opening with a capi
glass tube.

The actively motile vibrios become motionless, swell, dissolve, and
in twenty to thirty minutes are dead, or a few may still be alive.

According to the extensive publications of Dunbar (Z.
H. xxi, 295), Pfeiffer has the satisfaction of knowing that,
by the ‘experiments of himself, Dunbar, Sobernheim, and
others, cholera serum has been proved active against
eighty- six different true cholera cultures from all parts of
the world. With three cultures from cases in man which
were considered as cholera by clinicians, R. Pfeiffer ob-
tained negative results, and Dunbar, in subsequent exam- —
ination, obtained positive ones ; he ‘assumed that Pfeiffer.
had received different cultures. Two other cases could -
not be reexamined by Dunbar, since the cultures in Ham-—
burg had died. J

Negative results were obtained with Pfeiffer’s cholera
serum in nine cultures from suspected cholera stools (among —
them three were photogenic), in many vibrios (all photo-
genic ) from water isolated during the prevalence of cholera,
and in all varieties found in the Hamburg water since
cholera ceased. Dunbar concludes: One may now assert
that all varieties which do not react to cholera: serum
are not cholera vibrios, and it is hoped that we may also
some day declare that all varieties reacting to cholera
serum are true cholera vibrios.

Gruber and Durham (Miinch. med. Wochenschr., 1896,
206, 285) have taught how to make the diagnosis actually
more certain in cholera by means of observing the aggluti-
nating power of the serum. Serum is prepared as al-
ready described, and it is determined in what dilution
with bouillon it agglutinates known cholera vibrios. It
is usually still active when diluted from 100 to 200 times.
(See p. 105.)

Then it is determined whether the organisms which have
been isolated and are to be diagnosticated as cholera vibrios
are agglutinated by a similar concentration. Gruber and
Durham found the reaction rather strongly specific; only
a few cultures analogous to the cholera vibrio were agglu-

| bad
a Se
ea

VIBRIO NASALIS. 375

tinated, and of these, it is at least questionable whether
they may not be looked upon as cholera vibrios, as in the
_ ease of the Vibrio berolinensis.

Also here the negative result of the test (absence of
effect by a serum in dilution of 100, which produced a posi-
- tive effect against true cholera vibrios when diluted 120 to
- 150 times) allows an exclusion of cholera vibrios ; a posi-
_ tive marked result makes the diagnosis more sure. Witha
positive but weak reaction the diagnosis of cholera, on the
contrary, is not entirely certain ; for example, with a tardy

~ action in dilution of 50 times, while true cholera vibrios are
promptly agglutinated by dilutions of 80 to 100. It would
be best to place no dependence at all upon reactions which
_ only oceur in concentration above one in fifty and after
_ halfan hour. Compare also Mann (A. H. xxxtvy, 179).
: Occasionally also the testing of the agglutinating action
of the serum upon true cholera vibrios in a case of cholera
during the disease or convalescence may make the diag-
nosis more certain.

Some Other Vibrios Which Are Not to be Confounded
with the Vibrio cholere.

Vibrio spermatozoides. Loffler (C. B. vii, 637).

This remarkable variety, occasionally found in turnip-cabbage
_ infusion by Loffler, and photographed by him, is characterized by
_ powerful terminal flagella (56, v1) ; the latter disappear or are very
delicate upon turnip-cabbage gelatin, but return partially upon rein-
oculation into turnip-cabbage infusion. The organism presents Y-
shaped forkings! (See the note, p. 352.)

Vibrio nasalis. Weibel! (C. B. ii, 466; iv, 225).
(Plate 56.)

According to Weibel, a very interesting variety. We have not
studied it.
Microscopically : In nasal mucus, thick vibriones (56, II); in

1 Also of interest are the following closely related organisms, which
have been described by Weibel (1. c.) and grow upon gelatin with a
yellow color and without liquefaction: Vibrio flavus Weibel, aureus
Weibel, and flavescens Weibel. Regarding these varieties, which do
not come seriously into question in the differential diagnosis of the
Vibrio cholerz, the original must be consulted.

376 IMPORTANT VARIETIES OF FISSION-FUNGI.

bouillon, short, straight rods which stain like the chicken cholera
bacteria ; upon agar, beautiful screws and bizarre threads (56, 11) ;
upon gelatin, almost only the latter are produced (56, Iv). They are
always without motility. With further cultivation the tenacity of
the cultures is rapidly reduced. Upon gelatin plates, when magnified —
eighty times, there occur minute, yellowish-brown, finely granular
colonies with sharp borders. Gelatin stab cultures resemble Strept.
pyogenes, the surface growth being minimal. There is no liquefac-
tion. Upon agar the growth is somewhat more luxuriant, and little
characteristic; there is a luxuriant growth in nutrient bouillon and
bouillon-agar mixture. There is no growth upon potato, and no
marked odor. It has no decided pathogenic action. Found in nasal
mucus and coating on tongue.

Vibrio lingualis. Weibel (C. B. iv, 227).

According to Weibel, this variety corresponds to the former in ab-
sence of motility and liquefaction of gelatin.

Microscopically: Vibriones and threads wavy in one plane, spiral
forms do not seem to occur. Gelatin stab culture is somewhat more
luxuriant than in the preceding. Upon gelatin plates the deep col-
onies present a finely threaded border, the threads being coiled and
matted, and the colony resembling anthrax to a certain extent. In
bouillon there is a flocculent precipitate. It is distinguished from all
other known vibrios in that it stains by Gram’s method.

2. Spirillum. Ehrenberg, emend. Loffler (C. B.
Vii, 634).

Long cells, bent into spirals, corkscrew-like, rigid, with
usually a unipolar (often bipolar) bunch of flagella.?

For a long time only two true spirilla were obtained in
pure culture and easily cultivated: Spirillum rubrum
v. Esmarch and Sp. concentricum Kitasato. Kutscher
(Z. H. xx, 46, and C. B. xvi, 614) and Bonhoff (H.
R. vi, 351) have widened our knowledge of the spirilla
species very much, by cultivating from fluid manure and
the feces of swine an entire series of spirilla which were
already partially known through E. O. Miller, Ehrenberg,

1 Zettnow (Z. H. xxiv, 72, and C. B. L. Iv, 389) has made careful
studies regarding the structure of this organism, through which he
was led to entirely different results from those of A. Fischer and
Migula, which we related on page 20. His results, on the contrary,
correspond with those of Biitschli, founded upon many low o isms:
lack of a distinct membrane, honeycomb structure of the entire cell,
with numerous granules lying within.

a ee

—

F
}
3 SPIRILLUM CONCENTRICUM. 377

and F. Cohn, but were never previously cultivated.
"Kutscher himself described part of the same with a flat
bend to the spiral as vibriones, in spite of the fact that
he had stained the terminal, thick bunches of flagella.
The isolation was accomplished by means of agar plates,
after a preliminary culture had been prepared which fur-
- Pnished a surface pellicle containing spirilla, according to the
_ method employed in cholera diagnosis. The colonies sus-
_ pected of being spirilla were torn with a fine platinum wire
under the microscope and then it was noticed whether
_upon slight magnification motion could be observed in the
_ drop of fluid which collected in the rent. If this was the
case, it could be conjectured that spirilla (or vibrios) were
7 being dealt with, since the ordinary micro-organisms of
" manure were almost all non-motile.

3

_ Kutscher employed as nutrient medium, meat infusion agar, neu-
tralized with soda, without any further addition. Zettnow finds the
_ additions of 0.1% ammonium sulphate and 0.1% potassium nitrate
_ to be practical, and gives detailed descriptions for the preparation of
F “spirilla-agar ”? (C. B. xx, 393).

; ‘Spirillum concentricum.! Kitasato (C. B. iii, 72).
3
; (Plate 55, VI-Ix.)

_ Short, more or less winding spirals, 1-8 » long and 0.5 »
thick, actively motile,? staining by Gram’s method (55,
IX).
_ Upon gelatin plates delicate, transparent growths, finely
punctated (55, vit). In the gelatin and agar stab a
_ spindle-shaped growth below the surface, similar to the
Spirillum rubrum, but yellowish. Upon the agar plate,
_ delicate (according to Kitasato, firmly adherent) colonies,
_ opaque and yellowish in the center, and at the border trans-
_ parent and finely granular (55, v1). Bouillon is rendered

__ ?The name was given by Kitasato on account of the very charac-
_ teristic cockaded growth in gelatin cultures ; our plates show nothing
of it.

; 2 In spite of every precaution our cultures never showed the active
_ motility observed by Kitasato. We have never tried to stain flagella.
_ Lofiler has described terminal bunches of flagella.

378 IMPORTANT VARIETIES OF FISSION-FUNGI.

moderately turbid. Milk is not coagulated. There is
neither formation of gas, nor H,S, nor indol.

On one occasion it was cultivated by Kitasato from
putrid blood.

Spirillum rubrum. v. Esmarch (C. B. i, 225).
(Plate 55, I-v a.)

Beautiful threads, more or less elongated or winding,
like a corkscrew, often as long as 16”; on an average,
1-3.2 » long and 0.6-0.8 » thick (55, v). They are motile
because of terminal bunches of flagella, and stain by
Gram’s method. Upon gelatin plates the colonies are at
first roundish, almost smooth-bordered, and later they
usually have concentric rings with a yellowish-gray cen-
tral part. The peripheral zones usually appear greenish
or reddish. The gelatin and agar stabs grow below the
surface in a spindle or cylindric form, being at first gray-
ish-yellow, later rusty brown to red (55, 1). In the agar
streak there is a very scanty surface growth (55, 1).
Upon the agar plate the colonies are transparent and
slightly crumbly (55, m1). Bouillon is rendered faintly
cloudy. Gelatin is not liquefied. No formation of gas
nor of H,S. Indol is produced in traces.

On one occasion cultivated by v. Esmarch from a dead
mouse. At first it was preferably an anaerobe; after con-
tinued culture in bacteriologic collections, it now also
grows well at times as an aerobe.

Spirillum rugula. (Cohn.) Lehm. and Neum.?

We may add to our remarks upon page 126 in accordance with the
investigations of Bonhoff. It is a true spirillum, with thick threads,
8-16 u long and 1.5-2, thick, and is provided with terminal bunches
of flagella. Prazmowski’s ‘‘spores’’ could not be demonstrated with
certainty as such by Bonhoff. Zettnow is convinced that Prazmowski
was deceived. Gelatin plate colonies resemble very much those of an-
thrax; gelatin is never liquefied.

1 There appears to be a certain similarity in the cultures to the
Vibrio III of Kutscher, which is a thick vibrio provided with bunches
of flagella.

1h Bs

SPIRILLUM TENUE. 379

Spirillum tenerrimum. Lehm. and Neum.

Spirillum I Kutscher (Z. H. xx, 46). Description according to
_ Kutscher: Short S-forms, very fine and thin, as a rule with three or
_ four turns. Flagella have not been stained. Gelatin plates present

characteristic colonies with a compact center; then a finely granular,
thinner zone, which carries a row of anastomosing rays at the edge.
In the gelatin stab the growth resembles that of mouse septicemia,

' ; and also a gradual liquefaction occurs from above. Upon agar plates

the colonies are like dewdrops. Slight cloudiness of nutrient media
without pellicle formation.

Similar to this is the organism Kowalski has called Spirillum
hachaize.! It is a fine spirillum, sometimes seen in the intestine of
cholera cases, but also in human dejecta in masses (also often by our-
selves in the stools of cases of suspected cholera). Regarding it, there
is a large amount of literature, but it is not of much value. Kowalski
(C. B. Xvi, 321).

Spirillum serpens. (E. O. Miiller.) Zettnow (C. B.
x, 689).

(Vibrio serpens O. F. Miiller, emend. Cohn and Kutscher.)

Quite large spirilla, thin, with usually three or four slight, abrupt
turns (the length of two turns is 5-6 “), and with a terminal bunch of
flagella containing as many as fourteen. In the gelatin plate culture are
formed macroscopically small starlets which resemble somewhat micro-
scopically those of symptomatic anthrax, but the rays at the periphery
are arranged more in a radiating manner, and are only slightly
matted. The growth gradually settles down, and in the stab some-
times is accompanied by the formation of an air space. Both upon
potato and agar it resembles Bact. coli. The nutrient solution is ren-
dered very turbid, sometimes with a delicate pellicle. Vigorous indol
reaction. Our picture (56, 1), magnified 1000 times, copied from Zett-
now, makes the organism appear very much larger than Cohn’s
description indicates. Our own descriptions correspond to this.

Spirillum tenue. Ehrenberg, emend. Cohn and
Kutscher.

Thin (0.8), markedly winding threads, usually with two to five
turns (4-15 ), with terminal bunches of very delicate flagella. The

1 Bonhoff makes the very surprising communication that these fine
spirilla are degeneration forms (older forms) of a short organism which
grows upon gelatin exactly like the Bact. coli, and, in young cultures,
presents the picture of the Bact. coli when magnified 1000 times.
The rods have two flagella at one end, do not grow on potato, give the
nitroso-indol reaction, do not coagulate milk, and form no gas from
grape-sugar (Hyg. Rund. vi, 1896, 351). Further communications
regarding this interesting organism are expected, but have not yet

appeared.

380 IMPORTANT VARIETIES OF FISSION-FUNGI.

gelatin plates show the deep colonies as yellowish, round, finely
granular, and sharply outlined; the superficial are similar, but more
spreading, thin films. The gelatin stab culture presents’ a delicate —
growth in the stab, and yellowish, abundant surface growth, with
gradual liquefaction and formation of an air space. No growth
upon potato. Nutrient fluid rapidly becomes turbid with a thick —
pellicle. As Kutscher also remarks, Beijerinck’s descriptions of three

sa

Ni

Fig. 19.—Spir. tenue Ehr., after Migula.

forms of Sp. tenue (C. B. L. 1, 1) are not sufficient for identification.
Bonhoff found one form deviating somewhat from Kutscher’s descrip- —
tion; for example, with only two flagella on each side.

Spirillum undula. Ehrenberg, emend. Cohn and
Kutscher.

Relatively large threads; usually 4 to 1, rarely 1} to 3 turns;
height and diameter of each turn, 4-5 wu. After longer cultivation
there are often scarcely any except straight forms. With terminal
bunches of flagella, three to fifteen in number. In gelatin plates there
occurs only in the depth a slow growth of sharply outlined, finely
granular colonies, beneath which the gelatin sinks a little. In the
stab culture development takes place in the upper two-thirds of the
stab; the growth on the surfacé of the gelatin is thin, whitish, slightly
lobulated, and after ten days sinks slowly into a depression. Grows
on potato. Nutrient fluids uniformly cloudy, without pellicle.

Recently Zettnow and Kutscher have differentiated from this Spir.
undula minus also a Spir. undula majus, which is about one-third
larger and grows well on meat-infusion gelatin and agar (C. B. XVIII,
614; XIx, 393).

Spirillum volutans. Ehrenberg, emend. Cohn and
Kutscher.

Not only the largest spirillum, but one of the largest varieties of
bacteria. The threads are about 2-3 y thick and spirally wound, the
height of a turn being 6.6 “, length 13.2 ~; usually there are 23 to 33
turns. In cultures the forms are smaller, similar to the Spir. rubrum.
According to Cohn, they have one large flagellum at each end; accord-
ing to A. Fischer and Kutscher, a terminal tuft of three to eight long
flagella, which are often plaited together. The colonies in gelatin
plates at first resemble those of Bact. coli; later the gelatin sinks in,
and the peripheral parts of the colonies break up. Agar plates are

SPIROCH ATE OBERMEIERI. 381

ke those of the Bact. coli. In the gelatin stab there is a feeble
rrowth; the surface growth is porcelain white, markedly lobulated,

and after ten hours sinks into a depression. Upon potato a dry
_ growth. Nutrient fluid uniformly cloudy, without a pellicle or with
a scanty one.

’ Spirillum stomachi. (Salomon.) L.andN.

_ Salomon has described (C. B. xrx, 433, ) a very interesting
"beautiful spirillum, which has not been cultivated, and is
' never absent from dogs’ stomachs. It is also found in cats
and rats, and can be readily transferred to mice by feeding.
. It occurs especially in the glands of the stomach.

3. Spirochete. Ehrenberg.

The cells are flexible, and present long, pointed, spirally
bent threads. Flagella are unknown. Motility is assigned
to an undulating membrane.

_ A key for their differentiation may be omitted, since
_ only two or three species are known.

Spirochzte Obermeieri. F. Cohn.!

*
!
(Plate 56, vii and 1x.)

Literature.—Obermeier (C. f. med. Wiss., 1873, 145); Koch (Mitt.
a. d. Ges.-Amte, I, 167); Soudakewitsch (A. P. v, 545); Cohn (Bei-
trage, 1, Heft 11, 196); literature by Afanassiew (C. B. xxv, 415).
. The personal investigations of these authors are not adapted for use
- in a text-book.

_ Bacteriologically very little is known. Large, flexible,

_ motile threads, coiled like a corkscrew, with pointed ends,

14 to 26 times as long as the diameter of a blood-cell,
usually 20-30 4. Flagella and spores are not known.

7 1 Sakharoff discovered, in the blood of geese suffering from an epi-
_ zootic disease in Caucasus, a motile but not flexible spirochzte,—
_Spirochzte anserina Sakharoff (C. B. x1, 203),—through which the
_ disease may be transferred to healthy animals. Details are given re-
_ garding it by Gabritschewsky (C. B. xxi, 365). It was not culti-
vated. The following may be simply mentioned: Spirochzte plica-
_ tilis Ehrenberg from marsh-water and the Spirochete of the saliva,
_ which have been often seen but never cultivated. According to F.
Cohn (Beitrage, Bd. 1, Heft 11, and Heft m1, pp. 197, 199), these
_ varieties are not to be distinguished microscopically from the Spiroch.
_ Obermeieri.

382 IMPORTANT VARIETIES OF FISSION-FUNGLI.

It is typically found in the blood and spleen of recur-—
rent fever cases, hardly ever during the afebrile periods”
(an exception proved by Naunyn); demonstrated by Kar-
linski to be the cause of a part of the cases of febrile
icterus (C. B. x1, 26).

It stains readily with the usual anilin dyes. Giinther
recommends that the dried and fixed preparation be pre-
viously freed of part of the albuminous bodies by means
of a1% to 5% acetic acid solution. It is not stained by
Gram’s method.

No cultures have so far been successful. According to
Pasternatzky, the spirocheete may be preserved alive for
about ten days if a leech is allowed to fill itself from a
case of recurrent fever, and then is kept upon ice.

Inoculation experiments have succeeded only upon man
and monkeys. The monkeys become sick after about
three and a half days, but present only the initial attack
of fever and no recurrence. Extirpation of the spleen
makes the disease more dangerous for the monkey.

Em my tage

:

APPENDIX I.

Actinomycetes.

For the limitation of this group and its genera, see
page 127.

We have conscientiously recorded all that is known ie
“us in the literature regarding forking, branching, etc.,

other forms up to this time considered as true bantene:
thus, B. pyocyaneum, B. influenz, B. tetani, B. radici-

cola, Vibrios,—the cladothrix form of B. murisepticum

was ‘immediately retracted by Kitt himself,—and we must
naturally acknowledge that these observations make it

more difficult to perceive in the branching a distinguish-

ing peculiarity between actinomycetes and fission-fungi.

- Innumerable similar difficulties are, however, encountered

in the definition of higher plant families—some genera

_ are often placed with equal propriety in one or another
family. If at a later time, because of further investiga-

- tions, the significance of branching should be construed in

_a manner different from that of to-day, it will still remain

true in any case that the actinomycetes of to-day, which

_ we have collected in part upon the basis of branching, will
_ form a perfectly natural family, even if their family diag-
nosis should be essentially remodeled.

1. Corynebacterium. Lehm. and Neum.
Cultures having throughout the character of cultures of

: true bacteria; soft, lying flat and loose upon the nutrient
- media. Stain well with the ordinary bacterial stains, but
are not acid proof. Microscopically: Rods, which fre-

quently present clubbed swellings at the ends, appear
more or less distinctly composed of differently staining
383

384 ACTINOMYCETES.

segments, and in many cultures present constantly, an
undoubted, true branching.

Key to the Recognition of the Most Important Vari-
eties of the Genus Corynebacterium.

1. Plate cultures upon gelatin: Colonies like those of Bact. coli or
typhi—i. e., roundish, and when magnified sixty times, with distinet
lineal markings; upon agar and serum-agar, just like Bact. coli.
Potato culture brownish-red. Cause of glanders. Corynebact. mallei
L. and N., page 384.

2. Plate cultures pen agar and serum-agar with very characteristic
granulation (splintery!). Growth upon potato usually slight. |
Colorless to yellowish.

(a) Very luxuriant growth upon the nutrient media, even upon
potato. Gelatin gradually discolored brown. Growth often yellowish, ©
sometimes brownish. Not pathogenic for animals. Usually little
acid produced in bouillon. Usually no granules in the rods when
stained by Neisser’s method. Corynebact. pseudodiphtheriticum
(Hofmann-Wellenhof ) L. and N., page 404.

(6) Growth of medium intensity upon agar, and best upon serum-
agar; poor upon gelatin and potato. Vigorous production of acid in
bouillon. Usually granules in the rods when stained by Neisser’s
method. Pathogenic for man and animals. Corynebact. diphtheriz
Loéffler, L. and N., page 389.

(ce) Seanty erowth upon nutrient media. No production of acid in
bouillon. No granules staining by Neisser’s method. Not patho-
genic. Corynebact. xerosis (Neisser and Kuschbert) L. and N.,
page 406.

The close relationship of Corynebacterium diphtheriz with Coryn.
pseudodiphtheriticum and Coryn. xerosis permits no certain recogni-
tion from this key alone. (Compare p. 403. )

Corynebacterium mallei. (Loffler and Schiitz.)
L. and N.

(Plate 57.)

Common Names.—Glanders bacillus; German, Rotz;
Latin, malleus; French, morve; English, glanders. Bacil-
lus mallei Fliigge.

Principal Literature.—Loffler (A. G. A. 1, 141). Kranzfeld (C. B.
I, 273). Kitt (C. B. m1, 241).

Microscopic Appearance.—Slender rods (2-3 u long,
0.4 » thick), sometimes with brightly shining bodies
(metachromatic bodies), which may be shown very well

a CORYNEBACTERIUM MALLEI. 885

+

by means of Neisser’s staining method for diphtheria

granules. ‘True endogenous spores are never present ; all

“previous positive statements to the contrary are errone-

ous. In old cultures clubbed, vesicular enlargements

which create the impression of involution forms are often

seen; also long threads, which sometimes exhibit true

branching (57, x11) in great abundance. See Semmer (C.

| B. xvi, 68). Dissertation by Erich Wolf, Wiirzburg,
1898, and Marx (C. B. xxv, 274).

Staining Properties.—Somewhat difficultly with ordi-

“nary stains; does not stain by Gram’s method. For

_ staining the bacteria in sections, Nicolle’s method is to be

recommended (Technical Appendix).

Pie po

Requirements as Regards Composition of Nutrient

Media, Supply of Oxygen, and Temperature.—Grows

_ best at incubator temperature (minimum, 25°; maximum,

)

40°). Prefers glycerin-agar to ordinary agar, but is not

particular. Grows well aerobically, poorly or not at all

~ anaerobically.

Gelatin Plate.—(a) Natural size. Superficial and deep

; colonies: Small, whitish, punctiform; also after a long

time they do not become essentially larger. The super-

ficial ones have a delicate, transparent halo (57, v).

(b) Magnified sixty times. Superficial : Irregularly round-

ish; scalloped, wavy margin ; shining white, transparent,
_ with wavy elevations and marked reflex. Old colonies
_ are more yellowish, especially in the center, with linear,

depressed markings. They are very similar to colonies of

_ B. typhi and putidum in the early stages (57, vu, e).

Deep: Roundish or oval; sharply outlined ; in the center

_ delicately crumbly, at the outer part streaked. The

peripheral zone is sharply marked (57, vr).

Gelatin Stab.—Stab: Thread-like ; sometimes faintly
granular, sometimes like a string of pearls; gray. Sur-
face growth: Exceedingly delicate, perfectly transparent,
gray, with a ragged fringe, and of a dull luster (57, 1).

Agar.—Not distinguishable from Bact. coli; entirely
non-characteristic (57, Iv, vir). Fora year we cultivated a
form of the Cory. mallei, which occurred spontaneously and
which produces rusty-brown colonies upon agar. This is

25

386 ACTINOMUYCETES.

a counterpart to the rusty-colored streptococci mention
on page 137.

Bouillon Culture. — Almost clear, moderate homo-
geneous sediment, which rises up uniformly upon shaking.

Milk. —Slowly coagulated. 7

Potato.—At first there is a light yellow or brownish
growth, with a moist luster, scarcely at all‘or slightly ele-
vated, lighter at the edge, not sharply outlined (57, x).
After a longer time: brownish-yellow or brownish-red,
smooth, wavy border, sharp outline, the periphery still
being paler. The potato becomes discolored (57, 1x)z
The culture has much similarity to that of the Vibrio
cholere. Cultures upon carrots present a white growth
and were employed by Marx (being protected from dry-_
ing) for his studies upon branching.

Resistance to Drying.—Slight. At 25° dead in ten
days (Bonome). According to Bonome, it withstands 70°
for six hours without injury (!) ; 70°-75° kills in five or
six minutes ; 90°-100°, in three minutes. .

Chemical Activities.— Except for the formation of pig-
ment upon the potato and a trace of indol in bouillon,
nothing is known beyond the formation of mallein (bac-
terioprotein). Forms no H,S. No gas is formed from
carbohydrates.

Distribution.— ;

(a) Outside the body: Never has been found.

(b) In healthy body: Never has been found.

(c) In diseased human organism: Man is fairly suscep-
tible to glanders, almost always the transfer occurring from
horses. About 50% of the cases die. The bacteria are
found in the secretion from the ulcers of glanders and in the
glanders nodules. The principal places of infection are the
skin and mucous membranes. The glanders bacilli also
enter the uninjured skin through the hair follicles and
spread in the lymph-spaces. Chronic glanders also occurs
in man, although very rarely.

(d) In anwmals : Of our domestic animals the following
are attacked : Horse, ass, cat (and the wild canines of the
zoological garden); according to infection experiments,
also dogs (especially in the young), goats, sheep, rarely
swine are susceptible. Cattle and birds are immune. Ac-

CORYNEBACTERIUM MALLEI. 387

ording to Schiitz, there is no primary pulmonary glan-
ders ; on the contrary, the lungs are always affected secon-
a ily from the skin or mucous membrane. The primary
jort of entry in the skin or nasal mucous membrane is
xiten already healed when the pulmonary glanders begins.
According to Nocard, the transparent gray nodules in the
lung, which show a tendency to calcify, are due to glan-
ders infection. Schititz has (always?) found a small round-
worm in them, and denies that they are connected with
landers (C. B. xxi, 901).

_ Experimental Observations Regarding Pathogenic
Effects.1—(a) On animals: For experimental purposes
the guinea-pig is best, and next the field mouse (Arvicola
arvalis) (L6ffler). "Also the following may be used
Kitt): Mus sylvaticus (wood mouse) and Arvicola am-
phibius (water rat). The rabbit is slightly susceptible.
Immunity exists in the case of gray and white house
mice? (Léffler) and rats. Experiments upon cats and
dogs have more disadvantages than advantages.

_ The most important animal experiment is the injection
of 2c.c. (not too little) of a suspension of the pure cul-
ture or of the crushed, suspected organ through the median

guinea-pig (Straus, Arch. de Med. exp., 1, 1889, 460).
After forty-eight to seventy-two hours there is presented a
‘marked swelling, redness, and tenderness of the scrotum .
as a pathognomonic symptom of the successful transfer of
glanders. The swelling is dependent upon the formation
‘of numerous glanders nodules upon the tunica vaginalis
testis, the two layers of which are stuck together by a
purulent exudate; and glanders nodules also occur inside
the testicle. After twelve to fifteen days, sometimes even
four to eight days, the animals die, before which suppura-
tion in the testicle may have discharged externally. To
expedite the diagnosis, the diseased testicle may be exam-
‘ined even before the death of the animal by means of
potato cultures, etc. Subcutaneous injections are not to

1 Experiments are permissible only in well-equipped laboratories
and with most extreme precautions

_ # According to Shattock, they become sick only at a later time, and
die after two or three weeks (C. B. XXvV, 323).

388 ACTINOMYCETES.

be recommended in guinea-pigs, as the abscesses whick
form primarily imperil the experimenter by opening exter-
nally, and death occurs only after twenty-five to thirt

days (also here almost always the testicles are diseased).

(b) Upon man: No purposeful experiments have been
made with glanders bacilli in man. A number of fatal
laboratory cases indicate the great danger for man of the
pure culture.

Special Methods for Demonstration and Cultiva-
tion.—Acute cases of glanders in horses are usually noi
difficult to diagnose from the clinical symptoms. ‘
diagnosis in subacute and chronic cases is harder and ofter
very difficult, even after autopsy and with the additiona
help of bacteriologic aids. . 3

(A) In the case of living animals the following is recom
mended :

1. Mallein—the protein of the glanders bacillus—is
injected subcutaneously. While healthy animals remain:
afebrile or show only a slight fever of reaction, those
affected with glanders usually show a gradual elevation of
the temperature of 1.5°-2° ;! and after it has remained at
the highest point for a short time, it gradually falls. At
the point of the injection there remains a swelling for
several days if the animal is affected with glanders. The
method furnishes no absolutely certain diagnostic proof,
since sometimes the febrile reaction occurs in healthy indi-
viduals, or remains slight in diseased ones. Most authors
recommend it highly. ? ;

2. The suspected nasal cavity is wiped out with a
cotton swab and 1 c.c. of a suspension of the material
thus obtained is injected intraperitoneally into a guinea-
pig (see p. 387).

3. One of the swollen, paratracheal lymph-glands | is

1 The elevation of temperature is the more significant, the higher
the original temperature. An elevation of more than 2° with a high
initial temperature is fairly certain proof. An elevation of temper-
ature not over 1.1° indicates an absence of glanders ; 1.2°-1.9° is sus-
picious. See Eber (C. B. x1, 20).

2'The experiences of Prof. Schiitz make one especially skeptical.
This is particularly true in his latest results with 64 horses: 9 out
of 61 healthy horses reacted, while the 3 with glanders did not !

4

extirpated and smear cultures prepared from the same
(incubator) :
~ (a) Upon potato (brown color of the growth).

(6) Upon glycerin-agar.

Also a microscopic preparation is made, and, further, a
guinea-pig is injected.
- (B) In the case of living men: The secretions from
_ glanders ulcers are best examined by infections of guinea-
pigs.
j (C) In animals at the autopsy :
1. Cultures and animal investigation with fresh, crushed

glanders nodules.

2. Staining of sections of glanders nodules (difficult).

_ Kutscher (Z. H. xxt, 156) has described an interesting pseudo-

_gilanders bacillus. It grows similarly to cholera upon gelatin, lux-

uriantly upon agar, white and dry upon potato. Microscopically it

_ resembles the B. mallei absolutely, but stains by Gram’s method. It

is interesting that, if injected intraperitoneally according to the
method of Straus, it produces a swelling of the testicle in male
guinea-pigs, as the B. mallei does. The swelling is due more to

_ nodular swelling of the coverings of the testicle than to swelling of its
substance. The animals usually die after four or five days, when a
peritonitis (often hemorrhagic) dominates the picture. There are no
nodules in the other abdominal organs, but the omentum is always
rolled up and highly inflamed.

CORYNEBACTERIUM DIPHTHERLE. 389

Corynebacterium diphtheria. (Loffler.) L.andN.}
(Plates 58, 59, and 60. )

Synonym.—Bacillus diphtheriz Loffler.
Common Names.—Diphtheria bacillus, Léffler’s ba-
cillus. ‘‘ Léffler.’’ ;

Literature.—Loffler, Mitt. a. d. Ges. Amt., Bd. 11. Complete list

1 The statement of Zupnik (Berl. klin. Wochenschr., 1897, No. 50),
that the diphtheria bacillus may be separated into two varieties, could
not be verified by Slawyk and Manicantide (Z. H. xxix, 181). Zupnik
divides them as follows:

(a) Relatively large, flat, dull agar colonies, of irregular contour.
They stain by Gram’s method, are non-motile, and are fully virulent
for guinea-pigs. Bouillon is not cloudy and only presents the forma-
tion of a pellicle.

(b) Smaller agar colonies; circular, conically elevated, shining.

They do not stain by Gram’s method, are sluggishly motile (!), and
in guinea-pigs produce only infiltration and necrosis, and never death.

390 ACTINOMYCETES,

of literature up to 1894 is found in the thorough work of Escherich
Aetiologie und Pathogenese der epidemischen Diphtherie, Wien, 1894,
Latest literature: Heinersdorff, Arch. f. Ophth., Bd. 46, p. 1. Espe-
cially important also are: Neisser (Z. H. xxiv, 443) and Kurth (Z.
H. xxvii, 409). ©. Frankel(Berl. klin. Wochenschr., 1897, 1085).
Zupnik, J. e.

Microscopic Appearance.—Slender, rather long, rods, —
often a little bent and usually somewhat swollen at one or
both ends. Many times they are arranged in pairs. With —
Escherich, the following forms may be distinguished:

1. Wedge-shaped rods, about 1.5-2 » long, about
0.5 » thick (60, 1, Iv).

2. Long cylindric rods (especially upon agar and po-
tato) (60, 1), 3-4» long, 0.4-0.5 » thick.

3. Rods with clubbed swellings (especially upon serum),
as much as 6-8» long. The clubs reach a diameter of
1.0 » (60, 11). .

In 1 and 8 the thin ends are often long and drawn out
to a point. Thesame culture upon alkaline bouillon forms:
long clubbed rods; upon acid bouillon forms short, wedge- —
shaped rods. The short forms are more often parallel in
arrangement; the long, more at angles, and arranged in ©
rosettes like fingers, etc. — 7

According to Kurth, the probability of the form under ~
observation being pathogenic is increased if it can be ©
established that in contact preparations, from young cul-
tures (six hours at 35°) on Léffler’s serum, there are —
present at least a number of longer forms (seven times as —
long as thick) or V-shaped forms. Further, Kurth
attaches value to an appearance of the young rods being ©
so arranged as to suggest the fingers of two hands, spread —
out upon each other.

They have recently been often observed to grow into
unbranched threads (in part with clubbed swelling at the
ends), and even into branched threads (Babés, Klein, C.
Frinkel, C. B. xvi, 896). We have also possessed cul-
tures which presented striking branching forms in pre-

Bouillon is first rendered diffusely cloudy, then becomes clear beneath
the pellicle.

Slawyk and Manicantide found thirty completely investigated
pathogenic cultures to correspond to the plan, only many of them
presented more of the smaller, glistening, elevated agar colonies

|
*

CORYNEBACTERIUM DIPHTHERIA. 391

-ponderance (60, xm). The other forms represented in

: Plate 60, v-1x, also occur in true diphtheria, the short
forms especially in very young cultures.

_ Schiitz found the best formation of branches in different
cultures to occur sometimes upon albumin, sometimes on
glycerin-agar; also, in distinction to C. Frankel, he often
observed beautiful branching in bouillon (Berl. klin.
Wochenschr., 1898, 297).

Motility.—Never present. We have never seen any
motion at all.

Staining Properties.—Stains with all the anilin dyes,
especially in young cultures; also by Gram’s method.

_ Gram’s method, especially as modified by Czaplewski, is.

_ to be recommended for the study of smear preparations

_ from diphtheria material; carbol gentian-violet and Gram’s

solution of iodin are used. (See Technical Appendix. )

Carbol-fuchsin and anilin gentian-violet solution stain
very intensely, without revealing the finer structure.
Staining with warm Léoffler’s methylene-blue solution and
differentiating in water reveals a very characteristic struc-
ture in the bacilli. They consist of alternating sections of
intensely and faintly stained substance surrounded by a
delicate envelope of faintly stained material. This is most
marked in older cultures on blood-serum. Very young
bacteria stain uniformly blue.

Metachromatic Granules.—Max Neisser has pointed
out that the occurrence of metachromatic granules permits
the differentiation of the diphtheria bacillus from many
related forms. According to Neisser, cultures are employed
which are made upon Léffler’s serum and kept at 35° (not
warmer) for nine to twenty hours (in older cultures the
granules disappear in part). The dried preparation is
stained for one to three seconds (Auckenthaler found ten
to fifteen seconds to be often better) with acetic acid methyl-
ene-blue solution (Technical Appendix), washed in water
(tap-water should only be used if it does not contain much
free CO,), and then counter-stained three to five seeonds
with a weak solution of Bismarck brown (Technical
Appendix). There is then observed a blue granule at one
or often at both ends in a majority of the brown-stained
bacilli; not infrequently there are more than two such

392 ACTINOMYCETES.

granules (60, x, x1). Nevertheless virulent diphtheria
bacilli without granules occur, although very rarely (see —
Kurth, /. ¢.), so that a lack of the granules does not y
exclude the diagnosis of diphtheria. (See also p. 402.)

Relation to Oxygen.—Optimum growth with entrance _
of air; when oxygen is excluded, the growth is lessened. —

Requirements as Regards Temperature, Reaction, _
and Composition of the Nutrient Medium.—lIt grows _
well and abundantly at incubator temperature only. ~
Optimum temperature 33° to 37°; extremes, about 18°-20°
and 40°. Glycerin-agar is more favorable for its growth
than ordinary agar, but serum or ascitic nutrient medium
are much better. Ldoffler’s blood-serum mixture is much —
used (Technical Appendix); also Tochtermann’s and ~
(Deyke’s nutrient media are highly recommended (Techni- ~
cal Appendix). Since we have used glycerin-ascites-agar
almost exclusively instead of glycerin-agar, we have -
obtained excellent results, but one must become accus- —
tomed to the relatively luxuriant appearance of the growth.
Upon gelatin at 22°-24° the growth is so absolutely with-
out characteristics (no liquefaction), and so scanty, that —
such cultures are never prepared.

Gelatin Stab.—Along the stab canal only a slight
growth. The surface growth is yellowish-white, a little
elevated, with a smooth wavy border and in part lobulated.
It is faintly shining.

Glycerin-agar Plates.—(a) Natural size: Circular or
roundish colonies, white to dirty yellow. The border is
smooth, they are more or less elevated, and with a moist
or faint luster. Many cultures present more luxuriant
(58, vir a) and many more delicate growths (58, vm 0).

(b) Magnified sixty times: The colonies present their char-
acteristic form after twenty-four hours at 37°. They are
small, roundish, usually exceedingly transparent colonies
of a grayish-yellow or brownish color. At the periphery
they are usually split or torn, and almost without excep-
tion are markedly crumbly. Many colonies appear at the
periphery as if raveled out. Still, according to the cul-
ture, they are thinner or thicker, lighter or darker, coarsely
or finely granular (59,1 @ and 6). After two days the
colonies are thicker, somewhat irregular at the periphery,

CORYNEBACTERIUM DIPHTHERLZ. 393

and, when magnified a little more highly, distinct single

rods are seen to project at the edge. The center is opaque

-yellowish-brown (59, 1 a and 6). In still older cultures

dark irregular spots occur, the colonies become yet more
crumbly, the periphery more torn, and the inner part
more opaque (59, ur). Colonies occur, however, espe-

cially upon better nutrient media (ascites-glycerin-agar),

which are rounder, thicker, and therefore more opaque
from the first, and finely granular (59, via and 6). After
a longer time such luxuriant colonies resemble perfectly
those of cocci or sarcine (59, vit). Also all the other
forms reproduced in Plate 59 may occur as they are found

_ in closely related non-pathogenic forms.

Glycerin-agar Streak.—The same may be said of it as
of the growth upon glycerin-agar plates. There occur also
here more luxuriant and more delicate forms (58, I and
m1). Especially upon glycerin-ascites-agar the cultures
are sometimes so luxuriant that they resemble those of the
Bact. coli or micrococci. (Compare 58, m1 and Iv.) In
many cases after two to six weeks the agar shows a brown
discoloration.

Blood-agar Streak.—Very good growth.

In raw hens’ eggs there is abundant growth, and upon cooked
white of egg there is a relatively luxuriant growth.

Serum Culture.—Léffler’s coagulated blood-serum

_ mixture is often employed for cultures. It consists of the

serum of calves or sheep (or slightly alkalinized serum of
cows) to which is added one-third its volume?! of neutral
veal bouillon (containing 1% peptone, 1% grape-sugar,
and 0.5% sodium chlorid). We find this nutrient medium
to possess about the same advantages as glycerin-ascites-
agar.

Bouillon.—After twenty hours a cloud is deposited,
either in the form of fine, dust-like granules upon the
sides and bottom of the tube, or (and what most authors
give as most frequent, but Escherich only seldom found
in Gratz) fine flocculi form, which are easily precipitated,
and, upon shaking, rise again. Both types are connected

1 Escherich recommends one-fourth or one-fifth in order to insure
certain solidification of the serum upon heating.

“Fh
|
394 ACTINOMYCETES. .
by transition forms. Young cultures usually present
delicate, old ones thick pellicles.

Alkaline bouillon first becomes acid, then alkaline ©
again, the latter change being favored by passing air
through it. (See Chemical Activities.) The diphtheria
bacilli grow poorly upon bouillon which has been long
stored, and in such a case the nutrient value is increased
by boiling it (Escherich).

Milk.—Luxuriant growth usually occurs without coagu-
lation. They live a long time. Reaction amphoteric.
According to Schottelins, this is especially true of raw
milk, cooked milk being much less favorable (C. B.
897).

Potato.—Upon acid potato very poorly or not at all:
upon alkaline potato after eight to fourteen days, a very
scanty growth. It appears only as a delicate, shining,
sharply limited veil, which sometimes may be lifted with
a platinum needle. A more luxuriant growth of the
diphtheria bacillus upon potato occurs, although only
rarely (58, Ix).

eee ee ee

Special Nutrient Media.—In non-albuminous urine (Guinochet)
which has been sterilized and rendered faintly alkaline the diphtheria
bacillus grows slowly, but it is pathogenic. Schloffer (C. B. xiv, 657)
recommends urine-agar (a meat infusion-peptone agar—2%—is mixed
with fresh, sterile urine). According to Gamaleia, a good nutrient
medium contains glycerin, 40 parts; meat extract, 5 parts; sodium
chlorid, 5 parts; and water, 1000.

Spore-formation does not occur.

Viability.—(a) In the body: It is found in the throat
for weeks or even for two months after convalescence from
diphtheria in many cases (Léffler, Abel).

(6) In cultures: If kept cool and in the dark, for from
six months to one and one-half years. In the incubator
they usually die after one to three months because of
drying. In well-closed bouillon cultures they remain
alive also in the incubator for one year or longer.

(c) In water and foods: See Montefusco (C. B. xx1, 352). -

Resistance to: (a) Drying: Very considerable. Pure
cultures on silk threads in the room remain alive three or
four weeks, and under favorable conditions for months.
In dried diphtheria membranes they live as long as three

ee ee .-o

CORYNEBACTERIUM DIPHTHERIZ. 395

months. Even when dried so that it may be pulverized

into dust, the bacteria remain alive and infectious (Ger-

- mano).

(b) Moist heat: They are rapidly killed at 60°, and ina

_ few hours at 50°.

(c) Cold: When dried, many individuals bear the cold
of the German winter for two and one-half months with-

out reduction of virulence (Abel); according to Kasansky,
- cultures endure the Russian winter for months.

(d) Light: While germs suspended in water are destroyed —
in a few hours (two to eight hours) by direct sunlight,
agar and especially bouillon cultures stand the sunlight
for six hours very well.

Chemical Activities.—(a) Formation of gas and acids
from carbohydrates: From grape-sugar, even from the
minute quantity found in ordinary bouillon, easily demon-
strable acid is produced; also similarly from glycerin.

The increase of acid produced by typical diphtheria
cultures in 5 c.c. of non-saccharine bouillon after twenty
hours usually amounts to 1.2-1.5 c.c. of 1:40 normal
sodium hydroxid; after forty hours, 2.5-3.0 c.c., phenol-
phthalein being used as indicator. In 1% sugar bouillon
we found about twice as much acid formed : 2. e., 2.6-3.8
in twenty hours and about 6.0 in forty hours. Kurth,
like Spronck, proposes that 0.2% grape-sugar be always
added to bouillon, since he often obtained bouillon which
contained too little sugar.

(6 and ¢) Production of H,S is slight. Indol is always
produced.

_ (d) In older cultures there is some nitrite, so that the
‘*cholera-red reaction’’ is obtained with sulphuric acid
alone (Palmirski and Orlowski).

(e) Chromogenesis: Rarely there occur yellow to red
cultures. (Zupnik, Frankel. See p. 405.)

(f) Toxins: Old bouillon cultures filtered through clay
produce symptoms identical with those following inocula-
tion of the diphtheria bacillus itself 1! (Roux and Yersin).

1 The fibrinous exudate alone is lacking at the place of inoculation.
Often there occurs albuminuria, diarrhea, and very irregular action of
the heart. During the course or after the disappearance of the acute
symptoms paralyses occur, especially in the more resistant animals:

396 ACTINOMYCETES.

According to Dungeren, especially active toxins are ob-
tained by the addition of ascitic fluid to bouillon (C.
B. xix, 137). The addition of sugar to bouillon is to be
avoided (Sprouck, A. P., 1895, 758). So long as bouillon
cultures are of acid reaction they contain no toxin ; usu-
ally the toxic action corresponds to the increase in alka-
linity (Hilbert, Z. H. xxrx, 157), but not always (Mad-
sen, Z. H. xxvi, 157). According to Roux and Martin,
the formation of toxin is favored by the entrance of oxygen
(large surface of bouillon). Regarding this point, see
also Hellstrém (C. B. xxv, 217).

‘The toxins are precipitated by alcohol, and are scarcely
at all dialyzable. Precipitates of calcium phosphate
(from the addition of calcium chlorid to bouillon) carry
them down also. Temperatures above 60° rapidly reduce
the toxicity. With alcohol and vacuum apparatus the
toxins may be obtained asa powder. Toxins are produced
not only upon albuminous, but also upon non-albuminous
nutrient media, alkaline urine (Guinochet), and Uschin-
sky’s nutrient medium (p. 75). According to H. Kossel,
the diphtheria toxin is formed in the bodies of the micro-
organisms and at once secreted (C. B. x1x, 977).

The bodies of the bacteria contain no large quantity of
toxin. Regarding the chemistry of the toxins, see page 73;
also, regarding their resistance and other properties, see
Fermi (C. B. xv, 303). For the most recently advanced
division of the diphtheria toxins by Ehrlich—prototoxoid,
syntoxoid, epitoxoid—the original article must be consulted
(Deut. med. Wochenschr., 1898, 597). In distinction to
tetanus, the emulsion of the brain and spinal cord of sus-
ceptible animals has no antitoxic action against diph-
theria toxin (Bomstein, Aronson).

Distribution.—(a) Outside the body: Upon things used
by the diphtheria patient (linen, brushes, playthings, walls
and floor of room). On the hair of nurses. The air never

rabbits, pigeons, dogs, cats, rarely guinea-pigs. Most characteristic
are the paralyses which first appear after apparent recovery of the
animal from the acute symptoms of intoxication (post-diphtheritic
paralyses). The susceptibility of animals to the diphtheria poison is
much increased by hunger, exhaustion, ete. (Valagussa and Ranel-
letti, C. B. xxXIv, 752).

CORYNEBACTERIUM DIPHTHERLA. 397

contains living diphtheria bacilli (except from a moment-
ary contamination by the coughing of the patient—Fligge).

(6) In healthy body: Sometimes found in the mouth and
nasal cavities, also in the conjunctival sac of healthy per-
sons, especially in those coming in contact with diphtheria
eases. In a diphtheria epidemic in a barracks, Aaser
found diphtheria bacilli in the throats of 19% of the occu-
pants who were healthy.

(c) In diseased human organism: Are found without ex-
ception on the outer side (the side toward the cavity of
the mouth) of the diphtheritic membranes! of recently
affected men, and with more difficulty and less regularly
in chronic cases.

Principal localizations: Throat, nose, larynx, trachea ;
more rarely, stomach, defects in skin and muscle (wounds),
and vagina.

The wide-spread assumption that the diphtheria bacilli
are to be found only at the local seat of disease is un-
founded. Lately they have been found rather frequently
(also in man) in the blood and internal organs, especially
the spleen and kidney (Frosch, Z. H. xin, 49; Nowak,
C. B. x1x, 982). Recently also rhinitis fibrinosa, conjunc-
tivitis crouposa (severe and very mild forms), and many
middle-ear suppurations have been traced to the diph-
theria bacillus.

Almost regularly the Streptococcus pyogenes accom-
panies the diphtheria bacillus (Léffler) and in the patho-
logic process plays a synergistic role.

Regarding the significance of mixed infection, Bernheim
has ascertained :

1. The metabolic products of the streptococci favor the
growth of diphtheria bacilli and increase the virulence ;
also the production of toxin by the diphtheria bacillus is
increased (Hilbert, Z. H. xx1x, 157).

1 Diphtheritic angina also occurs without formation of membrane.
On the other hand, not rarely clinical ‘‘ diphtheria cases,’’ in spite of
a perfectly typical local symptom-complex, present no diphtheria
bacilli (according to Escherich, in Gratz about 25%). A number of
other organisms (for example, streptococci) can cause the symptoms of
diphtheria of mucous membranes. The mortality in these cases is
minimal. Also ‘‘ wound diphtheria ’’ may depend upon streptococci
or Bact. coli,

398 ACTINOMYCETES.

2. Mixed infection with streptococci and diphtheria
bacilli is more dangerous for the animal than pure diph-
theria infection.

Nevertheless the diphtheria bacillus alone may undoubt-
edly produce all the clinical symptoms of sepsis (Gener-
sich).

(d) In animals: Certain spontaneous disease produced
by Loffler’s bacillus has never been observed in any ani-
mal. The susceptible guinea-pig is immune to the diph-
theria bacillus introduced by feeding, by inhalation, or
by swabbing. Spontaneous disease (diphtheric broncho-
pheumonia) is said to occur in cats (E. Klein, C. B. vm,
7). Klein claims also to have observed spontaneous diph-
theria in milk cows, in which, moreover, the diphtheria
bacilli escaped in the milk. rie

The spontaneous diphtherias of hens, pigeons,+ and
calves always (?) have other causes. (Compare Léffler,
Mitt. G. A. m1; Ritter, H. R., 1896, 839). |

Still, certain of the causes of ‘‘animal diphtheria’’
appear to be transferred to man. Consult the well-known
observation of Gerhard (II. Kong. f. innere Med.), and
also Galli-Valerio (C. B..xxu, 500: extensive critical
review of literature).

Experimental Observations Regarding Pathogenic
Effects.—(a) Upon animals: The virulence of freshly iso-
lated cultures varies greatly; in general, severe cases fur-
nish virulent cultures and mild ones cultures with slight
virulence; still, there are exceptions. Experimental and
accidental (cultural) attenuation is often observed. Roux
and Yersin assert that there occurs a regular, striking re-
duction of virulence in the last few diphtheria bacilli
demonstrable during convalescence. It was not found so
by Escherich, and still other writers cultivated virulent
bacilli from convalescents long after the clinical symptoms
disappeared. A good standard for the virulence of a cul-

1 Gallez claims to have positively demonstrated in Belgium that,
besides the ‘‘ fowl diphtheria,’’ which has nothing to do with human
diphtheria, there is also a ‘‘ fowl glanders,’’ which is caused by attenu-
ated Loffler’s bacilli (H. R., 1896, v1, 472).

TP

CORYNEBACTERIUM DIPHTHERLE. 399

ture? consists in the toxicity of the filtrate of a culture of
_ a certain age.

In the interest of rapid work, Escherich recommends

for the estimation of virulence a statement of the quan-

tity, expressed in percentage of the body-weight, of feebly
alkaline twenty-four hours’ bouillon culture which just
suffices, when introduced subcutaneously, to kill a guinea-
pig with acute diphtheria. With 1.5 ¢c.c = 0.5% of the
body-weight, Escherich never obtained a negative result;

with his most virulent cultures, 0.1 to 0.3 c.c.—z72. ¢.,

about 0.05%—sufliced. Aronsohn has cultivated still
more virulent bacteria, of which 0.02% to 0.025% of
bouillon filtrate was certainly fatal.

Also, for infection experiments ? the best animal for use
is the guinea-pig. Death is caused by 0.02 c.c. of a viru-
lent culture in two days; by 0.01 c.c. in three or four days.

Usually 0.5 to 1c¢.c. is injected. About twenty-four hours

_ after the subcutaneous injection the following picture
_ develops: The animal is weak, without appetite, the hair
_ bristling, snout cold and bluish, respiration very harsh.
There is infiltration at the place of injection, and often

also for some distance beyond. Death occurs after twenty-
four to sixty hours. There may be entire absence of

special symptoms of disease except loss of weight.

Autopsy: At the point of injection a whitish infiltration
surrounded by hemorrhagic edema, and, in chronic cases,
callosities discolored by hemorrhage. The most important

_ changes in the internal organs are: Suprarenal capsules

hyperemic; exudate into pleuree, often also into pericar-
dium; spleen unchanged; often parenchymatous nephritis

and myocarditis. The upper part of the intestine is red-

ek i |

dened. Escherich observed cultures with which the in-
oculation was never followed by pleural exudate. In

1See De Martini (C. B. xxiv, 420) regarding occasional discrepan-
cies of toxin formation and infectiousness in the same culture.

2In order to recognize diphtheria bacilli of doubtful and very
slight virulence as still virulent, Trumpp injects them simultaneously
with a sublethal dose of diphtheria toxin. The animal must die, in
contrast to a control animal, and with reinoculation of definite quan-
tities into new animals the virulence must constantly increase, so that
finally the inoculated animals die without any additional diphtheria
toxin (C. B. Ts 721).

400 ACTINOMYCETES.

these experiments an increase of the bacteria occurs almost —
exclusively locally, and only rarely can they be cultivated —
from the internal organs. ;

Subchronic and chronic cases (death sometimes occur- —
ring only after months) present changes in the internal —
organs which are less marked, or no alterations at all are —
found. At the point of injection all changes may. be lack- ©
ing or ulcers may follow necrosis of the skin. The animals —
are always emaciated and very much reduced in weight.
Escherich never saw postdiphtheritic paralysis in experi- |
mental animals; other authors have occasionally.

Rabbits are much more resistant to subcutaneous inocu-
lation than guinea-pigs; white mice and rats are almost
immune. On the contrary, cats, dogs, and cows are sus- —
ceptible. Of birds, young pigeons and small birds ©
(finch, siskin, etc.) are especially susceptible; hens less,
and only when young. :

Diphtheritic diseases of mucous membranes analogous ~
to those observed in human diphtheria may be produced
by rubbing diphtheria bacilli into the slightly injured (not —
the uninjured) mucous membrane of the trachea and con-
junctiva of rabbits, of the throat of monkeys, of the
throat and larynx of pigeons and hens. The disease pro-
cess and membrane formation remains local. The best
results follow inoculations upon the vaginal mucous mem-
brane of guinea-pigs (Léffler): If one pulls apart the
vagina, which is always feebly adherent, and places a pin-
head-sized quantity of diphtheria bacilli upon the mucous
membrane, which has always received a minimal injury —
in the manipulation of separation, on the following day
there is marked redness and hyperemia, and after forty-
eight hours the formation of a thin, closely adherent cover-
ing can be demonstrated. This infection may terminate
in recovery or death.

Roger and Bayeux produced, by the injection of + to 1
drop of diphtheria poison into the trachea of rabbits, beau-
tiful diphtheritic membranes, while guinea-pigs die too
soon for it to appear.

(6) In man there have been no experiments.

Immunization.—Animals may be immunized against
diphtheria bacilli:

me Tee aii i ite eld ;

QR a= =a eo ee ee eee ee eC
a a see

CORYNEBACTERIUM DIPHTHERIA. 401

1. By treating first with slightly virulent and later with

highly virulent cultures of diphtheria bacilli.

2. By the injection of diphtheria toxins in small quan-
tities or toxins partially weakened by heat, and following
with larger quantities. This is repeated with increasing
doses.

3. By injection of serum from an animal immunized
against diphtheria.

Also, in man, prophylactic injection of immune serum
has been employed when there was danger of diphtheria,
in part with very good results. See, for example, Slawyk
(C. B. xxtv, 396). Regarding the almost universally
acknowledged success of the antitoxin injection for thera-
peutic purposes in cases of disease, it is not necessary to
enter into details here.

Special Diagnosis of the Coryn. Diphtheriz.!—
From the suspected material the following smear prepara-
tions are made :

1. Staining with methylene-blue or dilute fuchsin with
a little warmth.

2. A preparation stained by Gram’s method often pre-
sents the diphtheria bacilli more plainly, since the con-
taminating bacteria are in part unstained.

5. Granule staining by Neisser’s method.

If there are found, in this way, abundant and especially
long forms stained in segments with characteristic cross
arrangement and many granules, then the diagnosis of
diphtheria is to be considered as very.probable.

To render the diagnosis more secure, delicate smear in-
oculations are made upon ascites-agar, by drawing the
needle five or six times in succession over fresh parts of
the nutrient medium. The cultures thus obtained corre-
spond either to the typical picture of the diphtheria bacil-
lus, with its growth of moderate intensity, or we obtain
meager ‘‘ xerosis-like’’ or luxuriant ‘‘ pseudodiphtheria-
like’’ cultures.

1 Bruno (Berl. klin. Wochenschr., 1898, 1127) attempted to make
use of serum diagnosis here also. Diphtheria serum produced aggluti-
nation of certain diphtheria cultures, but not all. It was not sufficient
to separate diphtheria and pseudodiphtheria.

26

402 ACTINOMYCETES.

They are then tested further as follows:

4. Staining of granules in twenty hours’ serum cultures_
according to the method of Neisser. - This is very highly
recommended by C. Frinkel (Berl. klin. Wochenschr.,
1897, 1087).

5. Titration of the acid formed in 5 cc. of non-
saccharine bouillon in twenty and forty hours. If not
less than 0.7 and not more than 1.2—1.5 c.c. of 1:40 normal
alkali solution is required for neutralization, this speaks
in favor of diphtheria. Itis recommended that a parallel
observation be made with known diphtheria bacilli in
order to see whether a casual absence of acid production
does not depend upon the constitution of the bouillon.
(See p. 395.)

6. Animal experiment: If the injection of 1 c.c. of a
twenty-four hours’ bouillon culture produces the charac-
teristic symptoms and death in about forty-eight hours
(see p. 399), the diagnosis of diphtheria appears certain.
With lessened virulence only slight local symptoms occur,
and eventually only death from marasmus. (See p. 404.)

7. Demonstration of the protective action of antitoxin
against the infection in especially difficult or uncertain
cases.

By following this scheme, a typical diphtheria bacillus
is easily diagnosed, usually only the means given in 1 to
4, and perhaps also 5, are required.

However, there occur in the mouth in cases of diph-
theria, besides diphtheria bacilli which are typical in
every way, also most numerous variations. See, especially
Kurth (Z. H. xxvit, 409).

1. Non-virulent D. B., typical in all morphologic and ©

biologic peculiarities. Kurth found 3 non-virulent out of
39 typical cultures.

2. Virulent D. B., typical in everything, except that
they exhibit no granule staining (Neisser found 3 without
granules out of 39 typical cultures). We found a form
with very slight production of granules. This group
passes over into the following.

8. Virulent D. B., typical in every way, but without
the usual acid production. We found 1 out of 4 cultures
examined.

S

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.
'

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yd

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a a

CORYNEBACTERIUM DIPHTHERLAE. 403

4. Virulent D. B., typical in every way, but with very
little tendency to the formation of longer forms.

5. Virulent D. B:, typical in every way, but with such
luxuriant growths upon glycerin-agar and potato that they
cannot be distinguished macroscopically from the Coryne-
bacterium pseudodiphtheriticum. ~

In other words, we speak of a true diphtheria bacillus
whenever a bacillus stains in segments and presents a dis-
tinct, specific pathogenic action, without taking much ac-
count as to whether it corresponds exactly in one of the
peculiarities of length, granule staining, appearance of
cultures, and production of acid as given in the scheme
for the diphtheria bacillus. Even if several of these pecu-
liarities are found to differ from those in the scheme for
the diphtheria bacillus, still a typically pathogenic organ-
ism remains for us a Corynebacterium diphtheria, for clin-
icians have formed this species, and the single pathogenic
property appears so characteristic that we may build a
differential diagnosis upon it alone.

It is much more difficult, if pathogenesis fails, to pro-
nounce regarding the relationship to the true diphtheria
bacillus. If all the morphologic and biologic peculiar-
ities are present which belong to the true diphtheria
bacillus, and the pathogenic property only is lacking, then
it is safe to decide that one is dealing with a non-virulent,
true diphtheria bacillus.

It is more uncertain if, besides the virulence, still other
peculiarities fail; for example, the -production of acid.
Here the decision is doubtful, and the uncertainty in-
creases the more peculiarities are simultaneously lacking
—the more the organism approaches what are now cus-
tomarily called ‘‘ bacteria resembling diphtheria.’”? We
have devoted the following section to these.

The Pseudodiphtheria Bacilli of Writers.

Organisms resembling those of diphtheria, but not viru-
lent, are found in great numbers in the mouths of diph-
theritic and healthy persons, in the conjunctival sacs of
healthy and diseased eyes, etc.1 Proof has not been fur-

1 Schiitz very frequently found in the sputum in tuberculosis,
bacilli resembling those of diphtheria (Berl. klin. Wochenschr., 1898,

404 ACTINOMYCETES.

nished that these organisms, which in their extreme forms
differ widely from the diphtheria bacillus, are connected
with it genetically. Therefore there are no essential reasons
for regarding these forms simply as atypical diphtheria
bacilli in the broader sense.

On the other hand, it is not possible to separate them
naturally into definite varieties by the side of diphtheria
bacilli, any more than this is possible in the forms of the
Bact. coli and water vibrios. It is customary at present
to dispose of this by designating the luxuriantly, succu-
lently, and rapidly growing non-virulent forms as Coryne-
bacterium pseudodiphtheriticum (Hofmann-Wellenhof)
Lehm. and Neum., the scantily and delicately growing
forms as Corynebacterium xerosis (Neisser) Lehm. and
Neum., and the other non-virulent forms! are pressed
into this scheme as well as possible.

Corynebacterium pseudodiphtheriticum. (Léffler.)
L. and N.

(Plates 58-60, in part.)

Pseudodiphtheria bacillus of Léffler. Discovered by
von Hofmann-Wellenhof in 1887. Described in detail
by Escherich (Aetiol. der epid. Diphth.), Zarniko (C. B.
vi, 153), and Prochaska (Z. H. xxiv, 378).

Rods, which upon serum are shorter and thicker than
true diphtheria bacilli, show less ‘often a tendency to form
clubs and segments, but have a tendency to parallel group-
ing and are not virulent for guinea-pigs (Escherich). Upon
glycerin-agar it grows not.alone upon the inoculation line,
but in two to four days spreads out over the surface of the
agar. It varies from milky white to dirty yellowish or gray,
is succulent, and the border is slightly notched (58, m1).

297). R.O. Neumann (not yet published) found in every case of
catarrhal cold, but also in every healthy nose, often very abundant
diphtheria-like organisms, sometimes growing luxuriantly, sometimes
delicately, mostly producing a little acid and giving only slight
granule staining. The virulence has not been investigated.

1 These forms are usually not entirely non-virulent. C. Frankel
and others have seen animals die with marasmus a long time after the
injection of large doses of bouillon culture.

on ete ee ees Die ie el a

F
\
-
ow
id
™

eee ee Se oe a

CORYNEBACTERIUM PSEUDODIPHTHERITICUM. 405

The glycerin-agar plates appear correspondingly luxuri-
ant (58, vi1, a); when magnified sixty times, they present
dense, granular, dark colonies with ragged borders and
opaque centers (59, Iv, a and 6). Upon potato, fairly
abundant white growth. It is dry, elevated, lobulated,
often resembling the species of mycobacterium and actino-
myces (58, x). In bouillon the acid production (ex-
pressed in 1:40 normal alkali to 5 c.c. of bouillon),
according to all writers, is usually very slight (7. e., upon
ordinary bouillon after twenty hours, 0.3-0.7; after forty
hours, not more than 1.2; upon sugar bouillon after
twenty hours, 0.6—-1.4; after forty hours as much as 1.3
to 2.1), or there is none at all. After two to four days
the alkalinity increases perceptibly. We have observed
cultures, nevertheless, which produced upon sugar bouillon
in forty hours as much as 3.2.

Old agar tubes often exhibit a brownish-red to brown-
ish-black discoloration.1 This phenomenon is inconstant,
but, according to Escherich, is never present in diphtheria
bacteria.2, Upon gelatin there occurs a luxuriant growth,
even at 18°; bouillon shows a more rapidly forming tur-
bidity and ‘a denser and later forming sediment than
occurs with diphtheria bacteria.

In Gratz, v. Hofmann found this organism so frequently
(26 times ‘out of 45 healthy persons) in the mouth that
he considered it a normal inhabitant of the mouth. Other
writers found it much more infrequently. Escherich
never found it in healthy persons in Gratz, twice in 100
eases of diphtheria, and ten times in association with other
diseases of the throat. In Wiirzburg we found it not
infrequently in healthy and diseased eyes and noses.
Escherich admits the possibility that this organism may

1 An organism, obtained from Honl, of Prague, was very similar in
every particular (staining, clubbing, branching, luxuriant growth,
staining well by Gram’s method), but presented a reddish tint in all
cultures, especially intensely (rose) developed in the surface layer of
a milk culture. We have cultivated a similar one from the nose with
a luxuriant brownish-yellow growth (58, v).

* We obtained a brownish color of glycerin-ascites-agar in one of
our cultures in ten to fourteen days, and in three others after a longer
time (six weeks). F'or the cultures we are indebted to Dr. Silber-
schmidt (Ziirich).

406 ACTINOMYCETES.

sometimes be recognized as a form or descendant of the
diphtheria bacterium, yet it was impossible by the employ-
ment of most various means to render it virulent, not even
by the simultaneous injection of streptococci.

Important, but lacking confirmation, is the statement of
Hewlett and Knight that they have succeeded in the Lon-
don Institute of Preventive Medicine in converting the
Hofmann-Wellenhof organism into the virulent diphtheria
bacterium by passage through animals, and that typical
virulent diphtheria bacteria may be changed into the typi-
cal Hofmann-Wellenhof organisms by careful heating
(C. B. xxi, 793).

Corynebacterium xerosis. (Neisser and Kuschbert.)
L. and N.
(Plates 58-60 in part.)

Xerosis bacillus of Neisser and Kuschbert.

Grows especially in short forms, yet Heinersdorff, for
example, represents a number which differ in no way from
diphtheria bacteria, and we have also often observed such
forms. According to all writers, the growth on Léffler’s
serum is dry and more scanty than that of the diphtheria
bacterium, and still slower upon glycerin-agar (58, vil, ¢).
Upon potato no growth is to be seen. When magnified
sixty times, it is not distinguishable from feebly growing
forms of Coryn. diphtherie (60, vu1). When grown on
Loffler’s serum at 35° for nine to twenty-four hours, there
are none, or only a few, of Neisser’s granules.! Bouillon
always remains clear, and acid production usually is absent:
d. €., at most, 0.6 in twenty hours, about 1.0 in forty hours;
in sugar bouillon in twenty hours, 0.6—1.6; in forty hours
usually only 1-1.5, but may be as much as 3.2. In
numerous cultures from the eye and nose we usually
observed slight acid production parallel with the limited
growth, but sometimes, in spite of this, well-marked
granule staining. Pathogenesis is lacking, according to all
writers, and the organism appears to only accompany and
not cause the xerosis processes in the eye which are accom-

1 Not infrequently we found distinct granule staining, also in non-

virulent cultures which produced no acid and whose growths were dry
and scanty.

—_— — ee ae eS ee ee ee ey

CORYNEBACTERIUM XEROSIS. 407

panied by atrophy of the conjunctiva. The statement of
Spronk (Deut. med. Wochenschr., 1896, 571) that a dif-
ferentiation from diphtheria bacteria is possible from the
absence of effect of diphtheria antitoxin against the Coryn.
xerosis is doubted by most authors, especially since no
pathogenic action is observed in the latter (for example,
Heinersdorff, Archiv. f. Oph., Bd. 46, p. 1).

Kurth found in one-fifth of true diphtheria cases forms, probably
belonging here, which were absolutely non-virulent (Bac. pseudo-
diphtheriticus alkalifaciens, Kurth), and also three forms which pro-
duced acid as actively as the true diphtheria bacteria (Bac. pseudo-
diphtheriticus acidumfaciens). Gelpke (see below) found in his cul-
tures (in all?) less acid production in ordinary bouillon, nevertheless
much greater initial acid production in grape-sugar bouillon than with
diphtheria bacteria.

Gelpke (Bact. septatum, etc., Karlsruhe, 1898) has
recently regularly isolated an organism as the cause of
‘*catarrhal swelling,’’? which is a specific inflammation
of the eye characterized especially by bluish-red discolor-
ation and swelling of the fold of the conjunctiva, formation
of a fibrinous exudate, great pain and photophobia, to-
gether with general symptoms. He has called the organ-
ism Bacterium septatum Gelpke, considering it a new
variety, in spite of great similarity to the short xerosis
forms. So far as we see, aside from a not very great
pathogenic effect upon the human conjunctiva, as demon-
strated by Gelpke in some cases, there is nothing in the ex-
haustive description of the organism to distinguish it from
the Coryn. xerosis. Other authors appear to have obtained
the same impression.

What we have said in the preceding pages shows that, as
in the case of the virulent (‘‘true’’) diphtheria bacteria,
so also in the non-virulent, there is a long series of ex-
ceedingly closely related forms, which may be differen-
tiated by varying combinations of characteristics, —luxuri-
ance, length of rods, granule formation, production of acid,
etc.,—and which form a gradational series, into which the
true diphtheria bacterium also fits.

This is rendered still clearer in the following brief tabu-
lation of a part of our findings:

VARIETY AND

GLYC.-AGAR

sounce ov Gut-| atcnesnoete Fromage | Paraoeee ae
. SCOPICALLY.

1. | Corynebacte-| Slender rods, clubbed | Guinea- pigs | Whitish-yel-
rium diph-| at both ends.| injectedsub-| low, lux-
theriz from| Branching. Seg-| cutaneously | uriant,
exudate in| mentation distinct;| die after | “ moist.
throat (typi-| among them also| twenty-four
cal diphthe-| shorter organisms.| hours.
ria). Typical diphtheria.

2. | Corynebacte-| Much more luxuriant | Guinea- pigs | Grayish-
rium diph-| and thicker. The| injectedsub-| white, deli-
theriz from| swellings are more| cutaneously | cate, trans-
exudate in| irregular. Many] die after| parent.
throat(atyp-| short, thick, wedge-| forty-eight
ical diphthe-| shaped organisms.| hours.
ria). Segmentation.

Branching.

3. | Corynebacte- | Small, thick, wedge-| Not patho- | Grayish-
rium pseu-| shaped organisms,| genic. white, del-
dodiphther-| oftenin pairs. Part icate, trans-
it. from the| in formofovalcocci. parent ;
eye. Segmentation in the later more

middle. No branch- luxuriant
ing.

4. |Corynebacte-| Longer rods, club-| Not patho-| Yellowish,
rium pseu-| bing more to one| genic. luxuriant,
dodiphther-| side. Segmentation succulent ;
it. from the| present. Among later yel-
nose. them many short lowish-

forms. No branch- brown.
ing.

5. | Corynebacte-| Almost without ex- | Guinea- pigs | Grayish-
rium xerosis| ception small rods; notkilledby| white, deli-
from the| pointedattheends,| 5c.c. bouil-| cate, trans-
nose. arranged in pairs.| lon injected/ parent.

More rarely thick; intraperi-
forms with swell-| toneally.
ings. No branching.

6. | Corynebacte-| Regularly segmented | Infiltration at | Whitish,dry,
rium xerosis| slender forms with| point of in-| alittlemore
from the eye.| clubbed swellings.| jection in| luxuriant

Very often also| guinea-pigs| than pre-
shorter, wedge-| (subcutane- | ceding vari-
shaped rods. No} ous), but do| ety.
branching. not die.

408

Fe ‘TURES,GLYCER-

eh ae

a i alt eel

TTeroscohe. | “| SemowatEDNA.| TE Sutton. | “boutons
After | After | After | After
20 hrs. | 40 hrs, | 20 hrs. | 40 hrs,
Grayish- Slightly|Detached| 0.6 | 1.7 | 0.6 | 5.7}
brown; fair-| cloudy.| granulesat
ly transpa-| Abundant,} the poles.
rent; splin-| sandy pre-|} Atypical.
tery appear-| cipitate.
ance; torn,
_ tagged edge ,
Like 2 No. 1, | Very cloudy.| Regularly at} 1.2 | 2.3 | 3.8 | 5.8
butthicker! Sediment| the poles,
and only} homogene-| numerous,
slightly} ous,slight,} also de-
transpa-| easily dis-| tached ones
rent. tributed. in the mid-
dle.
Like No. 1. |Almost clear.,; Detached| 0.5 | 1.2 | 0.5 | 1.3
Sediment} granules.
slimy, easi-
ly distrib-
uted.
Like No. 2.| Clear. Sedi-/Granulesvery| 0.9 | 0.5 | 2.5 | 3.2
Interior! ment gran-| irregularly
verygranu-| ular, abun-| distributed,
lar. The} dant, easi-| not very
periphery; ly distrib-| few.
resembling| uted.
Like No. 1. | Like No. 2. | No granule} 0.6 | 1.1 | 0.5 | 0.7
staining.
' Like No. 2. | Like No.3. |Here and| 1.0 | 1.0 | 1.3 | 2.1
there a pol-
ar granule,
also isolat-
ed ones in
the middle
of rods.

ONE OER st ae a he gre

PLATE CuL-

BovUILLON CUL-

GRANULE For-

PRODUCTION OF 3; NORMAL ACID,

MATION ON

1 At first exceedingly slow acid production!

409

410 ACTINOMYCETES.

Supplementarily, we may here mention the following :

Bacillus pseudotuberculosis ovis. (Preisz.)
The rods are smaller and finer than diphtheria bacteria and stain

, a

well by Gram’s method. Grows only at incubator temperature, and —

upon agar and serum only scantily and dry. Upon bovine serum there
is often a striking orange-yellow color. Cultivated from the kidney
of a sheep. Injected intravenously into rabbits and guinea-pigs, it
produces pseudotuberculosis (A. P., 1894, 231).

Bacillus pseudotuberculosis murium. (Kutscher.)
Similar in many points to the preceding, but pathogenic for mice

only. Cultivated from the lung of a diseased mouse (Z. H. XVII, «

327).
The interesting ‘‘sporogenic’’ pseudodiphtheria bacillus of De
Simoni (C. B. xxIv, 294) scarcely seems to belong here, in spite of

:

3

:

certain similarities between it and the diphtheria bacillus (striped —

rods).
2. Mycobacterium Lehm. and Neum.

Cultures upon solid nutrient medium are elevated, more ©

or less wrinkled and dry. Microscopically: thin, slen-
der rods, often with typical dichotomous branching,
sometimes forming unbranched or branched threads.
When the rods have been stained with hot carbol-fuchsin,
they give up the stain from the action of acids with great
difficulty; they are ‘‘acid proof’’—. e., they behave
toward stains much like the spores of ordinary bacilli.

Mycobacterium tuberculosis. (R. Koch.) L. and N.
(Plate 61.)

Synonyms.—Bacillus tuberculosis R. Koch. Bacillus
Kochii Aut. nonnull. Sclerothrix Kochii Metschnikoff
(V. A. cxu, 70). See page 128.

Common Name.—Tubercle bacilli.1 T. B.

Most Important Literature.-—R. Koch (Mitt. aus. d. Gesundheitsamt
11, 1884); Nocard and Roux (A. P. 1, 19); Czaplewsky, Untersuchung
des Auswurfs auf Tuberkelbacillen, Jena, 1891; Fischel, Morphologie

1In the following we more often employ the common name of
tubercle bacillus (T. B.), but in spite of this we do not consider the
further application of the scientific name, Bacillus tuberculosis Koch,
to be proper.

al a fp tion Sh. « ‘ > .
cia i i ian tal AP e TES Rw oes

i i i i a

a

MYCOBACTERIUM TUBERCULOSIS. 411

und Biologie des Tuberkuloseerregers, Vienna, 1893; Coppen Jones
(C. B. xvu, 1); Hayo Bruns (C. B. xvi, 817); Cornet, Die Tuber-

kulose, 1899.

Microscopic Appearance.—In sputum and cultures
usually unbranched, slender rods, 1.5-4 » long, only
0.4 » thick, which often are slightly bent (61, vu, Ix, x).
More recently many writers have observed thread and ~

_ true branched forms—in sputum and in cultures, and in

the latter, with careful preparation, they are predominant
—which are injured and broken apart by only the rough-
est preparation. (Literature, history, and good illustra-
tions by Coppen Jones, J. ¢.) Lubinski obtained long
threads without branching upon acid potatoes (C. B.
xvi, 125). :

Inside of the tubercle bacillus from sputum and pure
culture there are sometimes found unstained vacuoles,
sometimes peculiar structures which give an especially in-
tense, dark red color with carbol-fuchsin. Still, these
latter bodies do not present the regular form of the true
spores of bacilli; also statements regarding resistance and
germination are not at hand. Coppen Jones compares
them to the chlamydospores of the mucorini.

In the same article the same author described very
remarkable forms from tubercular sputum resembling the
clubs of actinomyces, but which he recognized not as or-
ganized forms directly formed by the T. B., but (like acti-
nomyces-clubs) as secretions, concrements, etc.

Friedrich found T. B. resembling actinomyces—. ¢.,
clubbed, dense, radiating formations—in sections of or-
gans from animals which succumbed to a rapid tubercu-
losis infection (see p. 416).

Motility.—According to all authors motility is lacking.

’ Schumowski claims to have constantly seen a slow motion

of the T. B. (C. B. xxi, 838.)

Staining Properties.—The T. B. stains so difficultly
and imperfectly with the ordinary aqueous solutions of
anilin dyes that these are never employed. Also the stain
suggested by Koch, accomplished by prolonged action of
alkaline methylene-blue, has only a historical interest.

To-day two methods (Tech. Appendix), with innumera-
ble (insignificant) modifications, are almost exclusively

412 ACTINOMYCETES. 1

employed. Of these, we always use that of Ziehl-
Neelsen.

Also, Gram’s method is successful, but is not especially
recommended, since it does not possess the advantage of a
specific reaction.

Relation to Oxygen.—Without oxygen, no growth.

Requirements as to Temperature and Reaction of
Nutrient Media.—Growth occurs between 29° and 42°,
the optimum being 37°. Under all circumstances growth
is slow. ; |

Preliminary Remarks Concerning Cultures. — ~
Upon the ordinary agar and gelatin nutrient media the T.
B. grows scantily or not at all. For its cultivation, be-
sides solidified blood-serum, glycerin-agar is almost execlu-
sively employed (Nocard and Roux, C. B. 1, 404).

Glycerin-agar Plate.—Surface colonies like those on
the glycerin-agar streak.

Glycerin-agar Streak.— At first there are minute,
crumbly growths, irregular in form, white to yellowish-
white, fairly elevated, devoid of luster or faintly glistening
(61, 1). Later, after three to four weeks, the colonies grow
out and have lobulated sinuate borders. The peripheral
portions are still thinly transparent, and at intervals there
are formed elevations, like mountain ranges, running from
the border toward the center, which gradually converge to
form a mountain stem in the middle. The elevations are
usually yellowish to brownish in color; the depressions,
whitish to grayish-yellow. Still later the entire colony
becomes brownish (61, 11). We once obtained an orange
discoloration. Htippe reports that he has grown cultures
which presented a pronounced yellow to reddish-yellow
color. (See p. 480.) Kitasato cultivated. a luxuriantly
growing variety of Myc. tuberculosis. (Compare Mye.
tub. avium, p. 418.)

Blood-serum Streak.—A slight growth in the form of
light-colored, dry, crumbly scales becomes visible micro-
scopically after about six days and macroscopically after
ten to fourteen days. Blood-serum is never liquefied.
When magnified sixty times, the colonies, especially at
the borders, present S-shaped flourishes consisting of
nothing but parallelly arranged rods (61, v).

it i Ol eee a, B. = ni.

MYCOBACTERIUM TUBERCULOSIS. 413

Potato.—If potato is inclosed in an air-proof (7. ¢.,

_ protected from evaporation) reagent glass, there slowly
_ develops small, crumbly, yellowish, friable masses, devoid

of coherence, much elevated above the surface of the

potato, dull or with a faint luster (61, m1). The culture
is well developed after about three weeks. (See Pawlow-
sky, C. B. tv, 340). The growth is better if air can enter
and other precautions are taken to prevent drying of the
potato.

Fluid Nutrient Media.—If glycerin (up to about 4% )

- is added to the nutrient fluids, the T. B. will grow very

well upon most various mixtures; for example, bouillon,
potato water, and artificial non-albuminous nutrient
media. As an example of such a medium we may men-
tion: Mannite, 0.6; citrate of magnesium, 0.25; sulphate
of ammonium, 0.2; glycerin, 1.5; diphosphate of potas-

- sium, 0.5. See Proskauer and Beck (C. B. xvi, 974).

aie

7 ,

eS ee ee

ee Ee Oe ee ee

According to Rabinowitsch, the T. B. forms a thick film
upon all liquid nutrient media and gives off an odor of
flowers. Formation of endogenous spores does not occur,
and whether a form of arthrospore is produced is at least
very doubtful. (See p. 411 regarding chlamydospores. )

Resisting Powers Against:

(a) light: Pure cultures are very susceptible to direct
sunlight; are also injured by pale, diffuse daylight (accord-
ing to Koch, cultures on a window die in five to seven
days).

(6) Drying: According to Sawitzky (C. B. x1, 153),
human phthisical sputum retains its virulence, when
dried at room temperature, for two and one-half months;
also sunlight does not here produce injury. Obici (C. B.
xix, 314) obtained a series of similar results. On the
contrary, Migneco found them dead in the sun after
twenty-four to thirty hours if the dried sputum was not in
too thick a layer (A. H. xxv, 361). Tubercule bacilli
dried on cigars die in ten days; on the contrary, on paper
they may live as long as four weeks.

(c) Moist heat: 50° does not kill in twelve hours, 55°
kills in four hours, 60° in forty-five to sixty minutes, 70°
in ten minutes, 80° and 90° in about five minutes, 95° in
one minute (Forster, H: R. 1, p. 869).

414 ACTINOMYCETES.

(d) Cold: It is borne very well; for example, winter
cold for twenty-one days by bouillon cultures.

(e) Disinfectants : Injure slowly, especially T. B. which |
are found in sputum; 3% carbolic acid, for-example, kills —
T. B. only after twenty hours.

An extensive survey of the tenacity of the T. B. is given
by Schneiderlin, Dis. med., Freiburg, 1897.

Chemical Activities —(a) No chromogenesis or pro-
duction of odoriferous substances.

(6) Cellulose is formed in distinction to many other |
investigated bacilli.

(c) Indol and H,S production were not observed in our |
cultures.

(d) Regarding toxins, see page 417.

Distribution.—(a) Outside the body: So far, found only”
in living rooms (dust of railroad cars, street dust, etc.) In-
places where tubercular cases have deposited their sputum. —
In the air they are seldom found, and then are isolated. —

They are very frequently found in milk. A third of ©
tubercular cows furnish, even when the udder is healthy, ©
milk containing T. B. Still there is great variation. While ©
the butter of a large Berlin dealer contained T. B. in the
butter in 100% of the cases (Obermiiller, H. R., 1897,
712), thirteen other establishments in Berlin were proved
to furnish butter free from T. B. (Rabinowitsch,.C. B.
XXvV, 77).

(b) In the healthy body: Very many apparently healthy
individuals, men and animals (cows), present at autopsy
smaller or larger, often completely healed tubercular foci.
Of such men there are said to be 66% with latent or
healed tubercular foci; and of these, it is the principal dis-
ease in 58%, a secondary affection in 6%, and entirely
latent in 41% (Schlenker). Healthy nurses and physi-
cians of tubercular patients are said to often show T. B.
in the nasal mucus.

(c) In diseased human organism:+ It occurs as the ex-
clusive and essential cause of miliary tuberculosis, of bone,
gland, and joint tuberculosis (caries, fungous inflammation,
white swelling, etc.), of lupus (tuberculosis of the skin),

1 Regarding cases of men affected with fowl tuberculosis, see page
419.

eae a ee ee

r MYCOBACTERIUM TUBERCULOSIS. 415

f of intestinal, peritoneal, renal, and meningeal tubercu-
{ losis, of dry and serous pleuritis, 1 etc. All the organs
: may be affected with tuberculous disease.
: Part of the tuberculous affections of the lungs are de-
. _ pendent upon the T. B. alone; in phthisis, streptococci
_ play a very important secondary role as the cause of the
_ typical irregular temperature curve and as destroyers of
_ the pulmonary tissue with suppuration. ‘‘ Anatomical”’
tubercles are only in part caused by the T. B.
_ The port of entry of the T. B. may be in any part
_of the body (lung, intestine, skin, wounds of the skin),
and is said by many authors to be located especially often
in the tonsils.
7 Tuberculous mothers sometimes furnish tuberculous ova,
_ or tuberculous fetuses (eventually through placental tuber-
_ culosis); tubercular fathers, even with tuberculosis of the
- testicle, scarcely ever transmit T. B., but certainly do a
disposition to tuberculosis (Gartner, Z. HH. , xr, 101). In
the same place are also given many statements from the
literature.
(d) In animals: Tuberculosis is very frequent in cows
(‘‘Perlsucht’”’). In newly born calves tuberculosis (al-
ways miliary tuberculosis) is a rarity (according to
Klepp, with exhaustive examination, it occurs in about
3% of slaughtered calves!). In slaughtered cattle as high
as 35% have been found to be tuberculous; in old milk
_ cows, as high as 80%.
_ Inplaces tuberculosis occurs frequently also in swine,—
_ for example, in the slaughter-houses of Dantzic in 11% ,—
_ yet mistakes in connection with the necrotic areas of swine
"plague (‘‘Schweineseuche’’) have been observed. Sheep,
goats, horses, dogs, and cats sometimes, though rarely,
_ present very extensive tuberculosis. Rabbits and guinea-
_ pigs sometimes present tuberculosis rather frequently; yet
of 3000 guinea-pigs which were killed during 1890-96 in
_ the Department of Health of Berlin, not a single instance
_ of spontaneous tuberculosis was observed 2 (Petri).

>=

1 Pleural exudates, apparently free of bacteria, are very often of a
tuberculous nature.

2 Vagedes has isolated twenty-eight different cultures of tubercle
bacilli from man and two from animals (‘‘ Perlsucht’’), mostly from

— e

416 ACTINOMYCETES.

Experimental Observations Regarding Pathogenic
Effects.—(a) In animals: With T..B. from man it is
very easy to infect cattle, swine, horses, and especially
monkeys and guinea-pigs; also dogs are easily infected,
especially intravenously. Fowls are immune; in hens, —
at most, there occurs a small, local area from inoculation
in the comb. |

Infection follows the introduction of T. B. by all sorts
of methods (also inhalation and feeding), but most cer-
tainly by the intraperitoneal. At the place of infection
a caseous area is formed, and in the neighborhood (omen-
tum, peritoneum) an acute miliary tuberculosis. With ©
intravenous infection a general miliary tuberculosis de-
velops. Tubercle bacilli, attenuated by iodoform, cause —
in rabbits, sometimes the picture of chronic phthisis in
man, sometimes the typical pearly disease (‘‘ Perlsucht”’)
(Troje and Tangl, C. B. x1, 613). I

If rabbits are injected subdurally or into the kidneys,—_
according to Friedrich, also into the veins,—then areas
are often produced which in from fourteen to fifty days —
correspond throughout to pictures of actinomyees: 7. ¢., —
a central tangle of genuine branching threads, limited at_
the periphery by clubs. The central structure is acid
proof; the clubs are often only feebly so, and sometimes
are stained blue with a counterstain of methylene-blue.
Both the threads and clubs stain well by the Gram-Wei-
gert method, while in the actinomyces the clubs rarely
retain the stain in Gram’s method. .

The close relationship between tuberculosis and actino-
mycosis is constantly demonstrated by these investigations.
Details will be found in the literature cited above. For
the latest researches, with beautiful illustrations, consult
Schulze and Lubarsch (Z. H. xxx1, 153 and 187). In
the same place special staining methods are also described.

(b) Inman: Experimental tests are lacking. Of the
clinical experiences, some cases of disease following infec-

the pus of cavities and pulmonary nodules. The virulence for animals
proved to vary very much. If aculture was highly virulent for rabbits,
it made no difference whether an infection was produced in the eye or
subcutaneously or intravenously, and such cultures were also always
very virulent for rats.

yr, “am

MYCOBACTERIUM TUBERCULOSIS. 417

tion of a wound of the hand by sputum (injury by a

_ broken sputum glass) have the force of experimental

demonstration.

Toxins, Immunity, Immunization.—From old cul-
tures of the T. B. upon glycerin bouillon by means of
evaporation and precipitation with alcohol, an albumi-

nous body is obtained, formerly known as ‘‘ tuberculin,”

and now as ‘‘old tuberculin.’”’ When this is injected in
cases of tuberculosis (Koch), it exerts a peculiar influence
upon the tuberculous process. Very weak doses call forth
a moderate increase of inflammation at the seat of the
tuberculous disease, with fever, while healthy persons ex-
hibit neither fever nor noticeable local symptoms. As
pointed out by Buchner and Rémer, the proteins of other
bacteria have an exactly similar effect upon tuberculosis.

While Koch and some of his students obtained good, or
at least satisfactory, curative and immunizing results with
the old tuberculin in man and animals, most investiga-
tors, after a brief enthusiasm, abandoned the preparation
as very rarely useful, but also as very often injurious.
Then Koch sought to improve his preparation, and, under
the name of ‘‘ Tuberculin TR,’’ recommended a new pre- .
paration, prepared as follows :

Virulent T. B. are dried and then pulverized, suspended
in water, again pulverized, and then separated by centrifu-
gation into a sediment, and a supernatant fluid. The latter
is decanted and only the further aqueous extract is em-
ployed, which is obtained by pulverizing and by separa-
tion of the solid ingredients by centrifugation (Deut. med.
Wochenschr., 1897, 209).

H. Buchner, following the method of E. Buchner, has
obtained a ‘‘ tuberculoplasmine ’? by trituration and com-
pression of fresh tubercle bacilli, concerning which no
practical results appear to have been published.

Koch has completely immunized a series of guinea-pigs
with his TR by means of carefully but actively increasing
doses. Complete immunity was obtained about two or
three weeks after the administration of large doses. Also
Koch has obtained a cure in previously infected guinea-
pigs, but the treatment must be instituted not later than
eight to fourteen days after infection, because of the rapid

ee

418 ACTINOMYCETES.

course of the disease ia guinea-pigs. Also the absorption

of tuberculin in animals already infected is slower, and
therefore it acts more unsatisfactorily. It must not be dis-
guised, however, that Baumgarten and others arrived at
absolutely negative results with the new tuberculin in
guinea-pigs, as previously was the case with the old tuber-
culin (C. B. xxi, 587); small doses were worthless, and
the larger the doses, the greater the disappointment. Re-
garding the value of the new preparation in man, there is
no unanimity. Spengler (C. B. xxi, 523) gives a favor-
able judgment, but unfavorable or skeptical opinions are
in the majority. See, for example, Stempel (Mtinch. med.
Wochenschr., 1897, No. 48) and Bukovsky (C. B. xxim,
518). <A review of collected literature is furnished by
Bussenius (C. B. xxu, 621). Maragliano claimed to ob-
tain good results with a tuberculosis serum, but his results
have been but little verified from other sources (at least
such is the experience of Hager, Mtinch. med. Wochen-
schr., 1897, 853). Also Behring recently hoped to succeed
in reestablishing an antitoxic tuberculosis serum (Congress
of Hygiene, Madrid, 1898). Tuberculin now plays a
large part, entirely aside from its therapeutic use, as a
diagnostic aid in tuberculosis. For details, see page 437.
For the differential diagnosis of the T. B., see page 436.

Mycobacterium tuberculosis § avium. (Maffucci.)
Lehm. and Neum.

Synonym.—Bacillus tuberculosis avium Maffucci.
Common Name.—Bacillus of fowl tuberculosis.

Most Important Literature.—Maffucci (Z. H. x1, 445); Straus and
Gamaleia (C. B. x, 300); Courmont (C. B. xtv, 602); Kruse (C. B.
xv, 50 1); Pfander (histologic, C. B. x11, 264); Fischel (Untersuch-
ungen tiber die Morph. und Biol. des. T.-Erregers, Wien, 1893).

From the standpoint of our present knowledge this in-
teresting organism, first separated from the T. B. by Maf-
fucci, must be looked upon as only a form of the latter
which has become acclimated to the bird’s body (its high
temperature), but which is occasionally also pathogenic for
other creatures.

ie a Ly ee an

MYCOBACTERIUM TUBERC ULOSIS 8 AVIUM. 419

In favor of this are the observations of Kruse and
Pansini. From a guinea-pig, inoculated with the juice

from tuberculous organs of cattle, and from another in-

- fected with human sputum, typical fowl tubercle bacilli
_ were cultivated which were pathogenic for chickens.

In accord with this, Johne and Frothingham found
fowl tuberculosis in cattle (C. B. x1x, 564) and Nocard
in horses (C. B. xxi, 807).

After prolonged cultivation on artificial nutrient media

at ordinary incubator temperature, the bacillus of fowl

tuberculosis becomes pathogenic also for mammals
(Courmont, C. B. x1v, 602). Fischel (C. B. xiv, 632)
observed transition forms connecting the two diseases.
Finally, Cadiot, Gilbert, and Roger were able to invert
the pathogenic properties of both varieties by continued
transfer (C. B. xrx, 567).

The following are given as the points upon which the

_ differential diagnosis of the avian form rests.

OS ee ee ee a eee oe. a ee

Microscopic Appearance.—Like the T. B., some-
times a little longer and slimmer. Staining properties are
the same.

Requirements as to Nutrient Media.—Does not
grow on potato; otherwise the same as the T. B.

Requirements as to Temperature.—Limits, 35°-
45°. In contradistinction to the T. B., it grows very well
and without reduction of virulence at 48° (Straus and
Gamaleia). The T. B. generally does not grow above 42°.

Serum and Agar Cultures.—They are always softer,
more succulent, more luxuriant, and grow more on a
level. However, Kruse has observed a dry culture, also
cultures which, upon certain varieties of agar, take on a
reddish, blackish, violet color.

Fluid Nutrient Media.—The pellicle is not so firm as
in the T. B.

Cultures live for two years.

Pathogenic for birds! when introduced in every way
except by feeding, attacking especially the liver and
spleen. Also the embryos of chickens in incubated eggs

1 The very common tuberculosis of parrots is usually produced by
true T. B., the animals being infected by receiving food from the
mouths of tuberculous persons.

420 ACTINOMYCETES. q
can be infected. The course is very chronic; giant cells
are rare; bacilli are present in great abundance.

Immunity against avian tuberculosis exists in the dog, —
monkey, and guinea-pig; but the last-named animal after
injection dies with a slow marasmus (chronic intoxication). —
Rabbits are rarely susceptible.

Leray (H. R., 1896, 358) describes differences in the
pathologico-anatomic picture in rabbits which are inocu-_
lated with avian and mammalian tuberculosis. In the |
former caseation is lacking, but there are many giant
cells, with common inclusions of the organisms; in mam-
malian tuberculosis the reverse obtains, the caseation
being extensive, giant cells few, the T. B. usually free.

'
.

Mycobacterium tuberculosis var. ; piscicola. L. and N. —
(Plate 62, v-Ix.)

Literature.—Bataillon, Dubard, and Terre (C. B. xxi, 61); Kral Krél
and Dubard (Rév. de la tubere., 1898, No. 2). ;

The French authors cultivated the organism, which re- —
sembles the T. B., from a tumor of carp about the size of —
a pigeon’s egg. Tt is not motile, is acid proof, forms —
branches, and grows at 23°-25° as the optimum tempera- —
ture (minimum, 12°). In bouillon there are abundant
flocculi, which settle to the bottom; upon potato there
occurs a thin whitish growth; gelatin is not liquefied.

In order to prove that their organism is the T. B.,
acclimated to cold-blooded animals, fish and frogs were
inoculated and fed with cultures of human and avian
tuberculosis. From the organs of such fish the var. pisci- —
cola was in fact obtained.

In association with Dr. Kumulis we have thoroughly
studied a culture obtained from Kral, and have completely
confirmed the statements of the French investigators.
Growth stops at once at 37°, and at 20°-25° it is a little
more luxuriant than that of the ordinary T. B. in the in-
cubator. In the microscopic examination we saw no
branching, probably by chance. In gelatin plates (62, v1)
there occurs a rather tough, dry, whitish-yellow, wrinkled
growth, which upon being magnified sixty times corre-
sponds to the T. B. upon glycerin-agar (62, ym). In the

MYCOBACTERIUM LEPRZ. 421

gelatin stab the growth is scanty ; upon the surface a dry,
thin, wrinkled pellicle, with no liquefaction. Upon agar
just like the T. B. on glycerin-agar, only a little more
luxuriant and more thickly padded (62, v). Bouillon is
clear with a crusty pellicle. The growth on potato is
wrinkled, thick, sharply outlined, whitish-yellow. The
growth upon milk is strikingly different from all varieties
studied by us. It is not coagulated, and after one to three
months takes on a dark violet-gray color, the pigment being
soluble in alcohol.

Mycobacterium tuberculosis 6 ranicola. L. and N.

The efforts of various investigators to acclimate the T. B.
to the frog resulted in the information that also here the T. B.
is gradually transformed into a variety which grows at lower
temperatures. For details see Lubarsch (Z. H. xxx1, 187).

Mycobacterium tuberculosis « anguicola. (Moéller.)
L. and N.

Moéller, Therapeutische Monatshefte, Nov., 1898 ; Lu-
barsch (Z. H. xxx, 187).

Isolated from the spleen of a blindworm which was in-
fected one year previously with human_ tuberculosis
(sputum). Grows best at 22° (generally not at all at 28°
to 37°); upon agar it forms a moist, glistening white layer !
In dilute bouillon and non-albuminous nutrient media
plentiful branching occurs. It cannot be inoculated into
rabbits.

Mycobacterium lepre. (Armauer Hansen.) L.andN.
(Plate 62, I-Iv. )

Common Name.—Lepra bacillus.

Principal Literature.—Max Wolters, Der Bacillus lepre (C. B. X1mI,
469), and Finger in Ergebn. der allg. Aetiologie, 1896. Compare
also Mitteilungen und Verhandlungen der internation. Leprakonfer-
enz in Berlin, Oct., 1897, 2 Biinde; Babés: Der Leprabacillus and die
Histologie der Lepra, Berlin, 1898.

Since the communications of Armauer Hansen and
Neisser (Virch. Archiv, Lxxxiv, 514) there has been no
doubt that the cause of leprosy is a non-motile organism,

422 ACTINOMYCETES.

very closely related to the tubercle bacillus.

and often in enormous numbers.

within special ‘‘lepra cells,’’

endothelium, sometimes as lymph thrombi.
By means of staining reactions the L. B. cannot be cer-
tainly differentiated from the T. B. They stain by the

Koch-Ehrlich method just as well as the T. B., and, like —

them, are also stained by Gram’s method and by a suffi-
cient action of aqueous anilin dyes.

or seven minutes with an aqueous solution of fuchsin that
good preparations are obtained after washing with water,
while the T. B. is not; on the contrary, alkaline methyl-

ene-blue stains the T. B. quicker than the L. B. Compare —

the controversies upon this between Baumgarten and
Wesener (C. B. 1, 450, 573; 1, 131, 291).

These organ- —
isms, which are often a little shorter than the T. B., are ©
found in the specific new-formation (nodes and nodules) —
of leprosy in the various organs in cases of the disease, —
The bacilli are found in —
groups, the individuals being parallelly arranged, and lie ~
which have recently been —
explained, sometimes as confluent, proliferating lymph —

A difference is said —
to consist in this, that the L. B. is so well stained in six ~

Still, all authors are now agreed that the staining reac-

tion cannot help much in the differential diagnosis, no
more than the form of the bacilli,! from which it follows

1The following difference in sections is given by Spiegel from
Unna’s laboratory (C. B. xxi, 817):

LEPROSY. TUBERCULOSIS.
Number of bacilli: Exceedingly abundant | Very much less nu-
in all organs and se-| merous.
cretions.
Arrangement of the/|In heaps like a cigar | Moreas individuals or
bacilli: in form. in irregular bunches.

Form: Rod-shaped, straight, | Thready, curved, and
and plump. fine.
Angles: Sharp. Roundish.
Appearance of granules:) Coarse. Fine.
Widely separated. Close together.

Arrangement of gran-
ules:

These differences are naturally never so typical as here appears.

: ig

MYCOBACTERIUM LEPRZ. 423

that the separation of leprous and tuberculous affections
in the cadaver appears often impossible, since at least it is
made differently by different persons. Since, according to
Hansen and Looft (Bibl. med., 1894), the cause of death
in 40% of the cases of leprosy is tuberculosis, this uncer-
tainty is very unfortunate.

While for a long time efforts to obtain cultures of the
infective agent from cases of leprosy were frustrated, more
recently positive results have multiplied.

Almost all the cultures obtained present branched forms
closely related to the Myc. tuberculosis ;. the typical acid-
resisting power was rarely present (Bordoni-Uffreduzzi, Z.
H. m1, 178, with plate); sometimes there was a certain
limited acid-proof property, and at other times none at
all. Babés, who, if not the first to study this subject, has
studied it most extensively, holds the organism, for evi-
dent reasons, to be the cause of lepra in spite of the absence
of acid-proof quality (C. B. xxv, 125).

The growth upon artificial nutrient media (glycerin-
agar, glycerin-serum-agar, glycerin-potato) was found by
all writers to usually be delicate and slow; morphologically
and biologically their behavior was very similar to the
T. B. Our culture obtained from Kral exhibited a good,
although a slow, growth. Morphologically the rods re-
semble the T. B., but also the diphtheria bacterium.

An additional proof that we may recognize the culti-
vated organisms with the greatest probability as the Myco-
bacterium lepre has been furnished by Spronck (C. B.
xxv, 257). It is the demonstration of agglutination of
the questionable organisms by serum from many cases of
lepra, even in high dilution.

Animal experiments are said by some authors to have
succeeded (see Wolters), but no one has produced typical
leprous changes. The greatest number of writers observed

1 Czaplewski (C. B. xx1tI, 97), who has also isolated an organism
belonging here, says with entire truth that these forms constitute a
connection between the diphtheria and tuberculosis groups, or, accord-
ing to our terminology, the genus Mycobacterium and the genus
Corynebacterium, which, in the first edition of this book, we placed
side by side as closely related. See also Levy (A. H. xxx, 168).

424 ACTINOMYCETES.

a rapid death of the bacteria introduced, and ascribe the
positive results of others to tuberculous infection.

Because of the prolonged period of incubation, it is diffi-
cult to determine the way by which the infection in man
gains entrance. Generally an infection through the vari-
ous mucous membranes and slight wounds is assumed ;
there are said to be no ports of infection in the alimentary
and respiratory organs. Congenital lepra is at least very
rare. Abundant L. B. are found in the sperm and milk.
They have never been found in the ovaries. Except in
the secretion from ulcers, they are found most frequently
in the nasal secretion (128 times in 153 cases) (Sticker).
The nose is the most common location of the primary
affection, as well as the most dangerous source of contam-
ination of the surroundings (Sticker, Miinch. med. Woch-
enschr., 1897, Nos. 1063 and 1103).

A positive differential diagnosis of isolated and cultivated
lepra from tuberculosis organisms can at present only be
based upon the less developed acid-proof quality, the more
delicate growths, and perhaps the agglutination.

Organisms in Verrugas.- .

We may only mention briefly that in the Peruvian ‘‘ Ver-
ruga,’? an endemic disease characterized by rather large
cutaneous swellings (hypertrophy of the papillary bodies),
and formerly much confused with syphilis, organisms
have recently been found in all the organs which are very
similar to the T. B. morphologically and tinctorially. See
Letulle, Nicolle, Odriozola (C. B. xxiv, 889).

Mycobacterium smegmatis. L. and N.

Common Name.—Smegma bacillus.

Since the investigations of Tavel (C. B. 1, 673) and
Matterstock (Mitt. a. der mediz. Klinik Wiirzburg, 11) we
have known that still another mycobacterium is found in
the smegma of the prepuce and clitoris, and only recently
it has been cultivated a few times.

Laser (Mtinch. med. Wochenschr., 1897, 1191) first
succeeded in cultivating an acid-proof, non-motile bacillus,
corresponding in morphology to the smegma bacillus.

ed “
! "

es:
iy

MYCOBACTERIUM SMEGMATIS. 425

_ Upon agar smeared with blood the growth was scanty, like

| streptococci ; upon glycerin-agar, like dewdrops; upon

_ the ordinary nutrient media it grows poorly or not at all.

-_ The organism was not pathogenic. Czaplewski (eod. loco)

has observed a little better growth in a culture, and also

_ yellow, honey-like growths on potatoes. See also Griin-

| baum (eod. loco, No. 45, p. 1254).

Stolz (A. H. xxx, 156) has described a very closely

related, beautifully branching, but not especially acid-

_ proof organism, which grows very poorly in cultures.

In practical medicine more interest attaches to the ques-

tion whether the smegma bacillus can be differentiated from

|
3
4
i.
4
s
:
:
.

_ the T.B. microscopically from its staining properties, than to

its cultivation. This delicate question, with which we have
had no personal experience, has been the subject of very

; much investigation. According to Grethe (Fort. d. Med.,

1896, No. 9), a method of Weichselbaum is to be especially
recommended for differential diagnosis. The preparation
is stained with hot carbol-fuchsin, thus staining the T. B.
and smegma bacilli red ; then a saturated alcoholic solu-
tion of methylene-blue is allowed to act upon it (how long
is not stated), when gradually, even in the thickest parts
of the preparation, the smegma bacilli become blue, the
tubercle remaining red.

According to the careful studies of Bunge and Trauten-
roth, there are, as was to be expected, acid-proof smegma
bacilli which are quite different from one another, but none
of them retains the stain after the following process :
Absolute alcohol, three hours; 5% chromic acid, fifteen
minutes; carbol-fuchsin, two to three minutes; dilute sul-
phuric acid, two to three minutes; concentrated alcoholic
solution of methylene-blue, at least five minutes. Hon-
sell (C. B. xx1, 700) recommends for the same purpose: ~
Ordinary carbol-fuchsin stain, ten minutes in acid alcohol
(absolute alcohol 97 parts, HCl 3 parts), wash in water,
washing in alcoholic methylene-blue diluted one-half.
Only the T. B. remain red.

For differential diagnosis, after a preparation has been
stained according to the usual method for T. B. with
gentle action of acid, and acid-proof red rods are found,
then a second preparation must be stained by one of these

one
ry

496 ACTINOMYCETES.

methods, and if blue rods are obtained, the smegma bacil-

lus can be diagnosed. Finally, also cultures may be pre-
pared upon ordinary nutrient media to search for Myco-
bacterium lacticola and related organisms. -

Micro-organisms in Syphilis.
Lustgarten (Wien. med. Wochenschr., 1884, and Wien.

Jahrbuch, 1885) was the first to find micro-organisms in ~

syphilis by modern methods. He succeeded in staining,
by means of a special method, organisms resembling the
tubercle bacillus, in the interior of syphilitic guammas and

in the primary lesions (Technical Appendix). Doutrele- —

pond arrived at similar results.

After the discovery of very similar organisms in normal
smegma (see p. 424) had very much reduced the signifi-
cance of the syphilis bacillus in the secretion of the pri-
mary lesion, and after very many investigators had sought

in vain for Lustgarten’s organism in the interior of syph- —

ilitic bodies, it was the general opinion that Lustgarten’s
positive findings in gummas were to be explained by a
mixed infection with tuberculosis.

After ten years scarcely more than one article worthy of
notice upon the etiology of syphilis had appeared, when
van Niessen undertook to investigate anew this important
question. Unfortunately, he entered upon the work with
absolutely insufficient knowledge, and his first publications
(C. B. xx, 48) deserve in general no serious discussion,
as they were so full of technical errors.

Van Niessen has recently made further communications
(Wiener mediz. Presse, 1899, Nos. 11-18) of investiga-
tions, carried out in the St. Petersburg institute of Prince
Oldenburg, which indeed show that he has learned much
in the meantime, but which can in no way be accepted as
proof for his assertions. Reexaminations by known inves-
tigators are absolutely essential.

Van Niessen succeeded in cultivating an organism,
which resembles somewhat the Corynebacterium diphthe-
riz, from the blood of suitable cases of secondary syph-
ilis, and also from flat condylomas which were not
ulcerated (how often is not clear). It exhibits distinct
clubbing and branching, stains with the ordinary anilin

a ee ee a a ne Ne ee A TE my

MYCOBACTERIUM SMEGMATIS. 427

} dyes, and also well by Gram’s method, and often (not
always) by Lustgarten’s, but not by Ziel-Neelsen’s
_ method. Thestatements regarding spores sound improb-
able.

It grows slowly upon all ordinary nutrient media as a

whitish to yellowish or brownish formation, and it grows

very slowly upon gelatin. In the gelatin stab there are
very fine points and teeth, and often branches are pre-
sented.

All gelatin plates present pinhead-shaped, prominent,
yellowish-white, glistening, transparent colonies. Upon
agar the growth possesses a slimy tenacity. Upon serum
the organism thrives, although the growth is not much more
abundant. The color is often quite a pronounced yellow.

In bouillon (at 37°) there is first turbidity; later a sedi-
ment; also a delicate, cobweb-like pellicle and a ring on
the wall of the tube (optimum, 28°-32°).

~The animal experiments are given so absolutely super-
ficially that they are referred to with great difficulty. The
organism (only one culture appears to have been em-
ployed) proved pathogenic for monkeys and swine. The
three monkeys succumbed after a few weeks with a very
complicated clinical picture, in which there appeared ulcer-
ations at the point of inoculation, most extensive cuta-
neous eruption, nervous and cerebral disturbances, and
indolent swelling of the glands.

At the autopsies there were found, in the first animal,
ischemic alterations in the brain (areas of softening); in
the second, leptomeningitis and endocarditis; in the third,
hematocephalus internus. Of other disturbances there
were, especially, multiple swellings of lymph-glands.

Microscopically, periarterial processes like those in syph-
ilis appear, but the examinations are insufficiently pene-
trating throughout. The pathogenic organism was never
successfully isolated again from one of the inoculated
animals, and none of the animals was completely studied
bacteriologically; in short, scarcely anything can be con-
cluded from the animal experiments.

Van Niessen finally attaches value to the demonstration
of an agglutination of his organism by serum from syph-
ilitic patients.

428 ACTINOMYCETES.

Mycobacteria which Grow Luxuriantly at Room Tem-
perature.

Literature.—The real discoverer of these organisms in butter is Petri.
He pointed them out to R. Koch in July, 1896, who expressed his
belief that they were different from true tubercle bacilli, which was
also the opinion of Petri. A subsequently undertaken investigation
by Lydia Rabinowitsch at Koch’s suggestion appeared before Petri’s
work as a preliminary communication in the Deutsch. med. Wochen-
schr., Aug. 5, 1897, and in detail in Z. H. xxvi, 1897, 90. Petri’s
work was published (A. G. A. xiv, 1, 1898) after he had published a
note concerning it in the H. R., Aug. 15, 1897. ~

Further important communications are : Hormann and Morgenroth
(H. R., 1898, 217 and 1081); Moéller (Verh. d. Gesell. deut. Naturf. u.
Aerzte, 1898 ; Deutsche med. Wochenschr., 1898 ; and C. B. xxv, 369);
O. Schultze and Lubarsch (Z. H. xxxI, 153).

The most interesting discovery in the field of the mor-
phology of bacteria during the past three years is to be
considered that of the micro-organisms resembling the
tubercle bacillus. Three years ago every acid-proof bac-
terilum was a ‘‘tubercle or lepra bacillus’’; to-day we
know, through the harmonizing labors of many inyesti-
gators! (Petri, Rabinowitsch, Moéller, and others) that
acid-proof varieties occur not infrequently in the environs
of men and domestic animals. Still, their differential diag-
nosis from the Mycobacterium tuberculosis appears quite
easy, and yet the forms now known approach it in so
many characteristics that, after the experiences with diph-
theria and cholera, we must expect that still further diffi-
culties will arise after more extensive studies. The exceed-
ingly interesting forma piscicola of the Mycob. tuberculosis
described on page 420 shows us to what a degree the true
T. B. culture may change biologically (growth at room
temperature, formation of a violet pigment in milk).
Who knows how our newly discovered mycobacteria cul-
tures may change if they are cultivated at incubator tem-
perature or in the animal body for many generations?

To-day naturally it is not possible to consider the Mye.
tuberculosis, without further knowledge, as a form of one
of these organisms, which has been adapted to warm-

1Jn manure acid-proof bacteria had already been found by stain-
ing: Severin (C. B. L. 1, 98); Ferran (C. B. xx11) ; Capaldi (Z. H.
XXVI, 105).

MYCOBACTERIUM LACTICOLA a PLANUS. 429

blooded animals ; but we may hope that the study of these
latter will some time disclose to us much which now is
dark in the matter of tuberculosis.

In connection with Dr. Kumulis we have studied all the
representatives of this group which we could obtain; in all,
thirteen cultures. By detailed systematic comparison
they arrange themselves into two varieties, of which one
shows two or three forms.

Rabinowitsch has pronounced her organism (Myc. lac-

ticola) identical with Myc. phlei. We find certain rather

De a ee

Oe ae ee a Oy I ae Bite Pe Tee ge terme

,
3

a ee ee ee

important differences, so that we believe for the present
two species should be made. Lubarsch’s observations,
which we received after our own were entirely concluded,
correspond in general very well with ours. The patho-
genic properties are so similar that they may be treated
together.

Mycobacterium lacticola a planus. L. and N.
(Plate 64. )

Synonyms. — Organisms resembling tubercle bacillus
(Tuberculoseihnlicher) of Rubner-Obermiiller, grass or-
ganism II of Moéller+ (C. B. xxv, 369).

Microscopic Appearance. — Irregularly formed,
shorter and longer rods of exceedingly variable length,
straight or bent, partly acutely bent, often with clubbed
swellings; generally in older cultures they are rather thick
and often grown into threads of very irregular form.
Branching is present (64, x; 63, v1, xm a and b).

They are not motile, and stain with methylene-blue,
ordinary fuchsin, by Gram’s method, and exactly like the
true T. B. by the method for tubercle bacilli.2 Growth
occurs upon all nutrient media, taking place slowly at
room temperature and somewhat more rapidly at incuba-
tor temperature, but always perceptibly faster than the
T, B. Oxygen is indispensable. In agar shake cultures
there is exceedingly little or no growth-deep in the tube.

1 According to our observations, Moéller’s grass organism II is the
Myce. phlei.

2 In smear preparations the staining property is lessened by agents
employed in removing fat,

430 ACTINOMYCETES.

Gelatin Plate.—In young cultures the colonies are
very similar to the Bact. coli macroscopically; later the
colonies are more wrinkled. When magnified sixty times,
they present a wavy, scalloped border with more or less

wrinkling internally, being very similar to old colonies of —

the Bact. coli (64, v1, a). The deep colonies are not
characteristic (64, vi, 6).
Glycerin-agar Plate.—Macroscopically at first small,

crumbly colonies, which later become wrinkled and ele- —

vated, and still later usually present a delicate transparent
peripheral zone (64, x). Magnified sixty times the colo-
nies are usually pronouncedly granular, always becoming

more transparent toward the periphery, and in this latter ks

zone often present proteus-like markings (64, vr).

Glycerin-agar Streak.—After six days at 37° a dirty
grayish-white growth with a wavy smooth border, a fatty
luster, numerous more or less elevated folds, and trans-
parent in many places (64, 11). Sometimes the wrinkling
from the beginning is not so outspoken, the surface being
much more homogeneous, and after many weeks it is of a
yellow to a coppery red color. The consistency of the
growth at first is like butter, later like saliva, and not
crumbly and dry (64, m1). Upon ordinary agar there
later occurs a brownish-yellow coloration and little wrink-
ling (64, 1).

Gelatin Streak Culture.—The growth was somewhat
thinner and more delicate, the wrinkling more pronounced
and regular. A decided orange color did not develop.

Bouillon Culture.—At first turbid, later clear. A
pellicle is sometimes formed. The precipitate is moderate
and yellowish-white. In sugar bouillon growth is more
luxuriant, with almost constantly a thick wrinkled pelli-
cle on the surface, the sediment being abundant and dis-
sociated with difficulty. Milk is not coagulated, but after
a time it becomes transparent and sometimes gelatinous.
At the edge a bright orange pigment is deposited.

Potato Culture.—Slightly elevated, more or less wrin-
kled, often also a homogeneous growth, with notched or
smooth border; when older, there are knobby elevations,
the growth being bright to deep orange, with a fatty or
moist luster (64, v).

fay eg

i | a i

MYCOBACTERIUM LACTICOLA 8 PERRUGOSUM. 401

- Indol is formed in small amount and H,S in both
_ sugar bouillon and ordinary bouillon. Acid production
Fis limited; in 10 c.c. of 2% grape-sugar bouillon as much
as corresponds to 0.6 ¢.c. of decinormal sodium hydroxid.
Gas is not formed in nutrient media containing sugar.

_ 435. For pathogenesis, see page 434.
_ We can separate the following from this only as a
4

¥,
_ Mycobacterium lacticola @ perrugosum. L. and N.
. (Plate 63, I-v1l. )

_ Synonym.— Butter organism of Rabinowitsch. We
; obtained two cultures which corresponded perfectly.
It is very similar to the Mye. lacticola « planum, and
_ perhaps only a cultural form of the same, for Rabino-
_ witsch described the young cultures as moist, thick, and
creamy when cultivated direct from the animal body, and
_ only after multiple passages through animals do they
_ acquire the dry, early wrinkled character which this form
_ presents.

' Regarding the microscopic character, nothing special is
_ tobesaid. We report, in detail, regarding the growths of
_ these forms because of the interest which they now possess.
| Glycerin-agar Plate.— Macroscopically in a short
_ time there appear much wrinkled, irregularly dentate, dry
_ scales, which later increase perceptibly in size. They are
_ transparent, grayish-white, and are easily lifted from the
_ surface and broken up (63, Iv). When magnified sixty
times, the smallest and youngest colonies are very granu-
lar; being fringed and torn at the periphery, grayish-white
_ and transparent (63, nr). Later they become opaque,
_ brown to gray, with irregularly distributed markings,
_ often spotted like a panther’s skin (63, 11). The gelatin
_ plates are scarcely different.

_ G)lycerin-agar Streak Culture.—Similar to the growth
on the plates; there occurs here also an abundant, wrin- _
_ kled, dry formation, which is extraordinarily like the

432 ACTINOMYCETES.

growth of the true T. B. Especially when young, the
cultures are easily mistaken for it. Later the growth be-
comes colored somewhat orange to coppery red (68, 1)
(Rabinowitsch).

In bouillon cultures a_ thick, wrinkled pellicle is”
formed very early. Still, the fluid remains clear, with —
scarcely any precipitate, indicating aerobic growth. There _
is a disagreeable ammoniacal odor (Rabinowitsch). In_
sugar bouillon the pellicle-formation is still more pro-_
nounced. ;

Milk Culture.—Like those of Myeob. lact. a planum.

Potato Culture.—At first there is a whitish to bright —
orange growth, with some elevation. After a short time ©
it becomes wrinkled, and in older cultures this is especially —
prominent, similar to the appearance of elycerin-agar
streak cultures. The growth is dull, rather dry, scarcely
at all glistening. Indol is not formed; according to Rabi- —
nowitsch, there is a trace. HS is produced i in ordinary 3
bouillon only. Acid production: In 10 c.c. of sugar bouil- —
lon as much as corresponds to 0.8 ¢.c. of decinormal i
sodium hydroxid. There is no liberation of gas, and i
gelatin is not liquefied. . ¢

The Bacillus friburgensis described by Korn (C. B. xxv, 540)
stands about midway between Mye. lacticola a planum and B per-
rugosum, and, according to our nomenclature, may be called Myco-
bacterium lacticola y friburgense (Korn) L. and N. Upon glycerin-—
agar the streak growth at first is white, and more luxuriant than in
planum; later there form sturdy, wrinkled elevations, which in time,
especially at room temperature, become coppery red.

Korn gives the following as characteristic:

1. Stains by Ziehl-Neelsen’s method especially well and is little
influenced by acids.

2. Uniform growth—not interrupted—in gelatin stab.

3. The surface of the agar culture is depressed in the center, the
peripheral zone being elevated.

4. Upon bouillon it produces a disagreeable but not ammoniacal
odor. The characteristics are partly inconstant, partly unessential.

Inoculation with large quantities of pure culture and diseased
organs causes no disease in guinea-pigs, rabbits, chickens, and pigeons. —
White mice are readily infected by the intraperitoneal injection of 0.5
c.c. of a suspension. The animals die in from four to forty days, and
present massive nodules in all the abdominal organs,

153)

ee Pap =

eg ee ee a ee

MYCOBACTERIUM PHLEI. 433

Mycobacterium phlei.! (Moeller.) L. and N.

(Plate 63, VIII—XII.)

We here consider the micro-organisms described under the follow-
ing names: ‘‘ Moéller’s manure organism,’’ Moéller’s grass organism
I, from timothy grass, Petri’s butter organism. Judging from his in-
sufficient description, Petri appears to have often found also the Myc.
lacticola, but he did not distinguish the forms. We do not believe
any great value can be attached to the insignificant differences be-
tween these cultures (bouillon diffusely cloudy or only with a pre-
cipitate). The work of Dr. Kumulis will contain details regarding
this. Obviously the timothy organism of Lubarsch also belongs here.

Literature.—Petri (A. G. A. xiv, 1). Lubarsch (Z. H. XxXxXI,

Microscopic Appearance.—After growing for three
or four days the rods are strikingly short and thick, and
in this condition resemble the Corynebact. pseudophtherit-
icum very much. Later they become longer, sometimes
clubbed ; they also branch, and are then not distinguish-
able from the two preceding varieties (63, x, a and 6).
They are not motile. The staining properties, intensity of
growth, and relation to oxygen are just the same as in the
Mycobact. lacticola.

Glycerin-agar Plate.—After a few days the macro-
scopic colonies are orange-red, with a wavy smooth border,
without wrinkles and with a moist luster (63, x1). When
magnified sixty times, the colonies are transparent, with a
dark nucleus and markings like curls of hair. Toward the
periphery is a delicate, transparent, more or less crumbly
zone with a fringed, notched border (638, rx). Later the in-
terior of the colony becomes darker and more opaque ;
only at the edge is there a delicate, veil-like zone.

Glycerin-agar Streak Culture. — Luxuriant, succu-
lent, bright orange-red, homogeneous growth, which in
time presents knobby elevations, but yet later, and especially
in very old cultures, a considerable wrinkling appears, and
then, except by the color, it is indistinguishable from
Mycob. lacticola & perrugosum (63, vir). In the gelatin
streak culture the wrinkling is never so marked; in gen-
eral, the growth.on gelatin is somewhat less.

Bouillon Culture.—Sometimes there is a thin pellicle ;

1 Timothy-grass is scientifically called Phlewm pratense.
28

434 ACINOMYCETES.

in other respects it is rather variable ; the fluid is often
almost clear, with a slight orange sediment, which rises up
in columns upon shaking. At other times there is a slight

transitory cloudiness. In sugar bouillon the growths are

identical.
Milk culture exactly like Mycob. lacticola.
Potato Culture.—Like that of the glycerin-agar streak.
Indol is produced only as a trace, H,S not at all, and
also no gas is liberated. Acid production j is the same as
by Mycob. lacticola. Gelatin is not liquefied.

Pathogenic Effects of Mycobacterium lacticola and
phlei.!

According to the descriptions of all authors, the patho-
genic action of these varieties is so similar that it is not

worth while to treat them separately.

It is ageed that the organisms are essentially less patho-
genic without the simultaneous injection of butter, and even
that the resulting peritonitis (adhesions, membranous for-
mations, fibrinous and purulent exudates) may be depen-
dent upon the injection of butter alone (Hormann and
Morgenroth); ‘still, the intraperitoneal injection of large
quantities of pure cultures often produces an infection and
causes the formation of nodules in the abdominal organs,
yet the process often heals. If animals are killed three or

ew

anneal i ye inl dept Puce abies

four weeks after the injection of large quantities, the fol-
lowing conditions are found (Rabinowitsch): Slightly dis- —

tended abdomen, more or less severe peritonitis, the peri-
toneum and mesentery beset with nodules, numerous small
nodules beneath the intestinal serosa, mesenteric glands
perceptibly swollen and often caseated. Likewise the liver,
spleen, and kidneys show yellowish exudation in nodules
in variable degrees. The lungs exhibit, at most, numer-
ous transparent nodules, and are usually free from more
serious disease. The transfer of small pieces of the organs
to a new animal transfers the disease, according to Rabino-

1 Animal experiments appear to have been performed by Rabino-
witsch, Hormann, and Morgenroth exclusively, and by Petri in part,
with Myce. lacticola. Petri and Moéller have worked with Mye.
phlei. Also Korn’s experiments with his Myce. friburgense correspond
except in unessential matters.

rh A ee
—_—
"

ey Da

EFFECTS ON ANIMALS. 435

witsch; but according to Petri, and Hormann and Morgen-
roth, this only occurs if tuberculosis is simultaneously .
present.

When butter is experimented with (4 to 5 c.c. of the
previously thoroughly mixed mass, melted at 37° and

- containing butter-fat, watery part, and casein layers), if

2 EN NT gan PLAN TITS OR SN GTi Wether

the bacilli are present in abundance, a fatal result often
follows the injection after three to fifteen days. There
are then found changes similar to those described above,
only very much more intense, the abdominal organs being
covered with well-developed inflammatory, fibrinous mem-
branes which are swarming with the organisms.

Rabinowitsch found rabbits insusceptible in contrast to
guinea-pigs. It is generally admitted that guinea-pigs are
the most suitable experimental animals, although single
reports concerning infection af rabbits are not lacking.

Korn could infect only mice with the pure culture of
his Friburgensis (0.5 ¢.c. of a suspension or larger doses).

Whether injected into the blood-vessels, subdurally, or
into the kidneys, the organisms produce more or less
well-developed areas, such as described on page 416 in
true tuberculosis: 7. e., formations resembling actinomy-
cosis, but which disappear in the course of months. The
animal body destroys the organisms which are introduced
(Lubarsch and O. Schultze).

Also, in the structure of the accompanying miliary
tubercles, these authors can often detect no difference from
those of true tuberculosis.

Little has yet been learned concerning the distribution
of the organisms resembling the Mycobac. tuberculosis.
However, it appears very wide, for about 60% of butter

_ samples in Berlin contained such organisms, and the in-
_ vestigations of Moéller demonstrated their frequent occur-
rence in manure, on grasses, etc. Lubarsch and Dieu-

donné verified this.

To obtain the acid-proof bacteria from butter, about 4
gm. are injected intraperitoneally in two guinea-pigs with
a wide cannula, as indicated above. After about six to ten
days the animals, if not dead, are killed, and cultures are
prepared from the contents of the abdominal cavity which
yield acid-proof bacteria, growing at room temperature.

, Oe
Et
ed
- wy J
iy.

To cultivate the organisms from grass, the grass is coy-
ered with water (Phleum pratense is especially recom-
mended) and allowed to remain at 37° for thirteen to
twenty-four hours. When frequent (every two hours) —
examinations have demonstrated that acid-proof organ-
isms are present in quantity, plates are prepared.

It cannot be stated with certainty whether these new
varieties of mycobacteria may also be pathogenic for man.
So far as we know, no acid-proof organisms which grow
upon all nutrient media and at room temperature have
been cultivated from the sputum in cases of pulmonary
diseases or from human organs.

Ginsberg stained numerous bacteria from two cases of
eye disease, which were closely related to the T. B., but
they were not cultivated (C. B. xxu, 62).

Flexner described a Streptothrix pseudotuberculosa Fl.
from the lung of an old negro, growing with beautiful —
branching, staining by Gram’s method, but imperfectly —
acid-proof with the T. B. stain, and not certainly patho-
genic for guinea-pigs (Jour. of Exp. Med., m1, 435).

436 ACTINOMYCETES.

aie 7

of AB A, emt a halle gh Se cal

Opt Bl tees

Differential Diagnosis of the Mycobact. tuberculosis
(Tubercle Bacillus; T. B.).

1. If the T. B. is to be distinguished from bacteria
which are not acid-proof, simple preparations are stained
by the Ziehl-Neelsen method. Those which do not stain
can be left out of question. T. B. which do not stain by
the Ziehl-Neelsen method are unknown.

In the examination of sputum one proceeds as follows:
The sputum is to be obtained as free as possible from
foods and secretions from the mouth, and it is best col-
lected in a sterile dish after the mouth has been well
washed with water. Of the sputum, the more purulent
(not mucous) portions, which are in lumps, are selected,
spread upon the cover-glass, and stained (see Technical
Appendix).

If no bacilli are found in a few preparations, although
tuberculosis is suspected of being present, then one of the
more searching methods described in the Technical Appen-
dix must be employed in the attempt to discover isolated
bacteria.

one

DIAGNOSIS OF MYCOBACT. TUBERCULOSIS. 437

2. For the differentiation of T. B. from lepra and

_ smegma bacilli the present methods scarcely suffice. T.B.

are difficult to cultivate; lepra and smegma organisms are
only very rarely cultivated successfully. For the tincto-
rial differences, see pages 422 and 425.

3. There is no difficulty in differentiating the T. B.
from ‘‘ pseudotubercle bacilli’? which grow well at room
temperature, so long as only one variety is present. Agar
or gelatin plates are prepared and kept at 22°. True

_ tubercle bacilli do not grow at all, at least when they come

oo dhs cia een

from warm-blooded animals, while the false varieties grow
out well in two to four days. Reinoculations are made
upon glycerin-agar and into bouillon in order to differen-
tiate Myc. phlei and Myc. lacticola.

_ We are unable to suggest a method for recognizing cul-
turally the true T. B. when associated with large numbers
of bacilli resembling it ; in cultures the true T. B. would
be overgrown. According to the present state of our
knowledge, a guinea-pig must be injected intraperitone-
ally with a moderate amount of the mixture.

If the guinea-pig dies after the intraperitoneal injection
of small amounts of culture (one loopful) and without the
addition of butter, with well-marked tuberculous changes
in the abdominal cavity (enlargement of liver and spleen),
and with involvement of the respiratory organs, these speak
in favor of true tuberculosis. The histologic examination
must give a predominance of true, giant-celled tubercles ;
and from the nodes and nodules organisms must be culti-
yated which will not grow at room temperature and on ordi-

nary nutrient media, but at incubator temperature and upon

ascites-glycerin-agar in the case of true T. B. Since death

~ from tuberculosis in guinea-pigs usually occurs six weeks
_ after the infection, before that the few germs resembling

the T. B. are absorbed and have disappeared.

4. To determine whether a person or an animal is tuber-
culous, the injection of tuberculin is often made use of.
Although it does not lack in individual contradictory re-
sults, yet there is no doubt that the tuberculin reaction
constitutes a very important aid.

It is customary to inject cows subcutaneously with 0.3
to 0.5 c.c. of tuberculin, and to observe whether an eleva-

438 ACTINOMYCETES.

tion of temperature of 1.5° to 2° or 2.5° occurs after twelve “

to ‘fifteen hours. A French commission speaks especially
favorably of it (C. B. xix, 645). The reaction sometimes
is absent when the animal is extensively diseased, but for
these cases the reaction is not required. It scarcely ever

appears in healthy animals, and here it is to be remem- —

bered that small areas are often to be found with diffi-
culty at the postmortem. © The result is not influenced by
other diseases in cattle. Exceedingly rarely, latent tuber-

culosis is stimulated to new activity. It is of importance —

that an animal often fails to give a positive reaction a sec-
ond time for a month after a typical reaction was first ob-
tained.

The question does not appear to have been investigated
as to whether the tuberculin reaction occurs in animals in-
fected with Mycob. lacticola and Mycob. phlei.

Naturally it is much more difficult to come to a con-

clusion regarding the reliability of the tuberculin reaction

in man, since it cannot be controlled subsequently by
thorough postmortem examinations; at any rate the injec-

tion of tuberculin in man for diagnostic purposes is not —

employed to any great extent.

3. Actinomyces. Harz, emend. Gasperini.

Growths upon solid nutrient media are elevated, tough,
more or less wrinkled, often cartilaginous. Microscopi-
cally, they appear as long, thin, elongated mycelial threads,
the young ones with homogeneous contents without par-
tition walls, without a developed covering, and with abun-
dant, true branches. In older threads there can be dis-
tinctly recognized a delicate membrane, and within it the
colored contents, broken up into fragments. Positive cell
divisions are rarely observed in cultures of varieties of ac-
tinomyces. Some varieties, in the animal body, present
clubbed enlargements upon the ends of the radially ar-
ranged threads, which are to be explained as thickenings
of the sheath (see below). Many species form chains of
short colorless spores (conidia) upon thickened air hyphz
(two to ten times thicker than the threads) which rise
above the solid medium and compact culture film like a

14 MN OR eg Sh i Me SN I gi I

i Sag he

KEY TO GENUS ACTINOMYCES. 439

| | white mold. Not infrequently chains of conidia are formed
_ when the culture is submerged in river-water or 4% sugar
- solution ; yet here the spore chains are copiously branched,
and the segmentation may even encroach far upon the
threads (Gasperini, Lachner-Sandoval). Also some au-
thors describe the occurrence of spore-like formations in
the interior of the threads, but we have never certainly
seen it. According to Lachner-Sandoval, this is only a
fragmentation of the contents of the threads, which must
not be interpreted as spore-formation even if it can be
demonstrated that such fragments of threads cicatrize at
the ends and later grow out again. They are not stained
by the T. B. method, but always by Gram’s method. 4

Ne >

Key to Some of the More Important Varieties of the
Genus Actinomyces.

(A) Pathogenic varieties, with clubbed swellings of the ends of the
threads in the animal body. Upon artificial nutrient media the for-
mation of clubs is rare; conidia are sometimes produced in cultures,
sometimes not.

(a) No growth below 22°, no growth on potato, no air mycelium,
formation of clubs in artificial cultures very limited. Pathogenic for
rabbits. Actinomyces Hofmannj. (Gruber.) Gasperini. Page 447.

(b) Grow below 22° and upon potato; formations of clubs in cul-
tures scarcely ever observed.

1. Agar cultures, yellowish-orange, knobby, sometimes with air
mycelium. Gelatin slowly liquefied. Typical club-formation in the
body. Cause of the typical ray-fungus disease in cattle and man.
Actinomyces bovis. Gasp. Page 440.

2. Agar growth, dry, granular, scanty. Pathogenic for cattle.
Clubs have not been demonstrated in the animal. Actinomyces far-
cinicus. Gasp. Page 447.

3. Agar culture, a luxuriant, wrinkled, orange-yellow layer, with

1 For the limitations and naming of this genus, see Lachner-Sando-
val, Ueber Strahlenpilze, Bonn, 1898; Sauvageau and Radais (A. P.
VI, 242, Sur de genre Oospora); and our discussion on page 127. Re-
garding the species, the following articles are also important: Alm-
quist (Z. H. viii, 189, 1890), Gasperini (Annales de Micrographie, Bd.
It, 449, 1890), and Annal. dell’Istit. d’Igiene di Roma, m1, 1892,
166 (C. B. xv, 684). Rossi Doria (Annal dell’Ist. d’Ig. de Roma,
_ Bd. 1, 1892, 399). See also Berestnew (Z. H. xxix, 94).

2 While we ourselves naturally appreciate that this key is not satis-
factory, our information does not allow us as yet to prepare a better
; one,

a ee ee Te ae ee em en Aen my peer ts

440 ACTINOMYCETES.

air hyphe. Pathogenic for rabbits. Typical clubs formed in the
animal. Actinomyces asteroides. Gasp. Page 449.

4. Agar growth whitish-red. Conidiaare formed. Beautiful clubs
in the animal. Actinomyces madure. Lachner. Page 452.

(B) Non-pathogenic varieties :

1. Growth colorless, nutrient medium brown. Actinomyces
chromogenes. Gasp. Page 452.

2. Growth colorless, nutrient medium colorless. Act. chromogenes.
Gasp. falbaL.and N. Page 455.

3. Growth colorless, nutrient medium colored violet. Act. viola-
ceus. Gasp. Page 456.

4, For varieties with other colors, see Gasperini’s Act. carneus,
albido-flavus, citreus, etc., pages 451- 456.

Actinomyces bovis. Harz.
(Plate 65. )

Synonyms.—Actinomyces bovis Harz, Act. bovis sul-
phureus Gasp., Nocardia Actinomyces de Toni e Trevisan,
Streptothrix Actinomyces Rossi Doria, Oospora bovis
Sauy. et Radais.

Common Names.—Ray fungi, Actinomyces.

Literature.—Israél (Virchow’s Archiv, Bd. 74, 15; and 78, 421);
Bostrém ( Ziegler’s Beitrige, Bd. 1x, 1). «« Actinomykosis in Eulen-
as s Realencyclopedie, Bd. 1, i894, by Birch-Hirschfeld. Grill
(C. B. xvii, 181).

Microscopic Appearance.—In the body of men and
animals the organism forms sand-like masses, 0.2 to 0.6
or even as large as 1.2 mm. in diameter, of a gray, yellow,
red, sometimes also green color, and when young, of a
soft, and when older of a tougher consistency. The
masses are made up of a ball of threads, the threads
being radially arranged at the periphery ‘and provided
with characteristic, club-like formations, which are to be
considered as derived from the gelatinous membranes of
the threads (Bostrém). The threads terminate in the
clubs, either free or with slight bud-like enlargements
(Fig. 20, a, 6). The threads show true branching, are
thin (0.4—0.6 »), partly without division, partly apparently
composed of longer and shorter fragments. The sur-
rounding ‘‘membrane’’ is very delicate. In the interior
of the colonies, there are usually found between the
threads, cocci-like formations, which originate from fre-

Sa Ahly at! sand aS aS, FANE I A AER RA tA Rate ~

:

|

and later may be outside of the empty membranes (Fig.

ACTINOMYCES BOVIS. 441

quent fragmentation of the contents of the long threads,

_ 20, ¢). These are not endospores! Older clubs become
notched and cut, so that structures like an asparagus
head may occur (Fig. 20, a). Often branched threads
reach far beyond the zone with the clubs (Fig. 20, d).
_ Sometimes clubs are entirely absent. Many actinomyces

PE PPS Re a ET ad a ER NC

masses are dead when expelled in pus.
In cultures the branching mycelium is easily obtained

_ (65, 1x); the clubs are found only in the deepest layers

of the nutrient medium.

Staining Properties.—The threads, but not the clubs,
are best stained by Gram’s method; afterward the clubs
may be stained red with saffranin and diffusely staining
carmine. According to Berestnew (Z. H. xxx, 94), young
actinomyces clubs stain by Ziehl’s method, sometimes also
by Gram’s method.

Relation to Oxygen.—Grows aerobically and anaero-
bically, but better aerobically (Bostrém). The growth is
limited.

Chromogenesis.—The production of pigment is ex-
ceedingly variable; from white to various shades of yellow,
orange, rusty, and brown appear to occur upon the various
nutrient media; the darker tones at least predominate

- upon serum media, the brighter ones on gelatin.

Gelatin Plate.—(a) Natural size: After six days the
colonies have a very irregular outline, are yellowish-

_ gray, shining, sometimes fairly elevated above the sur-
_ face of the gelatin, sometimes growing deeply into it (65,

IV).
(6) Magnified sixty times: Dark yellowish-gray, homo-

_geneously shaded colonies, sometimes presenting more
_or less distinct concentric rings. The peripheral zone is
_ dark and beset with fine, curly hair (65, vir).

Gelatin Stab.—Surface growth at first is whitish-yellow,

flatly elevated, faintly shining, rather tough; later the

growth sinks into the gelatin with the limited liquefaction,
leaving an air-space above. In the stab at first there are
small yellowish-white clumps, which later have bristly
outgrowths (65, m1).

Agar Plate.— Macroscopically and microscopically

a. Various clubbed forms from b. Clubs with threads
fresh preparations. which contain fragments re-

sembling cocci.

c. Threads with fragments like cocci and d. Line of growth !
club-shaped swellings. with threads extending
beyond the clubs.

e. Part of a cluster with frag- f. Section through } of a perfectly

mentation in the interior. developed cluster.

Fig. 20—Formation of clubs in Actinomyces bovis. Harz (after
Bostrém). (a, b, and e are highly magnified—about 1000 to
2000 times ; d, e, and f, slightly magnified. )

442

—

;
E
|

+ ied

PG ee |

ACTINOMYCES BOVIS. 443

ayes

_ scarcely distinguishable from those in gelatin plates, ex-
cept that the colors are fainter.

Agar Streak.—At first delicate, like dewdrops; then
there slowly forms (after six to ten days) a whitish to
' whitish-yellow growth with an abruptly scalloped border,

faintly lustrous and fairly elevated. This gradually comes

to resemble a growth of Mycobacterium lacticola with its
_ elevated paddings and ridges. After a very long time
(thirty days) the growth gradually becomes dry, sinks in,
and the color changes from white to yellow or brown.
The culture appears to grow deeper into the nutrient
medium, and often becomes surrounded by a more deli-
cate zone, but in our cultures, in distinction to Bostrém’s,
no air hyphe and no downy appearance was formed. The
water of condensation remained clear.

Serum Streak Culture (after Bostrém).—At first the
colonies are like dewdrops, which first become a little
broader and thicker ; then, extending out from some places,
a whitish, velvety, dry covering is obtained. While the
surface of the colony which is turned toward the serum
gradually becomes colored from yellowish-orange to brick-
red,—as do the older, puffed portions of the growth,—a
delicate border of transparent bristling hairs is formed
about the growth, in which later there form anew little but-
tons and puffs, which are first whitish and then change to
yellowish or reddish.

Bouillon Culture.—The bouillon remains clear; at the
bottom ball-like masses form, which are broken up with
_ difficulty by shaking. Colonies upon the surface were
never observed by us and rarely by Afanassiew. Micro-
scopically the balls consist of threads with radially
arranged fibers. Even in old bouillon cultures we could
see no clubs.

Milk Culture.—Unchanged after eight days.

Potato Culture.—Slightly knobby, yellowish-white
layer, closely attached to the potato, strictly limited to the
streak. Often there occur distinct white, or yellow, and,
according to Bostrém, also red spots (65, vmr).

Special Nutrient Media.—<According to Bostrém, the
fungus also grows in non-albuminous nutrient media, and
even in sterilized water, as it does in bouillon. In an

444 ACTINOMYCETES.

eBe, Wolff and Israél obtained especially good dichotomons |
orms. -
Conditions of ‘‘Spore-formation.’’—In many threads ©
(not exclusively, but especially, with entrance of air)
there are formed by continued transverse fission, short —
cocci-like and roundish oval bodies, which sometimes lie _
in close, but usually in broken rows within the empty and _
finally torn membrane (Bostrém, Kruse). Before the dis-
charge of the ‘‘spores’’ the end of the thread is often some- _
what swollen. The ‘‘spores’’ stain like protoplasm, not

Fig. 21.—Bouillon culture of Actinomyces bovis.

like the endospores of bacteria. We have seen nothing of
these structures (Fig. 20, ¢) in either young or old cultures,
although we took much pains to look for them. Typical
conidia spores cut off in rows do not appear to be de-
scribed.

Viability and Resisting Properties.—Very old cul-
tures (nine months) are still alive.

Chemical Activities.—They have been but little
studied. The odor is very faint, disagreeable but not
moldy. From grape-sugar within eight days, neither gas
nor acid is formed, and no H,S in peptone bouillon.

athe: ene Sea Bat (cain alee Puliterialllte. Bhed<e ug paiaenldlin a

Le pete

2 ihe

ACTINOMYCES. BOVIS. 445

Distribution.—
(a) Outside the body: They have not been found, but

_ must frequently occur upon the beards of grains and grasses,

- because infection most often depends upon the penetration

_of a fungus-carrying barley barb, which is often found in

the actinomycotic swelling (Bostrém). (Compare Berest-

| new, p. 447.)

(b) In healthy body: Never found.
(c) In diseased human organism: It is the cause of acti-

7 nomycosis. Principal ports of entrance: (1) Mucous
membrane of mouth and throat; (2) respiratory tract;

ed Re oe ee

(8) intestine; (4) skin. Almost always beards and other
parts of grain are the vehicle; more rarely, wood. From

_ the primary areas the fungus is carried to all parts of the

body by means of wandering cells and emboli. The dis-
ease in man produces soft granulation tissue, which is not
encapsulated, and has a tendency to break down and to
spread slowly but extensively to the surrounding tissue
(chronic phlegmon). The formation of fistule favors the
extension. More rarely there are distinct tumors, as in
cows. In the actinomycotic pus the actinomyces bodies
are found. (See under Microscopic Findings.) There is
scarcely a tissue or an organ of the body in which the

_ actinomyces has not been demonstrated. Generalization

of the actinomyces throughout the body is rare (see Mess-

ner, C. B. x1x, 487).

In 1892, 421 cases in man were known. Recently acti-

- nomycosis has also been observed in America.

(d) In animals: Especially in cows (rarely in swine,
dogs, and horses). Formerly it was considered quite rare

_ (1 in 10,000 to 1 in 8000), but it evidently is much more

common, and simply overlooked. Sometimes it is epi-
demic. The localization is similar to that in man. Most

_ often its seat is in the marrow of the upper and lower jaw;
_ the marrow is traversed by soft granulation tissue and
_ denser connective-tissue masses, the medullary cavity is

enlarged, and new bone grows out from the periosteum
(bony tumor). In other cases the soft parts of the face

_ may be primarily attacked and the bones first involved
_ from without. Also the pharynx and the wall of the
stomach may be primarily attacked. The maxillary

446 ACTINOMYCETES.

swellings were formerly described as white swelling, sar- 2
coma of the jaw, spina ventosa, etc.; the disease of the —
tongue as ‘‘ wooden tongue’’; the growth in the lymph- —

glands as scrofula, ete.

Experimental Observations Regarding Pathogenic —
Action.—As opposed to many positive statements, Bos-—

trém takes the stand emphatically that, in experiments on

various animals, a multiplication of the introduced para-—
sites is never observed, but only an encapsulation of the
same. The most recent investigations of Wolff and Israél

demonstrate that sometimes the short rods, when intro-

duced intraperitoneally, grow out into threads and colo-
nies. They did not succeed in producing a grave, progres-—
sive disease in the experimental rabbits, and after about ~

seven weeks the organisms appear to die out. (Compare
Mycobacterium lacticola and phlei, p. 434. )

Special Methods for Diagnosis and Cultivation.— i

Diagnosis: In man very often by recognizing the actino-

myces clusters with the naked eye, or at least with the

microscope (unstained or with a double stain).

Cultwation: For diagnostic purposes it is usually unne-—
cessary. If aculture is to-be made, it is best accomplished ~
by means of numerous smears upon serum or ascites-agar, —

:

after the contents of the actinomycotic swelling have been

thoroughly rubbed up in a mortar. The tubes are to be ~

kept at incubator temperature and protected with rubber —

caps.

produce the clinical picture of actinomycosis; for example,
the ‘‘ Actinomyces albus Gasp.’’ (See Gasperini, C. B.
xv, 684, and p. 455.) The ‘‘ Cladothrix liquefaciens
No. 2’’ of Garten (C. B. xv, 287) also stands very close.
The Actinomyces musculorum suis Duncker (Zeitschr.

f. Mikrosk. und Fleischbeschau III, No. 3), found in Ber-

lin in the pale, watery muscles of quite a number of swine,

is also related, but it has not been cultivated. There is the

formation of clubs, but characteristic clusters are lacking.
The organism described by Kruse as the Strept. Israéli
is quite different.

While Bostrém isolated the organism here described —
from all cases in men and cattle, Italian investigators —
especially claim that other closely related varieties may —

ACTINOMYCES FARCINICUS. 447

-
:

Actinomyces Israeli. (Kruse.) L. and N.

Isolated twice from cases of human actinomycosis,
grows best anaerobically, exhibits no branching upon
ordinary nutrient media, and is readily inoculable into

animals.

_ Other ‘‘ atypical or pseudo ’”’ causes of actinomycosis
have been described (in part, incompletely) by Berestnew
(Z. H. xxrx, 94, etc.). According to him, organisms re-

_sembling actinomyces can be very easily obtained if par-
ticles of barbs, etc., from grain are stuck into nutrient

media which are subsequently kept in an incubator.

Actinomyces Hofmanni. (M. Gruber.) Gasperini.
Micromyces Hofmanni M. Gruber (A. H. xv, 35).

| This organism was obtained on one occasion from air in Vienna. It
_ forms no air hyphz, but the contents of the older threads of the fun-
_ gus break up into cocci-like fragments. Especially beautiful is the
formation of clubs exactly like those of the actinomyces and the
_ observation of their final calcification (after several months) in bouil-
lon cultures. It grows aerobically, and will only grow anaerobically
if sugar is added to the medium. It does not grow below 22°, 37°
being the optimum. Does not grow on potato and gelatin, and but
poorly on serum and agar, but, on the contrary, it grows well upon
most solid and liquid nutrient media with the addition of 0.5% to
3% of sugar. Sugar-agar cultures: Superficial colonies are elevated,
_ sharply outlined, wrinkled, lusterless; the deep ones show a radiating
_ structure with a delicate fringe. From sugar it forms acetic acid and
some alcohol.
| In animals, especially rabbits, it causes swellings filled with leuko-
cytes and coagulated exudate, which then soften and heal by encapsu-
- lation, and which contain beautiful clusters.

Actinomyces farcinicus. Gasperini.
(Plate 66.)

Synonyms.—Bacille du farcin du Boeuf (Annales de
- P Inst. Past., 1, 1888, p. 293). Nocardia farcinica Trevisan
et de Toni.
__ Microscopic Appearance.—Short, segmented (knotty)
threads with true branching. Nocard has photographed
_ true branchings, but has interpreted them as false ones
(66, x).

448 ACTINOMYCETES.

_ Spontaneous motion is lacking.
Staining Properties.—Stains with ordinary anilin
dyes, and by Weigert’s modification of Gram’s method, in 2

which anilin is used instead of alcohol after the iodin. oe

According to Nocard, it does not stain well with the common aaa
dyes. According to Berestnew, it stains by Ziehl’s method (Z. H
XXIX, 94); but according to Nocard, it does not. We could not
repeat the tests, as our culture proved to be dead. .

Requirements as Regards Temperature and Com--
position of Nutrient Media.—Not particular as to
nutrient medium; grows at room temperature, and espe-
cially at 37°. :

Gelatin Plate.—(a) Natural size: Scanty growth,
After ten days there are only minute, round, transparent,
shining colonies (66, v).

(b) Magnified fifty times: The superficial and deep col-
onies appear as smooth-edged, shining, gray to grayish-
green masses in which no finer structure is distinguishable
(60, v1).

Gelatin Stab.—Scanty; after twelve days the surface
growth is whitish and granular; that in the stab crumbly
(66, 11). :

Agar Plate.—(a) Natural size: The superficial colonies —
reach a size of 1 to 2 mm., are yellowish-white, irregular
in form, and shining. The deep colonies remain tinyg
(66, vit).

(6) Enlarged fifty times: The surface colonies are similar
to those on gelatin plates. The deep colonies are bright
yellow, delicate, distinctly filamentous, and curly in struc-
ture (66, vu).

Agar Stab.—About like the gelatin stab. Upon the
surface of the agar there forms a whitish, coarsely granular
mass, which is very irregular in shape because of fissures.
The faintly colored growth shows air mycelium in pn
(Nocard).

Agar Streak.—After eight days there is formed a gray
to yellowish- -white growth, consisting of transparent col-_
onies, with a rough, finely fissured surface. The colonies — :
are slightly or not at all connected. The water of conden-
sation is clear, with slight grayish-white precipitate. After

ACTINOMYCES ASTEROIDES. 449

cultivating it for years we obtained growths which were
_ actually more yellow and wrinkled, similar to our Act.
_ bovis. The consistency became tough, while earlier it was
~ crumbly.

Bouillon Culture.—Bouillon remains clear, with a
moderate, slimy to tough precipitate, which is not entirely
dissociated by vigorous shaking. Single colonies develop
at the top as dirty gray pellicles with a dusty surface.
Upon glycerin bouillon (according to Nocard) the pellicle

_ is tougher.

Milk Culture.—Casein is dissolved without coagula-
tion. Reaction alkaline.

Potato Culture.—Grows slowly (according to Nocard,
rapidly), is whitish-yellow and lusterless. The surface

_ appears as if beset with dry scales.

** Spores.’’—We have not observed them. Nocard de-
scribed non-staining spores.

Distribution.—Cause of ‘‘ Rinderwurms,”’ ‘‘ farcin du
beeuf,’’? occurring upon the island of Guadeloupe, and
rarely in northern France. The clinical picture resembles
that of cutaneous glanders, as also that of tuberculous affec-
tions of the cutaneous lymph-glands.

For animal experiments guinea-pigs are most suitable;
then cattle and sheep. Rabbits, dogs, cats, horses, and

_ the ass appear to be immune. In guinea-pigs intraperito-

neal and intravenous injection is followed by death in
nine to twenty days, with the clinical picture of mili-
ary tuberculosis, yet the nodules contain a ball of the
threads of the fungus (clubs?). Subcutaneous infection
produces a very chronic disease in all susceptible animals,
which answers to the picture of the spontaneous farcin du
boeuf.

Actinomyces asteroides. (Eppinger.) Gasperini.

Synonyms.—Cladothrix asteroides Eppinger ; Ziegler’s
Beitrage, 1x, 287, good illustrations. Strept. Eppingeri
Rossi- Doria.

Microscopic Appearance.—Rather sturdy, branching
threads. When stained by Gram’s method and faintly

decolorized, as also in the fresh preparation, they have no
29

450 ACTINOMYCETES.

distinct partition walls,’ but many threads show a frag- *

mentation into short, square (‘‘cocci-like’’) segments,

which (according to Eppinger), by opening of the thread :

membrane, may become free at the end (not observed by

us). The branching, as represented by Eppinger, and as —

always seen by ourselves, is true branching, although he
described it as false.

Spontaneous Motility.—The shorter threads exhibit a
sluggish, and the shortest threads and spherical forms a
very active motion (Eppinger). We have observed no
motility.

Staining Properties.—Stain with all the anilin dyes

Fig. 22.—Oospora asteroides Sauv. and Rad. (after Eppinger).

and by Gram’s method. According to Berestnew (Z. H.
xxx, 94), they also stain by Ziehl’s method.

Requirements as Regards Nutrient Media and Temperature.—
Grows best at 37° and upon all ordinary nutrient media, but most
luxuriantly on 2% grape-sugar agar. Upon gelatin at room tempera-
ture the growth is limited and the colonies are similar to those on agar.
There is no liquefaction. Old gelatin cultures are distinguished by an
orange-red color.

Agar Plate.—(a) Natural size: In the depth the col-
onies are round and slight. On the surface they grow
well, and are circular, with a yellowish-white, faint, finely
granular nucleus and a narrow, pale, peripheral zone.

1 By overstaining with hematoxylin and differentiating with glacial
acetic acid-glycerin mixture, and counterstaining with eosin, Eppinger
has convinced himself that the long threads consist of shorter and
longer rods in a sheath,

dette

eae | \

he Oe wees,

—<~— Ts
ay

a ty

au a

—e opel: te bith Ll anal

— a ear Ya

~~

2
i

ACTINOMYCES CARNEUS. 451

(6) Magnified fifty times: When very young they are
delicate, star-like, branching figures; later there gradually
- develops a denser, opaque center, with a delicate, ramifying

_ peripheral zone.

Sugar-agar.—Stab: After twenty-four hours there is a
small, whitish wart upon the surface, which gradually
grows into a slightly elevated disk ‘with a somewhat
wrinkled surface and brownish-yellow color. The wrink-
ling, elevation, and extension of the growth increase for a
long time; the periphery presents a delicate, flat, radially

_ wrinkled border. In the stab there is only slight growth

—_——_—

in the upper part. Upon ordinary agar the growth is more
feeble and paler in color.

Bouillon Culture.—Delicate surface pellicle with white
granules. The latter grow downward as tough masses (re-
sembling drops of stearin), and then fall to the bottom,
where a rich mass of the fungus gradually accumulates.
The bouillon always remains perfectly clear.

Potato Culture.—At first a coarsely granular fillet,
made up of snow-white nipples, and by degrees becoming
wrinkled and brick-red. After about fourteen days a deli-
cate, white, hairy covering develops at the periphery and
gradually covers the entire red growth.

Spores.—Nothing is known regarding the resistance of
the short segments known as ‘‘spores.”’

Distribution.—Found by Eppinger on only one occa-
sion in the lymph-glands, and especially in a brain abscess
and in the cerebral and spinal meninges of a glass-grinder,

where it apparently was the cause of the disease.

Pathogenic Effects.— W hen introduced in various ways,
it causes in animals (guinea-pigs, rabbits) a fatal disease re-
sembling tuberculosis. (Pseudotuberculosis cladothrich-
ica.) It has been shown by Lubarsch that it can form in
animals colonies which are deceptively like actinomyces.

' Actinomyces carneus. (Rossi Doria.) Gasperini.

Streptothrix carnea Rossi Doria. Closely related to the
Act. asteroides. However, ‘velatin and agar cultures show
distinct air mycelium, which gives the gelatin culture a
pink, and the agar culture from a flesh to a reddish-

452 ACTINOMYCETES.

orange color. It is not pathogenic for animals. The
actinomyces aurantiacus (Rossi Doria) Gasperini is
similar. oe

Actinomyces madure. (Vincent. ) Lehm. and Neum,

Streptothrix madure Vincent (A. P., 1894, 129).

There is great similarity to actinomycosis in the course
of the disease known as ‘‘madura-foot, madura-boil,
Dehli-boil”’ (first brawny, then nodular, usually perfor-
ating externally). It is a disease, affecting especially the
feet and hands, which is native to India, but also occurs
in northern Africa, Italy, etc. Our description is from
Vincent. In the pus from the fistule there are found
bodies (gray, yellow, black) similar to those of actino-
mycosis, which, according to Kanthack’s illustrations,
have the same structure as actinomyces granules.

/.. aoa

rt Ce eee

bibinat):

The organisms are obligate aerobes, and grow excellently upon de-
coctions of potato, turnip, etc., which have not been neutralized, with —
the production of alkali. As the best solid nutrient medium, Vincent
employed a decoction of hay or potato to which is added, for every
100 gm. of gelatin, 4 gm. of glycerin and 4 gm. of grape-sugar. Gela-
tin is not liquefied. Old gelatin cultures resemble a vaccine pustule,
being dry, closely attached to the nutrient medium, somewhat de- —
pressed in the middle, whitish, the periphery red. Upon potatoes —
whitish-red prominences, which often present an air mycelium with —
conidia ; also in other mycelium, spore-formation occurs. There is no —
musty odor. The spores die in three minutes at 85° and in five
minutes at '75°. The cultures with no spores die at 60° in from three
to five minutes. The threads and conidia stain readily with all the
anilin dyes and by Gram’s method. It is not pathogenic for animals
(rabbits, guinea-pigs, mice, cats).

3
4
D
7
Actinomyces chromogenes. Gasperini.
(Plate 67.)

Synonyms.! — Streptothrix chromogena Gasperini, .

Oospora Metschnikovi Sauvageau and Radais,? Strepto-
thrix nigra Doria. Cladothrix dichotoma Macé, Giinther

1 We describe a variety isolated by ourselves ; the synonyms we have
determined in part from comparison with the descriptions, in part from
the cultures. 3

2 Sauvageau and Radias could not find spores in their Streptothrix —
Metschnikoyii, ;

a ee ae

al ri i

La

ACTINOMYCES CHROMOGENES. 453

“non Cohn. Cladothrix odorifera Rullmann (C. B. xvuy,

884, and C. B. L. mn, 701). ‘‘ Brauner Hesse.’’
Microscopic Appearance.—True branching threads,

often with perceptible separation into longer and shorter

rods (67, x). There isno motility, but Rullmann has seen
motion in the youngest stages.

On the air threads (see below), by continuous trans-
verse division, short roundish members are formed (true
conidia), which very readily fall off, and, upon germina-

_ tion, form new branching mycelium.

Fig. 23.—Actinomyces chromogenes. Gasperini. (Upper side of a
bouillon pellicle magnified about 700 times. )

Staining Properties.—With all anilin dyes and by
Gram’s method.

Relation to Oxygen.—Grows better.aerobically. _

Requirements as Regards Temperature and Nutri-
ent Media.—Thrives upon all ordinary nutrient media at
room and incubator temperature, more rapidly at the
latter.

Gelatin Plate.—(a) Natural size: At first the colonies
are brownish, round, slightly elevated, tough, dull, and
begin first in the center, rarely at the periphery, to take
on a whitish, chalky condition. Hereupon are formed
concentric, wide, white rings; the drier (thinner) the nu-
trient medium, the more rapidly there occurs a more or
less complete overgrowth of the colony, with white air
hyphe, and therewith a chalky appearance. The gelatin
near the colony is colored dark brown and is slowly lique-

454 ACTINOMYCETES.

in shallow cups (67, v and v1).

fied, so that finally round, pea-sized, chalky crusts float

(b) Magnified sixty times: Very young colonies consist —

of a confused ball of threads; older ones appear but
slightly transparent, with zones having wavy, jagged boun-
daries, all of which are darker in their peripheral por-
tions. The edge of the colony is beset with delicate
threads, like a fringe, which extend outward into the dis-
colored gelatin (67, vm).

Gelatin Stab.—The surface growth is like that on the
gelatin plate. Sometimes drops of fluid (no oil!) areseen
upon the surface of the growth. The gelatin is very slowly
liquefied from above downward. In the stab the short,

radiating tufts of fibrils, which even at first develop, can —

be observed for a long time (67, 1).
Agar Plate.—(a) Natural size: As upon gelatin.
(b) Magnified sixty times: After about six days no struc-

ture is distinguishable in the dense colonies; they are —
dark and homogeneous and surrounded by distinct fringes —

(67, vit).
Agar Stab.—Upon the surface the growth at first is

rather moist, with a yellowish luster and elevated like the /

head of a nail; later it is drier, tough, and somewhat
puffed. The agar is discolored brown. In the stab there
are radiating, bristle-shaped branches (67, m1 and Ivy).

Agar Streak.—The growth spreads out only moder-
ately, presents (after four to six days) a brownish color,
and on the thinner parts of the agar a white, chalky,
peripheral zone. In the course of time the whole becomes
whitish and chalky. Upon the clear water of condensation
there later forms a tough, brownish film, which also de-
velops chalky, white air hyphe, especially on the glass
walls (67, 11). At other times a clumpy growth is pres-
ent at the bottom of the water of condensation without any
pellicle.

Bouillon Culture.—At first a delicate and later a dense
pellicle. . In grape-sugar bouillon there are thick, clumpy,
radially arranged masses on the bottom. The bouillon
becomes brown.

Milk Culture.—Dense, yellowish-brown to cinnamon-
colored layer on top. The milk is clarified and alkaline.

ACTIN. CHROMOGENES GASPERINI B ALBA. 455

Potato Growth.—Growth quite rapid and luxuriant.
As early as forty-eight hours in the incubator, a yellow,
yellowish-brown, greenish-brown, or brown layer, 8 mm.
wide, has formed. Im our cultures the chalky appearing
air hyphe always began at the edge. The potato is later
discolored intensely brown to black, and becomes strongly
alkaline.

Chemical Activities.—There is produced a dark-brown
pigment and an intense moldy odor upon all nutrient
media. According to Rullmann, the earthy smell is most
intense upon bread pap and other media composed of car-
bohydrates. There occurs a nitrogenous body, soluble in
water and ether. The odor is said to be the same as arises
from an unclean floor upon washing. Ammonia is abun-
dantly formed. According to Rullmann, in symbiosis with
fission-fungi it has considerable capacity for forming ni-
trate.

Distribution.—(a) Outside the body: In Wurzburg it is
not uncommon in the air, soil, and water, and appears
also otherwise distributed.

(6) In organism: We found it once in gastric contents.

Special Methods for Demonstration and Cultiva-
tion.—Agar plates in incubator. Observe brown halo,
chalky discoloration, odor.

Actinomyces chromogenes Gasperini @ alba. L. and N.

Streptothrix Foersteri Gasperini (Cohn?), Streptothrix
alba Rossi Doria, Streptothrix I and II Almquist, Oospora
Guignardi Sauvageau and Radais, Actinomyces albus Gas-
perini,! Oospora Doris Sauvageau and Radais.

According to Doria, it is especially frequent in Rome, but occurs
also in Wiirzburg. It does not color the nutrient media. It forms a
white cushion of circular form, and tends to produce abundant air-
spores. Gelatin is liquefied.

According to Gasperini, the cultures many times exhibit a sudden
production of pigment, like the Actin. chromogenes. According to
Rossi Doria, it also grows upon fucus (sea-weed) nutrient media with
a dark discoloration of the nutrient medium. From what we have
seen and learned from the literature, the only possible comprehension

1 The Cladothrix invulnerabilis Acosta y Grande Rossi (C. B. XIv,
14) appears closely related. It is said to bear heating for one-quarter
hour to 120°. The cultures have an earthy odor.

456 ACTINOMYCETES.

of this form appears to be as a variety of Act. chromogenes. According
to Gasperini, this form may cause actinomycosis in cattle.

Actinomyces violaceus. (Rossi Doria.) Gasperini.

Found many times in Rome by Doria. It liquefies gelatin. The
nutrient media are discolored. Gelatin becomes bright wine-red, agar
grayish-violet, potato reddish-brown.

Streptothrix aurantiaca, citrea, albido-flava have been described
by Rossi Doria (J. e.), and their standing as species must be proved
by further comparison. According to Gasperini, they are all to be
considered as actinomyces.

Lachner-Sandoval (J. c.) has studied in detail the Actinomyces
albido-flavus Gasp. He describes the germination of the conidia as
accompanied by elongation, many times following two directions simul-
taneously. The vegetative forms were killed by 70° in three minutes;
the conidia by 80° in five minutes.

A ‘‘streptothrix’’ which is not acid-proof, and which possesses
pathogenic properties for animals, was isolated by Rullmann from
sputum (Miinch. med. Wochenschr., 1898, 919). Upon Léffler’s serum
it furnished chrome-yellow growths, otherwise they were colorless.
Often the cultures give off a moldy odor.

Actinomyces erysipeloidis. (L. and N.) Lachner-
Sandoval.

Synonyms.—Oospora erysipeloidis L. and N., Strep-
tothrix Rosenbachii Kruse.

As the cause of the rare sporadic disease, chronic ery-
sipelas, erythema migrans, ‘‘ erysipeloid’’ of Rosenbach,
the latter author has described a true branching micro-
organism, related to the ‘‘ cladothrix,’’ but often occurring
in the form of short rods and spheres. The threads often
terminate in ‘‘a thick point.’’ The description of the
cultures reminds one most of mouse septicemia. In all
growths they become brownish. It grows best at about
20°, less well at incubator temperature. When inoculated
into man, it causes non-febrile, intensely itching, sharply
outlined redness, which spreads slowly.

Actinomyces necrophorus.! (Fliigge.) L. and N.
Bacillus of diphtheria in calves (Mitteil. des Kais. Ges.
Amts., 1). Bac. diphtherie vitulorum Fliigge and B.

1 Since Fliigge gave both the above names simultaneously, we are
free to choose between them. The species designation of ‘‘ necropho-
rus’’ seems to us to be especially happy.

/

od Riedi Sas Re

i, on . EO rie

rae ieee

HIGHER FISSION-FUNGI. 457

necrophorus Fliigge, Necrosis bacillus Bang (C. B. x1,
201). Streptothrix cuniculiSchmorl. Actinomyces cuni-
culi Gasp. (Deut. Zeitschr. f. Tiermed., xvi). The or-
ganism lies in the necrotic tissue (diphtheritic membrane,

_ ete.) on the side turned away from the surface as long,

_ often radially arranged meshes or bundles, separated from

healthy tissue by a narrow, necrotic zone. It is an obli-

_ gate anaerobe, growing best on blood-serum or blood-serum

1 See, HP = Bee

agar at incubator temperature, and has been incompletely

_ described as to its morphology (branching!). Itis of great

practical interest as the cause of numerous diseases in ani-
mals. It was cultivated by Schmorl from a destructive
epidemic in rabbits, and first described by Léffler as the
cause of calf diphtheria (mouth, larynx, nose). Accord-
ing to Bang, it also causes in young and old cattle, horses,
and swine the most various necrotic affections (panar-
itium, gangrenous pock, intestinal diphtheria, liver ab-
scess, vaginal and uterine diphtheria, etc.). We were un-
able to study this evidently important organism.

Regarding thermophilic varieties of actinomyces, see Kedzior
(C. B. L. 11, 154) and Tsiklinsky (C. B. xxv, 385).

After subcutaneous inoculation of mice, Loffler obtained the picture
of progressive connective-tissue necrosis. A layer of lardaceous infil-
tration extends subcutaneously from the point of infection and envel-
ops the kidneys, liver, and intestine with yellowish masses of exu-
date. According to Léffler, rabbits are not affected characteristically ;
but according to Schmorl and Bang, they are. All investigators found
other experimental animals to be immune.

APPENDIX II.

Higher Fission-fungi. (Higher Fission-
algze.)'
The close relationship with the chlorophyllaceous alge
is still more evident in the varieties of this section than in

1 We have had little personal experience with this group, and
limit ourselves partially to a critical review of the literature.

458 HIGHER FISSION-FUNGI.

all the forms previously discussed, and many investigators

consider them as true alge. On the other side, the con-

nection with the simple fission-fungi is still so close that —

at least a brief mention seems necessary.

In distinction to the true bacteria, the members of this’

group have this in common, that the threads can be recog-

nized as having a basal (not growing, often attached) and —

an apical (growing, free) end. The ends are often of
different thickness.

Key to the Recognition of Some of the More Impor-
tant Genera of Fission-alge.

T hreads without distinct sheaths :

(a) Without sulphur granules. Leptothrix Kiitzing, see below.

(b) With sulphur granules, motile, not attached. Beggiatoa Tre-
visan, page 461.

T hreads with sheaths :

(a) Without sulphur granules.

1. Without pseudodichotomous branching. Crenothrix Cohn, page
463.

2. With pseudodichotomous branching. Cladothrix Cohn, page
465.

(6) With sulphur granules. Thiothrix Winogradsky.

Leptothrix epidermidis Biz.
(Plate 69.)

Microscopic Appearance.—In our preparations there
are sturdy unbranched threads, distinctly jointed and
readily falling apart, with no evident distinction as to base
and apex. Young cultures show bacilli about like the B.
mesentericus (69, x1 and x1).

Motility.—The young rods present distinct motion like
that of bacilli. We were unable to stain flagella.

Staining Properties.—Stains with all anilin dyes and
by Gram’s method. With iodin and iodid of potassium
alone there is no blue stain.

1 We must certainly admit that the discernment of the two ends
has often caused greater trouble than was to be expected from the
statements of Biicher, for example, in the case of Beggiatoa.

2 Before the leptothrix of the mouth had been cultivated we could

place the L. epidermidis among the ‘‘true’’ leptothrices only with
reservation. (See p. 461.)

ee, ee ee ee ee en

LEPTOTHRIX EPIDERMIDIS. 459

Relation to Oxygen.—Grows better with the admission
of oxygen.

Requirements as to Temperature and Nutrient
Media.—Grows luxuriantly and rapidly at room and in-
cubator temperature upon all nutrient media.

Gelatin Plate.—(a) Natural size: At first, minute
white points which liquefy the gelatin as soon as they
become a little larger—after twenty-four to forty-eight
hours. The older colonies exhibit a small white flake in
the center of the liquefied areas; also the edges of the areas
present a whitish border (69, vimz).

(6) Magnified fifty times: Young, superficial colonies
present a pretty convolution of curly, wavy threads. The
center soon becomes dark and cloudy and sinks in, while
there remains a row of fine radiating hairs as a peripheral

a: y </
DS Sgrd

Fig. 24.—Leptothrix epidermidis Biz.

zone. As the liquefaction advances there is finally a flat,
gray saucer, which presents a delicate hairy border toward
the solid gelatin, and in the center of which is a curly
mass whose structure becomes more and more indistinctly
crumbly (69, 1x).

Gelatin Stab.— After twenty-four hours there is formed
a conical area of liquefaction with whitish flocculi, and at
the apex crumbly, yellowish-white masses of bacteria

gradually accumulate. After four or five days there forms

a grayish, tough pellicle on the surface (69, 1).

Agar Plate.—(a) Natural size. Superficial colonies:
White or yellowish white, sharply outlined growths with
smooth but irregularly notched borders. The deep colonies
remain small, dense, yellowish-white (69, v).

460 HIGHER FISSION-FUNGI.

(6) Magnified fifty times. Superficial colonies: The center :

is opaque, brownish-yellow, passing gradually into a yel-
lowish-gray peripheral zone which consists of very closely

set, fine, radiating hairs (69, v1). Deep colonies: Roundish,

nodular, yellowish-brown, here and there presenting single
or bunched projecting hairs (69, vi).

Agar Stab.—Surface growth: Yellowish-gray to brown,
rather smooth-bordered, slimy. Gradually it becomes dull,
with some whitish elevations. In the stab the growth is
like a grayish-white thread. After several months the
agar becomes colored dark brown (69, m1 and Iv).

Agar Streak.—Like the surface growth of the stab cul-

ture. Upon the water of condensation is a tough, wrinkled, ~

brownish pellicle. Where this passes on to the tube-wall
it is pure white. The water of condensation is clear
(69, 11).

Bouillon Culture.—Only on the surface there is a
tough, thick, wrinkled pellicle, which is firmly adherent
to the glass.

Milk Culture.—On the surface a dense scum. The
milk is not coagulated, and while the milk becomes trans-
parent, a limited white precipitate is formed.

Potato Culture.—There is rapidly formed an elevated,
reddish to grayish-brown, sharply outlined growth. In
time the surface develops wavy wrinkles. In old cultures
the peripheral zone presents a white, chalky discoloration
(69, x).

Spores.—There are no endospores.

Distribution.—Found by Bizozzero upon the skin of
healthy men. ;

Related Varieties.—This variety resembles in many
points the subtilis-mesentericus group. Wavy growth,
vigorous liquefaction, and air hyphze upon potato are
observed, in which respects its cultures are very similar to
those of the B. subtilis. The variety is more properly
called Bacillus epidermidis. To be sure, it must then be
taken for granted that accidentally a spore-free form of
bacillus is exemplified in this variety.

verge eee ee be

4 ee, Ae

Sam @. tw

‘ ~ o

BEGGIATOA ALBA. 461

Regarding the forms designated as ‘‘ Leptothrix buc-
calis,’’ which occur frequently in the mouth, especially
in deposits on the teeth, little can be said that is satisfac-
tory, since cultures have almost always failed.

Miller (Die Bakterien der Mundhohle, 11. Aufl., Berlin, 1894) ap-
to have cultivated no leptothrix. He cites the following uncul-
tivated leprothrices, briefly and very insufficiently characterized:

Leptothrix gigantea Miller. Threads fixed at one end, small to
very thick, with or without distinct septa. Iodin reaction?

Leptothrix maxima buccalis Miller. Jointed threads 1 to 1.3 uv
thick, without iodin reaction.

Bacillus maximus buccalis Miller. Like the preceding, but with
iodin reaction. It is notstated why this species is designated bacillus
while the former are designated leptothrix.

Leptothrix innominata Miller is said to present slender, 0.5 to 0.8
f thick, tangled, unsegmented, often wavy or bent threads, which
sometimes stain violet with iodin.

Arustamow (C. B. vi, 349) described two unnamed leptothrices of
the mouth, cultivated by him, both of which grow at incubator tem-
perature, No. 1 being an exquisite anaerobe, No. 2 an outspoken
aerobe. The agar growth corresponds in some measure to that of the
L. epidermidis; potato and gelatin growths are not described.

Dobrzyniecki (C. B. X XI, 225) has described in detail a Leptothrix
placoides alba, which was successfully cultivated aerobically on gela-
tin. The growth at first resembled anthrax, and then liquefied. The
growth on agar was slow—hard, dense colonies being formed. The
organism presents long, jointed threads, with a tendency to form tan-
gles, and it stains blue with iodin and iodid of potassium solution and
a little lactic acid. Stains by Gram’s method. Is not motile.

Flexner has isolated an interesting organism from a rabbit which
died of puerperal infection. It occurs in long threads, is always free
of spores, has no motion, and does not branch. It is pathogenic, but
very difficult to cultivate outside of the body. He names it Bacillus
(Leptothrix?) pyogenes filiformis Flexner (Jour. of Exp. Med.,

Vol. 1, 211, 1896).

Beggiatoa alba. Vauch.

Long and rather thick (1 to 5) unbranched threads,
without membranes, and quiet or with a gliding motion.
When fresh, there are in part no partitions, but often
numerous very highly refracting bodies may be recognized.
The granules consist of sulphur, which may especially be
recognized if the threads are previously allowed to dry,
and also by their solubility in H,S. In this way also the
previously indistinguishable transverse partitions become
apparent, According to Winogradsky, the breaking up of

462 HIGHER FISSION-FUNGI.

the threads into smaller pieces, which later grow out, is the —

only method of multiplication. The statements of Zopf

regarding other forms in the cycle of development of Beg-—

giatoa are opposed by Winogradsky. .

According to the old idea, Beggiatoa formed H,S and
sulphur from sulphates, and was the cause of H,S being
present in sulphur springs. According to Winogradsky,
on the contrary, it is dependent for its nourishment upon
the preexisting H,S, which it transforms into sulphur.

(See Untersuchungen tiber Schwefelbakterien, C. B. u,
590. )

Fig. 25.—Beggiatoa alba Vauch (after Zopf).

B. alba is found especially in foul slime and dirty water;
also sometimes as isolated individuals in pure water. If
abundant, they form whitish films.

B. nivea Rabenhorst is recognized in sulphur springs
as the principal constituent of the slime in the spring.

B. roseo-persicina. Zopf. (Die Spaltpilze, 3. Aufl.)
Bacterium photometricum Engelmann (Pfliig. Arch., Bd. 30, 95).
This variety is very striking because of its rose-color.

In the cooler parts of the year it spreads widely along the
banks of small streams, pools, etc. It is always a sign of

en ee ee fa ee f Wintec

eT Saree Se lh

F CRENOTHRIX POLYSPORA. 463

feontamination of water, but not specific (perhaps from
‘sulphite wood-pulp factories or the like). According to
Winogradsky, Zopf has improperly included in this variety
a large number of other rose-colored inhabitants of water.
Jegunow (C. B. m, 279) has reported many interesting
_ observations regarding another sulphur bacterium, whose
classification is still undetermined, but it most likely
_ belongs to the spirilla.

p Crenothrix polyspora. Ferd. Cohn.
(Cohn’s Beitrage, Bd. 1, H. 11, 130.)

Long, rigid, unbranched threads, consisting of a single
row of low cells, unpigmented, included by a membrane
which is very thin at the younger parts of the thread and
thick at the older parts. The membrane is a product of
the cuticle of the cell. In the membrane is deposited

ee ee ee ee tee a ny age AL 8 oe %

Fig. 26.—Crenothrix polyspora. Cohn.

some iron hydroxid or carbonate; which stains it brown.
Sometimes also the membranes for considerable lengths
are surrounded by a yellow, ferruginous mass with a luster
like oil, so that macroscopic, brownish flakes appear.

The thickness of the threads varies from 1.5 to 5.2 p, it
often being easily recognized that the older part of the

ee a ee ee ee ee,

464 HIGHER FISSION-FUNGI.

thread (where it is fixed) is wider and stronger. Alsothe —
height of the individual cells varies from one-half to four 2
times the thickness, square forms being most common
(Fig. 27, a and e).

Sometimes the terminal cell of a thread is large and 5
oval (like a spore), in which case a deeper cell grows out
laterally.

Propagation occurs through a peculiar breaking up of ‘
the cells at the end of a thread into fragments. Cohn dis- i
tinguishes two types: (a) Formation of microgonidia:
several individual cells in the thread break up by longi-
tudinal and transverse division into not less than 16 very

j | ene | Actas | a | ee | ee

ye |

r Ga \@
=)

\

oe Pct

Fig. 27.—Crenothrix polyspora. Cohn.

small plasma spheres, which later are freed from the some- —
what swollen ends of the threads and grow out into new
threads (Fig. 27, d). (6) Formation of macrogonidia: F
several of the cells in a thread near its end are transformed —
by infrequent division into larger, roundish, oval or diplo- —
cocci-like forms, which grow out into new threads (Fig. —
27, 6). One type passes over into the other. ;
The plant is widely distributed (especially) in waters —
containing iron (also in tap-water). A pure culture in the
bacteriologic sense has not been obtained. According to
Rossler, cultivation readily succeeds in well-water in which
pieces of brick are boiled and to which is added ‘‘some”’
ferrous sulphate. (Compare Ferd. Cohn, Beitrige zur

CLADOTHRIX DICHOTOMA. 465

Biologie, Band 1, Heft 1, p. 108, Breslau; Rossler, Arch.
f. Pharm., Bd. 233, 1895.)

Here also should be included the interesting Leptothrix
ochracea Kiitzing, which does not belong to the lepto-
thrices in a strict sense, because of its fully developed mem-
brane. Winogradsky, who describes it in detail, gives the
following characteristics: slender, jointed, fixed threads of
bacilli, with membranes. The membrane is thick below,
thin at the free ends, and the terminal rods are entirely
_ without any membrane. The bacilli in the membrane are
_ motile, but this motility is lost as soon as the membrane
has reached a certain thickness. This variety thrives only
in water containing ferrous oxid. In the metabolic pro-
_ cess hydrated oxid of iron is deposited in the membrane.
_ In hay decoction prepared from well-water and freshly
‘ precipitated hydroxid of iron the organism always grows
| easily and rapidly. It forms yellowish flakes and films,

1 OTM CH SOT alana mee, aA

and in nature gives rise to extensive ocher deposits. (Com-
pare Winogradsky, Bot. Zeitung, 1888, p. 261.)

Cladothrix dichotoma. Ferd. Cohn.
(Cohn’s Beitrage, Bd. 1, Heft. 11, p. 185.)

| Long, apparently non-segmented threads, with thick or
_ thin membranes, in part free, in part attached to putre-
_ fying alge. Thickness 1 to5 4. The pseudodichotomy
_ isespecially interesting. It is dependent upon the growing
_ of a lower segment of a thread along by the side of a higher
_ one (Fig. 30). Pure cultures of this-organism have been
_ but little studied; we have not possessed any. According to
Biisgen, the most recent investigator of this organism (Ber.
der deutsch. bot. Gesellschaft, 1894, p. 147), it grows
slowly and without perceptible liquefaction in gelatin con-
taining a little meat extract.

The surface growth consists of a ‘‘round white patch,”’
which is not elevated, and from which, as from the stab,
after a few days delicate threads grow out.

The threads have thin membranes when grown on gela-
tin and thick ones in dilute solutions of meat extract. The
membrane is patent at the end of the threads, and through
this opening, as also through irregularly occurring tears of

30

466 HIGHER FISSION-FUNGI.

the membrane, there pass (by means of unilateral bunches q

of flagella, 8 to 12 » long, according to G. Fischer, see Fig.

Fig. 28.—Characteristic appear- Fig. 29.—Characteristic appear-
ance of Cladothrix dichotoma nce of Cladothrix dichotoma
Cohn (after Migula). Cohn (after Cohn).

Fig. 30. — Pseudodichotomous Fig. 31.—Separation of flagel-
branching of Cladothrix dicho- lated organisms of Cladothrix di-
toma Cohn (after Cohn, reduced). chotoma Cohn (after A. Fischer).

31), short, actively motile bacilli, which, after wandering,
become fixed by one end and again form new threads.

eo veer hoe ee

ae Bes cate

| PHLYCTENULAR OPHTHALMITIS. 467
| There are no “‘spores’’ nor ‘‘sporangia’’ unless the occa-

7 sionally occurring expansions of the threads in which the
; rods lie in double rows are so called.

APPENDIX III.

Notes Concerning Insufficiently Elucidated
Diseases Which Perhaps Depend
upon Bacteria.

Of the diseases not yet mentioned in this book the fol-
lowing are outside of our consideration, because:

(a) Dependent upon higher molds: favus, herpes ton-
surans, the deep wound suppurations caused by hypho- .
mycetes, certain mold mycoses.

(6) Caused by yeast fungi: many tumors in man and
qpimals.

(ce) Dependent upon protozoa: malaria, dysentery (?),
Texas fever in cattle, Surra or Tsetse disease, variola.

Some diseases which are probably produced by fission-
fungi, as syphilis, are treated briefly in the text; some
others are suited only to a discussion in an appendix,
because that which is known regarding their etiology is
either very uncertain or so incomplete that the insertion
of the micro-organisms in a system is not possible.

Phlyctenular (Scrofulous, Eczematous) Ophthal-
mitis.

In contrast to the authors who would recognize in the
Micrococcus pyogenes alone the cause of the above-men-
tioned inflammations of the eye developing upon a scrofu-
lous substratum, a number of investigators have shown
that in carefully selected uncomplicated cases, in a major-
ity of the examinations, no micro-organisms were to be

468 DISEASES WITH UNCERTAIN CAUSES.

found i in the conjunctival phlyctenule and recent corneal
infiltrations. See Axenfeld (C. B. xxiv, 194).

Beri-beri.

Regarding this important tropical disease, the literature
contains the most heterogeneous statements. The cause of
the disease is recognized by Musso and Morelli (Compt.
rend. de la Soc. de Biolog., 1893, 18) in an organism
which is very closely related to the Micr. pyogenes a
aureus; by Hunter, in agreement with Pekelharing and
Winkler, in a white, motile staphylococcus (C. B. xxty,
537).

Besides, there are those who conceive of beri-beri as
a chronic intoxication (from sea animals), and place it
in the same class with pellagra, etc. See Grimm (C. B.
_ XXIV, 538).

Articular Rheumatism.

While some authors believe articular rheumatism is due
to the Micrococcus pyogenes (see p. 184), Bannatyne,
Wohlmann, and Blaxall (C. B. xx, 400) believe they
have found its cause in a short, fine bacillus (2 » long, 0.6
pw thick) which usually stains at the ends (eighteen posi-
tive cases!). In bouillon it slowly grows as small, fine
puncta and crumbly particles. From the bouillon scanty
growths upon agar and Loéffler’s serum may be obtained.
According to the English writers, the organism is con-
stantly found in the joint fluid; more rarely, and only in
severe cases, in the blood. Leyden has found in five
eases of endocarditis (C. B. x1x, 722) a fine diplococcus
which can hardly be cultivated at all, and which may
possibly be the cause of the disease.

Hospital Gangrene.

Vincent (A. P., 1896, 488) observed in Arabs, who were
infected in Madagascar, in the typical cutaneous ulcers,
very abundant, straight, rarely slightly bent, non-sporu-
lating bacteria which do not stain by Gram’s method. In
sections it is easily demonstrated in the characteristic loca-

wae se ee ee

+ tain

MEASLES. 469

tion. The organisms lie below the characteristic pseudo-
membrane in abundance. For their demonstration the
organs are first hardened in concentrated sublimate solu-
tion, then in alcohols of increasing strengths. The sec-
tions are stained ten minutes in cold phenolthionin solu-

_ tion, placed in alcoholic solution of iodin a few seconds

A
cs
4

;

(0.01 iodin in 200 alcohol), washed with alcohol, and
finally counterstained with safranin. The inoculation of
animals was successful only when streptococci; B. coli,
pyocyaneum, etc., were also inoculated with the special
bacterium. The organism appears strikingly similar to
those found in stomatitis ulcerosa (see below). Also
sometimes abundant spirochete are found in hospital
gangrene.

Pneumonia in Cattle.
(Péripneumonie des bovidées. )
It does not lie within the scope of this book to speak of

the cause of this disease in detail, while, on account of its
extreme minuteness, nothing definite can be said of its

- form even when magnified two thousand times. The cul-

tivation was primarily successful when small, thin, collo-
dion sacs, containing bouillon and a trace of the fluid from
the lung of a sick animal, were placed in the abdominal
cavities of living guinea-pigs. After fifteen to twenty days
the sacs were removed, and the fluid was found very slightly
cloudy because of the above-mentioned, most minute,
motile objects. By means of the organisms which have
been transplanted repeatedly upon artificial nutrient
media, cattle may be infected in the characteristic manner,
and the organism again be cultivated in vitro in peptone
solution to which a few drops of serum have been added.
(See Nocard and Roux, Annales de |’ Inst. Pasteur, 1898,
240. )
Measles.

Canon and Pielicke (C. B. xrvy, 287) claim to have found
constantly in fourteen cases of measles a bacterium which
is most variable in size (very minute to 3.4 »), and which
stains interruptedly with a mixture of 80 c.c. of saturated
aqueous solution of methylene-blue and 20 c.c. of 0.25%

470 DISEASES WITH UNCERTAIN CAUSES.

eosin solution (in 70% alcohol) after three hours at incu- F

bator temperature. Only from the sixth day of the disease

on, are the organisms found in preparations, which does —
not bespeak an etiologic significance for them. Still, the —
discoverers consider this organism, which does not stain by —

Gram’s method, and cannot be cultivated (only upon —

blood-bouillon many times a slight growth appears), to be
the cause of measles.

Recently Czajkowski (C. B. xvim, 517) has found sim- —
ilar organisms in the blood, which he has portrayed and —

cultivated upon glycerin-agar, but especially upon blood-
glycerin-agar. The growth is delicate, scanty, and like
dewdrops. The organism is pathogenic for mice. It is
motile and is not stained by Gram’s method.

Mouth and Foot Disease.

In opposition to numerous defective investigations which
have recognized the cause of this disease in most variable —

Ahh ely ara tt BN! tinal

sized and sometimes easily cultivated fission-fungi (coms

pare Stutzer and Hartleb, A. H., 1897, 372), Loffler and ©
Frosch have determined that the cause of the foot and
mouth disease is indeed - present in the contents of the

mouth and foot vesicles, but that it is so minute that —

it passes through dense bacterial filters and is invisible
with the best microscopes. Only after repeated filtration

through the densest filters does the lymph lose its infec-

tious properties (C. B. xxim, 371).

Myxoma Disease.

The cause is so far unrecognizable. The disease occurred —

spontaneously in Sanarelli’s rabbits in Montevideo. It

manifests itself in conjunctival catarrh, leonine swelling of —
mouth and nose, inflammatory swelling of the urinary and ~

genital organs and buttocks, especially in hyperplastic
changes at the places where the skin and mucous mem-
branes join. Also, myxomatous or gelatinous subcutane-
ous swellings which are very vascular are found in various
parts of the body. In most animals dying from the sub-
acute disease the postmortem reveals hypertrophy of the
lymph glands, orchitis, and swelling of the spleen. The

a

ee a ee

DISEASES OF PLANTS. 471

disease is easily produced in rabbits by inoculation with
the perfectly clear and apparently entirely sterile serum
from other cases. Also dogs and men react to the inocu-
lation (C. B. xx1, 865).

Noma.

Petruschky has found diphtheria bacteria together with
pseudodiphtheria bacteria in two cases of noma faciei,
which he cured with diphtheria antitoxin (Deut. med.
Wochenschr., 1898, 600).

Epidemic Parotitis.

Laveran (Compt. rend. de la soc. de Biolog., 1893,
95) found diplococci in the blood and organs in 67 out of
92 cases of mumps. They kill mice and cause in rabbits
and dogs a transitory orchitis, which often is associated
with mumps also. Mecray and Walsh (C. B. xxi, 68)
have made similar observations. The cultures were de-
scribed by the latter authors as very similar to those of
very poorly growing Microc. pyogenes.

Diseases of Plants.

In spite of the efforts of numerous investigators to de-
monstrate bacteria as the cause of diseases of plants, we
possess little knowledge which is free from objection in this
field. The botanist Alfred Fischer takes an especially
skeptical stand regarding the statements so far made (C. B.
L. v, 279), while Erwin Smith, who has himself done much
work in this field, is much more optimistic in his judg-
ment, and considers the connection of bacteria and disease
to have been demonstrated in a whole series of instances.
He enumerates some of the varieties of bacteria which are
most certainly pathogenic for plants (C. B. L. v, 271; in
the same place the literature is given). We refer to the
following: Bacillus amylovorus Burrill (cause of a dis-
ease of apple and pear trees), Bacillus olez Savastano
(tuberculosis of the olive tree), Bacillus hyacinthi-
septicus Heinz, Bacillus tracheiphilus Erw. Smith (in-
jurious to various cucurbitaceze), Bacillus solanacearum

472 DISEASES WITH UNCERTAIN CAUSES.

Erw. Smith (injurious to crucifere). Besides, there haye
been described bacterial diseases of vines, celery, and
sugar-beets, yet only exceptionally are the descriptions suf-
ficiently complete.

Rachitis.

Mircoli ascribes it to Microc. and Strept. pyogenes (C.
B. xx, 321).

Acute Rheumatism.

It may be mentioned that Sawtschenko, with Achalme,

has recently recognized the cause of acute rheumatism in

-an anaerobic, sporulating bacillus. Confirmation is awaited
with interest (C. B. xxiv, 794). )

Cattle Plague.

We only know that the causative agent does not pass
through a clay filter: the German investigators in South
Africa consider the statements, accompanied by illustra-
tions, of Nencki, Sieber, and Wyznikiewicz (C. B. xxi,
529) as delusions. They recognize the cause of the plague
in a small, spherical organism, but do not directly desig-

nate it a micrococcus. The organism is said to grow in—

peptone solution.

According to Abbe, the limit of the working capacity
of our microscopes lies at 0.1 to 0.2 pn.

Scarlatina.

Many of the older writers especially, and of the newer
ones;—for example, d’Espine (C. B. xvin, 132),—are of
the opinion that the specific cause of scarlatina resides in
the streptococcus which is very frequently present, but
this is almost certainly not true. Czajkowski (C. B.
xvi, 116) never failed to find in the blood of seven-
teen cases of scarlatina a diplococcus (according to the
illustrations, more like short diplobacilli) which grows to
a limited extent upon solid nutrient media (glycerin-agar,
blood-agar, serum), more luxuriantly in fluid nutrient
media, and is pathogenic for mice. It is not stated
whether the diplococcus appears as a streptococcus upon

ee Tat Pe ee

Sl Eppes 2;

TYPHUS EXANTHEMATICUS. 473

fluid nutrient media. The great tenacity of the cultures
_is striking.

Doehle (C. B. xr, 906) and L. Pfeiffer consider proto-
zoa to be the cause of scarlatina.

Ulcerative Stomatitis and Angina.

Bernheim (C. B. xxi, 177) described two organisms as
frequently or constantly present in the ulcers of the gums
and tonsils:

1. A hacillus resembling the B. diphtheriz (once no-
ticed to be motile), somewhat larger than it, with pointed
ends, often more or less curved, staining rather faintly
with Léffler’s methylene-blue, and decolorized by Gram’s
method if the alcohol acts a long time.

2. A fine spirochete, which does not stain by Gram’s
method, similar to the spirochetee of the teeth.

Neither could be cultivated.

Similar results have been obtained by many other
writers, as Vincent and Abel. (See Abel, C. B. xxtv, 1).
The results obtained by Vincent in hospital gangrene in
Madagascar are strikingly similar; also both organisms
were present. (Compare p. 468.)

J. Seitz has described as the Bacillus hastilis a widely
distributed (tonsils, etc.), long, slender organism with
pointed ends, which has not been obtained in pure cul-
ture. It appears to be closely related to Bernheim’s
organism of stomatitis ulcerosa. From non-saccharine
bouillon it forms foul-smelling gas (Z. H. xxx, 47).

Trichorrhexis Nodosa.

According to Marcusfeld, caused by a sporulating bacil-
lus, perhaps from the subtilis group. The relation to the
disease is uncertain (C. B. xxi, 230).

Typhus Exanthematicus.

Lewaschoff (C. B. xu, 635, 728; xvim, p. 132) claims
to have cultivated a characteristic Micrococcus exanthema-
ticus in pure culture upon ascites-agar from the juice of
the spleen or blood from the finger in 118 cases of typhus

A74 BACTERIOLOGIC TECHNIC.

fever. It is strikingly motile, always in the blood, and ©
grows anaerobically. Both in the blood and in cultures ~
many, but not all, of the individuals present one or two —
very long, motile, spiral appendages, which take the stain ©
for flagella. Lewaschoff calls this form of remarkable —
spirochete, exanthematica. According to his opinion, —
there is perfect agreement between his findings and the

newer investigations of Ljubimoff (cocci), Calmette and

Thoinot (A. P., 1892, 39) (egg-shaped bodies and spirilla), —
von Dubief and Bruhl (C. B. x1v, 17), Curtis, and Combe- —
male (diplococci). .

7 t

pues

-

APPENDIX IV.

6 OLA uel) decid Males

Essentials of Bacteriologic Technic.

The following directions and short explanations include about all
the technical material which is given in a thorough bacteriologic
course. We have introduced only those things which are essential
and, according to our experience, practical, without referring to the
literature. More details will be found in the books mentioned in the
preface.

I. Microscopic Examination of Bacteria.

1. Hints upon Microscopic Technic.

For bacteriologic examination we use almost exclusively the modern
microscope with Abbé’s illuminating apparatus, iris diaphragm, and a
low-power and an oil-immersion objective.

(A) Low magnification (60 to 100 times) with a narrow dia-
phragm is used in the minute examination of plate cultures. In this
examination either the cover! is removed and the colony examined
from above, or, if one does not wish to contaminate the plate by expos-
ing it, the dish is laid upon the cover and the colony examined from
below, but this does not give such characteristic pictures in all cases.

(B) High Magnification. Oil-immersion Objective (700 to
1200 times).—This finds its use in the examination of single indi-
viduals. A drop of cedar oil is placed upon the preparation (slide,
cover-glass) and the tube is lowered by means of the coarse adjust-

1 Our plate cultures are always poured into dishes.

MICROSCOPIC EXAMINATION. 475

ment until the lens just touches the oil; then it is accurately adjusted
upon the preparation with the micrometer screw.

(a) Unstained preparations. Narrowdiaphragm! They are exam-
ined in two ways:

1. A drop of pure culture in a fluid medium or a little drop of water
with a trace of pure culture mixed in it is placed between the slide
and cover-glass; or, better,

2. Ina hanging drop. <A drop of a pure culture in a fluid medium
or a drop of bouillon in which is mixed a minute quantity of a pure
culture is placed upon a cover-glass; the cover-glass is then turned over
and placed upon a hollow ground slide so that the drop is suspended
within the hollow. The cover-glass is now fixed to the slide by apply-
ing a very little water to each corner of the cover, or, if the observa-
tion is to be more prolonged, by means of vaselin.

(b) Stained preparations. Open diaphragm! Abbé’s illuminating
apparatus. In the examination of sections with a double stain, the
wide diaphragm is required for the bacteria, the narrow opening for
the tissues.

(C) Cleaning the Preparations and the Microscope.—The
immersion oil is always gently brushed off, and now and then quickly
cleaned with xylol and chamois skin; the setting of the lens is loosened
by prolonged action of the xylol. Also immersion oil dried upon the
cover-glasses of old preparations is readily removed by xylol.

2. The most Important Solutions for Use in Making
Preparations.
(A) Staining Solutions.

1. Aqueous Alcoholic Solutions of Fuchsin and Methylene-
blue.—A concentrated ‘‘stock-solution ’’ is prepared by pouring abso-
lute alcohol upon the pulverized dyes (fuchsin, methylene-blue) in

_ bottles, and after shaking and allowing them to stand a few hours,

they are filtered. Of this saturated solution 1 part is mixed with 4
parts of distilled water, and before using is filtered. In order to
obtain good preparations it is better to stain a longer time with weaker
solutions than for a short time with strong solutions.

2. Carbol-fuchsin (Ziehl’s Solution).—

eh Se Oded ON eee al en 1.0 gm
ee OO. LUNE Ss os eat ete se st ice
MEN er ohne ray eh} mane ee 10.0 ‘
Aq. dest. Ee OR ee Se ae 90.0, **

3- Anilin Fuchsin.—Four parts of anilin oil (anilin. pur.) are well
shaken several minutes with 100 parts of distilled water, and then fil-
tered until all the water has passed through clear (then the funnel is
removed, since otherwise oil will also pass through). In this anilin
water 4.0 gm. fuchsin are dissolved and it is again filtered.

4. Anilin Gentian-violet (Ehrlich’s Solution).—To 100 c.c. of
anilin water, 11 c.c. of a concentrated alcoholic solution of gentian-
violet (stock solution) is added. This solution does not keep long.

476 BACTERIOLOGIC TECHNIC.

Czaplewski and E. Frankel recommend that instead of anilin water,
2.5% carbolic acid water be employed. This has the advantage that
the solution does not decompose so soon as anilin mixtures.

5. Loffler’s Methylene-blue.—To 100 c.c. of water is added 1 ¢.c.
of 1% solution of potassium hydroxid, and 30 c.c..of concentrated
alcoholic solution of methylene-blue. The staining property of the
dye is intensified by the addition of the alkali.

6. Acetic acid methylene-blue, according to M. Neisser, for the
staining of granules:

(a) 1.0 gm. of methylene-blue is dissolved in 20 c.c. of 90% alcohol,
and to this is added 950 c.c. of distilled water and 50 c.c. of glacial
acetic acid. ;

(b) 2.0 gm. of Bismarck brown are dissolved in 1 liter of boiling dis-
tilled water (filter !).

7. Bismarck Brown.—Prepared like No. 1 (stains tissues, but stains
bacteria poorly ).

8. Alum Carmin.—In 100 c.c. of a5% solution of alum 2 gm. of

carmine are placed. It is boiled for an hour and filtered. It stains —

nuclei and tissues, but not bacteria.

g. Eosin.—For staining the tissues about the bacteria. Two gm.
of eosin are dissolved in 100 c.c. of distilled water.

10. Safranin.—Three gm. of safranin are dissolved in 100 c.c. of
hot distilled water.

(B) Differentiating Agents.

1. Distilled water,
. Absolute alcohol.
3- Iodin, iodid of potassium solution (Gram’s solution):

iS)

POU ER See. See rae BPP rag Stas ar 1.0 gm.
Poise ioc: + .< 4is Ys sede ee 2.9."
Distilled: water... 5 is 5 We le Ae 300.0 ‘*

4. Sulphuric acid, 25%.

5. Acetic acid, 3%.

6. Acid alcohol:
Alcohol (90%) 2. es Sew a Pat -. 100c.¢.
Piatillod wales os, Ses Ss ds so ees 200 ‘
Pure hydrochloricacid .....+...-, 20 drops.

(C) Mordants for Staining Flagella.

1. Loffler’s mordant: 10 ¢.c alcoholic solution of fuchsin ; 50 c¢.c.
cold saturated solution of ferrosulphate ; 100 c.c. 20% solution of
tannic acid.

2. Bunge’s mordant: 25 c.c. of officinal solution of ferric chlorid,
diluted twenty times ; 75 c.c. of a saturated aqueous solution of tannic
acid. Just before using, enough of a 3% solution of hydrogen peroxid
is added to give a reddish-brown color, and it is then filtered. (We
have always dispensed with this last addition. )

we Bt me yo

Se

a Tie aes Ar eperes

ee ee a ee

ae ae

MICROSCOPIC EXAMINATION. 477

(D) Clearing and Mounting Agents.

1. Xylol.
2. Canada balsam.
3. Dammar varnish.

3. Preparations of Stained Specimens of Bacteria.

(A) Smear Preparations.

1. Simple Stain with Fuchsin or Methylene-blue.—This may be
used for all bacteria except the tubercle bacillus.

A drop of distilled water is placed upon a cover-glass (cover-glass
preparation) or upon a slide (slide preparation), and in it is mixed a
trace of pure culture (preferably from a solid nutrient medium), and
the drop is then spread out in a very thin layer. After the fluid has
evaporated, the preparation is passed quickly through the flame three
times with the film up in order to fix the bacteria to the glass (avoid
burning !). The film of bacteria is then covered with the staining solu-
tion. After a short time (a few seconds toa minute), perhaps with
gentle heat, the preparation is washed with water and allowed to
dry (perhaps again with gentle heat). Finally the dry cover-glass is
fixed upon a slide by means of a drop of Canada balsam, the bacterial
side being down.

It may here be remarked that slides and cover-glasses upon which
the drop of water does not adhere or does not spread out evenly are
best cleaned with soap. Alcohol and ether, which are much used for
cleaning purposes, do not remove the particles of fat so completely.

. Gram’s Stain.—

. The smear preparation is made as above.

. Stain with Ehrlich’s solution three minutes.

. Wash with water.

. Differentiate with Gram’s iodin solution one minute.

. Decolorize with absolute alcohol until no more color is removed
(usually one or two minutes).

If the alcohol does not decolorize rapidly enough, it can be accele-
rated by placing a drop of anilin-xylol upon the preparation and then
again washing with alcohol.

6. Dry and mount.

According to our experience, the current idea that every variety of
bacterium either stains well or not at all when treated by this method
is false. Thus, we observed, in the case of the Bact. fluorescens, for ex-
ample, which is usually said in the literature not to stain, that three
out of twelve different cultures, of twenty-four hours’ growth, stained
very beautifully. According to Zimmermann, all the varieties of
Bact. fluorescens stain in young cultures.

In like manner we found the bacillus of symptomatic anthrax from
a culture to stain, although it has often been said not to stain. The
contradictory results may be easily explained in part as due to vary-
ing age or youth of materials experimented with, and to variations in
the differentiating process with alcohol. The Bacillus tenuis, which
has been designated above as failing to stain by Gram’s method, upon

COR WORN

478 BACTERIOLOGIC TECHNIC.

a later test with the same culture and technic stained very well. In
every case whenever the test is to be made, a fresh anthrax preparation
should be simultaneously stained, and all preparations should be sub-
jected to the action of alcohol for the same length of time (one or two
minutes). Then we can judge very well whether a variety of bacte-
rium retains or gives up the stain. A separation of the individual

genera and varieties by Gram’s stain now seems scarcely at all possi- —

ble, since there are found within a single genus all stages, from those
organisms which stain well to those that stain poorly.

The following may usually be stained by Gram’s method:

Micrococci (not the Micr. gonorrhez).

Sporulating bacilli (symptomatic anthrax and malignant edema
are uncertain).

Of the non-sporulating bacilli: Proteus, mouse septicemia,
swine erysipelas, lactic acid bacterium. (Uncertain: fluorescens,
some bacteria not unlike the coli Bact. )

Genus Corynebacterium, Mycobacterium, and Actinomyces.

The following usually do not stain:

Micr. gonorrhez, Bact. influenze, Bact. coli and typhi, Fried-
lander’s Bacillus pneumoniz, bacteria of pest and glanders, the
vibrios and spirilla.

3- Demonstration of Capsules.—The following is Johne’s method:

(1) Cover the preparation with 2% solution of gentian-violet and —

warm until it steams; (2) wash in water; (3) moisten with 2% acetic
acid for six to ten seconds; (4) wash with water.

By this method also in varieties which are not regarded as ‘‘ cap-
sule-bacteria,’’ a distinct membrane may often be demonstrated about
the intensely stained bacterial cell. The capsule is seen most beauti-
fully when examined in water.

4. Staining of Flagella.—The flagella, which are almost always
invisible when unstained, are usually demonstrated according to
Loffler’s method:

1. Making the preparation (rubbing up a trace of young agar
streak culture—not bouillon culture—in a very small drop of water,
spreading well, and drying rapidly).

2. Heating the preparation with the mordant (p. 476) until it”
steams (not boils!) for one-half to one minute.

3. Wash with a vigorous stream of water.

4. Wash in alcohol to remove the remains of the mordant adhering
at the edge.

5. Cover with staining solution (a few crystals are dissolved in
10 c.c. of anilin water, and to this is added, drop by drop, 0.1%
sodium hydroxid solution until the clear fluid just begins to become
opaque—precipitation) and warm until it steams for one minute.

6. Wash with water, dry, and mount in Canada balsam.

Extreme cleanliness is essential in the manipulations, especially
very thorough cleaning of the cover-glasses with acid and alcohol, or,
still better, with soap. Also the cultures must be young, although it
is not essential, as some authors claim, to employ cultures which are
only twenty-four hours old. We have often obtained very good prepa-

MICROSCOPIC EXAMINATION. 479

‘rations even after twelve days. We usually use freshly prepared

According to Léffler, the addition of a very definite amount of acid
or alkali to the mordant is required for most kinds of bacteria in order

_ to obtain well-stained flagella. Loffler recommends that to 16 c.c. of

_ the mordant there be added:
Cholera vibrios ....... 3- 1 drop 1% NaHO.
Spirilum rubrum ..... 9 drops ‘‘ as
Bacterium typhi ...... be Se a oS
Bacillus subtilis ...... a tere ay
Bacillus cedematis maligni. . 36-37 ‘S ‘ = «
Bacterium pyocyaneum ... 5-6 ‘ equivalent H,SO,.

Our results show that, in the majority of cases, very satisfactory
pictures are obtained with the original mordant, and that the addition
of alkali or acid is by no means essential. Similar results have been
obtained by other writers; for example, Lucksch, Giinther, A. Fischer,
Nicolle, and Morax.

Recently Bunge has employed a somewhat different method, which
has given us good results also, but, like Loffler’s process, it has also
sometimes left us capriciously in the lurch: (1) The preparation is
made as for Loffler’s ; (2) warming with Bunge’s mordant (p. 476)
until it steams for one minute ; (3) careful washing with water and
drying ; (4) staining with slightly warm carbol-gentian-violet or car-

_ bol-fuchsin ; (5) washing with water, drying, mounting in Canada

balsam.

The silver method of van Ermengem is also much employed for
staining flagella. The cover-glass preparations are stained with a mix-
ture of 1 part 2% osmic acid and 2 parts 10% to 25% solution of tan-
nic acid, to 100 c.c. of which 4 to 5 drops of glacial acetic acid are
added. It is then washed with water, and afterward with absolute
alcohol, and the preparation is next moistened for a few seconds with
0.5% to 2.5% silver solution. The preparation, without washing, is
brought into a solution of tannic acid 3.0, gallic acid 5.0, sodium ace-
tate 10.0, and water 350, for a few moments,.and again back to the
silver solution until it begins to get black. Finally it is again washed
in water.

Most of our preparations have been made with Bunge’s mordant
when it was several months old.

5. Staining of Endospores.!—<According to Hauser :

1. Preparation of the film. (It is recommended to pass it through
the flame ten times, instead of three.)

2. Stain with watery fuchsin or carbol-fuchsin ( Ziehl’s solution ), the
preparation being covered with abundance of the staining solution and
warmed above a flame for one or two minutes until it begins to simmer
(not boil). The fluid which evaporates is constantly replaced by fresh
staining solution.

1 Arthrospores possess no undisputed tinctorial reaction. Regarding
metachromatic bodies, Ernst’s and Bunge’s granules, preliminary stages
of spores and their demonstration, see page 21.

480 BACTERIOLOGIC TECHNIC.

3. Wash with acid alcohol? (p. 476) until the preparation no ;

longer appears red.
4. Counter-stain with methylene-blue (a few seconds).
The spores remain red, the bacilli appear blue.
6. Staining of Tubercle Bacilli—This is accomplished under thal

same principles as spore staining. The preparation is treated with a —

hot, actively staining solution, and afterward with some acid solution. —

Everything except the tubercle bacilli is decolorized.

(a) The preparation is stained (according to Ziehl-Neelsen) ex-
actly the same as spores, except that it is passed only three times
through the flame. We employ this method exclusively.

ad

Another favorite method is the one recommended by A. Friinkel —
and Gabbet, in which decolorization and counter-staining are aecom-
plished at the same time. Then the preparation, after being stained with —

hot carbol-fuchsin and washed with water, is ‘placed in the following
solution: Sulphuric acid, 1; distilled water, 3; pulverized methylene-
blue, enough to produce a most intense blue color. It is then again
carefully washed in water, dried, and mounted in Canada balsam.

Although this method is convenient, it is better for those without
much experience to carry out the staining, differentiation with acid,
and counter-staining separately, since in this way success is more cer-
tain.

(6) Ehrlich-Koch’s method is also often employed. ‘The prepara-_

Sree rr eo ee ee

tion, after being dried and fixed in the flame, is treated with hotanilin —

gentian-v iolet solution for one to two minutes, and then with acid —
(usually 30% nitric) for one to four seconds, and for a few seconds —

with 60% alcohol. It is then placed in an aqueous solution of Bis-

marck brown for a few minutes and washed in water. The tubercle —

bacilli now appear violet upon a brown background.

-

These methods are suitable for cover-glass preparations made from —
pure cultures and from tuberculous sputum containing many tubercle —

bacilli. If very few or notubercle bacilli are found in the first prepa-
rations, then some method must be employed for concentrating them.
We give two of the innumerable ones which are recommended:

(a) According to Strohschein : 5 to 10 ¢.c. of sputum are mixed with —

three times the quantity of Wendriner’s borax-boracic acid solution, ?
and, after vigorous shaking, the mixture is set aside for four or five
days to settle. The mixture becomes fluid and the bacilli settle to

*

the bottom. Such sputum may be used for examination after years.

(b) According to Dahmen, modified by Heim: The entire sputum is—
boiled in a steam chamber for fifteen to twenty minutes and allowed —
tocool. The opalescent fluid is poured off and the crumbly sediment
used for smear preparations.

1 In place of acid alcohol, 30% nitric acid or 5% to 25% sulphuric
acid may be employed, but they must be allowed to act for a shorter
time.

acid are added, and then again 4 gm. of borax; after crystallization it
is filtered.

2 Eight gm. of borax are dissolved in hot water, 12 gm. of boracic _

YY eRe

i i i at lia OE here i be
yr, ‘ ™

MI CROSCOPI C EXAMINATION. 481

7. Neisser’s Stain for Diphtheria Granules.--The requirements
for good success with the stain, according to Neisser, are as follows:
The preparation is stained one to three seconds with acetic acid
methylene-blue, rinsed off, counter-stained two to five seconds with
Bismarck brown.
; 1. The cultures must be grown upon Loffler’s serum, solidified at
00°.
2. The cultures should not be under nine nor over twenty to
twenty-four hours old.
ce The cultures must be kept in an incubator at 34°-35°, not above
8. Stain for the Organisms of Syphilis. —According to Lustgarten
the preparations are stained for twenty-four hours in anilin gentian-
violet, placed a few seconds in 10% permanganate of potassium solu-
tion, and quickly decolorized in dilute sulphuric acid. The syphilis
organisms are dark violet to steel blue; the tissues are decolorized.

(B) Preparation of Sections.

1. Universal method according to Loffler, adapted to almost all
bacteria:

The sections are carried upon a German silver or glass spatula from
alcohol to Loffler’s alkaline methylene-blue solution, where they re-
main for five to thirty minutes. They are then placed for a few
seconds in 1% acetic acid, and after differentiation are passed through
alcohol and xylol and mounted in Canada balsam. It must be deter-
mined how long the acetic acid may be allowed to act, and the dehy-
dration in alcohol must not be prolonged any more than is essential.
The bacteria should be blackish-blue, the nuclei blue, the protoplasm
bluish.

2. Nicolle states that by the following method he has obtained
satisfactory staining in sections in the case of bacteria which stain
with difficulty; for example, glanders, typhoid, ete. :

Loffler’s blue one to three minutes; wash in water; 10% solution
of tannic acid a few seconds; wash in water; absolute alcohol, oil of
cloves, xylol, Canada balsam.

3. Gram’s method: (1) Ehrlich’s solution, three minutes; (2)
Gram’s solution of iodin, two minutes ; (3) alcohol, one-half minute ;
(4) alcohol containing 3% hydrochloric acid, ten seconds ; (5) alcohol
several minutes until maximum decolorization ; (6) xylol, mount in
Canada balsam.

If it is desirable to have a contrast stain of the tissue, after max-
imum decolorization in alcohol, the sections are placed in an aqueous
solution of Bismarck brown (10 : 100) for a few minutes, then again
for fifteen to twenty seconds in absolute alcohol, then in xylol, and
finally mounted in Canada balsam.

4. Botkin maintains that Gram’s stain is facilitated by washing
preparations which are stained with anilin gentian-violet in anilin
water. The preparations, when removed from the iodin solution, sub-
sequently bear the action of alcohol much better. In this way it is
possible to stain the Bac. ceedematis maligni and Bacterium pneumoniz
Friedlander.

31

482 BACTERIOLOGIC TECHNIC.

5. Gram-Weigert method (stain for fibrin, also good for bacteria): :
The sections are stained with anilin or carbol gentian-violet, washed —

in 0.6% sodium chlorid solution, and dried upon the slide with filter-
paper. The iodin solution is then applied and the excess again removed —
with filter-paper. The section is then decolorized with anilin oil until —
no more color is given up, placed in xylol, and mounted in Canada —

balsam.
6. Kutscher’s modification of Gram’s method: There is prepared

a concentrated solution of gentian-violet in a mixture of anilin water —

1 part, alcohol 1 part, 5% carbolic acid water 1 part. Of this concen-

trated solution a drop at a time is added to a watch-glassful of water
until a shimmering layer forms upon the surface. In this the sections —
are placed, and after ten to fifteen minutes are washed in water, then —

placed in the iodin solution for a minute, then in alcohol, xylol, and
mounted in balsam. By this method malignant edema and symptom-
atic anthrax are also stained.

7. If tubercle bacilli are to be stained in sections, carbol-fuchsin or
anilin gentian-violet are used as for cover-glass preparations, except that
the heating is omitted, and instead the stain is allowed to act from
fifteen to thirty minutes.

4. Preparation of Sections.

At the autopsy small pieces of the organs are placed in an abun- —

dance of absolute alcohol and allowed to remain there two or three —

days, the alcohol being changed twice. Usually they can then be cut.
For this purpose the firmer parts of the kidneys, liver, muscle, ete.,
are stuck on corks with liquefied commercial gelatin,’ and then, with

the cork, again immersed in absolute alcohol. After another twenty- :

four hours the objects may be cut by means of a microtome. In order

to obtain sections of more delicate organs, they must be embedded in —

celloidin or paraffin. Before staining the paraffin is completely
removed by several changes of oil of turpentine or xylol, and the
preparation carried from xylol to absolute alcohol.

II. Cultivation of Bacteria.
1. Nutrient Media.
(A) Non-albuminous (according to C. Frankel and Voges).

Sodium chlorid, 5 gm.; neutral commercial sodium phosphate,
2 gm.; ammonium lactate, 6 gm.; asparagin, 4 gm., are dissolved in 1
liter of distilled water. We may add 10% gelatin or 1% agar, and
thus obtain a non-saccharine nutrient medium which is suitable for

most bacteria. By the addition of milk-sugar a milk-sugar nutrient —
medium is obtained which is free from dextrose. (Lehmann and —

Neumann. )

To produce bouillon free from sugar according to Th. Smith’s

? One part of gelatin is dissolyed in two parts of water,

— Son Sr ORS

TS Sok Saeed Aa eR ie ee

la iene i lg ens

CULTIVATION OF BACTERIA. 483

method, beef-extract is inoculated with Bact. coli, allowed to stand
twelve hours in the incubator, and sterilized after the addition of pep-
tone and sodium chlorid. Such a non-saccharine bouillon, which is
also, after so short a time, free from indol, is better for testing indol
production than that prepared in the ordinary manner.

(B) Albuminous.

1. Peptone Water.—In a liter of water 10.0 gm. of dry peptone
and 5.0 gm. of sodium chlorid are dissolved and it is then sterilized.

2. Milk.—Fresh, preferably freshly centrifugated, milk is placed in
test-tubes and sterilized in the steam chamber for one-half hour on
two successive days. Milk which contains the spores of the subtilis
group (see p. 53) can often not be sterilized in this way.

3- Litmus Whey (Petruschky).—The casein is cautiously precipi-
tated from milk by producing a very feeble acid reaction with dilute
hydrochloric acid. The filtrate is boiled, filtered and neutralized, and
mixed with some litmus. The preparation is not very easy. It can
be bought.

4. Hay Decoction.—About 10 gm. of dry hay are boiled in a liter
of water. The filtrate is filled into tubes and sterilized for two hours
on three successive days, the tubes being kept overnight in the incu-
bator, in order to destroy the very resistant spores.

5. Beer wort (not neutralized) is allowed to settle for some time,
preferably several weeks, after being sterilized, and then it is poured
off clear into test-tubes and again sterilized.

6. Nutrient Bouillon.—(a) From meat: 500 gm. of lean beef are
boiled in 1000 c.c. of water in an enamelled pot over a flame for one-
half hour and filtered. The filtrate (meat infusion) is brought up to
1000 ¢.c., and to this is added 10 gm. peptone and 5 gm. sodium
chlorid. This i is placed in the steam chamber until dissolved, and the
whole then carefully neutralized with normal sodium hydroxid (phe-
nolphthalein being used as indicator!) (see p. 36). It is then filtered,
filled into tubes, and sterilized.

(b) From extract of beef: 10 gm. of beef-extract are dissolved in .
1000 ¢.c. of water, and 5 gm. of sodium chlorid and 10 gm. of peptone
added. The solution is then neutralized and well sterilized several

times.

7. Potato Water for Tubercle Bacilli—Five hundred gm. of
peeled potatoes are rubbed upon a grater and allowed to stand over
night in a refrigerator in 500 ¢.c. of water. The fluid is then decanted
and brought up to 1000 ¢.c., boiled for an hour in the water-bath, fil-
tered, 4% glycerin added, filled in tubes, and sterilized.

8. Gelatin Nutrient Media. —(a) Meat infusion-peptone gelatin
(ordinary ‘‘ gelatin ’’ or ‘‘ nutrient gelatin ’’ of laboratories): To 1000
c.c. of meat infusion (see nutrient bouillon) are added 100 gm. gelatin,

1 For example, 10 c.c. of bouillon required 2.2 ¢.c. of ;45 normal
sodium hydroxid solution for saturation; 1000 ¢.c. of bouillon requires
220 c.c. of the same, or 22 ¢.c. of normal solution. We usually add
only 20 to 21 ¢.c.,—i. e., a little less,—so as to be sure of having no
free sodium hydroxid i in the nutrient medium.

484 BACTERIOLOGIC TECHNIC.

10 gm. peptone, 5 gm. soda chlorid. It is warmed in the steam
chamber until all is melted, then neutralized with normal sodium
hydroxid, filtered, and sterilized. After the liquefied gelatin is ae '
into tubes, it is again sterilized.

(b) Meat infusion gelatin: The same as a, but without the addition
of peptone and sodium chlorid.

(ce) Beer-wort gelatin: Is obtained by the addition of 10% gelatin to
the wort. It is not neutralized.

(d) Plum decoction gelatin: 500 gm. of dried plums are boiled in —
500 c.c. of water ; the fluid is then poured off, and the plums again —
boiled in 500 ¢.c. of water. Both fluids are then mixed, filtered, and ~ '
10% gelatin added. It is not neutralized.

(e) Herring gelatin: Two salt herrings, unwashed, are boiled in 1
liter of water, and to the filtrate 10% gelatin is added. It is not neu- :
tralized. 4

( f ) Potato-water gelatin: according to Holz, for Bact. typhi: g
gm. of potatoes are carefully washed, peeled, finely grated, oni’
squeezed through a linen cloth. The turbid juice may be allowed to
settle for twenty-four hours and is then filtered, or, as we always do,
it may be filtered at once through animal charcoal, and, after heatings@
if necessary the filtration is repeated. After heating for an hour in the —
steam chamber, 10% gelatin is added to the clear fluid, when it is” 3
again heated in the steam chamber, filtered, filled into tubes, and —
eel on three successive days. It is not neutralized. (H. K. ;
Lang. y

(g) Potassium iodid potato-water gelatin (Elsner): 1% potassium iodid
is added to prepared gelatin. This is best done by adding the required —
amount of strong sterilized solution to prepared gelatin just before — :
using it.

9g. Nutrient Agar.—To 1000 c.c. of meat infusion 10 gm. of fine cut ;
agar are added and the mixture is boiled in a glass flask over an open
fire for one hour until the solution is complete; then the evaporated
water is replaced and 10 gm. peptone and 5 gm. sodium chlorid are
added. After again heating in the steam chamber the fluid is neutral-—
ized and filtered by means of the hot-water funnel, filled into tubes, —
and sterilized.

10. To obtain grape- or milk-sugar agar, 2% of either substances
is added simultaneously with the peptone and salt. Since meat-infu-—
sion agar usually contains traces of grape-sugar, we have for some time
prepared a milk-sugar agar which is free from grape-sugar according”
to the method described under A.

11. Glycerin-agar.—To the prepared nutrient agar 5% of glycerin”
is added, when it is filled into tubes and sterilized.

12. Sugar- chalk Agar.—Usually finely pulverized, dry, sterilized —
carbonate of calcium is added to liquefied sugar-agar in ‘sufficient quan-—
tity to render it cloudy and opaque. It is then inoculated with the
bacteria and poured into plates. .

13. Potatoes.—After thorough washing and rinsing the potatos
are peeled, cut into slices 1 em. thick, and sterilized several times in~
deep Petri dishes. The peeled potatoes may also be perforated with a—
large cork borer and the cylinders be divided by an oblique cut into

i ee eee ———

Fade

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CULTIVATION OF BACTERIA. 485

two parts. The pieces are then placed in test-tubes with a little dry
cotton at the bottom (to absorb the water of condensation) and steril-
ized several times in the steam chamber.

14. Blood - serum.— Blood, obtained from slaughtered animals
under proper precautions, is allowed to stand in a refrigerator in
well-cleaned glass cylinders for twenty-four hours. Then the serum is
removed with large sterile pipets. It is then placed in flasks with the
addition of 1% of chloroform and allowed to stand several weeks,

' being occasionally shaken. Before using, the tubes, into which the

serum has been filled, are placed in the incubator for a few days to
insure complete evaporation of the chloroform. It may be used asa
fluid or after being solidified at 65°.

15. Loffler’s serum mixture for diphtheria bacilli: Three parts of
beef- or sheep-serum are mixed with 1 part of veal bouillon which
contains 1% grape-sugar, 1% peptone, and 0.5% NaCl.

16. Ascitic Fluid, Fluid from Ovarian Cysts.—The fluid obtained
by puncture has added to it a little chloroform (30-50 gm. to liter) and
is stored in a dark place for several weeks or months, being frequently
shaken. If the fluid is clear like water it is drawn into a sterile pipet
and filled into test-tubes. The tubes are placed in a water-bath for
half an hour at 30°-35° to drive off the chloroform.

Glycerin-ascites-agar is prepared by mixing equal parts of the
above fluid and of 2% nutrient agar which has been liquefied, cooled
down to 40°, and which contains 5% glycerin. This medium re-
places blood-serum in most cases, and upon it we have observed a very
good growth of the organisms of gonorrhea, pneumonia, tuberculosis,
whooping-cough, and diphtheria, and of streptococci and Bact. duplex
(Morax’s diplobacillus).

17. Silicic acid nutrient medium is very different from the other
media. It was first devised by Kiihne, and has been modified by
various authors. Gelatinous silicic acid, which is merely mixed with
certain salts, is an important nutrient medium for some organisms
(for example, the nitrate producers) because of the lack of organic
nutrient substances. For the rather complicated preparation consult
Stutzer and Burri (C. B. L. 1, 722).

18. As a substitute, Beijerinck has recommended a water agar, pre-
pared by extracting for a long time with distilled water, from which
the nutrient substances are removed by decomposition and diffusion
and to which the salts may be added (C. B. x1rx, 258).

1g. Cerebral Nutrient Medium (vy. Hibler).—Human brain from
fresh cadavers is cut up finely with a chopping machine, placed in
small flasks, and stirred up with enough distilled water to form a semi-
fluid infusion. The masses are then sterilized. Before using, the
nutrient medium is again boiled for half an hour.

20. Nutrient Medium for Differentiation of Species of Actino-

MMR UIE as 05 Gog ay eee ale ee nt ke
GONE EE > a eae a Ee ean ae eA 1000
TS” cb ei igri ieee see eae a 0.5
MPeM METER no oS e's Se eee te 1.0
CCIE 66S 6 eke ae We se, See

486 BACTERIOLOGIC TECHNIC.

2. The Employment of the Different Nutrient Media 4

Depends upon the Following Points of View:

I. Fluids (Bouillon, Sugar-bouillon, Milk, Non-albuminous
Nutrient Media)
are employed:
1. To produce culture en masse.
2. To obtain suspensions of bacteria in which the number can be
accurately determined (counting by means of plates).
3. For the study of the formation of pellicles and sediments.

4. For the study of the metabolic products (compare p. 58 and ~

what follows).

II. Solid Nutrient Media.
1. Gelatinous Nutrient Media.—The gelatinous, transparent nu-

trient media (agar and gelatin) are most extensively employed for the :

following reasons:

(a) They may be employed as fluid and solid nutrient media: as :
fluids, allowing a separation of the bacteria; and as solid substances, —

a fixing of the isolated germs and their separate growth into colonies.

(b) On account of their transparency they allow a macroscopic as —

well as microscopic observation of the cultures; they allowa thorough
differential diagnosis of varieties and an early recognition of any con-
taminations.

They are especially used: (a) For plate cultures—i. e., for demon-
stration, for accurate separation and counting of the individuals and
varieties.

(b) For obtaining characteristic, macroscopic cultures which serve
in differential diagnosis.

(c) For permanent cultures, or collections of living bacteria.

The special advantages of agar and gelatin are:

(a) Gelatin. — Advantages: Easily prepared, readily made into
plates (at 25°); the property of being liquefied by many bacteria is of
great diagnostic value. Disadvantages: Since it melts at 25° it cannot
be used in hot weather nor at incubator temperature.

(b) Agar.—Advantages: It may be used at incubator temperature
(i. e., for the rapid growth of bacteria—spores—and especially thermo-
philic bacteria). Disadvantages: Difficulty of preparation, more diffi-
cult to make plates from (the agar, melted at 80°, must be cooled
to 40° before being inoculated). Colonies are often not characteristic.

2. Blood-serum, glycerin-agar, and glycerin-ascites-agar. —
Employed especially for growing pathogenic varieties, which thrive
poorly or not at all on other nutrient media. It is only possible to
make plate cultures with glycerin-agar and mixtures of agar and serum.

3. Potato.—(a) To obtain macroscopically characteristic cultures
of great durability and for differential diagnosis.

(b) Sometimes for spore-formation.

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CULTIVATION OF BACTERIA. 487

3. A Few Words Regarding the Technic of Ordinary
Cultures.

The platinum needle must be heated red-hot each time before it is
used and before putting it down.

(a) Fluid culture media are inoculated with a loopful of pure
culture.

(b) Gelatin and agar stab cultures are made with a straight
needle, only a single stab being made in each tube, but it should ex-
tend almost to the bottom of the tube.

(ec) Agar and gelatin streak cultures and potato cultures
are inoculated by a gentle superficial stroke over the surface with the
platinum loop. It is sometimes necessary to rub the culture into the

potato.

(d) Gelatin plate cultures:

1. To isolate certain bacteria in pure culture: The gelatin in three
tubes is melted, and after it is cooled down to 30°, a loopful of a fluid
or a trace of a solid pure culture is introduced into one of them and
well mixed. From this first tube one or two loopfuls of gelatin are
carried to a second tube, and from this, after mixing, two or three
loopfuls are again transferred to the third tube. After anything which
may be upon the edge of the tubes has been burned off, the contents
of each tube are poured into separate sterile plates, the cover being
quickly raised for this purpose, and the plate inclined gently to and fro
in order to distribute the gelatin as uniformly as possible. During
the transferring from one tube to another it is reeommended that they
be held inclined, to prevent the falling into them of foreign germs.
The plates thus prepared are then placed in a culture chamber with
a constant temperature of 22° (or room temperature is used), and after
two or three days the individual colonies which have developed are
studied macroscopically and with slight (fifty times) magnification.
Usually, of the three plates, only two are useful; at least one has been
sown too thick or too thin.

2. If one wishes to ascertain the number of bacteria, for example,
in water, 1 c.c., 0.5, and 0.1 ¢.c. of the water is placed in three tubes
of liquefied gelatin, well mixed, and poured into plates. To ascertain
the number of germs, if they are very numerous, the Wolfhitigel count-
ing plate is used; if only a few colonies appear, then the plate is in-
verted, the bottom divided into sextents with ink, and each visible
colony marked with a dot. Plates made to determine the number of
bacteria in drinking-water must be counted several times (on the »
second, third, and fifth days). In the case of fluids with very many
germs (sour milk, canal-water, etc.), 1 ¢c.c. is first placed in 100 ¢.c.
of sterilized water, and this then treated as above. Solid bodies are
first rubbed up in water. In the examination of air a definite quan-
tity is drawn through a tube filled with sterilized sand, the sand then
being washed in sterilized water and plates prepared from it.

(e) Agar plate-cultures are prepared in the same way. The agar
must not be too cool when poured into the dish or it will solidify at
once, forming an uneven surface. On the contrary, if it is too hot, the
bacteria are killed by the temperature. Recently it has been much

488 BACTERIOLOGIC TECHNIC.

advised that, in making agar (partly also gelatin) plates, the nutrient
medium be first allowed to solidify in the dishes, and then the surface
be superficially smeared over with the material to be examined by
means of a platinum loop, strips of filter-paper, or a platinum brush.
Only characteristic surface colonies are obtained in this way.

(f) Sugar-agar shake cultures: The contents of a tube are
melted in the water-bath and cooled down to about 40°. A loopful of
the pure culture is then introduced and thoroughly distributed, and
after the medium solidifies the tube is placed in the incubator.

4. Anaerobic Cultures.

We have employed almost exclusively the method of H. Buchner:
Absorption of oxygen by pyrogallic acid in the presence of potassium
hydroxid.*

(a) For Stab Cultures.—At the bottom of a glass cylinder, which
must be a little longer than the test-tube, is placed a heaping teaspoon-
ful of pyrogallic acid and 20 c.c. of 3% potassium hydroxid solution.
The inoculated stab culture is then placed in the cylinder, which is
closed at one end with a soft rubber stopper or a ground-glass stopper
which is sealed with paraffin. According to Kitasato, anaerobes
which are less sensitive to oxygen may be cultivated in high stab eul-
tures in sugar-agar without pyrogallic acid. A stab 8 to 10 em. deep
is made in sugar-agar with a small loop and the needle turned upon its
long axis before being withdrawn. 7

(b) For Plate Cultwres.—Instead of the ‘glass cylinder, a wide exsic-
cator with a ground cover is used. The lower part is filled with sand
(Ar onl Sinezaaes acid mixture, and then the manipulation is as above

Arens).

If it is desirable to obtain the most perfect anaerobiosis, the pyro-
gallic acid method is combined with either the pumping out of the air
with a water-pump or the displacing of the air with hydrogen, so that
only a slight trace of oxygen remains to be taken up by the pyrogallic
acid. We have employed the latter method many years. The cultures
are placed in a roomy exsiccator with sufficient pyrogallic acid and
potassium hydroxid, and then, by means of a double perforated rubber
cork, hydrogen is allowed to flow through for one-half hour. After
closing the opening, we sink the whole apparatus, weighted with lead,
in water.

Kabrhel recommends (C. B. xxv, 555), as a control for the absence
of oxygen, that a tube be introduced which contains liquefied nutrient
gelatin, to which is added, just before use, 0.3% to 1.0% grape-sugar,
and which is rendered a transparent blue with a strong alcoholic solu-
tion of methylene-blue. Such an uninoculated tube is completely
decolorized in twenty-four to thirty-six hours only in a chamber
entirely free from oxygen. This indicator will also point out how
essential it is to remove covers, corks, etc., in the case of anaerobic
cultures.

1 Sensitive varieties are said to thrive better in an atmosphere of
hydrogen.

uly:

_ mere ire see ele ee ne cnet i

ne -r

ANIMAL EXPERIMENTS. 489

III. Animal Experiments.

(A) Infection.

1. Subcutaneous Inoculation.—After the skin in some part has
been washed with 1 : 1000 corrosive sublimate solution, a shallow
incision is made with scissors, and inoculating material is introduced
beneath the skin by means of a stout platinum wire with a loop.
Mice are usually inoculated above the root of the tail, they being
simply held by the tip of the tail and allowed to hang into a glass
which is covered in great part by a piece of board. Guinea-pigs and
rabbits are inoculated on the side of the thorax.

2. Subcutaneous injection is usually carried out with Koch’s
rubber-ball injection syringe or with Strohschein’s syringe. A fold
of skin is picked up upon some part of the body, and the needle intro-
duced in the direction of the fold. If several cubic centimeters are to
be injected, it may be simply done as follows : Upon a graduated pipet
is fastened a short piece of rubber tubing provided with an injection
needle, and the whole sterilized. The pipet is sucked full, and the
fluid forced out with the mouth or a rubber bulb.

3. Intraperitoneal injection is made by perloratine the ab-
dominal wall at a single thrust with a sterile hollow needle ; then,
cautiously advancing the needle, the fluid is injected.

Regarding infection by feeding, inhalation, etc., consult more exten-
sive works on technic.

(B) Observation.

Mice may be kept in sterile glass vessels provided with cotton and
closed with wire gauze. Larger animals must be kept in sterilized
cages or stalls.

(C) Autopsy and Disposition of the Body.

‘Autopsies must be made at once after death; at least, the animal
must be placed on ice after death. The animal is placed upon a board
on its back and nailed or tied by its four legs. The abdomen and chest
are thoroughly moistened with sublimate solution and then the abdom-
inal cavity first opened with a sterile knife. The abdominal walls are
separated, and from the spleen, liver, and kidneys, some blood (or tis-
sue juice) is obtained with a sterile platinum loop and smeared at
once upon previously prepared agar plates. The organs are carefully
cut out, avoiding contact with the intestines, and placed in absolute
alcohol for further examination. Then the thorax is opened with
scissors, and blood removed from the heart and also the lungs. These
organs are also placed in alcohol. Before each operation the instru-
ments must be carefully heated to a glow or thoroughly burned. It is
better to have numerous sterilized instruments ready. The hands

- must be perfectly clean.

In interpreting the findings at the autopsy it is to be remembered
that often very soon (sometimes during the death agony ) micro-organ-

490 RECOGNITION OF BACTERIA.

isms migrate into the organs from the intestine. If living bacteria are
injected into the abdominal cavity or trachea of cadavers, they can
very often be found in the organs after a time (C. B. x x1, 418).

After the autopsy the body is best burned. If this is not practica-
ble, the body is wrapped in coverings wet with sublimate and buried
at least 0.5 meter deep, and quicklime filled in about it.

APPENDIX V.

ee

Brief Guide to the Recognition of Bacteria. .

(Illustrated with an example. )

The case is one of eczematous conjunctivitis in which a
number of the bacteria occurring in diseased eyes are
present. Purulent or serous material removed from the
conjunctival sac or edge of the lid with a platinum loop is
made use of.

I. Microscopic Examination (Smear upon Slide
or Cover-glass).

(a) Stained with fuchsin, we see:

1. Cocci, especially diplococci in heaps, usually dis-
tinctly ‘‘ biscuit-shaped,’’ many times within cells (per-
haps gonococci).

2. Cocci, single or united in irregular clusters (probably
Micro. pyogenes).

3. Short chains, of two or three links, of lance-shaped
cocci, some with capsules (probably Streptoc. lanceolat. ).

4. Rods, larger or smaller, often very irregular in form,
staining in segments, ends rounded or pointed, often of
the size of cocci (true diphtheria, pseudodiphtheria, or
xerosis bacillus).

5. Rods regular, rather thick, but small (perhaps coli
group).

6. Rods, often in pairs, quite large, the ends not rounded
(perhaps, although at the time without spores, a bacillus
or Bacterium duplex).

ore es 2, ee

1
!

PLATE CULTURES. 491

- (b) Gram’s stain: All the organisms in the prepara-
tion are stained except the biscuit-shaped cocci and the
small, plump regular bacteria. The loss of color speaks in
favor of the cocci being gonococci, and the rods Bact. coli.

(c) Stain for tubercle bacilli with carbol fuchsin: In
the differentiation the preparation is completely decolor-
ized with sulphuric acid. After counter-staining with
methylene-blue only organisms which are stained blue are
seen. Thus, in our case the tubercle bacillus and those
resembling it are excluded.

If, as sometimes occurs, no micro-organisms can be seen
in the fuchsin preparation, then a preparation is also
stained by Gram’s method because the stained cocci and
bacilli are more readily seen after the mucus and coag-
ulum have been decolorized. In any case if the examina-
tion of the slide is negative, the plate method is always
employed.

II. Plate Cultures.

In examining an animal body for micro-organisms the
nature of which we do not know, we employ ‘‘the best
nutrient medium ”’’: 7. e., serum or ascitic fluid-agar, and,
as a substitute, glycerin-agar. !

The usual plate method consists in placing the material
to be examined in liquefied gelatin or agar in various dilu-
tions and pouring it out into double dishes. This is not
especially suitable for the examination of materials which
contain relatively few germs. In our case we prefer
to pour the nutrient medium into plates, and, after it is
solidified, to carefully make several streaks over the sur-
face with a platinum loop which carries the pus or mucus,
etc., to be examined. The double dishes are then turned
upside down (so the agar will not dry so rapidly) and
placed in the incubator.

After forty-eight hours there appear upon the plate:

1. Moist, white, yellow, and orange, roundish, slightly
elevated colonies,? which, when magnified sixty times, are

1 On the contrary, many varieties from soil, water, etc., grow only

upon nutrient media poor in nutrient substances, like the ordinary
nutrient media (see p. 200).

2 The colonies here described are always such as lie on the surface
of the medium.

492 RECOGNITION OF BACTERIA.

finely granular. In stained preparation, magnified a thou-
sand times, they are micrococci (probably Micrococeus
pyogenes albus, citreus, and aureus). The examination
must be carried further, as indicated on page 163.

If there are only one or two colonies,—especially yellow
ones,—one may often recognize it as a contamination of
the plate by germs in the air, most often sarcine. The
edges of the sarcina colonies, when magnified sixty times,
are coarsely granular or jagged. When magnified one
thousand times, packets of micrococci are seen.

2. Most minute, scarcely perceptible colonies, not ele-
vated, half a millimeter in diameter. When magnified
sixty times, extremely delicate, transparent, very delicately
punctate. The edges practically smooth (recalling gono-
coccus, Streptococcus lanceolatus, and Streptococcus pyo-
genes). In the last, one often observes upon ascites-agar
that chains of cocci grow out from the edge of the colony
in the form of the finest curling threads! When the
stained preparation is examined under a magnification of
1000, the examination is to be continued according to
page 163, if cocci; page 134, if streptococci ; page 195, if
bacilli.

In order to render still more secure the diagnosis founded
upon morphologic and biologic peculiarities, several such
small colonies are taken up with a platinum loop and in-
troduced beneath the skin of a mouse, or this may be done
by employing more abundant infecting material (bouillon
culture). If we are dealing with a Streptococcus lanceo-
latus, we find in the blood and organs characteristic forms
of this variety with capsules. Smears from the blood and
organs are to be examined for the characteristic organisms
(Strept. pyogenes, Strept. lanceolatus), and also new
smear inoculations made upon nutrient media.

3. Tiny white to yellowish-white points, rather dense, and
just visible with certainty after twenty-four to forty-eight
hours. If the plates are kept longer, there is usually only
a slight increase in size, up to about 0.5 mm., and then,
with very few exceptions, they become no larger. They
are, however, always distinguished from those named
before by the tougher consistency. When magnified sixty
times, the border is ragged, often as if: gnawed away, and

ry pre ee ee ee cee

Wt aey «

a a a ee ee re a aa eh _

at ey

PLATE CULTURES. 493

splintery (see Plate 59, 1) and of a yellowish color. Mag-
nified 1000 times : Stained in segments, highly polymor-
phous, short, long, thick, thin, clubbed, pointed, also.
with the form of cocci (apparently diphtheria or pseudo-
diphtheria or xerosis bacilli). In the pseudodiphtheria
bacillus the border is often coarsely granular, similar to
sarcinee. The further examination is conducted according
to page 384.

4, Larger, moist, sometimes slimy, luxuriant colonies,
somewhat elevated, whitish to gray, with transmitted
light somewhat iridescent. When magnified sixty times,
the edge is smooth. Microscopic preparation magnified
1000 times: Small, plump or more slender rods, perhaps
also isolated short chains. Not stained by Gram’s method.
Belongs to the group of non-sporulating bacteria. Perhaps
or probably the Bact. coli or a closely related variety. It
is to be further studied regarding motility, gas-formation,
indol, coagulation of milk, according to page 169, ete.
When transferred to gelatin plates, the colon group pre-
sents, upon slight magnification, the characteristic, wavy,
smooth-edged, transparent colonies with intersecting lines.

5. Macroscopic: Similar*to the colonies described under
4, but never slimy; grayish-white, often gray. When
magnified sixty times, the border is matted or curly.
Microscopic preparations magnified 1000 times show
sturdy bacilli, of equal length, the ends not rounded,
staining by Gram’s method, often united in chains (very
probably sporulating organisms of the subtilis, anthrax,
and mesentericus group). To be further studied accord-
ing to page 304.

6. Essentially the same as under 5, but the periphery
is exceedingly delicate and transparent. There is neither
observed formation of curls nor irregular breaking up of
the periphery into a felty structure. The microscopic
preparation magnified 1000 times shows bacilli similar to
those described under 5, but usually arranged in pairs
(probably Bacterium duplex).

It may here be again stated for the beginner that the
diagnosis, especially the separation of the bacteria into
separate groups, may be much facilitated by paying atten-
tion to the periphery of the colonies. In the follow-

494

RECOGNITION OF BACTERIA.

ing table are entered the appearances occurring in the

most important varieties.

Exceptions occur, of course:

NUTRIENT ME-

BORDER OF SUPERFICIAL COLO-

DIuM.? VARIETY, NIES MAGNIFIED SIXTY TIMES,

Agar. Streptococcus pyo-|Smooth or only extremely
genes. finely granular. Excep-

Streptococcus lan-| tions: many Streptococci
ceolatus. pyogenes on ascites-agar.
Micrococcus gonor-
i rhee.
Bacterium influenze.

Agar. Micrococcus pyo-| Finely granular.
genes and all luxu-
riantly growing mi-
crococci.

Agar. Sarcine. Coarsely granular, often as if
eaten away. In many varie-
ties individual packets are
distinctly seen at the peri-
phery.

Gelatin. Bact. typhi and coli,| Wavy, smooth. In young
and related ogee stages, fine lines as if cut in,
isms, passing from border toward

center.

Gelatin. Liquefying air and/ Beset with most delicate little

water bacteria.

hairs.

Agar and gela-
tin.

Subtilis group and
anaerobic bacilli.

Periphery broken up into ir-
regular, tangled locks.

Agar, less in| Anthrax and closely | Regular, beautiful formation

gelatin. related organisms. of curls and locks.
Gelatin. Vibrios, especially Scalloped to finely lobulated,
cholera. in the interior moruloid.

Later the periphery is
crumbly, until it is finally
entirely disintegrated.

Gelatin and
agar.

Diphtheria bacilli
and its relatives.

Similar to sarcine, but irreg-
ularly cut and fringed.

Glycerin-agar.

Tubercle bacilli.
Actinomyces
their relatives.

and

Smooth, very wrinkled, car-
tilaginous, distinguished by
strong reflex.

1 The nutrient media are cited upon which the variety concerned
grows characteristically.

a dc

INDEX.

Asscrss, 184

Acetic acid bacteria, 197, 260
formation, 86
methylene-blue, 476

Acetone formation, 86

Acid addition to Léffler’s mor-

dant, 479
formation, 69
Acids from alcohols, 91
from carbohydrates, 85
isolation, 86
obtaining, 86
Actinobacter polymorphus, 173
Actinomyces, 127, 128, 438
albido-flavus, 440, 456
albus, 446
asteroides, 440, 449
bovis, 439, 440; Pl. 65
carneus, 440, 451
chromogenes, 440, 452; Pl. 67
alba, 440, 455
citreus, 440
‘diagnosis, 439
erysipeloidis, 456
farcinicus, 439, 447; Pl. 66
Hofmanni, 439, 447
Israéli, AAT
madure, 440, 452
media for differentiating spe-
cies, 485
musculorum suis, 446
necrophorus, 456
violaceus, 440, 456
Actinomycetes, 383
Adenin in bacteria, 30
Aerobes, 304
common characteristics, 306
facultative, 42

Aerobes, further sporulating va-
rieties, 330
new, 259
obligate, 41
spore-formation, 51
Aerotaxic figures, 56
Agar, advantages, 486
as medium, 484
plate-cultures, 487
Agglutination, 105
Gruber and Bordet’s theory,
108
in typhoid, 240
phenomena, 109
test in cholera, 374
Agglutinin, demonstration, 105
Albumin, effect on antisepsis,
39
on asepsis, 39
in bacteria, 29
Albumin-dissolving ferments, 59
Albuminous bodies, hydrogen
sulphid from, 76
in bacteria, 29
peptone from, demonstra-
tion, 60
media, 483
Alcohol, acids from, 91
butyl, formation, 88
ethyl, formation, 86
from carbohydrates, 85
Aldehyd, formation, 86
Alexins, 97
Algee, fission, higher, 457
Alinit, 84
Alkali addition to L6ffler’s mor-
dant, 479
formation, 69

495

496

Alkaloids, putrefaction, 71
Alum carmin, 476
American swine plague, 252
Amido-acids, 72
Amins, 72
Ammonia, demonstration, 78
formation, 69
Anaerobe, new pathologic, 344
Anaerobes, 305
as agents in ripening of cheese,

carbon dioxid and, 43

facultative, 42

fermentation and, 351

in retting of flax and hemp,
351

obligate, 41

spore-formation, 51

sulphuretted hydrogen and,
44

Anaerobic cultures, 488
producers of butyric acid, 345

Angina, diphtheric, 397
ulcerative, 473

Anilin fuchsin, 475
gentian-violet, 475

Animal experiments, 489
serum, collection, 105

Anthrax, 305, 307; Pls. 34-36
spores, resistance, 52, 53
symptomatic, 340; Pl. 45

Anti-bodies, 94

Antiricin, action, 100

Antisepsis, albumin and, 39
concentration necessary for,

definition, 37
Antitoxic sera, value, 102
Antitoxins, 99
action, 101
and toxins, mutual action, 99
normal, 102
origin, 101
Aroma-producing bacillus, 320
Aromatic metabolic products,
79
Arthrospores, 25
staining, 479
Ascitic fluid as medium, 485
Ascococcus Billrothii, 179
cantabridgensis, 179

INDEX.

Asepsis, albumin and, 39
Behring’s test, 38

concentration necessary for, 38

definition, 37

test, 38
Attenuation, 37
Autopsy, 489

Baciuuvs, 124, 125, 304
acidi levolactici, 224
acidi paralactici, 224
aerobic, 304
common characteristics, 306

further sporulating varie- ;

ties, 330
new, 259

aerogenes sputigenus capsu- —

latus, 228
vesice, 255
alvei, 306, 345
amylobacter navicula, 351
v. Tieghem, 351
amylovorus, 471
amylozyme, 348
anaerobic, 305. See also An-
aerobes.
annulatus, 264 3
anthraci similis, 315
anthracis, 305, 307; Pls. 34—
36

demonstration, 314 .
diagnosis, differential, 314
polar germination in, 27
_ spores, 312
sporulation, 26
without spores, 311
anthracoides, 315

aphthosus, 252 .

aquatilis communis, 264
arborescens, 271
argenteophosphorescens, 370
liquefaciens, 370
aroma-producing, 320
aterrimus, 305, 328
bernensis, 320
botulinus, 306, 337
butyricus, 305, 322, 346; PI.
38 3

cadaveris butyricus, 345
capsulatus aerogenes, 344

hit di, (thie ee i

cow

INDEX. 497

Bacillus capsulatus chinensis,

capsule, 228
carcinoma, of Scheurlen, 326
Chauveei, 306, 330, 339; PL. 45
cholere, 353
gallinarum, 210
claviformis, 310
coccineus, 272
colon, 243
comma, 353; Pls. 47-51
constrictus, 268
eyaneophosphorescens, 370
cyanogenes, Pls. 27, 28
devorans Zimmermann, 267
diphtheriz, 389
columbarum, 255
dysenteriz Shiga, 251
eclampsia, 300
« nphysematis maligni, 343
e..teritidis, 251
~ 1orescens albus, 288
aureus, 288
liquefaciens, 285; Pl. 25
lon
friburgensis, 432.
chsinus, 277
.uscus, 271
gallinarum, 211
gongrene pulpe, 305, $28; Pl.
68

istrophilus, 323
( austadt, 255
geniculatus, 304, 326
gracilis, 337

-gummosus, 326

hastilis, 473

hay, 317; Pls. 39, 40

hyacinthisepticus, 471

implexus, 320

indigoferus, 280

indigogenes, 252

key to diagnosis, 304

lactic acid, Pl. 14

lacticus, 224

lactis erythrogenes, 268
niger, 328

lepra, 421

leptosporus, 320

liodermos, 305, 328

lividus, 279

32

Bacillus loxiacida, 259

luteus, 268, 305
malariz, 320
mallei, 384
maximus buccalis, 461
megatherium, 305, 321; Pl. 41
melanosporus, 328
membranaceus amethystinus,
279
mobilis, 279
mesentericus, 305, $26; Pl. 43
niger, 328
panis viscosi, 325
ruber, 327
vulgatis, Pls. 38, 42
mycoides, 305, 316; Pls. 37, 38
cedematis maligni, 306, 330,
341; Pl. 46
differential diagnosis, 343
new, 344
of blue milk, 289
of ferret plague, 251
of frog-spawn disease, 23
of greenish-blue pus, 281
of grouse disease, 259
of intestinal diphtheria, 254
of Marseilles swine plague, 252
of mouse plague, 259
of pigeon plague, 255
of pneumonia in rabbits, 204
of spontaneous rabbit septi-
cemia, 252
of tuberculosis, 128
of yellow fever, 256
milk, 267
olez, 471
orthobutylicus, 348
oxalaticus, 305, 323
plasmolysis in, 21
phlegmonis emphysemtose,
306, 344
piscidicus, 345
plicatus, 270
pneumoniz, 225, 228; Pl. 15
potato, 323; Pls. 38, 42
pseudanthracis, 315
pseudodiphtheria, 403
pseudodiphtheriticus acidum-
faciens, 407
alkalifaciens, 407
pseudo-edema, 343

498

Bacillus pseudoglanders, 389

pseudo-influenza, 202
pseudo-cedematis maligni, 344
pseudopneumonicus, 228
pseudotetanus, 337

pseudotuberculosis murium,
410
ovis, 410
pyogenes filiformis, 461
foetidus, 259

quercifolius, 322
radicicola, 83
radicosus, 316
rosaceus metalloides, 277
rubiginosus, 272
saccharobutyricus, 348
sessilis, 320
smegma, 424
solanacearum, 471
sporogenes, 346
sputigenus crossus, 228
Pansini, 228
subflavus, 268
subtilis, 305, 317; Pls. 39, 40
suipestifer, 252
tenuis, 304, 320
termoidhnlichen, 285
tetani, 306, 330, 332; PI. 44
tracheiphilus, 471
tuberculosis, 410; Pl. 61
fatty acids i in, 29
in sections, 482
in sputum, staining, 480
potato water for, 483
staining, 480
wax in, 29
typhosus, 197, 282; Pls. 16,
See also Bacterium
typi
eus, 279
Tales, 295
vulgatus, 305, $323; Pls. 38, 42
temperature for, 44
water, Kiel, Pl. 22
water-red, 277
zymogenic varieties, 306

Bacteria, acetic acid, 197

activities, 54
chemical, 56, 58
from undulating membrane,

INDEX.

Bacteria, activities, optical, 56 7

mechanical, 55
thermic, 58
adaptation in, 43
adenin in, 30
aerobic, 41, 42
albumin in, 29
alkali formation by, 69 |
ammonia formation by, 69
anaerobic, 41, 42

aromatic metabolic products, |
79

ash, 30

attenuation, 37
black-growing, 68
branching, 19
Brownian motion, 55

capsule, 22

cell, 20

cellulose in, 29

central fluid, 20

chemical composition, 29

cholestearin in, 29

classification, difficulties, 116

cultivation, 32, 482

definition, 17

determination of number, 487

dextran in, 30

distilled water and, 40

dried, life, 40

dry substance, 30

effects of other bacteria on, 48

electrical influences, 46

enantobiosis, 49

envelope, 22

extractive substances in, 29

fat-decomposition by, 80

ferments, 59. See also Fer-
ments.

fission, 24

flagella, 23

forms, 18

gases and, 41

grape-sugar in, 29

guanin in, 30

guide to recognition, 490

hemicellulose in, 29

inconstancy of species, 117

increase, 32

injury to, by chemicals, 37

isolation of, in culture, 487

> fe 5 Alin he ec oll

INDEX.

Bacteria, lecithin in, 29
life of, conditions, 32
duration, 32
on media, 40
temperature and, 44
light and, 46
mechanism of action, 48
testing sensitiveness to, 47
mechanical influences, 46
mesophilic, temperature for,44
microscopic examination, 474
molecular motion, 55
morphology, 17
motility, 23
nomenclature, 119
rules, 119
nonmotility, 24
nourishment-deficiency and,
40
nucleus, 21
of acetic acid, 260
outer surface, 23
oxygen and, 41
pathogenesis, 92
pathogenic action on, 92
peripheral appearance of col-
onies, 493
phosphorescence, 56
photogenic function, 56, 57
pigment production, 66-69
plasmolysis in, 20
preparation of stained speci-

mens, 477
pseudodichotomy in, 19
psychrophilic, | temperature

tor,

ptomain formation, 71

quantitative composition, 30

resembling diphtheria, 403

resistance, 103

rod, 124, 193

Roéntgen rays and, 46

salts in, 29

screw, 125, 352

shaking and, 46

spherical, 122, 133

spore-formation in, 25

staining with Gram, 478

sulphur in, 30

sulphuretted - hydrogen pro-
uction , 76

499

Bacteria, sunlight and, 46
symbiosis, 49
thermophilic, temperature for,
44
toxalbumins, 73
toxins, 71, 73
triolein in, 29
tripalmitin in, 29
tristearin in, 29
urea-fermentation by, 69
vegetative multiplication, 24
virulence of, variation in, 94
water content, 29, 30
water-deficiency and, 40
weakening of, by chemicals, 37
well-water and, 40
xanthin in, 30
Bacteriacee, 124, 193
critical remarks, 125
flagella classification, 125
Bacterial cell, granules, 21
membrane, 22
swelling, 22
metachromatic bodies, 21
sporogenic granules, 21
structure, 20
metabolism, chemical activ-
ity, 64
Bactericidal bodies, origins, 108
in sera, demonstration, 107
Bacteridium, 125
Bacterio-fluorescein, 68
Bacteriologic technic, 474
Bacterioplasmin, 73
Bacterioprotein, 73
Bacterium, 124, 125, 193
aceti, 261, 262
lactici, 196, 220, 229; Pl. 14
egyptiacum, 195, 204
aerogenes, 196, 221; 229
agile, 265
alcaligenes, 257
aurescens, 272
aureum, 272
avicidum, 210
bipolare multocidum, 210
brassice acide, 251
bruneum, 271
brunificans, 199, 292
butyri colloideum, 225
fluorescens, 286

500

Bacterium ceruleum, 198, 280
canicule, 197, 260
carnosum, 270
caucasicum, 223
cavicida, 223
cholerz suum, 197, 252
chrysoglea, 198, 272
cloacee, 265
coli, 197, 248; Pls. 18, 19

differentiation of typhosus
form, 239
dysentericum, 251
polaris, 252
serum diagnosis, 249
cremoides, 198
nobis ad interim, 267
cuniculicida, 210
denitrificans, 289
disciformans, 197, 263
duplex, 195, 206
egregium, 272
erysipelatos suum, 200, 302;
Pl. 33
erythrogenes, 198, 268
ferrugineum, 199, 292
fluorescens, 199, 285; Pl. 25
non-liquefaciens, Pl. 26
putidum, Pl. 26
foetidum liquefaciens, 265
Fraenkelii Hashimoto, 129
fulvum, 270
Guillebeau, 256
Giintheri, 196, 223
hemorrhagicum, 212; Pl. 20
helvolum, 198, 268
Hessii, 231
icteroides, 197, 256
in melzna neonatorum, 259
indicum, 277
indigonaceum, 198, 280
influenza, 195, 202; Pl. 68
janthinum, 279
key to recognition, 195
Kiliense, 276; Pl. 22
Kiitzingianum, 261
lactis acidi, 224
saponacei, 198, 269
viscosum, 196, 230
latericium, 198, 272; Pl. 20
levans, 255
luteum, 198

INDEX.

Bacterium miniaceum, 277
morbificans bovis, 255
multocidum, 210
murisepticum, 200, 300; PI.

33

mustelicida, 251

mycoides roseum, 271

neapolitanum, 223

nitrobacter, 195, 200

nitrosomonas, 195, 200

nubilum, 198, 269

ochraceum, 198, 270

of Barbone in buffalo disease,
210

of brick-pock, 304

of calf dysentery, 256

of dermatitis epidemica ex-
foliativa, 223

of Giard, 231

of red pus, 277

of septicemia, 223

ozenze, 228

Pasteurianum, 261, 263

pestis, 196, 213; Pl. 13

Pfliigeri, 231

phosphorescens, 196, 231

piscatorum, 277

plymuthicum, 277

pneumoniz, 196, 225, 229;
Pl. 15

prodigiosum, 198, 272; Pl. 21

pseudomelanosis, 266

pseudotuberculosis rodentium,
196, 213 ;

punctatum, 197, 264

putidum, 199, 287; Pl. 26

pyocyaneum, 199, 281; Pl. 24

rancens, 263

ranicida, 287

rhinoscleromatis, 229

rosaceum, 277

salmonicida, 197, 266

septicemizse hzemorrhagice,
196, 208; Pl. 12

Stutzeri, 258

suicida, 209

suisepticus, 209

syncyaneum, 199, 289; Pls.

28

synxanthum , 267
tholoeideum, 225

Me ee es eR Ee eee

INDEX.

+r

Bacterium tremelloides, 271
turcosum, 197, 267
tussis convulsive, 195, 205;
ey 232; Pls. 7
t 197, 16, 1
demonstration, 237
differentiation from bacte-
rium coli, 239
murium, 258
serum diagnosis, 240
preparation, 241
ulceris cancrosi, 196, 207; PI.
68
violaceum, 198, 277; Pl. 23
viridans, 285 ~
vitulinum, 197, 264
vulgare, 199, 200, 295; Pl. 31
mirabilis, Pl. 32
xylinum, 263
Zopfi, 199, 293; Pls. 29, 30
Beef bouillon, 483
Beer wort, 483
Beggiatoa alba, 461
nivea, 462
roseo-persicina, 462
Trevisan, 458
Behring’s asepsis test, 38
test for serum, 102
Beijerinck’s water agar, 485
Beri-beri, 468
Bilineurin, 72
Bipolar germination, 27
Bismarck brown, 476
Black pigment, 68
Black-growing bacteria, 68
Bleeding host, 276
-: dele as medium, 485
mployment, 486
Blue itmus, reduction, 77
milk, Pls. 27, 28
bacillus, 289
pigment, 67
Body, disposition after autopsy,
489

Botkin’s method for sections, 481
Botulism, 337
prophylaxis, 98
Bouillon, 483
cultures, descriptive
used, 131
employment, 486

terms

501

| Bouillon, Smith’s preparation,
82

| | Brain as medium, 485

Branching, 19

Brick-pock, 304

Brieger’s isolation of ptomains,
72

Broncho-pneumonia, diphtheric,
398
Brownian motion, 55
Bubonic plague, 213
Buffalo disease, 210
Bunge’s granules, 22
method for flagella, 479
mordant, 476
Butter organism, 431, 433
Butyl alcohol, formation, 88
Butyric acid, anaerobic pro-
ducers, 345
bacillus, Pl. 38
formation, 86, 88

CADAVERIN, 72
Capsule bacillus, 228
Capsule-bacteria, 22
Capsules, demonstration, 478
Carbohydrates, acids from, 85
alcohol from, 85
gas-production from, 89
Carbol-fuchsin, 475
Carbon dioxid, bacteria and, 43
Carbonic acid from cellulose, 89
from fermentation, 90
Carcinoma bacillus of Scheurlen,
326
Carmin, alum, 476
Carotin pigments, 66
Catarrhal swelling, 407
Cattle plague, 472
pneumonia in, 469
Cellulose decomposition, 88
in bacteria, 29
Cerebral nutrient medium, 485
Cerebrospinal meningitis, 148;
Pl. 68
Chancre, soft, 207
Cheese, ripening, 349
Chemical activity of bacteria, 58
Chemotaxic figures,
Chemotaxis, negative, 56

502

Chemotaxis, positive, 56
Chicken cholera, 208, 210; Pl. 12
Cholera, 353; Pls. 47-51
agglutination test in, 374
chicken, 208, 210; Pl. 12
culture, preliminary, 372
hog, 252
laboratory, 363
nostras, 368
serum, obtaining, 373
serum reaction in, 373
toxin, 75
vibrio, life, on glass, 41
Cholera-red reaction, 79
Cholestearin in bacteria, 29
Cholin, 72
Chromogenic function, fluctua-
tions, 68
Cladothrix, 128, 458
dichotoma, 465
invulnerabilis, 455
liquefaciens, 446
Clearing agents, 477
Clostridium, 125
licheniforme, 349
Coccacez, 122, 133
Collidin, 72
Colon bacillus, 243
Colorless growths, 69
Comma bacillus, 353; Pls. 49-51
Conjunctivitis, 207
epidemic, 205
Corynebacterium, 128, 383
diagnosis, 384
diphtheriz, 384, 389; Pls. 58-
60

immunity against, 400

metachromatic granules,391

mixed infection, 397
special diagnosis, 401
toxins, 395
mallei, 384; Pl. 57
pseudodiphtheriticum, 384,
404; Pls. 58-60
xerosis, 384, 406; Pls. 58-60 >
es eet ee
yspora, 46
tures, anaerobic, 488
colorless, 69
descriptive terms used, 130
fluid, 487

INDEX.

Cultures, plate, 487
stab, 487
streak, 487
technic, 487

Cytoryctes variole Guarnieris,
188

DAHMEN’s concentration of tu-
bercle bacilli, 480

Davaine’s septicemia, 211

Deforming synovitis, excitant,
146

Denitrification, 82

Dextran in bacteria, 30

| Dextrorotatory lactic acid, 87
_ Diastatic ferments, 62

Diblastic theory, 50
Dichotomy, 19
Differentiating agents, 476
Diphtheria, 389; Pls. 58-60
animal, 398
bacteria resembling, 403
diagnosis, 401
granules, Neisser’s stain for,
481
ce lf against, 400
intestinal, 254
mixed infection, 397
toxin, 75, 395
wound, 397
Diplococcus, 123
albicans tardissimus, 168
definition, 19

intracellularis meningitidis,

148

pemphigi acuti, 188

pneumoniz, 143; Pl. 2

roseus, 190; Pls. 58-60
Disinfectants, definition, 37
Distilled water, bacteria and, 40
Druse, 142
Drusestreptococcus, 142
Dungern’s specific serum reac-
ie tion, 107

ysentery, 251

calf, 256

Ecrampsta bacillus, 300.
Eczematous ophthalmitis, 467
Edema, malignant, 341; Pl. 46

Me

> ae! MP Te
ee a.

i gem a ee ee, -

sn eyes,

INDEX.

Ehrlich-Koch’s method for tu-
bercle bacilli, 480
Ehrlich’s solution, 475
test for serum, 103
Elsner’s gelatin medium, 484
Emulsin, 63
Enantobiosis, 49
Endocarditis, 186
Endospores, 25
investigation, 26
staining, 479
59. See also Fer-

Bacin, “476
Equatorial germination, 27
Ernst’s granules, 21

si swine, 302; Pl. 33
thytalechol. formation, 86

Faminy, definition, 115

Farein du Boeuf, 447

Farcy, Pl. 57

Fats, decomposition, 80

Fatty series, gas-production by,
89

acids in tubercle bacilli, 29
Fermentation, 86

anaerobes and, 31

definition, 64

essential for, 64

in sugar media, 64

lactic acid, 87

oxidation, 66

products, 59

splitting, 65

tube, 90

urea-, 69
Ferments, albumin-dissolving,

definition, 59
diastatic, 62
inverting, 63
proteolytic, 59
fluctuation of production, 61
rennet, 63
Fermi’s test for proteolytic fer-
ments, 60
Ferret plague, 251
Fibrin, stain for, 482
Fischer’s bacteriacez classifica-
tion, 125

503

Fission, 24
Fission-fungi, higher, 457
diagnosis, 458
Flagella, 23
mordants for, 476
staining, 478
Flax, retting, 351
Fluorescent pigments, 68
Foaming liver, 344
Foot-and-mouth disease, 149,
252, 470
Formic acid formation, 86
Fowl tuberculosis, 418
Frankel and Gabbet’s tubercle
bacilli staining, 480
Frankel’s pneumococcus, 143
Friedlinder’s bacillus, 225; Pl.
15
Frog-spawn disease, bacillus, 23
fungus, 150
Fuchsin and methylene-blue,
aqueous alcoholic, 475
anilin, 475
carbol-, 475
for smear preparations, 477
Fungi, classification, 115
description of varieties, 129
families of, formation, 122
fission, higher, 457
phosphorescent, 56
frog-spawn, 150
genera of, formation, 122
ray, 439, 440
Furuncle, 184

GaBBET and Frinkel’s tubercle
bacilli staining, 480
Galtcoccus, 142
Gangrene, hospital, 468
Gas analysis, 91
phlegmons, 344
Bae production from carbohy-
drates, 89
Gaustadt bacillus, 255
Gelatin, advantages, 486
as medium, 483
liquefaction, 61
plate cultures, 487

-Gelatinous media, employment,

486

504

““Gelbe Galt,’”’ 142
Genera, biologic, 119
Gentian-violet anilin, 475
Genus, definition, 115
Glanders, 384; Pl. 57
fowl, 398
Glycerin-agar, 484
employment, 486
Glycerin-ascites-agar, 485
employment, 486
Gonococcus, 164; Pl. 10
Gonorrhea, 164
Gonotoxin, 167
Gram’s method for sections,
481
Kutscher’s modification,
482
solution, 476
stain, bacteria staining with,
478

for smear preparations, 477
Gram-Weigert method for sec-
tions, 482
Granules, 21
Grape-sugar agar, 484
in bacteria, 29
Grass organism I, 433
Grass organism II, 429
Greenish-blue pus, bacillus, 281
Gruber and Bordet’s agglutina-
tion theory, 108
Gruber-Durham agglutination
test in cholera, 374
demonstration of agglutinin,
105
Guanidin, 72
Guanin in bacteria, 30

HAFFKINE’s cholera toxin, 364
prophylaxis, 219

Hair, falling, 189

Hanging drop, 475

Hauser’s method for endospores,
479

Hay bacillus, 317; Pls. 39, 40
decoction as medium, 483

Heat-resisting substances, 98

Hemicellulose in bacteria, 29

Hemp, retting, 351

Hepatitis, 185

INDEX.

Hog cholera, 252

Human serum, obtaining, 102

Hydrogen from fermentation, 9
peroxid from light action, 48

Hyphomycetes, 126, 127

Hyposulphites, hydrogen sulphic
from, 77

Immunity, acquired, 98
active, 98
congenital, 96
causes, 97
increase in, 98
passive, 99
relative, 96
' specific, 98
bacterial, 103
poison, 99
Immunproteidin, 111
Indigo, reduction, 77
Indol, 72, 79
demonstration, 79
reaction, 372
Infection, 92
animal, 489
disease of cattle, new, 210
Inflammation, 184
Influenza, 202; Pl. 68
Injection, intraperitoneal, 489
subcutaneous, 489
Intestinal diphtheria, 254
Inverting ferments, 63
Iron in bacteria, 30

JANTHIN, 67
Johne’s method for capsules, 475§

Kepuyr fermentation, 223

Kiel water bacillus, Pl. 22

Kiuhne’s silicic acid medium, 48

Kutscher’s modification 0!
Gram’ section method, 482

Lactic acid, dextrorotatory, 87
fermentation, 87
levorotatory, 87
production, 86

INDEX.

Laschtschenko’s serum diagnosis
of typhoid, 240
Lecithin in bacteria, 29
Legume-tubercles, 84
Leprosy, 421; Pl. 62
Leptothrix, 458
buccalis, 461
epidermidis, 458; Pl. 69
gigantea, 461
innominata, 461
maxima buccalis, 461
ochracea, 465
placoides alba, 461
pyogenes filiformis, 461
Leuconostoc, 123
lagerheimii Ludwig, 151
Levorotatory lactic acid, 87
Lieben’s iodoform reaction, 86
Light, bacteria and, 46
Liquefaction of gelatin, 61
Litmus, blue, reduction, 77
in titration, 35
whey, 483
Léffler’s bacillus, 389
method for flagella, 478
for sections, 481
methylene-blue, 476
mordant, 476
serum mixture, 485
_ Lophotrichia, 24
_ Lysogenic material, 107

Mapura-Foor, 440, 452
Magnification, 474

Malignant edema, 341; Pl. 46
Mallein, 73, 388

Malta fever, 169

Manure organism, 433
Marmorek’s serum, 140
Marsh-gas from cellulose, 89
Maststreptococci, 142
Measles, 469

Meat bouillon, 483
Mechanical activity of bacteria,

55
Media, albuminous, 483
essential constituents, 33
fluid, employment, 486
influence of, on liquefaction,
61

505

Media, nitrogen production in, 89
nonalbuminous, 482
nutrient, 32, 482

acid, 36, 37
alkaline, 35
containing sugar, 37
employment, 486
neutral, 35, 37
reaction, 35
solid, employment, 486
sugar, fermentation in, 64

Meningitis, Pl. 68
cerebrospinal, 148

Mercaptan, 77

Merismopedia, 123

Merista, 123

Mesophilic bacteria, tempera-
ture for, 44

Metabolism, bacterial, chemical
activity, 64

Methane from fermentation, 90
Methylene-blue, acetic acid, 476
and fuchsin, aqueous alco-
~ holic, 475
for smear preparations, 477
Léffler’s, 476
reduction, 77
Micrococcus, 123, 163
acidi lactis, 175
paralactici, 142
liquefaciens Halensis, 224
agilis Ali-Cohen, 192
albicans amplus, 168
aquatilis, 163, 171
ascoformans, 179
aurantiacus, 164
Cohn, 189
badius, 164, 178
bicolor, 164, 189
biskra Heydenreich, 188
cerasinus, 164, 193
citreus agilis, 178
concentricus, 163, 174
candicans, 163, 169; Pl. 9
candidus, 171
corallioides, 175
coronatus, 164, 175
cremoides, 189
cyaneus, 164, 193
cyanogenus, 193
erythromyxa, 164, 198

506

Micrococcus flavus, 164, 178
conjunctive, 181
Freudenreichii, 174
galbanatus, 177
gonorrheee, 163, 164; Pl. 10
halensis, 224
in variola, 187
key to recognition, 163
latericius, 191
liquefaciens conjunctive, 181
luteus, 164, 176; Pl. 6
melitensis, 163, 168
of bitter milk, 174
pyogenes, 180
albus, 163, 180, 187; Pl. 9
aureus, 181, 187; Pl. 8
citreus, 180, 187; Pl. 9
quadrigeminus, 188
radiatus, 164, 176; Pl. 5
roseo-fulvus, 192
rosettaceus, 163, 174
roseus, 164, 190, 192;
typicus, 192
sordidus, 178
Sornthalii, 142
subflavus, 168
sulfureus, 164, 178
tardigradus, 178
tetragenus, 163, 171; Pl. 7
albus, 173
aureus, 173
mobilis ventriculi, 173
subflavus, 173
ures, 171
viticulosus, 163, 174
zymogenes, 144
Microscope, cleaning, 475
Microscopic examination for rec-
ognition of bacteria, 490
technic, 474
Microspira, 126, 352
Migula’s bacteriaces classifica-
tion, 215
Milk as medium, 483
employment, 486
bitter, micrococcus, 174
Milk-sugar agar, 484
Molecular motion, 55
Monotrichia, 24
Mordants for flagella, 476
Mounting agents, 477

Pl. 11

INDEX.

Mouse plague, 259
septicemia, 300; Pl. 33
Mouth-and-foot disease, 149, 252,
Muscarin, 72
Mycobacteria growing at room
temperature, 428
Mycobacterium, 128, 410
lacticola friburgense, 432
pathogenic effects, 434
perrugosum, 431; Pl. 63
planus, 429; Pl. 64
lepre, 421; Pl. 62
phlei, 433; Pl. 63
pathogenic effects, 434
smegmatis, 424
tuberculosis, 410; Pl. 61
anguicola, 421
avium, 418
differential diagnosis, 436
piscicola, 420; Pl. 62
ranicola, 421
toxins, 417
Myxoma disease, 470

NEGATIVE chemotaxis, 56
Neisser’s stain for diphtheria
granules, 481
Neuridin, 72
Nicolle’s method for sections, 481
Nitrates, free nitrogen from, 82
reduction, 78
Nitrification, 81
Nitrites, demonstration, 78
free nitrogen from, 82
Nitrobacter, 81
Nitrogen, 84
assimilation, 83
from nitrites and nitrates, 82
production in media, 89
Nitrosomonas, 81
europcea, 200
Noma, 471
Non-albuminous media, 483
employment, 486
Nuclein in bacteria, 30
Nucleus, 21
Nutrient acti 32, 482. See
also M

Ot a

wi} Sem

INDEX. 507
OBLIQUE germination, 27 Plague, 213
Oil-immersion objective, 474 cattle, 472
Old tuberculin, 417 ferret, 251
Oospora, 128 mouse, 259
Ophthalmitis, phlyctenular, 467 pigeon, 255
Optical activity, 56 swine, 252

Osteomyelitis, 184

Ovarian cysts, fluid from, as
medium, 485

Oxidation fermentation, 66

Ozena, 228

PackEtT, definition, 151
Panaritium, 277
Paraplectrum, 125
foetidum, 349
Parasites, obligate, 32
Parotitis, epidemic, 471
Parvolin, 72
Pasteuria, 25
Pediococcus, 123
flavus, 177
Pemphigus, 184, 188
Pende’s ulcer, 188
Peptone from albuminous bod-
ies, demonstration, 60
water, 483
Pericarditis, 184
Periostitis, 184
Periphery of colonies, 493
Peritrichia, 24

~ Perlsucht, 415, 416

Pest, 213
Pfeiffer’s cholera-serum test, 373
demonstration by bacterial
bodies in sera, 107
preparation of typhoid serum,
241

Phagocytosis, 97
Phenol, 79

demonstration, 79
Phenolphthalein in titration, 35
Phlegmon, 184

chronic, 445
Phlyctenular ophthalmitis, 467
Phosphorescent bacteria, 56
Photobacterium, 57

javanicum, 231
Pigeon plague, 255
Pigment production, 66-69

Planococcus, 123
Planosarcina, 123
Plants, diseases, 471
Plasmolysis, 20
Plate cultures, 487
anaerobic, 488
descriptive terms used, 131
for recognition of bacteria,
491
Pleuritis, 185
Pneumococcus, 143; Pl. 2
Pneumonia, 143, 185, 196, 225
in cattle, 469
in rabbits, 204
Poison, normal, 102
resistance, 99
Polar germination, 27
Polyvalent serum 140
Porcelain coccus, 171
Positive chemotaxis, 56
thermotropism, 56
Potato as medium, 484
employment, 486
bacillus, 323
cultures, descriptive
used, 131
water for tubercle bacilli, 483
Predisposition to infection, 96
Preparations, examination, 475
smear, 477
solutions for, 475
Prodigiosin, 275
pigments, 67
Propionic acid, formation, 86
Proteolytic ferments, 59

Proteus, 199
Bordoni-Uffreduzzi,

terms

hominis
300
capsulatus, 228

mirabilis, 300; Pl. 32

vulgaris, 295; Pl. 30

zenkeri, 300
Pseudodichotomy, 19
Pseudodiphtheria bacilli, 403,

404

508

Pseudo-edema bacillus, 343
Pseudoglanders bacillus, 389
Pseudo-influenza bacilli, 202
Pseudomonas, 125
Pseudotuberculosis, 410
Psychrophilic bacteria, tempera-
ture for, 44
Ptomains, 71
isolation, 72
Putrefaction, 80
alkaloids, 71
Putrescin, 72
Pyocyanase, 110
Pyocyanin, 68
Pyridin, 72

Racuitis, 472

Rauschbrand, 340

Ray fungus, 439, 440

Red milk bacillus, 268
pigment, 66
pus bacterium, 277
water-bacillus, 277

Reduction processes, 77

Rennet ferments, 63

Resistance, 96. See also Jmmu-
nity.

Retting of flax and hemp, 351 -

Rheumatic tetanus, 335

Rheumatism, acute, 472
articular, 184, 468

Rhinitis, atrophic, 228

Rhinoscleroma, 229

Ricin, action, 100

Rickets, 472

Rod bacteria, 124, 193

Roéntgen rays, effect on bacteria,
46

SAFRANIN, 476
Saliva spirochete, 381
Salts in bacteria, 29
Sanarelli’s bacillus, 256
Saprophytes, 33
Sarcina, 123, 151
alba, 153, 168
aurantiaca, 154, 160; Pls. 4, 5
aurea, 162
aurescens fusca, 162
fuseescens, 162
canescens, 153, 159; Pl. 5

INDEX.

Sarcina cervina, 154, 162; Pl. 5
compacta, 158
definition, 19
diffuens, 158
equi, 154, 158
erythromyxa, 154, 162; Pl. 5
flava, 154, 159; P13
fulva, 154, 156
key to recognition, 153
livido-lutescens, 154, 159
lutea, 154,157; Pl. 5
mobilis, 154, 160
pulmonum, 153, 155; Pls. 5,6
rosea, 154, 162,192; Pl. 5
typica, 158
variabilis, 159
ventriculi Goodsir, 155
Scarlatina, 472
Schizomycetes, 17
Schweineseuche, 209, 252
Sclerothrix, 128
Kochii, 128
Screw bacteria, 125, 352
Scrofulous ophthalmitis, 467
Sections, preparation, 481, 482
Sepsin, 72, 298
Septicemia, 223
hemorrhagic, 254
mouse, 300; Pl. 33
rabbit, 208, 211; Pl. 12
spontaneous, 252
vibrio, 366
Septicopyemia, 184
Serum, animal, collection, 105
effect of oxygen on action,109,
110
human, obtaining, 102
immune, demonstration of
bactericidal bodies in, 107
polyvalent, 140
reaction in cholera, 373
Shake cultures, sugar-agar, 488
Silicic acid as medium, 485
Silver method for flagella, 479
Skatol, 72, 79
Smear preparations, 477
Smegma bacillus, 424
Smith’s preparation of bouillon,
482
Species, definition, 115
Spherical bacteria, 122, 133

ew a

~<_ *,*

Se id

Te ay ee er. Ae

INDEX.

Spirillacee, 125, 352

- Spirillum, 126, 352, 376

cholerze, 353; Pls. 47-51
concentricum, 377; Pl. 55
endoparagogicum, 126
hachaize, 379

of nasal mucus, PI. 56
rubrum, 378; Pl. 55

rugula, 378

stomachi, 381
tenerrimum, 379
tenue, 379
undula, 380; Pl. 56
volutans, 380
Spirochete, 126, 352, 381
anserina, 381
Obermeieri, 381; Pl. 56
of saliva, 381
plicatilis, 381
Spirosoma, 126
Splitting fermentation, 65
Spore-formation, 25
conditions, 50
of Actinomyces bovis, 444
temperature for, 51
Spore-germination, conditions,

Spores, antecedents, 22
attenuation, 95
bipolar germination, 27
degeneration forms, 28
demonstration, 28
equatorial germination, 27
growth, 50
involution forms, 28
mature, 26, 27
oblique germination, 27
polar germination, 27
resistance, 51, 52
against chemicals, 53
test for, 52
to gases, 53
to light, 53
Sputum, tubercle bacilli in,
staining, 480
Stab cultures, 487
anaerobic, 488
descriptive terms used, 130
or specimens, preparation,

509

Stains, 475
Staphylococcus, 123, 180
albus, Pl. 9
aureus, Pl. 8
bovis, 189
cereus albus, 170, 187
flavus, 187
citreus, 178, Pl. 9
definition, 19
pemphigi neonatorum, 188
pyogenes albus, 180, 187
aureus, 181
citreus, 180, 187
salivarius pyogenes, 181
Sterilization, definition, 37
Stomatitis, ulcerative, 473
Streak cultures, 487
descriptive terms used, 131
Streptococcus, 123, 133
acidi lactici, 142
agalactize, 142
aggregatus, 142
albidus, 143
brevis, 140, 141
definition, 19
cinereus, 143
conglomeratus, 141
equi, 142
gracilis, 134
granulatus, 143
intracellularis, 148
involutus, 134, 149
key to recognition, 134
lanceolatus, 134, 148; Pl. 2
liquefaciens, 177
longus, 141
definition, 19
magnus, 143
mastitidis sporadice Guill.,
142
meningitidis, 148
cerebrospinalis, Pl. 68
mesenterioides, 135, 150
palleus, 143
pallidus, 143
phneumoniz, 143
pyogenes, 134, 135; Pl. 1
stramineus, 143
turbidus, 141
tyrogenus, 143
viscosus, 141

510

Streptothrix, 128
albido-flava, 456
aurantiaca, 456
citrea, 456
Streptotrichee, 127
Strohschein’s concentration of
tubercle bacilli, 480
Sugar, hydrogen sulphid pro-
duction and, 77
liquefaction of gelatin and, 62
Sugar-agar shake cultures, 488
Sugar-chalk agar, 484
Sulphates, hydrogen sulphid
from, 77
Sulphites, sulphid
from,
Sulphur in bacteria, 30
powder, hydrogen
from, 76
Sulphuretted hydrogen, anaer-
obes and, 44
production, 76
Suppuration, 184
Susceptibility, 96
Swine erysipelas, 302; Pl. 33
plague, 252
Symbiosis, 49
Symptomatic anthrax, 340; Pl.
45
Syncyanin, 67
Synovitis, deforming, excitant,
146
Syphilis, 426
stain for, 481

hydrogen

sulphid

TEMPERATURE, effect on bacterial
life, 44
Termoahnlichen bacillus, 285
Tetanus, 330, 332; Pl. 44
prophylaxis, 98
rheumatic, 335
toxin, 74
toxicity, 75
Tetrad, definition, 19
Thermic activity of bacteria, 58
Thermophilic bacteria, tempera-
ture for, 44
Thermotropism, positive, 56
Thiothrix, 458
Tongue, wooden, 446

INDEX. 1

Toxalbumins, 73

obtaining, 74
Toxin-binding group, 101
Toxins, 71, 73

and antitoxins mutual action,

obtaining, 74
Toxophoric side chains, 101
Trichorrhexis nodosa, 473
Triolein in bacteria, 29
Tripalmitin in bacteria, 29
Tristearin in bacteria, 29
T R tuberculin, 73, 417
Trypsin, 60
formation of, influence of me-
dia on, 61
Tubercles, lezume-, 84
Tuberculin, 73, 417
Tuberculoplasmine, 417
Tuberculosis, 128, 410; Pl. 61
fowl, 418
placental, 415 te
toxins, 417 .
Tumor, bony, 445
Typhoid bacillus, 197, 232. See
also Bacterium typhi.
fever, 232. See so Bacter-
ium typhi.
Typhus exanthematicus; 43
Tyrosin, 79
Tyrothrix, 304

Utcer, 188

Universal method for sections,
481

Urase, 71

Urea-fermentation, 69

Urine, nitrite reaction i in, 82

Uschinsky’ s solution, 33

Van ERMENGEMW’s silver method
for flagella, 479
Variola, micrococci in, 187
Verruga, 424
Vibrio, 126, 352, 353
albensis, 353, 370; Pl. 54
aquatilis, 369; Pl. 53
aureus, 375
balticus, 371

- INDEX.

ibrio berolinensis, 369; Pl. 53
cholere, 353; Pl. 47-51
demonstration, 371
} >. Sin evacuations, 371
in water, 373
| resistance, 358
_ toxins, 360
__-varieties related to, 364, 365
viability, 357
vibriosnot to be confounded
with, 375
virulence, 363
danubicus, 369; Pl. 53
Fischeri, 371
flavescens, 375
flavus, 375
helzogenes, 368
_ indicus, 370
_ key to diagnosis, 353
lingualis, 353, 376
; lissabonensis, 368
luminosus, 371
Metschnikovii, 353, 366; Pl.
Y- ok
_nasalis, 353, 375
proteus, 353, 367;
_romanus, 365
-rugula, 126
sa, ~ophiles, 371
serpens, 379
spermatozoides, 375; Pl. 56
terrigenus, 353, 371
tonsillaris, 353
tyrogenes, 368
apayie olin, 72

Pls. 51, 52

511

Violet pigments, 67

Virulence, 94
reduction, 94
restoration, 95

WATER agar, 485

bacillus, Kiel, Pl. 22

red, 277

bacteria and, 40

cholera vibrio in, 373

in bacteria, 29
Wax in tubercle bacilli, 29
Well-water, bacteria and, 40
Whey, litmus, 483
Wooden tongue, 446
Wound diphtheria, 397

XANTHIN in bacteria, 30
Xerosis bacillus, 406
X-rays, effect on bacteria, 46

YELLOW fever, 256
milk, bacillus, 267
pigment, 66

ZIBHL-NEELSEN staining of tu-
bercle bacilli, 480

Ziehl’s solution, 475

Zooglea, 23

Zymase, 65

Zymogenic bacilli, 306

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Adopted by As an indication of the great practical value
U. S. Army of the atlases and of the immense favor with
which they have been received, it should be
noted that the Medical Department of the U. S. Army has adopted
the ‘‘Atlas of Operative Surgery ’’ as its standard, and has ordered
the book in large quantities for distribution to the various regi-
ments and army posts.

SAUNDERS’ MEDICAL HAND-ATLASES

Preiswerk and Warren’s
Dentistry

Atlas and Epitome of Dentistry. By Pror. Gusra
PREISWERK, of Basil. Edited, with additions, by GrorGE W
WarrEN, D.D.S., Professor of Operative Dentistry at the Penn
sylvania College of Dental Surgery. With 44 lithographic plate
in colors, 152 text-cuts, 343 pages of text. Cloth, $3.50 net.

JUST READY

- Preiswerk’s atlas will be found invaluable to the practicing dentist, for th
numerous excellent lithographs make very easy of comprehension those pro
cedures that would be but imperfectly understood from description alone.

The Dental Review

** Nowhere in dental literature have we ever seen illustrations which can begin to com
pare with these exquisite colored plates.”

Hecker, Trumpp, and Abt &
on Children

Atlas and Epitome of Diseases of Children. By Drs.’ R
HECKER and J. TRumpp, of Munich. Edited, with additions, b
Isaac A. Ast, M.D., Assistant Professor of Diseases of Children
Rush Medical College. With 48 lithographic plates in colors
147 text-cuts, and 453 pages of text. Cloth, $5.00 net.

JUST READY

It is a recognized fact that the Germans lead the world in the treatment o
children’s diseases, and this magnificent atlas fully maintains this! reputation
The lithographic plates are wonderfully accurate, and the accompanying tex
is particularly full on treatment. Dr. Isaac Abt, the editor, has greatly im
proved the work by the addition of all the latest methods of treatment an
diagnosis.

Each volume contains from 50 to 100 colored plates

+ SAUNDERS’ MEDICAL HAND-ATLASES

Zuckerkandl and DaCosta’s
Operative Surgery

Second Edition, Revised and Greatly Enlarged

Atlas and Epitome of Operative Surgery. By Dr. O.
ZUCKERKANDL, of Vienna. Edited, with additions, by J. CHatL-
MERS DaCosta, M. D., Professor of the Principles of Surgery
and Clinical Surgery, Jefferson Medicai College, Philadelphia.
With 4o colored plates, 278 text-cuts, and 410 pages of text.
Cloth, $3.50 net. .

ADOPTED BY THE U. S. ARMY

In this new edition the work has been brought precisely down to date.
The revision has not been casual, but thorough and exhaustive, the entire
text having been subjected to a careful scrutiny, and many improvements and
additions made. A number of chapters have been practically rewritten, and
of the newer operations, all those of special value have been described. The
number of illustrations has also been materially increased. Sixteen valuable
lithographic plates in colors and sixty-one text-figures have been added, thus
greatly enhancing the value of the work. There is no doubt that the volume
in its new edition will still maintain its leading position as a substitute for
clinical instruction.

OPINIONS OF THE MEDICAL PRESS

Philadelphia Medical Journal

‘©The names of Zuckerkandl and DaCosta, the fact that the book has been translated
into 13 different languages, together with the knowledge that it is used in the United States
Army and Navy, would be sufficient recommendation for most of us.”

. Munchener Medicinische Wochenschrift

** We know of no other work that combines such a wealth of beautiful illustrations with
clearness and conciseness of language, that is so entirely abreast of the latest achievements
and so useful both for the beginner and for one who wishes to increase his knowledge of
operative surgery.”

Each volume is edited, with additions, by a leading specialist

SAUNDERS’ MEDICAL HAND-ATLASES 5

Helferich and Bloodgood’s
Fractures and Dislocations

Atlas and Epitome of Traumatic Fractures and Dis-
locations. By Proressor Dr. H. HELFericu, Professor of
Surgery at the Royal University, Greifswald, Prussia. Edited,
with additions, by JosEpH C. BLoopcoop, M. D., Associate in
Surgery, Johns Hopkins University, Baltimore. vom the Fifth
Revised and Enlarged German Edition. With 216 colored
illustrations on 64 lithographic plates, 190 text-cuts, and 353
pages of text. Cloth, $3.00 net.

SHOWING DEFORMITY, X-RAY SHADOW, AND TREATMENT

This department of medicine being one in which, from lack of practical
knowledge, much harm can be done, and in which in recent years great
importance has obtained, a book, accurately portraying the anatomic rela-
tions of the fractured parts, together with the diagnosis and treatment of the
condition, becomes an absolute necessity. This present work fully meets
all requirements. As complete a view as possible of each case has been
presented, thus equipping the physician for the manifold appearances that
he will meet with in practice. The illustrations show the visible external
deformity, the X-ray shadow, the anatomic preparation, and the method of
treatment.

OPINIONS OF THE MEDICAL PRESS

Medical News, New York

This compact and exceedingly attractive little volume will be most welcome to al!
who are interested in the practical application of anatomy. ‘The author and editor have
made a most successful effort to arrange the illustrations that the interpretation of what
they are intended to present is exceedingly easy.’

Brooklyn Medical Journal

** There are few books published that better answer the requirements for illustration
than this work of Professor Helferich. . . . Such a collection of illustrations must se the
result of much labor and thought.”

They are Satisfactory Substitutes for Clinical Observation

6 SAUNDERS’ MEDICAL HAND-ATLASES

Sultan and Coley’s
Abdominal Hernias

Atlas and Epitome of Abdominal Hernias. By Privar-
DOCENT Dr. GrorG SuLTan, of Gottingen. Edited, with addi-
tions, by Witt1Am B. Cory, M. D., Clinical Lecturer on Sur-
gery, Columbia University (College of Physicians and Surgeons),
New York. With 1rg illustrations, 36 of them in colors, and
277 pages of text. Cloth, $3.00 net.

DEALING WITH THE SURGICAL ASPECT

This new atlas covers one of the most important subjects in the entire
domain of medical teaching, since these hernias are not only exceedingly
common, but the frequent occurrence of strangulation demands extraordi-
narily quick and energetic surgical intervention. During the last decade the
operative side of this subject has been steadily growing in importance, until
now it is absolutely essential to have a book treating of its surgical aspect.
This present atlas does this to an admirable degree. The illustrations are
not only very numerous, but they excel, in the accuracy of the portrayal of
the conditions represented, those of any other work upon abdominal hernias
with which we are familiar. The work will be found a worthy exponent
of our present knowledge of the subject of which it treats.

PERSONAL AND PRESS OPINIONS

Robert H. M. Dawbarn, M. D.,
Professor of Surgery and Surgical Anatomy, New York Polyclinic.

**T have spent several interested hours over it to- day, and shall willingly recommend
it to my classes at the Polyclinic College and elsewhere.’

Boston Medical and Surgical Journal

“* For the general practitioner and the surgeon it will be avery | useful book for reference.
The book’s value is increased by the editorial notes of Dr. Coley.”

They have already appeared in thirteen different languages

SAUNDERS’ MEDICAL HAND-ATLASES 7

Bruhl, Politzer, and
MacCuen Smith’s Otology

Atlas and Epitome of Otology. By Gusrav Bruut, M. D.,
of Berlin, with the collaboration of Professor Dr. A. PoLiTzeEr,
of Vienna. Edited, with additions, by S. MacCuren SmirH,
M. D., Professor of Otology in the Jefferson Medical Col-
lege, Philadelphia. With 244 colored figures on 39 lithographic
plates, 99 text-illustrations, and 292 pages of text. Cloth, $3.00
net.

This excellent volume is the first attempt to supply in English an illus-
trated clinical handbook to act as a worthy substitute for personal instruction
in a specialized clinic. This work is both didactic and clinical in its teach-
ing, the latter aspect being especially adapted to the student’s wants.

Clarence J. Blake, M. D.,
Professor of Otology, Harvard University Madial School, Boston.

“* The most complete work of its kind as yet published, and one commending itself to
both the student and teacher in the character and scope of its illustrations.”’

Griinwald and Newcomb’s
Mouth, Pharynx, Nose

Atlas and Epitome of Diseases of the Mouth, Pharynx,
and Nose. By Dr. L. GRUNWALD, of Munich. Edited, with
additions, by James E. Newcomp, M. D., Instructor in Laryng-
ology, Cornell University Medical School. With 200 illustra-
tions on 42 colored lithographic plates, 41 text-cuts, and at9
pages of text. Cloth, $3.00 net.

Journal of Ophthalmology, Otology, and Laryngology

“A collection of the most naturally colored lithographic plates that has been pub-
lished in any book in the English language. . . . Very valuable alike to the student, the
practitioner, and the specialist.”’

They are offered at a price heretofore unapproached in cheapness

8 SAUNDERS’ MEDICAL HAND-ATLASES

Sobotta and Huber’s
Human Histology

Atlas and Epitome of Human Histology. By Pr. Dr. J.
SopoTta, of Wiirzburg. Edited, with additions, by G. CARL
Huser, M. D., Professor of Histology and Embryology, Univer-
sity of Michigan, Ann Arbor. With 214 colored figures on 80
plates, 68 text-cuts, and 248 pages of text. Cloth, $4.50 net.

This work combines an abundance of well chosen and most accurate illus-
trations with a concise text, and in such a manner as to make it both atlas and
text-book. The colored lithographic plates have been produced with the
aid of over thirty colors, and particular care was taken to avoid distortion and
assure exactness of magnification.

Boston Medical and Surgical Journal

‘In color and proportion they are characterized by gratifying accuracy and litho-
graphic beauty. . . . May be highly recommended to those who are without access to his-
tologic collections.”’

Haab and deSchweinitz’s
Operative Ophthalmology

Atlas and Epitome of Operative Ophthalmology. By
Dr. O. Haas, of Ziirich. Edited, with additions, by GEORGE
E. DE ScHweEInitz, M. D., Professor of Ophthalmology in the
University of Pennsylvania. With 30 colored lithographic
plates, 154 text-cuts, and 377 pages of text. Cloth, $3.50 net.

RECENTLY ISSUED

The colored illustrations in this work exhibit the same perfection of art
and accurateness of detail which are found only in these invaluable atlases.

Johns Hopkins Hospital Bulletin

** The descriptions of the various operations are so clear and full that the volume can
well hold place with more pretentious text-books.”

Unsurpassed for accuracy, pictorial beauty, completeness, cheapness

SAUNDERS’ MEDICAL HAND-ATLASES 9

Haab and deSchweinitz’s
Ophthalmoscopy

Atlas and Epitome of Ophthalmoscopy and Ophthal-
moscopic Diagnosis. By Dr. O. Haas, of Ziirich. vom the
Third Revised and Enlarged German Edition. Edited, with
additions, by G. E. DEScHWweEINITz, M. D., Professor of Oph-
thalmology, University of Pennsylvania. With 152 colored
lithographic illustrations ; 85 pages of text. Cloth, $3.00 net.

Not only is the student made acquainted with carefully prepared oph-
thalmoscopic drawings done into well-executed lithographs of the most

important fundus changes, but, in many instances, plates of the microscopic
lesions are added. It furnishes a manual of the greatest possible service.

The Lancet, London

**We recommend it as a work that should be in the ophthalmic wards or in the library
of every hospital into which ophthalmic cases are received.”’

Haab and deSchweinitz’s
External Diseases of Eye

Atlas and Epitome of External Diseases of the Eye.
By Dr. O. Haas, of Ziirich. Edited, with additions, by G. E.
DESCHWEINITZ, M. D., Professor of Ophthalmology, University

of Pennsylvania. 98 colored illustrations on 48 lithographic
plates and 232 pages of text. Cloth, $3.00 net.

SECOND REVISED EDITION—RECENTLY ISSUED
In this thorough revision the text has been brought up to date by the addi-
iion of new matter, including references to some of the modern therapeutic
agents. There have also been added eight chromolithographic plates.

The Medical Record, New York

“The work is excellently suited to the student of ophthalmology and to the practising
physician. It cannot fail to attain a well-deserved popularity.” (Review of previous ed.)

They are convenient in size and uniformly bound

Io SAUNDERS’ MEDICAL HAND-ATLASES

Durck and Hektoen’s
General Pathologic Histology

Atlas and Epitome of General Pathologic Histology.
By Pr. Dr. H. Durcx, of Munich. Edited, with additions, by
Lupvic Hexroen, M. D., Professor of Pathology, Rush Medical
College, Chicago. 172 colored figures on 77 lithographic plates,
36 text-cuts, many in colors, and 453 pages of text. $5.00 net.

JUST ISSUED

Many of the magnificent illustrations required lata six colors to re-
produce them.

W. T. Councilman, M.D.,

Professor of Pathologic Anatomy, Harvard University. -

**T have seen no plates which impress me as so truly representing histologic appear-
ances as do these.’

Durck and Hektoen’s
Special Pathologic Histology

Atlas and Epitome of Special Pathologic Histology.
By Dr. H. Dirck, of Munich. Edited, with additions, by
Lupvic HeKxTroen, M. D., Professor of Pathology, Rush Medical
College, Chicagu. In Two Parts. Part I.—Circulatory, Respira-
tory, and Gastro-intestinal Tracts. Part I1.—Liver, Urinary and
Sexual Organs, Nervous System, Skin, Muscles, and Bones. 243
colored figures on 122 plates, and 350 pages of text. Per part:
Cloth, $3,00 net.

William H. Welch, M.D.,
Professor of Pathology, Johns Hopkins University, Baltimore.

*€T consider Diirck’s ‘Atlas of Special Pathologic Histology,’ edited by Hektoen, a very
useful book for students and others ‘The plates are admirable.’

They represent the best artistic and professional talent

SAUNDERS’ MEDICAL HAND-ATLASES II

Lehmann, Neumann, and
Weaver’s Bacteriology

Atlas and Epitome of Bacteriology: incLUDING a TextT-
Book OF SPECIAL BACTERIOLOGIC Di1AGNosis. By Pror. Dr.
K. B. LEHMANN and Dr. R. O. NEuMANN, of Wiirzburg. vom
the Second Revised and Enlarged German Edition. "Edited,
with additions, by G. H. Weaver, M. D., Assistant Professor
of Pathology and Bacteriology, Rush Medical College, Chicago.
In two parts. Part I.—632 colored figures on 69 lithographic
plates. Part Il.—511 pages of text, illustrated. Per part:
Cloth, $2.50 net.

INCLUDING SPECIAL BACTERIOLOGIC DIAGNOSIS

This work furnishes a survey of the properties of bacteria, together with
the causes of disease, disposition, and immunity, reference being constantly
made to an appendix of bacteriologic technic. The special part gives a
complete description of the important varieties, the less important ones being
mentioned when worthy of notice. The lithographic plates, as in all this
series, are accurate representations of the conditions as actually seen, and
this collection, if anything, is more handsome than any of its predecessors.
As an aid in original investigation the work is invaluable.

OPINIONS OF THE MEDICAL PRESS

American Journal of the Medical Sciences

** Practically all the important organisms are represented, and in such a variety ot
forms and cultures that any other atlas would rarely be needed i in the ordinary hospitai
laboratory.”

The Lancet, London

‘We have found the work a more trustworthy guide for the recognition of unfamiliar
species than any with which we are acquainted.”

There have been 82,000 copies imported since publication

12 SAUNDERS’ MEDICAL HAND-ATLASES

Schaffer and Edgar’s
Labor and Operative Obstetrics

Atlas and Epitome of Labor and Operative Obstetrics.
By Dr. O. SCHAFFER, of Heidelberg. Svom the Fifth Revised
and Enlarged German Edition. "Edited, with additions, by
J. Cuirron Epcar, M. D., Professor of Obstetrics and Clinical
Midwifery, Cornell University Medical School. 14 lithographic
plates in colors; 139 other cuts; 111 pages of text. $2.00 net.

The book presents the act of parturition and the various obstetric opera-

tions in a series of easily understood illustrations. These are accompanied
by a text that treats the subject from a practical standpoint.

Dublin Journal of Medical Science, Dublin
“One fault Professor Schaffer’s Atlases possess. Their name, and the extent and

number of the illustrations, are apt to lead one to suppose that they are merely ‘ atlases,’
whereas the truth really is they are also concise and modern epitomes of obstetrics.”

Schaffer & Edgar’s Obstetric
Diagnosis and Treatment

Atlas and Epitome of Obstetric Diagnosis and Treat-
ment. By Dr. O. ScHArrer, of Heidelberg. rom the Sec-
ond Revised German Edition. "Edited, with additions, by J.
CiirTon Epcar, M. D., Professor of Obstetrics and Clinical
Midwifery, Cornell University Medical School. 122 colored fig-
ures on 56 plates; 38 other cuts; 315 pages of text. $3.00 net.

This book treats particularly of obstetric operations, and, besides the

wealth of beautiful lithographic illustrations, contains an extensive text of
great value. This text deals with the practical, clinical side of the subject.

New York Medical Journal -

“The illustrations are admirably executed, as they are’in all of these atlases, and the
text can safely be commended, not only as elucidatory of the plates, but as expounding the
scientific midwifery of to-day.”

These are the famous ‘‘ Lehmann medicinische Handatlanten ”’

SAUNDERS’ MEDICAL HAND-ATLASES 13

Mracek and Stelwagon’s
Skin

Atlas and Epitome of Diseases of the Skin. By Pror.
Dr. Franz Mracexk, of Vienna. Edited, with additions, by
Henry W. Stetwacon, M. D., Professor of Dermatology in
the Jefferson Medical College, Philadelphia. With 77 colored
plates, 50 text-cuts, and 288 pages of text. Cloth, $4.00 net.

JUST ISSUED—NEW (2d) EDITION

This volume, the outcome of years of scientific and artistic work, con-
tains, together with colored plates of unusual beauty, numerous illustrations
in black, and a text comprehending the entire field of dermatology. The
illustrations are all original and prepared from actual cases in Mracek’s clinic.

American Journal of the Medical Sciences

“The advantages which we see in this book and which recommend it to our minds are:
First, its handiness; secondly, the plates, which are excellent as regards drawing, color,
and the diagnostic points which they bring out. We most heartily recommend it.”

Mracek and Bang’s
Syphilis and Venereal Diseases

Atlas and Epitome of Syphilis and the Venereal Dis-
eases. By Pror. Dr. FRANZ MRAceEK, of Vienna.’ Edited, with
_-additions, by L. Bo_ron Bancs, M-»D., late Prof. of Genito-
Urinary Surgery, University and Bellevue Hospital Medical
College, New York. With 71 colored plates and 122 pages
of text. Cloth, $3.50 net.

According to the unanimous opinion of numerous authorities, to whom
the original illustrations of this book were presented, they surpass in beauty
anything of the kind that has been produced in this field, not only in Ger-
many, but throughout the literature of the world.

Robert L. Dickinson, M. D.,
Art Editor of ** The American Text-Book of Obstetrics.”’

“ Ybe book that appeals instantly to me for the strikingly successful, valuable, and
Pople character of its illustrations is the ‘ Atlas of Syphilis and the Venereal Diseases.’
know of nothing in this country that can compare with it.”

The lithographs, all made in Germany, are unrivalled

14 SAUNDERS MEDICAL HAND-ATLASES

Schaffer and Webster’s
Operative Gynecology

Atlas and Epitome of Operative Gynecology. By Dr.
O. SCHAFFER, of Heidelberg. Edited, with additions, by J.
CLARENCE WEBSTER, M. D. (Epin.), F. R. C. P. E., Professor of
Obstetrics and Gynecology in the Rush Medical College, in affili-
ation with the University of Chicago. With 42 lithographic
plates in colors, many text-cuts, a number in colors, and . 138
pages of text. Cloth, $3.00 net.

RECENTLY ISSUED

The excellence of the lithographic plates and the many other illustrations
in this atlas render it of the greatest value in obtaining a sound and practical
knowledge of operative gynecology. They are based on hundreds of photo-
graphs taken, from nature, and faithfully reproduced. —

Medical Record, New York

“The volume should prove most helpful to students and others in grasping details
usually to be acquired only in the amphitheatre itself.”

Shaffer and Norris’
Gynecology

Atlas and Epitome of Gynecology. By Dr. O. SHAFFER,
of Heidelberg. vom the Second Revised and Enlarged German
Edition. Edited, with additions, by RicHarp C. Norris, A. M., ©
M. D., Gynecologist to Methodist-Episcopal and Philadelphia
Hospitals. With 207 colored figures on go plates, 65 text-cuts,
and 308 pages of text. Cloth, $3.50 net.

The value of this atlas will be found not only in the concise explanatory
text, but especially in the illustrations. The large number of colored plates,
reproducing the appearance of fresh specimens, will give the student a knowl-
edge of the changes induced by disease that cannot be obtained from mere
description.

Bulletin of Johns Hopkins Hospital, Baltimore

“The book contains much valuable material. Rarely have we seen such a valuable
collection of gynecological plates.”’

These books are next best to actual clinical work

—

SAUNDERS’ MEDICAL HAND-ATLASES 15

Jakob and Eshner’s
Internal Medicine & Diagnosis

Atlas and Epitome of Internal Medicine and Clinical
Diagnosis. By Dr. Cur. Jaxon, of Erlangen. Edited, with
additions, by Aucustus A. EsHNER, M. D., Professor of Clin-
ical Medicine in the Philadelphia Polyclinic. With 182 colored
figures on 68 plates, 64 illustrations in black and white, and
259 pages of text. Cloth, $3.00 net.

In addition to an admirable atlas of clinical microscopy, this volume
describes the physical signs of all internal diseases in an instructive manner

by means of fifty colored schematic diagrams. As a means of instruction
its value is very great; as a reference handbook it is admirable.

British Medical Journal

“Dr. Jakob’s work deserves nothing but praise. The information is accurate and up
to present-day requirements.”’

Grunwald and Grayson’s
Diseases of the Larynx

Atlas and Epitome of Diseases of the Larynx. By Dr.
L. GRUNWALD, of Munich. Edited, with additions, by CHARLES
-P. Grayson, M.D., Clinical Professor of Laryngology and
Rhinology, University of Pennsylvania. With 107 colored
figures on 44 plates, 25 text-illustrations, and 103 pages of text.
Cloth, $2.50 net.

This atlas exemplifies a happy blending of the didactic and clinical, such
as is not to be found in any other volume upon this subject. The author

has given special attention to the clinical portion of the work, the sections
on diagnosis and treatment being particularly full.

The Medical Record, New York

“This is a good work of reference, being both practical and concise. . . . It isa valu-
able addition to existing laryngeal text-books.”’

For ‘‘ Special Offer ’’ regarding these atlases see page I

16 SAUNDERS’ MEDICAL HAND-ATLASES

Hofmann and Peterson’s
Legal Medicine

Atlas of Legal Medicine. By Dr. E. von Hormann, of
Vienna. Edited by FREDERICK PETERSON, M. D., Clinical Pro-
fessor of Psychiatry, College of Physicians and Surgeons, N. Y.
120 colored figures on 56 plates, 193 text-cuts. $3.50 net.
The Practitioner, London

“The illustrations appear to be the best that have ever been -published in connection
with this department of medicine, and they cannot fail to be useful alike to the medical
jurist and to the student of forensic medicine.’

Jakob and Fisher’s
Nervous System and its Diseases

Atlas and Epitome of the Nervous System and its
Diseases. By Pror. Dr. Cur. Jakos, of Erlangen. vom the
Second Revised German Edition. "Edited, with additions, by
Epwarp D. FIsHErR, M. D., Professor of Diseases of the Nervous
System, University and Bellevue. Hospital Medical College, N. Y.
83 plates and copious text. Cloth, $3.50 net.

Philadelphia Medical Journal

“We know of no one work of anything like equal size which covers this important and
complicated field with the clearness and scientific fidelity of this hand-atlas.”

Golebiewski and Bailey’s
Accident Diseases

Atlas and Epitome of Diseases Caused by Accidents.
By Dr. Ep. Govestiewsk1, of Berlin. Edited, with additions,
by Pearce Baitey, M. D., Consulting Neurologist to St. Luke’s
Hospital and Orthopedic Hospital, N. Y. 71 colored illustrations
on 4o plates, 143 text-cuts, 549 pages of text. Cloth, $4.00 net.
Medical Examiner and Practitioner

“It is a useful addition to life-insurance libraries, for lawyers, physicians, and for every
one who is brought in contact with the treatment or ‘consideration of ae or diseases
growing out of them, or legal complications flowing from them.’ “a

The ‘‘ Atlas of Operative Surgery’’ has been Tae U. S_ Army

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