LIBRARV

OF THE

UNIVERSITY OF CALIFORNIA

GIFTT OK

l>e University of

KOTXDKI) 1IY JOHN I). ROCKEFELLER.

t COMPMUTIVE m EXPERIMENTtL STODV OF OiCILLI
PRODUCING BED PICMEHT.

A DISSERTATION

SUBMITTED TO THE FACULTIES OF THE GRADUATE SCHOOLS OF ARTS,

LITERATURE, AND SCIENCE, IN CANDIDACY FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BACTERIOLOGY

MARY HEFFERAN.

JENA,

GUSTAV FISCHER.
1904.

Contents.

Page

A. Introductory 1

B. His tori cal and Descriptive 3

C. Comparative and Experimental Stud y (Tables I

-VIII) 20

I. Color determination of bacterial pigment 20

II. Variability in the Prodigiosus group 23

1. Introductory "23

2. Discontinuous variation or mutation 24

3. Range of normal variation 27

a) Growth and pigment on ordinary culture media . . 27

b) Growth and pigment on special solid media ... 29

c) Growth and pigment in non-proteid media .... 35

d) The effect of light upon pigment production ... 40

4. Summary 42

D. Notes on Groups of Red Chromogenic Bacilli . . 43

I. The Prodigiosus group 44

II. The Lactis erythrogenes group 44

III. The Rubricus group 44

Table IX 46

Table X 48

Bibliography 45

Reference List of Other Red Chromogenic Bacteria .... 54

1 si non

A. Introductory.

The mechanical difficulties of observing structures so minute
as bacteria have led to the accumulation of much differential detail,
and to an insistence on all points of unlikeness in physiological
characters or cultural reactions. Further, the lack of uniform
methods and standards of comparison has tended to produce an
overwhelming multiplicity of inadequate species-descriptions, in
which any slight variation from existing descriptions, e. g. as regards
formation of colonies on gelatin, has been erected into a character
of specific importance. But with the publication of Marshall
Ward's series of studies on Thames bacteria, of F u 1 1 e r and John-
ston's work on the bacteria of the Ohio River, and of Jordan's
paper on some 600 germs found in the Mississippi River, a check
has been put to indiscriminate multiplication of bacterial "species".
Recently , again , the demonstration of linking intermediate para-
typhoid and enteritidis forms in the colon-typhosus group, and of
the marked range of individual variation occurring in other orga-
nisms, has rendered especially important the questions of varia-
bility and of the actual lines between "species", "varieties", and
"races".

The chromogenic bacteria afford, from the fact of their pig-
mentation, a particularly suitable field for observation; and the
non-parasitic nature of the special group here considered is another
factor favorable for comparative and for variation study. The
warping effect of parasitic life upon the physiological and morpho-
logical character of organisms is well known, and more characte-
ristic results are to be anticipated from the study of variation
in a group of saprophytic organisms than in a group of pathogenic
organisms.

The agreement, among a number of bacteria, in a characte-
ristic so marked as is pigment production, might conceivably place
a series of red or yellow chromogenic germs in a category by
themselves, and raise the question whether this agreement in color
production be paralleled by agreement in biochemical and other
features, and whether the pigmentation be a fixed, a variable, or
a vital character of the organisms. So far, however, attention has
not been directed to a series of chromogenic germs, with the
exception of the comparative work done byThumm, byRuzi^ka,
and by Jordan on strains of B. pyocyaneus, and of the chemical
studies on bacterial pigments in general, referred to below. The
best known of the red saprophytes, B. prodigiosus, has been

quite fully discussed since its first descriptions; but no more than
two or three of its nearest relatives have at any time been in-
cluded with it in the study.

The parallel treatment attempted in this paper possesses certain
points of value; but comparison of a relatively large] number of
organisms has its obvious disadvantages as well as its advantages.
The number brought under discussion is plainly too great to per-
mit, in limited time and space, of extending the work on each
germ into detail, or of carrying out all possible tests, chemical,
spectroscopic and physiological. But, on the other hand, the com-
parison of so many red chromogenic bacteria must throw some
light on general features of relationship and variability which are
apt to be obscured or lost sight of when attention is concentrated
on a single organism or on a small group.

With these considerations in mind, I have attempted compara-
tive study of the following series of forty cultures of red chromo>
genie bacilli. A few of these, often referred to in bacteriological
literature, have been more or less completely studied as individuals,
e. g. B. prodigiosus, B. kiliensis, B. lactis erythro-
genes; but the majority, though frequently mentioned, have
received only the most cursory descriptions. I have therefore
prefixed to my comparative notes a brief description of each
"species" here discussed. A list of red chromogenic bacilli not
obtainable for study will be found at the close of the paper, where
are also tabulated the data regarding my series.

As an aid in determining the specificity of some members of
the series, I obtained from various laboratories eight cultures of
B. prodigiosus; the range of variability thus demonstrated for
that species and the other parallels or differences between the
allied cultures have suggested my closing note on grouping and
differentiation.

Red chromogenic bacilli described in this paper.
B. prodigiosus (Ehrenberg) I VIII

I, from University of Chicago.

II, from Rush Medical College, Chicago, 1902.

III, from Board of Health, Chicago, 1902.

IV, from University of Minnesota, 1903,

V, from University of Minnesota, 1903.

VI, from Yale University, 1903.

VII, from Ontario, Board of Health, 1903.

VIII, from University of Michigan, 1903.

'iber

indie us (Koch) I, from KraTs Laboratorium, 1900.

i, II O AJMAUMMMIIUIlj -LiTWS.

II, from Rush Medical College, Chicago, 1902;
obtained by them from Parke, Davis & Com-
pany, Detroit, 1899, obtained by the latter from
the University of Michigan (?) several years before.

B. ruber plymouthensis (Fiacher) I, from KraTs Laboratorium, 1899.
* * II, (B. No. 18), from air at Cold Spring

Harbor, L. I., I'.iu).
III, (II. No. ID), from air at Cold Spring

HarUr.
B. ki liens is (Breunije) from Rush Medical College, Chicago, 1902; obtained by

them from Parke, Davis & Company, 1&9.
B. ruber balticus (Breunig, Kruse) from Krai'* Laboratorium, 1900.

s B^
*/&.

B. miniaceus (Zimmermann) I, from Krals Laboratorium, 1901
II, from Hoagland Laboratory, Brooklyn, 1899.

HI, from Rush Medical College, 1902; obtained

by them from Hoagland Laboratory, Brooklyn.
^Y. rutilus (n. sp.) from the water of the Mississippi River, 1899.
"K amyloruber (n. sp.) from the water of the Mississippi River, 1901.
B. fuchsinus (Boekhout and de Vries) from Krai's Laooratorium, 19QO.
B. ruber (Miquel) from Krai's Laboratorium, 1900.
f^B. rubricus (n. sp.) from the water of the Mississippi River, 1901.
^B. rufus (n. sp.) from the water of the Mississippi River, 1901.
B. ruber (Zimmermann) from Krai's Laboratorium, 1900.
B. havaniensis (Sternberg) from University of Chicago.
yt&. 1 act is ery thro genes (Hueppe) 1, from Krai's Laboratorium, 1900.
II, from the Mississippi River, 1901.

B. rubefaciens (Zimmermann) from Krai's Laboratorinm, 1900.
B. lactorubefaciens (Gruber) from Gruber, 1902.
B. ru til esc ens (n. sp.) from the water of the Mississippi River, 1901.
B. my co ides roseus (Scholl) from Krai's Laboratorium, 1900.
/BC mycoides corallinus (n. sp.) from the water of the Mississippi River, 1899.
B. latericeus (Adametz) from Krai's Laboratorium, 1900, 1903.
B. rubropertinctus (Grassberger) from Krals Laboratorium, 1902; not

named by Grassberger.

B. rosaceus metalloides (Tataroff) from Krai's Laboratorium, 1900 1 ).
B. mesentericus ruber (Globig) I IV

I, from Krai's Laboratorium, 1900, 1903.

II, from the water of the Mississippi River,

1901.

/<f III, IV, from the water of the Mississippi

River, 1901.

B. Historical and Descriptive.

B. prodigiosus is usually taken as a type of red pigment
producing bacteria, and is said to be the earliest chromogenic
bacterium known. Considering, however, the frequency with which
there are found in air different varieties of red pigment germs
not identical with, but much like Prodigiosus, the history usu-
ally connected with the name of Prodigiosus probably extends
to many varieties of this type.

The frequency with which certain of the red chromogenic bac-
teria appear upon food-stuffs has been a matter of observation
for centuries. L u c i a n , in one of his dialogues (2 d century A. D.),
makes Pythagoras give, as reason for forbidding his disciples to
eat beans, the fact that white cooked beans, if placed in the moon-
light, change into blood. Since the forbidding of beans as food is
common to various sects of ancient times, e. g. to the Egyptian
priests and to the Zoroastrians , from which latter Pythagoras
doubtless obtained the notion, the recognition of this pigmentation
appears to be of extreme antiquity. In the year 332 B. C. the so-
called "blood-miracle" was of service to Alexander the Great
in the conquest of Tyre. The bread of his besieging army was dis-
covered to be reddened when broken; but the priests quieted the
terrified soldiery by interpreting the omen to mean, that as the
"blood" was inside the bread, a bloody fate would fall upon those
inside, not outside, the city. For the story see Curtius Rufus,
Hist. Alexandri, chap. 2, bk. 4.

1) Previously described by the writer, cf. An unusual bacterial grouping.
(Centralbl. f. Bakt. Bd. VIII. 1902. p. 690.)

The phenomenon of the "bleeding host", so often regarded as
a miracle in the Middle Ages, was due to a similar cause. The
composition of the sacramental bread, rich in starch and poor in
acid, was well adapted to the rapid growth of Schizomycetes ; but
the popular explanation of the phenomenon was that the host had
been stabbed by unbelieving Jews. The number of executions
and murders due to this belief was so great that Scheurlen, in
alluding to it, remarks that "dieser Saprophyt mehr Menschen um-
gebracht hat als mancher pathogene Bacillus".

Three nineteenth century appearances of this "blood miracle"'
as epidemic in a town or neighborhood are of interest. In the
year 1819, at Legnaro, near Padua, Italy, the whole district was
set in commotion by the appearance of red spots upon food. An
investigation was undertaken by physicians and professors of the
University of Padua, the fungus nature of the growth was dis-
covered, and two separate names given to it. B i z i o (23) called the
organism Serratia marcescens, the Serratia in compliment
to the savant who first propelled a boat by steam on the Arno.
His generic description is of interest: - -"Funguli acaules, senri-
sphaerica, capsulis contortis. Serratia marcescens. Vesicula tenuis-
sima, latice primo roseo, dehinc rubro repleta." Bizio reprinted
his paper of 1823 in volume I of his Opuscoli chimico-fisico,
1827, and several times later asserted his claim to priority in the
investigation of B. prodigies us. Meanwhile, the same epidemic
of Legnaro had been reported by Sette (25), and the name of
Zoogalactina immetropa given to the organism. More than
twenty years later, in January 1844, there appeared in the Jour-
nal de Pharmacie a report by various members of the French
Academy of Sciences, who had been commissioned to investigate
a reddening of the munition bread which was exciting among the
French soldiery much the same agitation as had prevailed among
the troops of Alexander. This report, and the treatment of the
phenomenon as new, brought out a letter from Bizio (24), emphasi-
zing his work of twenty-four years earlier, and giving the reference
to it. However, the next noteworthy epidemic of the sort in
Berlin in 1848, investigated by Ehrenberg (26), has not only
fixed upon the organism the name Prodigiosus then given to
it, but has connected the name of the German scholar with its
discovery.

Since Ehrenberg 's monograph many investigators have dealt
with the biological characteristics and the pigment of B. prodi-
giosus. A differentiation among the red germs was introduced much
later; thus, B. ruber indicus was first described in 1884,
B. ruber plymouthensis by Fischer in 1887, B. mesenteri-
cns ruber (Globig) in 1888, and B. lactis erythrogenes
(Hueppe) in 1889, etc.

B. prodigiosus (Ehrenberg).

This most common of all the pigment forms has been so com-
pletely described within the last few years that I can add little

except in confirmation or contradiction of the results already ob-
tained. The following description of my culture B. prodi-
g i o s u s I , believed to be typical, will be used as a standard for
comparison of a series of similar forms.

Morph ology.

Ehrenberg described his organism as a monad. Erd-
mann (28), when it appeared in the cholera epidemic of 1866,
called it a bacillus. Schroeter (29) 1872) named it Bac-
terium prodigiosum, meaning by his use of the term Bac-
terium that it was non-motile. It was first called a Micrococcus
by Cohn (30) in 1872, and was known as such until Schotte-
lius (35) in 1887 described it as a motile rodlet. Wasserzug (37)
(1888) thought he could produce the coccus or the bacillus form
at will by growth on alkaline or acid agar. In 1896 Scheur-
1 e n (43) made a careful description of the morphology of B. p r o -
digiosus; a two day culture in neutral bouillon showed a rod
1,5 2 times as long as broad, with rounded ends. On the long
side were 24 flagella. On a two-day potato culture, the rods
were smaller, ellipsoid, and capsulated. Scheurlen ascribed this
variation to the production of unstable alkali (ammonia) by the
organism itself. The same thing was true for alkaline media.
Finally, Migula (10) (1900) adds, that the bacillus is 0,5 ji
broad and from 0,5 1,0 JLI long, sometimes occurring in chains of
26 bacilli. Motility increases and the rodlike form becomes more
marked in slightly acid media. Flagella are peritrichial , but vary
in number and length.

Cultural features 1 ).

Gelatin. Investigators have not differed as to the growth
of B. prodigiosus on gelatin and agar. On a gelatin plate
the growth takes place quickly ; after twentj -four hours at 20 25 C,
the deep colonies appear round, sharply contoured, granular and
gray. The surface colonies are thinner and more granular, white,
and round or slightly irregular on the edges. On the following
day the gelatin is completely liquefied, a red color appears, and
later the whole liquid becomes red and cloudy.

In a vigorous gelatin stab culture a deep funnel of lique-
faction appears in 24 hours. Pigment appears in 48 hours, and the
gelatin is rapidly liquefied to the bottom of the tube and colored
red throughout. Usually no pellicle is formed. The rapidity of
liquefaction as well as of pigment production varies according to
the quality of the medium and the degree of vigor of the cultures.

Agar. The character of the colonies on an agar plate is
quite uniform. In 24 hours the colonies appear in the depths,
ellipsoid, granular, and reddish. On reaching the surface they
spread out round, thinner, and granular, becoming bright red. The
color appears first in the center, and under low power the edges
are thin, transparent, and finely granular. The diameter reaches
37 mm in five days.

1) Unless otherwise specified, observations were made at room temperature,
18-24 C.

On agar slant the growth appears first as a white smooth moist
layer, in 24 hours becoming bright red. In three or four days a
green fuchsin-like luster appears on the surface. Old cultures grow
darker in color (cf. table in section on pigment).

Potato. This was the culture medium used by the early
investigators. The growth and pigment production are here most
beautiful, particularly if the potato is fresh and slightly acid. A
white line appears in twelve to eighteen hours, which rapidly turns
red. The growth is luxuriant and the surface takes on a green
metallic luster in a few days, while the red color spreads over the
potato by means of the film of moisture. If the potato is dry,
growth and pigmentation are limited to the needle track, and the
superficial green appears as an abundant, dull, granular layer.

Blood serum. The growth is like that upon agar, but
slighter. The medium later undergoes liquefaction.

Bouillon. Ordinary neutral bouillon becomes cloudy in 12
to 18 hours. Later a ring of red color appears at the surface and
a flocculent red and white sediment accumulates. The surface
sometimes has a slight red pellicle and the whole liquid may be
tinged red.

Milk. In milk the red color appears at the surface, the pig-
ment, according to Fliigge (3) and Migula (10), adhering to
the fat droplets. A soft solid coagulum^s formed in 48 hours by
the simultaneous formation of "Milchsaure und Labferment"
(Gorini (41)). In ten days the coagulum shrinks to half its size,
leaving a clear watery serum. Little or no peptonization takes
place.

Gas production. The question of the production of gas
in sugar solutions by B. prodigiosus has occasioned some dis-
cussion. Liborius (34) (1886) described gas development by
B. prodigiosus in dextrose gelatine. Schottelius (35) (1887)
stated that this organism possessed in a marked degree the power
of converting sugar solution into alcohol and C0 2 , a statement
which has been embodied in Fraenkel's text book (4). S c h e u r -
len, however, disagrees with this conclusion of Schottelius.
He found that, in a peptone solution to which 2 % sugar had
been added, a gas bubble appeared upon inoculation with B. pro-
digiosus, did not increase, and, because of its absorption by
NaOH, was proved to be C0 2 ; but he believed this to be the
result of the action of succinic acid (which he had shown to be
a product of B. prodigiosus in potato culture) upon the sodium
carbonate used to neutralize the bouillon. When he tried a sugar
peptone bouillon neutralized by sodium-phosphate, or one to which
no alkali had been added, or an asparagin-sulphate-phosphate sugar
solution, he obtained no gas bubble. The bubble appeared, however,
when B. prodigiosus was grown in any medium which had
been neutralized by Na 2 C0 8 , even when the medium contained no
sugar. He also obtained gas without the presence of B. pro-
digiosus merely by the addition of a little succinic acid to the
medium. Ritter (47) (1900) confirms Scheurlen, and says

that the gas of Liborius and Schottelius was only a chemi-
cal product of the action of succinic acid, which B. prodigiosus
forms from sugar upon Na 2 C0 3 .

My results differ from those of Scheurlen and of Ritter.
In standard bouillon, freed from muscle sugar by Theobald
Smith's method, neutralized by NaOH instead of by Na. 2 C0 3 ,
and then having added to it iy 2 / dextrose, B. prodigiosus
I produced at the end of a week a bubble of gas in the fermen-
tation tube. Contrary to Scheurlen's observation, this did not
remain stationary but increased daily.

1) 7th day 4 mm 2) 7th day bubble

8th 7 8th 12 mm

10th 5 10th 19

12th 5 llth

17th 5

19th

31 mm 34,5 %

26 mm 34,5 %

All of the gas was absorbed by NaOH, showing that the gas
formed was C0 2 . Fermentation of the carbohydrate must have
taken place, for in this case no sodium carbonate had been added
to the medium, from which succinic acid could set C0 2 free. The
end reaction of the medium was acid to litmus.

In lactose and sucrose bouillon no gas was produced, nor did
any appear in asparagin-sulphate-phosphate solution to which 2 %
dextrose was added.

Oxygen relations. Liborius says (loc. cit. p. 172) that
such facultative anaerobes as Bac. crassus sputig, Bac.
pneumoniae, and Proteus vulgaris, which possess the
power to ferment sugar, do this equally well in the presence or in
the absence of oxygen. "Der B a c. p r o d i g i o s u s bietet den einzigen
Ausnahmsfall, daB Garung nur in den sauerstofffreien Kulturen
eintritt; aber auch hier ist nicht etwa die Garung ein Mittei,
dessen sich der Pilz bedient, um bei dem SauerstoffausschluB leben
und wachsen zu konnen, sondern er vermag ebensowohl in luft-
freiem Raum zu gedeihen, wenn gar kein garfahiges Material in Nahr-
substrat vorhanden ist. a My results on the anaerobic life of B. pro-
d i g i o s u s differ from those of Liborius and agree with those
of Ritter. In sugar bouillon B. prodigiosus grows, without
pigment, in the absence of oxygen. In sugar free bouillon no
growth takes place, although upon admission of air after fifteen or
twenty days development will proceed. Liborius' results can be
explained by the probable presence of muscle sugar in his so-called
sugarfree media. As I have shown above, absence of oxygen is
not necessary for gas production.

Temperature relations. B. prodigiosus develops at
37 1 / 2 C, but without pigment production, which is impeded at
a temperature of 35. Development is prevented by a tempera-
ture of 42 C.

Nitrate reduction. B. prodigiosus, when grown in
nitrate solution , produces a marked reduction of nitrates into
nitrites. Grown in the fermentation tube, in sugarfree bouillon to
which 1 / KN0 3 has been added, further reduction is shown by
the appearance of gas bubbles at the end of the week.

Odor. The cultures of B. prodigiosus are characterized
by a strong odor of trimethylamine, although , S c h e u r len states
that this substance is not formed. It has also been stated that
the odor is absent in colorless races (Schottelius). It is absent
in non-proteid media.

B. prodigiosus II VIII.

Seven so-called B. prodigiosus cultures were obtained from
various sources (cf. prefatory list) for comparison with B. pro-
digiosus I. Cultures II and III agreed with the type in all
respects and were probably derived from it. B. p r o d i g i o s u s IV,

VI and VII were vigorous cultures showing characteristic lique-
faction of gelatin , coagulation of milk , red pigment with green
luster on agar and potato etc., but causing gas evolution in sucrose
as well as in dextrose bouillon. Agar colonies of B. prodigiosus

VII were sometimes spreading and proteus-like. B. prodi-
giosus V and VIII gave a less brillant pigment of a violet red
color without metallic luster. No. V produced a heavy orange red
membrane-like pellicle in sugar free bouillon, liquefied gelatin
more slowly, beginning with a cup-like depression, coagulated milk
only in 72 hours, but showed gas bubbles in dextrose, sucrose,
and even in lactose fermentation tubes sooner than any other
Prodigiosus culture, i. e. in 48 hours. B. prodigiosus VIII
produced a viscid growth in ordinary media, which formed long
threads when touched with the needle. It coagulated milk in
48 hours and liquefied gelatin rapidly, but produced no gas in
sugar bouillon.

B. ruber indicus (Koch, 50).

Isolated by Koch in India from the stomach of an ape, and,
according to K r u s e (52), again found byPasqualein Massua. I was
unable to obtain the descriptions of either of these investigators,
but that ofFraenkel (51) must correspond closely to the original,
since he relates how the organism was sent to the Berlin Labo-
ratory by Koch, between two pieces of filter paper in a letter.
It was subjected to all the fumigation which the sanitary police
deemed necessary for documents leaving a cholera country, was
"durchlocht, gechlort und geschwefelt", but survived. F r a e n k e 1
states that it differed from B. prodigiosus in developing pig-
ment at 37, and in its toxicity for guinea pigs and rabbits.

My two cultures, although evidently distinct for several years
(cf. prefatory list), were alike in every respect. They differed from
the Prodigiosus type as follows :

Gelatin stab, the liquefied portion colored red only at the
surface. Agar, colonies pink in 72 hours, 3 mm in diameter, edges

slightly serrate, later spreading and red with green iridescence.
On slant agar and potato, pigment production usually poor,
growth luxuriant but dirty white. Unlike any other of the red
forms, B. ruber indicus produces pigment more readily on al-
kaline than on acid or neutral agar (cf. section on special media).
Bouillon, pellicle and sediment usually white, but in a peptone
and water solution vivid red pigment is formed. Milk, coagulum
completely peptonized in 10 days at 37 C. Gas, all C0 2 , is
formed from sucrose as well as from dextrose bouillon, but none
from lactose. Development with pigment takes place at 37 C.
Nitrate, reduced to nitrite and further to free nitrogen or
ammonia. Odor, not characteristic.

B. plymouth ensis (Fischer, 54).

Isolated from water and described by Fischer (1887). The
complete description is as follows: (Ein) "beweglich(er) Bacillus,
den ich in der Wasserleitung von Plymouth entnommenem Trink-
wasser fand und der sich nicht nur durch seine Gestalt (kleine,
dicke Stabchen mit abgerundeten Enden, kurze Faden bildend),
sowie durch die karmoisinrote Farbe des Pigments von den bisher
bekannten, einen roten Farbstoff bildenden Bakterien (Micro -
coccus prodigiosus, Bacillus ruber indicus Koch, und
Bacillus ruber Frank) unterscheidet, sondern auch durch eine
stark fadenziehende Beschaffenheit der Kulturen , sowie durch leb-
hafte Gasproduktion gut charakterisiert ist." Voges (55) (1893)
also made some observations on this form, which my results con-
firm and amplify.

The pigment formed by B. plymouthensis on ordinary
media could be distinguished from that of B. prodigiosus only
by its less vivid color and tendency to deteriorate into a violet
pink. Freshly rejuvenated cultures sometimes showed metallic
luster on agar, but, contrary to V o g e s' observations, not on potato.
The "fadenziehende" character, mentioned above, seemed constant
in B. plymouthensis I during two years' observation , after
which it disappeared. This character also appeared in a culture of
B. prodigiosus I, and is present in B. prodigiosus VIII
(cf. section on discontinuous variation). The main points of distinc-
tion between the Prodigiosus type and B. plymouthensis
are (1) slower liquefaction of gelatin beginning with a cupshaped
depression (cf. B. prod. V), (2) a vigorous (contrary to Voges) pro-
duction of gas, 7078 % of it C0 2 , in dextrose, lactose and suc-
rose bouillon. Gas, 42 % of it C0 2 , is also formed in a standard
asparagin sugar solution. Cultures ofB. plymouthensis I have
a strong fecal odor.

B. kiliensis (Breunig), B. ruber balticus (Kruse).

Isolated from water and described by Breunig (56) (1888).
A culture from Krai under the name B. ruber balticus corre-
sponds to the description of Breunig and to that of Laurent
(57), who worked on the variability of the "Bacille rouge de Kiel".

A second culture, B. kiliensis, was atypical only in lack of the
characteristic pigment, which was revived on special media to the
violet red color without the usual green luster. These cultures
differed from B. prodigiosus as follows :

Morphologically larger, rodlets of a young potato culture
2,5 5 // in length, 0,60,8 /< in diameter; rodlets from an old
potato culture may reach 8,0 /i in length. Motility appears in
4 hours after inoculation on potato at 35 C, in 24 hours at room
temperature. Gelatin is rapidly liquefied, with a thick orange red
surface pellicle. B r e u n i g described the growth on potato as at
first sealingwax red and later like that of B. prodigiosus. My
cultures showed in 24 hours a slight growth of a red violet color,
which became luxuriant and darker until, in 10 days, it was heavy,
corrugated, and looked like iron rust. Green luster is often
seen. Bouillon cloudy, thick orange red pellicle, and red sedi-
ment. The production of gas by this form, so far as I can find,
has not been previously determined. It occurs readily in dextrose,
lactose, and sucrose bouillon, and unlike that of the forms so far
described, is only in small part (2028 ; ) C0 2 . In standard
asparagin solution with 1,0 / dextrose, gas to 14,3 of the tube
length was formed, none of it C0 2 . Development with pigment
occurs at 37 " C. The pigment is violet red and lacks the orange
red surface growth seen at room temperature. Nitrates are reduced
to nitrogen gas. Odor not characteristic.

B. miniaceus (Zimmermann).

Isolated in 1889 from water, by Zimmermann (59), who
has suggested that the organism seems identical with Dowdes-
well's B. rosaceus metalloides. Zimmermann described
it, however, and gave it the above name. Migula, commenting
on this form, said: "Zimmermanns Vermutung, dafi diese Art
identisch mit B. rosaceus metalloides ist, diirfte nicht rich tig
sein. Er schliefit sich eher an den Kieler Bacillus an."

Hoagland Laboratory, Brooklyn, furnished a culture, isolated
from water, which seemed identical with the B. miniaceus from
Krdl, except for a tendency to lose power of pigment production.
A second culture from Hoagland Laboratory, evidently identical
with the first, came into my hands as "B. rubrus", was colorless,
and could not be made to regain the chromogenic power. This
culture produced the typical amount of gas, 4045 / .

My cultures of B. miniaceus are more like B. ply mou-
th en sis than like B. kiliensis. Contrary to Zimmermann,
I find the bacillus motile in young cultures. Liquefaction of gelatin,
slower than B. plymouthensis, but not so slow as stated by
Zimmermann, i. e. begins within 5 days, and is complete in
three or four weeks. The liquid is red. Potato and agar, like
B. plymouthensis, metallic luster rarely seen and only on
glucose agar. Gas production like B. plymouthensis. Nitrates
reduced to nitrites only. No fecal odor.

B. rutilus (n. sp.).

This organism was isolated in November, 1899, from the Illinois
River. One ccm of water was incubated for 24 hours in 0,55 carbol
broth, then plated in litmus lactose agar, where such a production of
pigment occurred that the plate was brilliantly colored. The red pig-
ment form grew luxuriantly upon isolation, arid, since it differed from
B. prodigiosus and from the others of the series, is here de-
scribed as a new species. Its vigor of growth and pigment pro-
duction have been slightly lessened by two years of cultivation
upon laboratory media. Its points of difference are as follows:

A short, actively motile bacillus, slightly larger on all media
than B. prodigiosus, but smaller than B. ruber balticus.
Gelatin, growth and liquefaction rapid, little pellicle, the whole
liquid vivid red. When first isolated, agar plate colonies always
showed pseudopodia-like ramifications; later, branching more rare,
or only on plates from old cultures. Growth and pigment best on
acid agar. No metallic luster ever seen on agar or potato. In
acid bouillon, the whole liquid colored red, no pellicle. The pre-
sence of dextrose and lactose increases the pigment, that of sucrose
does not. Milk, acid and coagulated in 24 hours, later peptonized
at 35 C. Gas production, in dextrose and sucrose bouillon, when
first isolated, 90 | of tube length, 65 % of this C0 2 . Later, 50 /
of tube length, all C0 2 . Nitrates reduced only to nitrites. Odor,
like B. prodigiosus.

B. amyloruber (n. sp.).

An organism which differed from B. prodigiosus, B. ruber
balticus etc., in pigment and in some other characters, was iso-
lated from Mississippi River water in 1901 ; because of its ability
to grow actively upon starchpeptone media it has been given the
above name. After passing through a summer upon neutral agar
the character of its growth was somewhat changed, tending to a
thin crusty or granular growth instead of one soft and luxuriant.
The pigmentation on ordinary media has undergone no deterioration,
remaining deep violet red. The chief points of deviation from the
forms already described are as follows:

Upon ordinary media the pigment is deep violet red, taking
orange color only on alkaline agar. Sugar free bouillon, little pig-
ment; sugar bouillon, colored deep violet red. Milk, peculiar; at
first, no change; later, a violet red coloration; after 15 days, a
tine red and white sediment of pigment granules and precipitated
casein, but no coagulation and no peptonization. No gas is evolved
in any sugar bouillon. Nitrates, reduced to nitrites.

B. fuchsinus (Boekhout and de Vries).
This name has been given to two different organisms; 1) to
a "new chromogenic bacillus" described by Boekhout and
de Vries in 1898 (60), and 2) to the red bacillus described but un-
named by Lustig in 1893 (8), named by Migula in 1900 (10).
Lustig's organism, which he described very completely, differs
morphologically from B. prodigiosus in that it is a rodlet two

- 12

or three times as long as broad, and contains pigment granules
in the cell body. Its cultural features are like those of B. pro-
digiosus, with the exception of pigment production in the absence
of oxygen. This organism is called by Kruse (3) B. ruber
aquatilis.

Krai informed me that the B. fuchsinus sent out by him
was obtained from Boekhout and de Vries. According to the
description of these authors, their bacillus differs from B. pro-
digiosus in the following points: It is slightly longer, i. e.,
1 1,5 u long. They describe it as non-motile, but later state that
in "Maizagar" it is longer and shows motility. It does not show
the metallic luster on ordinary agar, but does on potato and on
sodium tartrate agar. (B. prodigiosus often does not show it
on agar.) It peptonizes the casein in milk, and does not produce
gas in any sugar bouillon.

The culture which came to me from Krai showed no pigment
upon arrival , nor was I able to induce any pigment production
by various methods of rejuvenation, such as alternating series of
bouillon and gelatin cultures, growth on potato or on sodium
tartrate, agar etc., although the growth was rapid and luxuriant
on all media. After cultivating the organism for over a year,
I had begun to suspect that this form either was not B. fuchsinus,
or had lost its power of pigment production permanently, when I dis-
covered on a culture made on old dry agar a trace of red at the
top of the agar slant, where the agar was dryest By careful trans-
ference through a series of old dry agar tubes, I was able to
increase the pigment until it showed on the upper third and all
around the edge of the white agar growth, and also at the top of
growth on dry potato. This pigment was promptly lost, however,
when inoculation was made upon fresh media, and never showed
in gelatin or bouillon. My culture differed from the description
of Boekhout and d e Vries only in absence of pigment, and in
non-peptonization of casein 1 ). Nitrates are reduced to nitrites and
to free nitrogen gas. Gas is not formed from sugar bouillon.

B. ruber (Miquel).

This organism was sent to me from Krai's Laboratorium ;
the only description of it is contained in the following very kind
answer by Miquel to an inquiry concerning the organism: -
,,Je n'ai jamais public* de travail sur le Bacillus ruber que vous
mentionnez .... Le bacille, qui possede les principaux caracteres
du Bacille rouge de Kiel, mais qui en differt par sa faculte
negative de liquifier la gelatine, fut donne" comme curiosit6 a 1'in-
stitut Pasteur d'ou il a Emigre un peu partout jusqu'au Labora-
toire de Dr. Krdl sans etre precede' d'un signalement quelqu'un.
Si le hazard me le faisait de nouveau rencontrer dans les eaux,
peut-etre en ferai-je Thistoire en raison de la beaute" de son pig-

U this paper is appearing in the Ccntralbl. f. Bakt. I receive through
the kindness of Dr. Boekhout a culture of B. fuchsinus showing luxuriant
red pigment.

ment moidore; mais actuellement je n'en saurais dessiner une mo-
nographie precise: mes recherches sur ce micro-organisme datant
de plus de 10 ans." (1901).

I have completed Miquel's description as follows:
Morphology, a rodlet 23 /LI in length, often in chains of
several bacilli, non-motile, sporeless. Gelatin colonies, 48 hours,
small, irregular, finely granular, with well defined edges ; 72 hours,
show a peculiar corrugated, overlapping growth; the next day pig-
ment appears at the centre, and later they become deep violet red.
In gelatin stab, a red surface colony and a white needle track
growth; no liquefaction. 24 hours, surface colonies on agar
show a peculiar lined and cracked appearance under low power;
in 48 hours they are tinged red, and later look like B. prodi-
giosus colonies. Agar streak shows green luster and is like B.
prodigiosus or B. ruber kiliensis. Potato violet, red line
in 24 hours, later darker, often green luster. Does not spread
on potato. Blood serum not liquefied. Milk, no change except
pigmentation. Gas, 45 | of tube length in dextrose bouillon only,
79 / of gas C0 2 . Oxygen and temperature relations, like B. pro-
digious. Nitrates reduced to nitrites. No characteristic odor.

B. r ubricus (n. sp.).

In the autumn of 1901 three cultures were isolated in this
laboratory (University of Chicago) from the Mississippi and the
Missouri Rivers, which produced red pigment but showed marked
differences from B. prodigiosus and the cultures previously
described. These cultures do not grow rapidly, and the pigment
appears very slowly, the color beginning as pink or salmon pink,
and not attaining the characteristic brilliant red for some weeks.
Morphology, a small, slender, non-motile bacillus. Colonies, gela-
tin and agar, slow growing, small, round, non-characteristic, under
low power finely granular; color, yellow orange, deepening to red.
When first isolated, began to liquefy gelatin very slowly in ten
days. After a year's cultivation, the power of liquefying ordinary
gelatin had entirely disappeared. Agar slant, when dry and
growth limited, may develop bright red pigment in a few days;
when moist and spreading, may remain white or light pink for
weeks, gradually deepening in color. Potato slight or no growth.
Bouillon, cloudy, with pink pellicle. When first isolated, milk un-
affected; later, litmus milk cultures showed marked alkalinity. No
gas, nitrate not reduced, aerobic. May grow at 37 C, but better at
room temperature.

B. rufus (n. sp.).

Two cultures, much like B. r ubricus, were isolated at about
the same time and place. In pigment and in manner of develop-
ment this organism was hardly distinguishable from B. r ubricus,
but differed in showing luxuriant growth on potato, and in not
losing its power of liquefying gelatin slowly but completely. Milk,
unchanged or slightly alkaline in 15 days.

B. ruber (Zimmermann).

This form was isolated from the Chemnitz water supply and
described by Zimmermann in 1890(61). Migula gives it the
name B. pseudoruber, since, he says, the name ruber had al-
ready been used by Frank, whose organism, he thinks, is not
identical with that of Zimmermann. Zimmermann himself
had mentioned Frank's form, but remarked, that identity with
his was impossible to determine because of the incomplete descrip-
tion of the former; he thinks that his organism is more likely to
be identical with Ei sen berg's red bacillus.

My culture of B. ruber Zimmermann, from Krai, differs
both from the original description and from that of Eisen-
b e r g 's bacillus (cf. Table X). It is non - liquefying, non-gas pro-
ducing, and non-luxuriant on potato. It differs from B. rubri-
cus and B. rufus in being an actively motile bacillus. Bouillon
shows a peculiar growth; no development is visible for three or
four days, then a thin pink pellicle forms on the surface which
sends floating cobwebby streamers down into the clear liquid.
This may still be seen after 26 days. Some pin-point pink colonies
attach themselves to the wall of the tube. Potato shows a slow
clear red growth, limited to the needle track. Litmus milk
becomes blue through alkali production. No development occurs
without oxygen , nor at 37 C. Blood serum not liquefied, nitrate
not reduced.

B. havaniensis (Sternberg).

Sternberg has unfortunately given this name to two different
organisms ; one, the color producing, he termed B. havaniensis (62),
the other, colorless, B. havaniensis liquefaciens. Kruse
and Migula describe the latter under the name B. havaniensis,
which properly belongs to the red form. Sternberg's descrip-
tion of the red form is as follows : A short oval bacillus, usually
in pairs, about 0,4 0,5 (j. in diameter. The cells are nearly sphe-
rical. It is an aerobic, non-liquefying, chromogenic bacillus, which
grows slowly at room temperature. On gelatin plate the colonies
are spherical, translucent, of a blood red color; on gelatin stab
there is an opaque carmine layer, with a scanty colorless growth
in the depths. On agar the growth is slow but continuous, of a
glistening red color, with wavy outlines. The organism frequently
fails to grow on acid potato, but sometimes develops on old dry
potato. Pigment is found only in the presence of free oxygen. -
Migula, in describing this organism, does not include it among
the red forms; he says that its growth on agar is brown, and that
it shows no growth on potato.

The culture here described is of unknown origin, having been
in the laboratory (University of Chicago), some years under the
name B. havaniensis. It agrees exactly with Sternberg's
description as far as that goes. Growth in bouillon is similar to
that described for B. ruber Zimmermann. Litmus milk is un-

changed except for orange pigment at the surface. No gas is pro-
duced ; growth but no pigment occurs at 37 . It differs very little
from B. rubric us, i. e., only in bouillon and in milk.

B. lactis erythrogenes (Hueppe).

It was long ago observed in dairies that so-called bacterial
44 red milk" occured in one of two ways ; either red flakes appeared
on its surface, or the whole mass of liquid was colored red. The
former phenomenon is ascribed to B. prodigies us; the second
is usually due to an organism discovered in 1886 by Hueppe,
and described in 1889 by Grotenf eldt, (63) as B. lactis
erythrogenes. This organism, according to Grotenf eldt,
is a small rodlet, 11,4/4 long, non- motile and sporeless.
The gelatin colonies are small, round, and at first grey-
white, later yellow, as the gelatin becomes liquefied. Around
them the gelatin has a rose tint. A gelatin stab cul-
ture liquefies slowly from the top. After 1012 days the
liquefied portion is red, with a yellow sediment, while the solid
portion below is a weak rose color. The growth on potato
and agar is yellow, and after 6 8 days at 37 C, the pigment be-
comes a deep golden color. Bouillon is cloudy and yellow. Milk
undergoes a slow precipitation of casein, leaving a clear serum
which becomes blood red; the reaction is alkaline. The organism
is not pathogenic; the cultures are characterized by a sweet odor.

My culture of B. lactis erythrogenes, from Krai, agreed
closely with this description. Another culture, isolated in Novem-
ber 1901 from Mississippi River water, corresponded to the Krai
culture in all characters except that the color of the yellow pig-
ment was a shade lighter, with a tendency to appear much later
in the growth. The following details may be added to the descrip-
tion of these cultures: Growth on agar is moist, luxuriant and
yellow, while the medium becomes rose colored as in gelatin, and
even wine red. The yellow growth on potato shows often a slight
pink tinge, the potato becoming dark and discolored. Blood serum
not liquefied; no gas in sugar bouillon; no development without
free oxygen ; development with pigment at 37 C ; nitrates reduced
to nitrites. The sweet odor of the cultures is very noticeable.

B. rubefaciens (Zimmermann).

Isolated from the Chemnitz water supply and described by
Zimmermann in 1890(67). Characterized by a rose red color
which diffuses throughout solid media such as gelatin or agar.
Morphologically, a thin rodlet, 1 1.6 u long, often in chains of
two or three. Actively motile, no spores. Gelatin colonies ap-
pear as minute white dots in 48 hours; under low power, pearly,
thinner at the edge, which is well defined, finely granular. In gelatin
stab, small spherical white colonies occur along the needle track,
giving a characteristic appearance. The surface colony is yellowish,
and in old cultures a red tinge may be seen in the medium. No

- 16 -

liquefactioo. Agar colonies, not characteristic in ten days, small,
white, with fragmented edges. An agar slant culture shows a
white, smooth, shining, somewhat luxuriant growth. In old cul-
tures the medium takes on a distinct wine-red color, clear and
transparent. Zimmermann does not specify this as true for
agar, only for gelatin and potato. A heavy cream white layer
forms on potato, which later becomes yellow brown. I observed
no rose color here, but a brown discoloration of the potato.
Bouillon becomes cloudy with a white surface pellicle; after
6 8 weeks it may show a slight reddish tinge. Litmus milk is
decolorized and acid, but coagulates at the end of ten days only
on boiling. No gas is formed, no growth at 37 C, nitrate reduced
to nitrite, facultative anaerobe.

B. lactorubefaciens (Gruber) (68).

Received from Gruber in November, 1902. Upon examination
it agreed closely with the original description. I found however,
that the deep growth in gelatin was sometimes arborescent, and
that the medium was tinged red. On agar and potato, a white
spreading growth. Bouillon is turbid, with pellicle and sediment.
The colonies are coli-like. Milk is made rose colored and very
slimy; litmus milk shows slight acidity, but does not coagulate at
room temperature. The organism does not develop at 37 C, which
fact, together with its behavior in milk, its lack of yellow pigment,
and its motility, distinguish it from B. lactis erythrogenes.

B. rutilescens (n. sp.).

Isolated from Mississippi River water in 1901. Its characters
connect it both with the Prodigiosus group and with the Lac-
tis erythrogenes group. Morphologically, an actively motile
bacillus, like B. rutilus and B. r ubefaciens, unlike B. lactis
erythrogenes. No spores. Gelatin plates show white, non
characteristic colonies which soon liquefy the gelatin. A stab cul-
ture is liquefied rapidly, with a white cumulus sediment, and a
white pellicle. Later the liquid portion becomes a beautiful clear
rose, and the floating pieces of sediment take on a pink tinge.
Agar colonies are often spreading, like those of B. rutilus or
B. ruber indicus, without pigment. Agar slant shows a luxu-
riant, smooth, moist, white growth, much like the pigmentless
culture of B. fuchsinus. Potato, luxuriant, white, and spreading.
Bouillon, marked turbidity, with white sediment and thin pellicle.
Litmus milk, at room temperature slightly acid, coagulating in
three days. At 37 C, coagulation in 48 hours, followed by partial
peptonization. Gas production, negative; facultative anaerobe, ni-
trates reduced to nitrites. Unlike B. rubefaciens, grows rapidly
at 37 C.

B. mycoides roseus (Scholl).
What seems to be the original description of this organism is

a footnote to Grotenfeldt's article on B. lactis erythro-
genes (69) (1889), mentioning a bacillus isolated from earth by
Scholl, and studied in the laboratory of Hueppe at Wiesbaden.
The bacillus formed red felt-like colonies on gelatin, causing li-
quefaction. The gelatin had a red pellicle, but was not colored
throughout. Agar cultures were red in the dark. It grew quickly
at room temperature, and was morphologically like the anthrax
bacillus.

Ei sen berg (70), Kruse(3), and Migula(71) repeat this
description, the latter two authors adding only data as to solution and
spectrum analysis of the pigment as determined by Schneider (19).
Mace" (9 p. |256) mentions the organism. These are the only
descriptions that I have found.

My cultures, I and II, were identical in all reactions tested.
Morphologically B. mycoides roseus is usually smaller than
the anthrax bacillus, but like it variable in size, rodlets from
2 10 ju long occurring in the same culture. In very old agar
cultures, short, thick, often coccuslike, non-motile, no spores. Ge-
latin colonies are characteristic. They appear in three days as
minute white dots, under low power showing an opaque center and
edges broken or fringed as though crystallized; later the edges
lose their fringing and become smooth. In ten days the colonies
are 35 mm in diameter, somewhat creased and corrugated ; they
form slight depressions in the gelatin, but do not actually liquefy
it. Gelatin stab culture develops slowly a slight needle growth and a
dry, thick, corrugated pink colony on the surface. The needle growth
is often finely arborescent, like that of the colorless B. mycoides
figured by Lehmann and Neumann (7). Agar colonies appear
in 48 hours; under low power they show the characteristic fringed
edges seen in gelatin colonies. At five days they are pink and
small to the eye, less than 1 mm in diameter, edges smooth and
granular. Agar slant cultures show little development before
48 hours or three days. Then a salmon pink growth is seen, which
becomes dull, dry, and wrinkled, deepening in color as it gets
older, finally becoming vermilion. Potato, growth very slow, no
development for three or four days; at the end of ten days small
orange dots, or if the potato is kept wet, an elevated, warty, moist,
salmon pink growth. In 25 days a thick raised layer is seen,
which is more orange in color than on agar. Bouillon, slow de-
velopment, no turbidity. On the third day small round pink co-
lonies appear at the surface; at the end of ten days the surface
is covered with a thick agglomeration of salmon pink colonies,
some of which sink to the bottom as sediment. Milk, at the end
of ten days, shows only the characteristic salmon pink colonies
floating on the surface; after 20 days, decidedly alkaline. No gas,
no growth in the closed arm; growth and pigment nearly as good
at 37 C as at room temperature; nitrates reduced to nitrites.

B. mycoides corallinus (n. sp.).

This organism was isolated from Mississippi River water in

18 -

1900, and has since been kept in stock in this laboratory (Uni-
versity of Chicago). Two years of artificial cultivation have some-
what changed its character, but it continues to show several
seemingly constant differences from B. inycoidesroseus, its
resemblance to which has led to the adoption of the name.

When first isolated, young cultures showed a large anthrax-
like bacillus, non- motile, sporeless; later the organism became
somewhat diminished in size. Gelatin colonies are characteristic,
but differ from B. mycoides roseus. They appear in three days
as minute points, which under low power have a peculiar woolly,
wispy look ; as they develop, they become pink, smooth, and raised.
Gelatin stab, slow development; a ten day growth is very charac-
teristic, a fine feathery development along the needle track, and a
raised, smooth, shining pink surface colony. The minute agar colo-
nies of 48 hours show microscopically the characteristic wispy fib-
rillar branching; high power shows that the branching is made up
of transparent beady ramifications extending in all directions. The
colonies are larger than those of B. mycoides roseus of the
same age. Agar slant culture is unlike B. mycoides roseus
in being smooth, moist, and salmon pink in color. Growth on potato
differs in the same way, the color deepening in 20 days to red.
In bouillon cloudiness appears in 24 hours; later the turbidity
increases, and a pellicle of small separate pink colonies covers the
surface, falls as sediment on shaking, and is re-formed. Litmus
milk shows strong alkali production and characteristic pigment.
Otherwise like B. mycoides roseus.

B. later ice us [?J (Adametz, 72).

I could not obtain the original description of this organism.
Migula (10) describes it as a non-motile rodlet, 35 times as
long as thick, slow growing and non-liquefying. Chester (1) adds
that the growth on agar is limited, moist, glistening, and reddish-
brown to yellowish-brown; bouillon is clear and alkaline, with
stringy sediment; potato, thin, moist, and reddish; litmus milk,
decolorized and coagulated; indol, negative; no growth at 36 C.
Lehmann and Neumann (7), who give a fuller description,
with plates, say that milk is not coagulated, the growth is from
vermilion to reddish brown ; no gas nor acid is formed from sugar,
but traces of indol occur. Their culture was isolated from air and
identified from Eisenberg's description (2).

My culture of B. latericeus, obtained from Krai, does
not agree with any of the above. Dyar (73) also notes the aber-
rancy of the culture which he received from the same source.
Dyar's culture, to which he gives the new name, B. fuse us
pallidior, agrees with that here described.

A short, non-motile bacillus, 11,3 n long, and 0,50,7 (i
broad, occurring single and in chains. Gelatin colonies after four
days small, granular, with slightly irregular and ragged edges.
Stab cultures show a slight needle growth, and on the surface a

UN!

slow growing dry white colony, which after weeks lines a cup-like
cavity with a thin dry spreading cream pink growth. No other
evidence of liquefaction occurs than the cup shaped depression.
Agar colonies are dry and shining to the eye; under low power
the edge is ragged, showing the individual rodlets pushing out
into the surrounding medium, Agar slant and potato show at
first a dry shining whitish layer, which later becomes "crusty",
wrinkled, and dull pink. Bouillon remains nearly clear, develop-
ment taking place in a thick surface pellicle of paraffin-like con-
sistency, from which flakes fall to the bottom of the tube on
shaking. Litmus milk, not coagulated, but shows intense alkaline
reaction and peptonization after a few weeks at 35 C. Aerobic,
non-gas producing, nitrates reduced to nitrites, slight growth at
37 C.

B. rubropertinctus.

In his experiments with acid-resisting organisms, Grass-
berger (74) made, in 1899, from butter and from guinea pigs inocu-
lated with butter, six isolations of the bacillus described below.
My culture, obtained from Krai in 1901, agress in biological
characters with Grassberger's original notes. As he gave it
no name, I have called it on account of its staining reaction,
B. rubropertinctus.

Grassberger describes a 24 hours agar culture as showing
rods 1,53,0 ^ long, having no trace of acid resistance when
stained. Older cultures occasionally show longer bacilli, which
retain the stain slightly when treated with 3 % ac id alcohol, but
never evince the true tubercle bacillus reaction. I found that a
ten day potato culture, when stained on a cover glass together
with B. c o 1 i by hot carbol fuchsin , washed several times with
10 % H 2 S0 4 , and counter stained with methylene blue, resisted
the acid perfectly, remaining bright red in contrast to the blue
B. coli. The bacilli could not be mistaken for B. tuberculosis,
being much shorter and thicker.

Gelatin plate shows characteristic colonies, the deeper ones
ovoid in shape and granular, ttye superficial colonies irregular, with
crenate edges, folded surface, and red color. Stab culture, slight
needle growth and orange red surface colony as above, no lique-
faction. Agar colonies, very slow, being just visible to the naked
eye in five days; uniformly granular, and irregularly triangular.
On agar slant, growth lustreless, dry, and spreading, later wrinkled
and vermilion red. Grassberger records one culture that was
moist. Potato development, slow but constant; at the end of ten
days orange red, rough, granular, and moist. Bouillon, clear save
for slight cloudiness at the top; a salmon pink pellicle is formed,
which is constantly renewed as it sediments on shaking. Litmus
milk shows no change except for the development of a pink pellicle;
B. rubro pertinctus is not gas-producing, grows at 37, is
aerobic. Grassberger notes slight indol production and cauli-
flower-like odor. My culture did not show these characters.

B. mesentericus ruber (Globig).

Isolated by Globig, 1888 (75), from potato. Described as
a slender, motile, bacillus, with oval, very resistant, spores. Gela-
tin colonies, round and yellow in the depths; on the surface
showing a fringe or halo of fine network, which breaks down in
34 days and liquefaction begins. Gelatin stab, funnel shaped
liquefaction. Agar colonies are non - characteristic. Agar streak,
dirty white and slightly wrinkled growth. On potato a dry growth
of a beautiful pink color. Globig described bouillon as clear,
with a thick pellicle; my cultures showed cloudiness and pellicle.
Litmus milk, curdled and acid. No gas is produced, nitrates are
reduced, growth with rose color at 37 C.

Two cultures from Krdl, 1900, 1903, differed from the above
description in not showing spores. Three cultures isolated from
the Mississippi River water at different times were used as the
basis of this description.

C. Comparatire and Experimental Study.

I. Color determination of bacterial pigment 1 ).

The increasing tendency towards the adoption of definite terms
of positive, negative, or quantitative value in bacterial descriptions
has not been generally extended to the determination of color
produced by bacteria. The pigmentation of an organism still
furnishes an opportunity for a more or less lax and undiscrimina-
ting nomenclature. Thus, among the red series one whole classi-
fication has been made on the basis of such divisions as these :
"Pigment carmine", "pigment flesh-colored," "brick-red", "reddish",
"rose-colored", "salmon-pink", "yellowish-reddish", "brownish-red",
"pinkish", "reddish-pinkish", "blood-red", "red-brick-red". These
distinctions may call up well defined color pictures to individual
workers, but it is evident that there are difficulties in the way of
fitting a new chromogenic organism to such an imaginative scheme.

In default of a more exact method, statistical biologists have
for some time employed a color top 2 ), upon which discs of the
primary colors may be arranged, showing sectors of definite pro-
portions ; when the top is rotated rapidly the colors blend and give
the intermediate tints. Any color may thus be matched, and the
percentage of primary colors which go to make it up very closely
determined. This method is easily applicable to bacterial colors,
and has been adopted in this study. Other color-terms appearing

1 ) The chemical nature of bacterial pigment, its solubility, and its spectrum
analysis has been discussed at length by Schroter (29), Cohn (30), Griffiths
(39), Scheurlen (43), Schneider (19), Rosenberg (45), Kraft (49), and
others.

2) The Milton Bradley color top has standard colors of the following wave-
lengths: Bed, 656061; orange, 606-611; yellow, 577582; green, 514519;
blue, 467472; violet, 419 424. For method cf. Davenport , C. B., Statistical
Methods. New York, 1899, p. 9.

Vermilion
Orange red
Salmon pink
Pink
Violet red
Ked
Dark red

30
45
50
50
80
90
100

70
55
25
25
15
10

here are either quoted or applied to the culture media in distinction
from the bacterial pigments.

One difficulty, however, in determining bacterial pigment, is
the possible range of variation in any one organism under influence
of preliminary cultivation, reaction of media, and age of culture;
so that one color-determination may be insufficient for general
description. Neither can the mean of several determinations be
specified as the typical color of a given organism. But, allowing
for occasional degenerate or pigmentless cultures, if an organism
has been put through the regular course of rejuvenation and then
grown on slant agar of definite composition and reaction, the pig-
ment of young and of ten day old cultures can be fairly well charac-
terized. The appended tables will give an idea of the results of
this method; and the terms used in this paper to designate the
different reds will have definite meaning as follows :

Bed Orange White Blue

22 3

(see Table I p. 22.)

Of the cultures producing "insoluble" pigment on agar, which
are studied here, a division may be made into three groups, accord-
ing to color of pigment.

I. The B. prodigiosus group, including

B. prodigiosu s,

B. ruber indicus,

B. ruber plym outhensis,

B. kiliensis (B. ruber balticus),

B. miniaceus,

B. rutilus (n. sp.),

B. arayloruber (n. sp.),

B. fuchsinus,

B. ruber miquel.

The members of this group develop on agar a pigment in
mass, which shows a large percentage of pure red, with varying
quantities of orange and sometimes a small amount of blue, which
gives a violet tinge, or of black. They are characterized by the
fact that the amount of orange diminishes as the cultures age;
at the same time there is an addition of black, i. e., the cultures
grow darker. This group includes the "carmine", "blood red",
and "violet red" cultures.

II. The B. rubricus group, including

B. rubricus,

B. ruber zimmermann,

B. ha vaniensis,

B. rufus (n. sp.),

B. rosaceus metalloides.

Since these cultures are slower in development, the pigment
appears somewhat later than in the organisms of group I. In
young cultures a large percentage of orange is present, and the
cultures never grow black, but keep the orange red tone. Here
are included "orange red", and "yellowish reddish" cultures.

Table I.

ROW Blue Blk V
Agar slant, 48 hrs. (1,5 % 4- ) 30 70

5 da. 75 25 (met. lus.)

,, 10

Potato 48 hrs.
10 da.

Gel. stab (liq.)
Agar slant, 48 hrs. (1,5 /
,, ,, 10 da.
surface
depth

Potato 48 hrs.
6 da.

Gel. stab. 10 da. (liq.)

10 da. (pellicle)
Agar slant, 24 hrs. (l,5<>/o +) 50 40
48 80 20

10 da. 68 14

nearly black
90 10

B. prodigiosus.

B. ruber balticus.

90 10
85 15
60 10
90 10
+) 85 15 (met. lus.)

30 (m. 1.)

B. rutilus (n. sp., 1900)

65 35
100
80 15
95 2 1 /,
90 10
70 30

2V,

(m. 1.)

3 15

10
Gel. stab. 10

B. ruber miquel.

(liq.)

Agar slant, 48 hrs. (1,5 % +) 80 20 (met. lus.)

B. ruber indicus.

Potato
Gel stab.

85 10
62 26
68 22
80 20

10 da.
48 hrs.
10 da.

10 (colony)
Agar slant, 48 hrs. (1,5 / ) 70 25 5

10 da. 80 10

Potato 5 30

Gel. stab. 10 (pellicle) 75 25

i, 10 (liq.) 100

B. ruber ply mouthen s is. Agar slant, 48 hrs. (1,5 / +) 57 35

10 da. 75 20

Potato 48 hrs. 30 22 23

10 da. 33 35 10

Gel. stab. 10 (pellicle) 80 20

>. , 10 (liq.) 100

B. rubricu s (n. sp.) (slow) Agar slant, 10 days 6330 2

B. havaniensis 10 55 45

B. rosaceus metalloides 10 5743

B. mycoides roseus 3 (1,5 / c +) 17 18

5 15 35

Potato 10

B. mycoides coral, (n.sp.) Agar slant, 10

Potato 10

12
10

(m.l.)

25
22

42 35

,. (wrinkled) f>7 33
(1,5%-) 50 25
, (smooth) 28 42

III. The B. mycoides roseus group, including
B. mycoides roseus,
B. mycoides corallinus (n. sp.),
B. rubropertinctus,
B. latericeus (?).

- 23 -

This group differs from the last in the presence of a con-
siderable percentage of white , with sometimes a trace of yellow.
Here would belong the so-called "salmon pink", "coral pink",
"rose", or "flesh colored" cultures.

Cultures producing "soluble" red pigment may be classed as
the Lactis erythrogenes group, including
B. lactis erythrogenes,
B. rutilescens (n. sp.),
B. rubefaciens,
B. lactorubefaciens.

B. mesentericus ruber is peculiar in producing its pink
or red pigment only on potato, not upon agar.

The term "Prodigiosus group" as used below, refers then to
the series of Group I as described.

II. Variability in the Prodigiosus group.
1. Introductory.

In the discussion of variability or variation as regards bac-
teria, several complicating factors are present. In the first place,
the facility with which bacteria, more than any other class of or-
ganisms, respond and adapt themselves to changes in the nature
of their environment is a constant character in their biology.
Again, a bacterial type, as we assume it, is more or less artificial,
developed and maintained by our methods of culture, which are so
arranged as to reduce or eliminate variation. And the old simple
distinctions between typical varieties are now complicated not only
by the natural variability of the organisms, but by the conception
of intermediary paratypes, each with its own possibilities of
variation.

The variability of bacteria, whether manifested spontaneously
or under compulsion, seems to find its expression principally in
the loss of certain characters or in their return after loss. If, for
instance, B. anthracis be exposed to a temperature of 42,
B. tetani to a gradual admission of oxygen, orB. prodigiosus
to the action of light, the organisms will all live and develop, but
one will lose its power of sporulation, another its virulence, the
other, its power of pigment production; and unless the abnormal
conditions be maintained for many generations, and the process
fostered by artificial selection, the organisms will, upon restoration
to their usual environment, revert to their original "normal" type.
These and many similar observations indicate that bacteria have a
certain number of biological characters, without which they may
continue to live; that these characters may be lost as a result of
environmental modification, but are so far typical that they tend
strongly to return.

This "tendency to return" finds its nearest explanation quan-
titatively, that is, in considering as a factor in variation not only the
effect of the environment upon the organism , but also the nature
of the compound of characters which we term a type. The aber-

24 -

rant or white forms among the red, for example, may be considered
as due to sudden or irregular predominance of characters always
present, though latent or much in the minority; and the problem
before the student of variability is the determination of the "lowest
terms" to which an organism can be reduced, the discovery of the
minimum and the maximum of characters which can be found to
belong to each type.

The only means at our command for such investigation
is the modification of the nutritive medium. Such culture media
as agar, the composition of which is uncertain, do not give
definite results; and I have employed, as set forth below,
compounds whose elements and arrangement are more accurately
known. Whatever modifications of the organism, i. e., what-
ever response, by disappearance of characters, has followed change
in the environment, I have set down under the general head of
range of normal variation, reserving the term discontinuous
variation or mutation for the sudden appearance of by-forms with-
out any apparent cause. For such sports I do not offer expla-
nation. They may be due to a phenomenon parallel to what Wei s -
mann, speaking of budding, calls "abnormal differential nuclear
division", or they may arise in response to physical external causes,
confined to a very limited area, and invisible to us. But along
with the cases of "discontinuous variation" in the higher organisms,
they offer an interesting problem in the origin and differentiation
of varieties.

2. Discontinuous variation or mutation.

Variations of bacterial cultures, apparently spontaneous in
character, have been frequently noted. D y a r (65) considered that
when tubes were filled with media from the same flask, inoculated
at the same time from the same culture, and grown on the same
shelf, variation resulting in such a series was "discontinuous". The
possibility of contamination in bacterial cultures which may thus
suddenly vary is always a question, which can only be decided by
testing the new variety through a sufficient number of unchanged
differential characters, as was done by Dyar in studying a "crusty"
variety of B. lactis erythrogenes. The tendency, upon plating
such a variety, for some of the colonies to revert to the parent
type may be viewed as proof of the true nature of the variation.

Among chromogenic cultures the most frequently observed
"sport" variation is in the appearance of colorless colonies upon
a plate where the majority of colonies are normal. Such colonies
are of course more numerous in plates made from old or dege-
nerate cultures, and decrease gradually with successive transferences.
Thus, five B. prodigiosus cultures which were obtained from
different laboratories showed the following variations in the early
platings. All the plates were made in the same way, and were
second dilution. The original agar cultures were evidently young,
were all growing well, and were all pink or red in color except

No. V, which showed no pigment, and, as I was informed, had not
for at least two months back. The first result on 48 hour agar
plate was:

B. prod. IV, 200 small colonies, apparently all orange red.

B. prod. V, 2 pink colonies, 48 white.

B. prod. VI, 300 to 400 colonies, all red.

B. prod. VII, 35 soft spreading white colonies, 3 pink.

B. prod. VIII, 5 smooth round colonies, all violet red.

All the agar streak inoculations from red colonies, and some
from white ones, gave ordinary red cultures of varying intensity;
and it was evident that the number of abnormal white colonies which
appeared as "sports" on the plates were in each case only an indi-
cation of the degree to which the colorless variations of the original
cultures had gained an ascendency over the red type through con-
tinued unfavorable conditions, probably because of long intervals
between transferences. That this was the case was shown by a
second plating after rejuvenation, fifteen days later. The results of
this were:

B. prod. IV. 400 brilliant vermilion colonies.

B. prod. V, 50 violet red colonies, 5 white ones.

B. prod. VI, all red.

B. prod. VII, 683 colonies, some spreading, all vermilion.

B. prod. VIII, 26 smooth violet red colonies, 1 white.

There are, however, exceptions to the good results of rejuve-
nation, unless plating and careful selection of colonies is a part of
the process. It sometimes occurs that an old culture on neutral agar
which has stood in the stock case two or three months will give a
brillant pigment on the first transference to neutral agar again, while
successive inoculations will seem to diminish the pigment production.
The first vigor may be the result of natural selection, but I have
no explanation to offer for the later deterioriation in this case.

Light colored or colorless colonies which appear as discontinuous
variations upon plates often give rise to apparently constant varie-
ties. Such a colony of B. ruber miquel produced a luxuriant
white agar streak, which showed only a few pin-point dots of red.
This was allowed to grow for several weeks, and the next and
several future transfers gave pure white cultures with all the other
characters of B. ruber miquel. Davis 1 ) notes "sports" of B. r o s a -
ceus metalloides which gave rise to dark and to light colored
varieties.

Among my series several instances of discontinuous variation in
mass cultures have appeared. A "crusty" variety of B. amylo-
ruber occurred, as in the case of Dyar's B. lactis erythro-
genes, after a summer's storage. The original culture had not
become contaminated, the pigment of the wrinkled crusty culture was
identical in violet red color and rapidity of development with that

1) Davis, N. F. (Science, Vol. XIII. 1901. p. 324.)

of the original, and all the other characters were true. Upon plating,
some colonies gave rise to the original soft smooth growth ; a series
of cultures made after exposing the variety for varying lengths^ of
time to the sunlight also showed one tube like the original, a tube
made after 30 minutes' exposure. This result is explicable on the
theory of the selective action of sunlight, as noted below.

Variations in colony contour, noted in the case of B. ruber
indicus, B. rutilus, B. fuchsinus, B. amyloruber, and
rarely for B. kiliensis, where proteus-like and round surface
colonies appeared on the same plate, are to be explained partly
through physical conditions of the media, and partly by spontaneous
variations which arise in the viscidity of the capsular envelope or
in the motility of the organism. Agar streaks from the proteus
colonies were sometimes slightly more spreading, but the next plate
might show total reversion to the round type.

B. plymoutheusis is recorded in the original description as
differing from B. prodigiosus in marked viscosity on agar and
potato cultures. Dyar (loc. cit.) uses viscidity of B. plymouthen-
s i s to distinguish it from B. prodigiosus and B. rosaceus
metalloides. My culture also differed in producing gas in lactose
and sucrose bouillon and in standard asparagin dextrose solution,
as well as in a strong fecal odor. During a two years' obser-
vation of this culture, viscosity seemed as constant a character of
B. plymouthensis as any other of these differences. But the
next year, upon revival of the cultures after two months' summer
storage, B. prodigiosus I was found to evince the agar culture
viscosity of B. plymouthensis. Contamination naturally suggested
itself as first explanation, but plating and examination showed the
prodigiosus culture to be true in all other respects as noted
above, and it was necessary to ascribe the viscosity to a variation
which had arisen in the old summer culture and become dominant
accidentally in the first plating. Three different cultures now show
this peculiarity, the third, B. prodigiosus VIII, presenting it when
received at the laboratory. These observations make it necessary
to drop the character of viscosity or capsule formation as differen-
tial for B. plymouthensis.

In general, what we are in the habit of regarding as important
biological characters are not subject to sudden or spontaneous vari-
ation; i. e M the power of liquefying gelatin, of producing gas, or
of coagulating milk, does not appear or disappear abruptly with no
apparent cause. As has been observed in varieties appearing in the
same culture of B. coli 1 ), the variants are chiefly due to a
morphological change, such as the production of more or less of the
capsular substance upon which often depends the configuration of
surface colonies, or to change in an easily disturbed physiological
character such as excretion of pigment.

1) Smith, T., and Reagh, A. L. The agglutination affinities of rela-
ted bacteria parasitic in different hosts. (Journ. ofMed. Research, Vol. IX. 1903,
p. 270.)

3. Range of normal variation.

a) Growth and pigment on ordinary culture media.

Morphology.

All the cultures of the Prodigios us group, except B. kilien-
sis and B. ruber miquel, are small, actively motile bacilli.
B. kiliensis is distinctly larger than B. prodigiosus, while
B. ruber miquel is larger still and non-motile. None have spores.
A gelatinous capsule is often present in B. prodigiosus and
B. ruber plymouthensis. All of these organisms tend to be
somewhat larger on solid media, especially on potato.

B. prodigiosus and B. kiliensis showed peritrichial flagella ;
the others were not examined for flagella.

Cultural features.

Gelatin. Plate colonies vary but slightly beyond the diffe-
rences in appearance due to variation in the viscosity of the medium.
In general the colonies are all like those described for B. pro-
digiosus, with slight variations as to time and manner in which
pigment and liquefaction appear. B. ruber miquel only, does not
liquefy gelatin. The same variations in time and degree of lique-
faction are seen in gelatin tube cultures, even in parallel inocu-
lations made at the same time, into the same lot of gelatin and
from the same culture. This is, however, not an unusual variation
in a series of tubes inoculated from one colony, and is probably due
in part, as shown by W hippie 1 ), to slight physical disturbances
in the action of the proteolytic enzyme.

A gar. Variation in the agar colonies, principally in contour
and in coloration, is due to the same causes as in the gelatin colo-
nies. Proteus- like colonies often occur, i.e., in B. ruber indicus,
B. rutilus, B. fuchsinus, and rarely in B. kiliensis, but the
usual Prodigiosus form is round. Pigment usually appears in
granular masses throughout the colony, but often the colonies show
fine concentric rings of pigmentation, with darker or lighter centres.
My attempts to make accurate colored reproductions of these proved
futile, since they are quite inconstant. One plate of B. prodigio-
sus IV may be normally and uniformly pigmented; on another all
colonies may become red from the edges inward, leaving at first a
white centre; other cultures may have violet red centres and white
edges. It is noticeable in such variations that they do not occur
on the same plate, i. e., they are regular responsive, and not sport
variations.

Potato. The tendency to consider potato as a rejuvenating
medium for B. prodigiosus is borne out by the vigorous growth
and pigmentation of B. prodigiosus I VIII on this medium,
cf. also B. kiliensis, rutilus, amyloruber, and ruber
miquel. For B. ruber indicus, B. plymouthensis, and
B. miniaceus, however, potato cultures were unsatisfactory for
chromogenesis.

Bouillon. Dense turbidity is always produced in bouillon by

1) W hippie, G. C., On the physical properties of gelatine, etc. (Techno-
logy Quarterly. Vol. XV. 1903. p. 159.)

** f> -

all of these cultures, but great variation is shown in the amount of
pigment elaborated and in the formation of pellicle. The amount of
sugar present in the medium has an effect upon pigment production,
as noted in the descriptions of the separate cultures; the reaction
of the bouillon is also a feature, more pigment being produced ir.
bouillon of slightly acid reaction. Usually in sugar free neutral
bouillon B. prodigiosus I IV, VI, VII, and B. ruber miquel
show slight coloration of the liquid and a red surface ring, but
little true pellicle ; B. k i 1 i e n s i s and B. prodigiosus V give thick
orange red surface membranes, while B. plymouthensis,B. raini-
aceus, and B. prodigiosus VIII show color only in a pink or
violet surface ring, and B. ruber indicus usually lacks pigment
entirely in ordinary meat bouillon but produces it in abundance iu
a peptone solution. On the other hand B. rutilus and B. amylo-
ruber color the entire liquid deep red, in observing which fact
we may remember their recent isolation.

Milk. All except B. amylo ruber and B. ruber miquel
acidify milk in 24 hours and coagulate it in from 24 (B. rutilus)
to 72 hours at room temperature. Some of the cultures show pep-
tonization of the casein. B. amyloruber does not coagulate, but
precipitates the casein, while B. ruber miquel produces no change
in milk except that of red pigmentation. Almost no variation is seen
in these reactions in milk.

Gas production. The appended table shows that great vari-
ation is evinced in this respect, not only in the group, but in the
same organism at different times, with different stocks of bouillon.
The table gives only the limits of numerous determinations made
with neutral 1,5 /o dextrose bouillon.

Table II.

Dex

Per cent,
of Gas

trose

Per cent.
ofC0 2

Lac

Per cent,
of Gas

:tose

Per cent.
ofC0 2

Sacci

Per cent,
of Gas

larose

Per cent.
ofCO 2

B. prodigiosus I, II, III

034,5

100

10-20

VII

1020

VIII

B. ruber indicus I, II

30-70

100

2025

100

B. kiliensis (B. r. bait.)

30-40

26-28

B. plymouthensis I, II

7078

38-42

7072

2530

7075

B. miniaceuB I, II, III

4000

40-05

36-40

3557

30-53

2070

B. rutilus (n. sp.)

2891

100-65

6-88

100-60

B. ruber miquel

3446

7779

B. amvloruber (n. sp)

___

and B. fuchsinus

It is to be remarked that, except in the case of B. rutilus,
th relation of C0 2 to the total gas produced remains fairly con-
stant for each organism. B. rutilus produced 6065 %> C0 2 at
first, but with gradual loss, after isolation, of the power to produce

a large amount of gas, it seemed also to lose the power to form
anything but C0 2 . B. prodigiosus I usually failed to produce
gas in 1/ dextrose, 1,5 2 | being more favorable.

Oxygen and temperature relations. All of this group
are facultative anaerobes, but grow without pigment in the absence
of oxygen. Only B. ki lien sis and B. ruber indicus produce
pigment at 37 C, although all are able to grow at that temperature.

Indol production, nitrate reduction, odor. No
indol is formed by the members of this group. Nitrate is reduced
to nitrite in each case and often to free gas. The trimethylamine
odor is often present in cultures of the Prodigiosus group, and a
strong fecal odor is characteristic ofB. plymouthensis, B. pro-
digiosus V, and VIII.

B. ruber indicus only is said to be pathogenic for labora-
tory animals. 2 ccm of a 48 hour bouillon culture inoculated intra-
peritoneally killed a mouse in 48 hours. Unfortunately I was unable
to make an autopsy. 20 ccm of a 48 hour agar suspension failed
to produce any effect upon a guinea pig 1 ).

b) Growth and pigment on special solid media.

The earlier investigators of red chromogenic bacteria, Ehren-
berg (26), Fresenius (27), and Cohn (30), concerned them-
selves chiefly with the systematic position of B. prodigiosus; Erd-
mann (28), and Schroeter (29) worked with the chemical nature
of the pigment; Schottelius (35) was the first to pursue ecolo-
gical studies upon the pigment production, without, however, much
attention to the composition of the cultural media. His conclusions
as to the conditions necessary for pigmentation were 1) a sufficient
supply of atmospheric air, 2) a suitable temperature. Wasser-
zug (37) experimented upon the effect of alkaline and of acid media,
obtaining colorless races in alkaline bouillon. K uebler (38) repeated
Wasserzug's procedure , but contradicted him and confirmed
Schottelius in asserting the non-permanency of white cultures
obtained by high temperature and alkaline media.

The next important paper on the subject was that of Gale-
otti (15), who studied eight chromogenic organisms, among them
B. prodigiosus, L us tig's "red bacillus", and B. lactis ery-
throgenes. He found that B. prodigiosus gave less pigment
in liquid media than in solid, but that this was not due, as Wasser-
zug had thought, to lack of oxygen, since an atmosphere of pure
oxygen produced no better chromogenesis. He decreased the amount
of peptone in the agar, and inferred that a scarcity of proteid did
not prevent pigment production. B. prodigiosus was the only
organism of his series in which pigment production could be impeded

1) Subcutaneous inoculation of 2 ccm. 48 hr. agar slant culture, suspended
in 5ccm 0,85 % Nacl solution, causes illness in rabbits. A large abscess forms
at the site of inoculation, from which a vigorous and pigmentforming culture of
B. ruber indicus was isolated at the end of two weeks. Abscesses are also
formed by B. prodigiosus 1 and VII, B. rutilus (n. sp.) , and B. amylo-
ruber (n. sp.). Further investigations upon the pathogenicity as well as upon
the agglutinative properties of these organisms are in progress.

by a high temperature without interfering with the luxuriance of
development. White light had a limiting, red light very little effect
upon the pigment; lack of oxygen and also pure oxygen were both
detrimental 1 ). Galeotti thus concluded 1) that the power of
chroinogenesis in bacteria is not connected indissolubly with the life
of those bacteria; such microorganisms may be able to live without
producing their characteristic pigment. 2) that the conditions of life
which affect the chromogenic power are generally those which have
an unfavorable influence upon the bacteria themselves in all their
functions. 3) that, given conditions unfavorable to the production
of pigment by any special chroraogenic microorganism, that organism
will, in a longer or shorter period of time, reacquire the power of
pigment production by adapting itself to the unfavorable conditions.

G a 1 e o 1 1 i ' s first conclusion is supported by most investigators
of bacterial chromogenesis, and by all the observations which are here
cited as to the ease with which the power of pigment production is
lost by some microorganisms. His second and third generalizations
are, however, debatable.

Noesske (18) says, speaking of B. pyocyaneus and
B. prodigiosus, "Nicht trotz, sondern infolge zu uppiger
Vegetationsbedingungen sistiert mauchmal die Farbstoffbildung auf
unserer gebrauchlichen Bouillon." Noesske is supported by
Wool ley (21), who concludes that B. pyocyaneus, B. jan-
thinus, and B. prodigiosus show better development but less
pigment in sugar media as compared with sugar free media; pig-
ment is produced more easily in 1 / than in 2 / sugar media,
with the exception of B. prodigiosus, which is alike in both
cases, but better in glucose than in lactose or saccharose.

The presence of sugar in nutritive media was, according to
Wasserzug, detrimental to the pigment production of B. pro-
digiosus; but Laurent (57) found that the influence of sugar
could be counteracted by the addition of alkali, i. e., that the in-
jurious effect was only indirect, through the acid formed from the
sugar by the bacteria.

Although no very definite conclusions can be drawn from media
containing so many unknown elements as our ordinary bouillon or
agar, a few preliminary experiments were made with agar as to the
effect following on the elimination of some of its constituents. Cul-
tures which had been 4 months on neutral sugar -free agar were
transferred to similar fresh agar slant tubes. After rejuvenation by
the bouillon, gelatin, and agar plate method, sugar -free and 1

1) cf. Pfeffer, W. Ber. der Kgl. Sachs. Gesellsch. d. Wissensch. Leipzig,
math.-phys. Klasse. 1896, p. 379, re Baumgartens Jahresb. 1896, p. 705.
,,Farbstoffbildende Bakterien verraogen den Sauerstoff locker zu binden (ahnlich
wie das Hamoglobin) und ihn im sauerstofffreien Raum wieder abzugeben. Der
Trager dieser Erscheinung ist der Farbstoff, der die gleiche Wirkung auch iso-
liert im alkoholischen Extrakt zeigt, wahrend bei farblosen Bakterien em Gleiches
noch nie beobachtet ist. Die Farbstoffbildung, die bisher mehr als eine Luxus-
produktion erscheint, erscheint hiernach vieljeicht in einem fiir die Art un-
gleich zweckmassigeren Sinne, indem sie vielleicht die Bedeutung hat, dem betr.
Bakterium eine stete bereite Sauerstoffreserve zu sichern."

31 -

dextrose agar slant tubes were inoculated from the same colonies,
with the following results:

Table III.

5 days,

Rejuvenated

Sugar-free
neutral

24 hrs.

5 days

agar, after 4
months on

Sugar-
free

Glucose

Sugar-
tree

Glucose

neutr. agar

agar

Gr.

Pigm.

Gr.

Pigm.

Gr.

Pigm.

Gr.

Pigm.

Gr.

Pigm.

B. prodigiosus I

lux.

red

lux.' deep

lux.

pink,

lux.

red

lux.

red and

pink

trace

white

B. ruber indicus I

pins

pink,

pink and

trace

white

B. II

pink,

pink and

trace

white

B. ruber balticus

orange

1 7J

pink,

orange

red at

red

trace

red

top 1 )

B. ruber plym. I

pink

red,

thin

red,

violet red,

trace

luster,

trace

lustre

B. ruber miquel

red

thin

orange
red

pink

orange

violet red

B. rutilus (n. sp.)

pink

red

red and

white

B. amyloruber

wrink-

violet

deep

violet

dark

(n. sp.)

led

red

pink

red

violet red

B. plym. II

lux.

pink

red,

red, luster

luster

B. kiliensis

red,

fair

red, trace

trace

B. miniaceus

pink

red,

thin

pink,

red, luster

trace

luster

trace

A comparison of the five-day sugar- free agar growth shows
B. plymouthensisl and B. rutilus' to have been apparently
greatly benefited by the rejuvenating process, B. kiliensis and
B. plymouthensis II as adversely affected, and the others as
not affected at all. This may be due in part to the fact that no
particular attempt was made to wait for colonies showing the
highest pigmentation, but only to inoculate both tubes from the
same colony.

When we compare the results of 24 hours for the two agars,
we find some interesting differences; the cultures are divisible at
once into two classes. B. prodigiosus I, B. ruber indicus I
and II, B. ruber balticus, B. ruber miquel, B. rutilus, and
B. amyloruber, all show more pigment on sugar free agar. With
the exception of B. ruber miquel, which was thin on sugar free
agar, the development was of about the same degree of luxuriance
on all. Three other cultures , B. plymouthensisl and II and
B. miniaceus, gave a surprising result in comparison with the others,
maximum pigmentation with green luster upon the sugar medium,

1) Later, more luxuriant and darker pigment on glucose agar.

and very little or none at all upon the sugar free agar. B. kill en sis
is also of interest; it is a degenerate culture of feeble pigmentation,
and still shows a lingering tendency to return to normal if oppor-
tunity affords, i. e., upon a fresh transfer from an old culture, or
upon transference to a new (here a sugar) medium.

The five day growth presents the same differences, though less
distincfly. The mosaic appearance of B. ruber indicus and
B. rutilus is a beautiful expression of the existence of some pig-
ment-producing individuals among the mass of those not producing
pigment. This seems to argue that, here on solid media at least,
the presence of sugar has the effect not of modifying the pigment,
but of either permitting it unmodified or of inhibiting it. That the
acid formed from the sugar is the inhibiting influence may be ques-
tioned because of the early appearance of the differentiation. The
effect of acid directly upon the pigment may explain the more violet
red color of B. ruber miquel after five days, and the darker
color of B. a m y 1 o r u b e r. The 'late and luxuriant appearance of pig-
ment inB. ruberbalticuson sugar agar is a peculiar result entirely
inexplicable on the acid theory. It is to be noted that the three
cultures which produce early luxuriant pigment on sugar agar are
among those, of the whole series, which produce the most active fer-
mentation of sugars in the fermentation tube.

It seemed possible to obtain some light upon the question of
growth-luxuriance and pigment-luxuriance by eliminating the bouillon
from the ordinary agar medium. Accordingly a series was grown
upon agar 1,5 %, peptone 1 | , and water. This medium was first
tried of different reactions, 1,5 | acid, neutral, and 1,5 | alkaline
to phenolphthalein. The same medium, neutral, was used with the
addition severally of 1 | pure dextrose, lactose, and saccharose.
Two differences between the "meat-free" medium and the ordinary
nutrient agar were noticeable, though hardly measurable. The growth
is more limited, i. e., less spreading and "massy", and the pigment
is in general more intense in the former. Some of this depends, without
doubt, upon the vigor of the culture, a particularly vigorous strain being
able to overcome slight differences in the media, expressed, in feeble
strains like B. kiliensis, by a series showing distinct gradation.

(See Table IV p. 33.)

This 48 hour table brings out several points :

1) The tendency to violet red pigment on the more acid, to
orange red on the more alkaline media.

2) The similarity of pigment color on dextrose and saccharose
agar to that on sugar free agar of acid reaction. That is, acid is
probably formed from assimilation of these two sugars.

3) The similarity of pigment color on lactose agar to that formed
on sugar free neutral agar; i. e., lactose is probably not easily
assimilated.

This and the last named point seem to afford evidence of the
activity of sucrase but not of lactase.

33
Table IV.

48 hours.

1,5
Gr.

Agar 1
Pigm.

r\ 01
.,<J /

Gr.

Pept<

Pigm.

Dne 1
1,5
Gr.

Pigm

Agar 1
1 L Dex.
Gr.|Pigra.

,5 %, Peptone 1
1 % Lact. |1 %
Gr. |Pigm. Gr.

Sacch.
Pigm.

B. prod. I

+ +

violet

_l_

violet

si.

violet

red

violet

red

B. II

+ +

violet

si.

+ +

violet

orang

violet

red

B. III

violet
red

si.

violet
red

r r| g

violet
red

B. IV

+ +

red

+ +

ver-

_J__I_

ver-

+ +

dalrk

+ +

orang

+ +

dark

+ +

milion

red

re?

red

+ +

luster

B. V

+ +

red

si.

violet
red

orang

violet
red

B. VI

+ +

violet

red

violet

+ +

orang

+ +

dark

red

violet

luster

+ +

red

lustre

B. VII

+ +

ver-

+ +

ver-

+ +

ver-

+ +

violet

+ +

orang

_!__!_

violet

milion

red

+ +

lustre

+ +

lustre

B. VIII

_l-

violet

_j_

violet

_^.

violet

_l_

violet

red

B. r. ind. I

orange
red

dry

orange
red

dry

orange
red

dark
red

dry

orange

dry

orange
red

B. II

+ +

orange

pale

dark

orange

dry

red

dry

red

dry

orange

red

j
red

red

-j- 4.

_(-

red

4. 4.

_l_ 4.

luster

B. r. bait.

f -f

orange

+ +

violet

orange

dark

red

+ +

luster

+ +

B. r. miquel

red

sl.

dark

red

B. r. ply. I

sl.

si.

+ +

dark

pink

+ +

dark

red

duster

luster

B. rutilus

[-4-

violet

red

si.

orange

_l_

orange

_}_

violet

red

B. amylo-
ruber

violet
red

red

orange

si.

pale
violet

violet
red

_j__|_

si.

_j_

_i_i_

B. ply. II

[- +

-|-

.4.

dots

+ +

r +

dots

red

B. kiliensis

deep
pink

pale
pink

+ +

dark
red

+ +

pink

- 34

4) The orange red color of B. ruber iudicus I and II on
acid agar is probably due to alkali-production. Query: - - Is the
large amount of acid formed byB. ruber indicus in bouillon and
on ordinary meat agar, as evinced by its better growth on alkaline
meat agar. a result of the meat albumins?

5) The range of color in B. amyloruber, as follows:

Red Orange Blue

1,5 / acid 87 10 3

Saccharose 70 20 10

Dextrose 50 30 20

Neutral and Lactose 65 15 20

1,5 % alkaline 45 55

6) the inhibition of pigment by dextrose in the case of B. ru-
tilus, although the saccharose tube shows evidence of assimilation,
i. e., is like 1,5 / acid.

After five days' growth some of these cultures were slightly less
characteristic, as would naturally result from the constantly increasing
complexity of the metabolic products. For example, B. pro-
digiosus IV had become vermilion on 1,5 % acid agar, no doubt
as a result of the alkali produced by the vigorously growing culture.
Other of the more slowly developing cultures bore out the results
of the first 48 hours, e. g., B. prodigiosus I IV had produced
orange red pigment on 1,5 alkaline agar, and B. plymouthen-
sis I slight violet red color on acid agar. Metallic green luster
had appeared for several cultures, noticeably for B. ruber balti-
cus on 1,5 % acid agar, and for B. plymouthensis I and II
on dextrose and on saccharose agar. Orange red color had
changed to red on lactose agar for B. prodigiosus V and for
B. rutilus, but excepting these and the colorless lactose agar cul-
tures of B. ruber miquel and B. plym. II, the orange tone still
held for the series on this medium, i. e., the alkali produced by the
bacteria was not neutralized by acid formed from the sugar. It
would seem that since B. ruber balticus, B. plymouthensis I,
and B. miniaceus ferment lactose with gas production, these cul-
tures would show a decrease of the orange tone.

Although the relative results arrived at on the above media are
of some value, their absolute value is lessened because of the
unknown factors present in agar and peptone. Agar itself contains
a certain amount of carbohydrate, galactose (Bauer, Jour. Prakt.
Chemie, B. XXX. p. 367), which is probably assimilable by the organisms.
A further reduction of the medium, by leaving out the peptone,
resulted in very feeble white growth in five days. Three cultures
only showed a trace of pigment, B. ruber indicus II,
B. rutilus, and B. prodigiosus VII. The last named showed
a green iridescence on the thin pink layer.

In the endeavor to do away with agar and still have a solid
medium for pigment production, the following mixtures were employed,
and gave interesting comparative results. The flour and starch were
cooked, poured into Petri dishes, sterilized by discontinuous method,
and inoculated.

- 35

Table V.

Eice flour 35 %,

Starch 10 %,

Starch 10/ ,

Pept. 1 /.,

Pept. 1 %,

water

Gr.

Pigm.

Gr.

Pigm.

Gr.

Pigm.

B. prodigiosus I

good

red

good

red

slight

slight,

pink

B. ruber balticus

fair

dark red

luxuriant

intense

thin

pink

B. kiliensis

good

red

spreading
good

red luster
red

spreading

pink

B. ruber miquel

fair

red

luxuriant

violet red

slight

red

B. rutilus (n. sp.)

good

dark red

cream

white

B. amyloruber

good

violet red

luxuriant

intense

spreading

pink

(n. sp.)

spreading

violet red

luster

Rice flour, which gave luxuriant growth when used with bouillon
by Schneider (19) and P e t r o w (58), gives good pigment, but rather
a thin dry layer, when bouillon is omitted.

The similarity between B. ruber balticus and B. kiliensis
was brought out by their development on starch and water. On
the other hand B. prodigiosus, B. ruber miquel, and
B. rutilus scarcely developed at all on cooked starch, but with the
addition of 1 % peptone gave a luxuriant growth. B. rutilus was
peculiar here in its absence of pigment, while B. amyloruber for
the first time produced, in addition to its usual vivid dark red color,
a distinct green metallic luster. The development of this organism
was evidently at its height of luxuriance on the starch-peptone
medium. Considerable liquefaction of the solid starch also took
place, an evidence of the production of a diastatic ferment by
B. amyloruber. After two weeks the semi-solid mass on this
plate was rubbed up in distilled water, filtered germ free and tested.
12 ccm peptonized 5 ccm of 10 % gelatin and water in two hours
at 37 C. A half inch cube of 10 % cooked starch was not liquefied
by the filtrate in 24 hours at 37 C, and I am unable to adduce
any evidence, other than that of the first observation, for the presence
of diastase. Fermi (22), and G o r i n i (41) report negative results as
regards diastatic ferments from B. prodigiosus and "B. ruber".

c. Growth and pigment in non-proteid media.

The value of the above experiments as to the affect of sugars
could only be tested by their repetition with non-proteid media,
where all the elements which go to make up the nutritive supply of
the organisms are definitely known. The only investigations of this
sort hitherto undertaken for red chromogenic forms have been upon
B. prodigiosus and B. kiliensis. Those upon the latter orga-
nism, by Laurent (57), were carried out mainly to test the effect
of acid in the medium, and led Laurent to the conclusion that an
alkaline reaction was most favorable to growth and pigmentation. But

since in every case Laurent used media which contained either
saccharose or glycerine, and in which the initial alkaline reaction
had only the effect of neutralizing the acid produced by the organism
from these substances, his results prove nothing definite as to the
reaction best for non-fermentable media. As, also, the results of
Kuntze (46), Noesske (18), Luckhardt (16), and Sulli-
yan (20) upon B. prodigiosus in non-proteid media have dis-
agreed, the following tables of experiments on the Prodigiosus
series will be of interest 1 ).

Table VI.

Sol. A.

Sol. B.

Sol. C.

Sol. D.

Sol. E.

Sol. F.

Asp.

0,2 /

Asp. 0,2 o/o
MgSO 0,lo/o
KjHP0 4 0,lo/

Asp.
0,2 o/o
KHP0 4

0,1 o/o

Asp.

0,2%
MgS0 4

Asp.
0,2%

Asp. 1,0 /
MgSO, 0,1 %
<HP0 4 0,lo/

K ^

Glycerine

2,0 %

Or. Pigm.

Pigm.

Or.

Pigm.

Gr. |PIgm.

Or.

Pigm.

Gr. |pigm.

B. prod. I

days

-j- -}-

-f

~H~i~

5 da.

~\~ ~\~

tr.

B. II

4.4.

-f

+ +

5 da.

_j__j_

tr.

B. III

+ +

5 da.

+ 4-

tr.

B. IV

+ -|_

+ +

B. V

_|__{-

+ +

_j_ i

B. VI

_!__{_

_^_

B. VII

+ +

48 hrs.

+ +

48 hrs.

+ +

3da.+ +

f
3da.+ +

B. VIII

+ +

6 da.

+ +

tr.

B. rub. ind. I

48 hrs.

+ +

+tr.

+ +

3da.+ +

B. 11

48 hrs.

-f +

+ 4-

+tr.

_}._}-

48 hrs.

+ +

~T~

3da.+ +

B. rub. bait.

-f

+ +

3 da.

+ +

B. plym.I
B. .. miquel

+ +

-r

+sl.

+_ +

-f si.

+ +

si.

B. rutil. (n. sp.)

-j_

_|-

-j-gl.

_l__i_

B. amylo-
ruber (n. sp.j
B. plym. II

+ +

15 days

-r

+8l.

^-~

si.

15 days

1) The water used in these solutions was redistilled in glass. The inocu-
lations were made in flasks of Jena glass previously cleaned in acid and rinsed
repeatedly in distilled water. The reaction was neutral to phenolphthalein.

37 \ '

These series bring out interesting results in the behavior of the
cultures. In standard aspafagin solution (Sol. A), B. prodigiosus I,
tested five times, and B. prod. II VI and VIII, tested twice, grew
luxuriantly, i. e., showed dense white cloudiness, but showed no trace
of pigment. B. ruber balticus, B. plymouthensis I and
II and B. r u t i 1 u s gave the same results , except that B.
rutilus developed less cloudiness. On the other hand B. prod.
VII, B. ruber indicus I and II, B. ruber miquel, and
B. amyloruber produced a good red coloration of the medium,
but none grew luxuriantly, except B. ruber miquel and B.
prod. VII.

In the next series (Sol. B), MgS0 4 was eliminated from the
medium in an attempt to determine whether this substance be neces-
sary for the elaboration of red bacterial pigment, as has been shown
for the fluorescent pigment. Nearly all the cultures developed,
although less readily than in Sol. A; three of them, B. ruber
indicus I and II, and B. prodigiosus VII, although showing
scarcely any cloudiness, colored the solution a beautiful red in
48 hours. With these three cultures which gave pigment in the
absence of MgSO 4 further tests were made. A pure 0,2 / solution
of asparagin was prepared, and flasks were inoculated by touching
the surface of an agar plate colony with a fine needle, or from a
growth in Sol. B. In each case B. ruber indicus I and II produced
no distinguishable cloudiness of the solution, but slowly and gradually
colored it as deep a red as they did the standard solution. B. pro-
digiosus VII failed to show pigment here, and control cultures of
B. ruber balticus and B. ruber miquel also remained perfectly
clear and colorless.

These results point toward one of two conclusions. Either the
pigment of some cultures of what is here designated as the Prod i-
giosus group has a different chemical basis from that of others of
the group, or on the other hand, great variation occurs among these
cultures in their ability to elaborate the same pigment out of the
same synthetic material. The results of chemical and spectroscopic
analysis of the pigments of B. prodigiosus, B. ruber balticus,
and B. ruber indicus (19), (45), (49) give us no reason to believe that
they are essentially different. Further, the fact that some cultures
do not produce pigment in a 0,2% asparagin solution even in the
presence of MgS0 4 , while others beside B. ruber indicus have
this power, indicates a continuous, rather than a discontinuous variation
of the ability to elaborate pigment. Whatever the cause of this may
be, it appears from my results to the present time that the different
strains or varieties are very constant in their ability or non-ability
to form pigment in the above solutions, whether the latter be in-
oculated from young or old cultures on various media.

In his work on B. prodigiosus, Kuntze used 12 %
asparagin and 0,1 0,2 / K 2 HP0 4 , obtaining pigment if MgS0 4 were
added in smallest crystals. In order to determine whether lack of
pigmentation in my series was due to an insuffiency of the organic
compound, I increased the asparagin content to 1 %. The first trial

added B. ruber balticus only to the list of color producers. A
second set of flasks was inoculated from five day potato cultures,
with the result that B. prod. I III developed a slight trace of
pigment. Growth was most luxuriant for all the cultures in this
solution, but on the other hand B. ruber indicus I lost its power
of producing color by the concentration of the medium.

Standard asparagin solution with the addition of 2,0 / glycerine
allowed luxuriant growth, but of the whole series B. plymouthensisl
only showed a slight trace of pigment.

It seems evident from these experiments that the conclusions
drawn by Kuntze and Noesske regarding the necessity of MgS0 4
for pigment formation, and of phosphorus for growth, in the case of
B. prodigiosus, do not hold for all of the various strains going
under this name. Out of eight strains three did not form pigment
even under these conditions, although all were cultures in the height
of vigor; one culture produced pigment in the absence of one, and
two strains of a closely related form in the absence of both the
above substances.

The more luxuriant development of some of the non-pigmented
cultures in comparison with the slighter growth of other colored ones,
notably B. ruber indicus, seems to confirm the statement made
by Noesske and quoted previously in this paper. Further light
upon the subject of correlation of chromogenesis and growth, as well
as upon the effect of the presence of carbohydrate in the nutritive
medium, was obtained by the following experiments.

Table VII.

Sol. fl.

Sol. I

Sol. K

Asp. 0,2 %
MgSO A 0,1 %

Dextrose l'o/

Asp. 0,2%
MgSO 4 0,1 %
K,HP0 4 0,1%
Lactose 1,0%

Asp. 0,2%
MgS0 4 0,1%
K 2 fiP0 4 0,1 %
Saccharose 1,0%

Gr.

Pigm.

Gr.

Pigm.

(ir.

Pigm.

B. prod. I.

4-4-

tr. 4 da.

4-4-

+ +

B. prod. II

4-4-

tr. 8 da.

4-4-

+ +

B. prod. Ill

.j_.|-

tr. 3 da.

_j_ _).

4- +

tr. 3 da.

B. prod. IV

+ +

4 da.

+ +

+ 4-

+ 15 da.

B. prod. V

+ +

+ tr. 3 da.

+ +

4- tr. 3 da.

tr. 15 da.

B. prod. VI

_l__j_

tr. 8 da.

+ +

4-4-

+ 8 da.

B. prod. VII

4-4-

+ tr. 4 da.

+ +

+ 3 da.

_l__j_

+ 4 da.

+ + 15 da

+ tr. 15 da.

+ +15 da.

B. prod. VIII

+ +

+ tr. 4 da

+ +

+ tr. 8 da.

B. rub. ind. I

_^._j_

+ 4-

_l__^.

B. rub. ind. II

_j__^_

+ 8 da.

_l__j_

B. rub. bait.

4-4-

+ 3 da.

+ +

4-4-

+ tr.

B. rub. pty. I

+ +

si.

_^_^_

B. rub. miquel

+ +

+ 3 da.

si.

+ 4 da.

+ +

+ + 8 da.

B. rutilus (n. sp.)

4. _|_

+ 8 da.

,|_

+ 4-

B. amyloruber (n. sp.)

+ 4-

B. plym. II

4-+

+ +

- 39 -

When this table is compared with Table VI the similarity of
results with Sol. E and Sol. H is at once noticeable, the presence
of dextrose producing much the same effect as the concentration of
the medium by increasing the asparagin content. \Vith the addition
of dextrose to a standard asparagin solution, growth is luxuriant in
all cases, but although there is an increase of pigment production
over Sol. A, where in most cases there was none at all, still the
amount of pigment for B. prod. I VI and VIII is at most only
a trace. On the other hand, B. prod. VII shows a development of
pigment, which at the end of fifteen days is as strong as in the
standard solution in three days; and B. ruber indicus I and II
show, as in Sol. E, more luxuriant growth with a lessening of pigment
production.

Comparing the effect of the different carbohydrates, we see that
saccharose behaves on the whole like dextrose, although in some
cases the pigment failed to appear in Sol. K, where it did appear
in Sol. H. Lactose gives quite different results. In fact, Sol. I con-
taining lactose behaves exactly like Sol. A with no carbohydrates,
i. e. pigment is produced with B. prod. VII, B. ruber indicus I
and II, B. ruber miquel and B. amyloruber only, these, with
the exception of B. prod. VII, showing less luxuriant growth than
the rest of the series. This seems an exceedingly interesting result,
and falls in line with the conclusion drawn from Table IV, where the
color of the pigment gave evidence of the peculiar lack of effect of
the presence of lactose.

It is difficult, even here, to arrive at any general conclusion in
regard to the relation of growth luxuriance and pigment luxuriance.
As has just been stated, the majority of the cultures showing pigment
in Sol. I have grown less luxuriantly than the others. The same
thing was true with Sol. A. Again, B. ruber indicus I and II
tended to lose entirely their power to form pigment when the growth
luxuriance was increased by concentrating the medium or by adding
glycerine or sugar.

These facts, taken in connection with that of less massy growth
and more vivid pigment on peptone agar as against the more com-
plex meat peptone agar, seem to argue in confirmation of Noesske's
and Wool ley's views. On the other hand, Sol. H, in which dextrose
induces luxuriant growth, shows pigment, though only traces of it, in
fourteen cases, against four cases in Sol. A without dextrose. It may
be that here the sugar contributes chemical or physiological aid to
pigment formation as well as to vegetative luxuriance. The contrary
effect of glycerine points to this conclusion, since here we have
luxuriant growth without pigment.

In this connection also, some of the atypical cultures which have
virtually lost the power of forming pigment are interesting. Both
B. fuchsinus and B. miniaceus III are among the most
vigorous strains of the series in rapidity and amount of development.
The latter and B. kiliensis, although colorless, have lost none
of their vigor in the fermentation of sugar to gas formation, or in the

liquefaction of gelatin or coagulation of milk. These facts do not
support, then, Galeotti's second conclusion, that the conditions of
life which affect the chromogenic power are generally those which
have an unfavorable influence upon the bacteria themselves in all their
functions.

d) The effect of light upon pigment production.

Following the methods of Buchner and of Marshall Ward,
Dieudonn6 (13) confirmed the simpler experiments ofGaleotti (15)
regarding the effect of light upon bacterial chromogenesis. He used
B. prodigiosus and B. fluorescens, and found that the direct
sunlight of March, July, and August hindered development in
half an hour, while 48 hours of exposure entirely prevented pigmen-
tation and trimethylamine production. B. prodigiosus also lique-
fied gelatin more feebly. Eleven hours of incandescent electric light
killed both organisms. According to Dieud onus's investigations,
these effects were produced by the light and not by the heat rays,
the violet and ultra violet rays being the destructive agents, and the
red and yellow rays having no effect. The injury is chiefly to the
germs themselves, the chemical change produced in the medium being
a very small factor. Similar results were obtained with B. c o 1 i ,
B. typhosus, and B. anthracis, confirming those of Ward, who,
however, limits the injurious effect to the violet end of the blue, the
actinic rays.

Beck and Schultz (14) criticised Dieudonn6's methods, and
observed no injury to the development of B. prodigiosus, B.
pyocyaneus etc., from exposure to colored light, though in some
cases there was an influence upon chromogenesis (2 3 days). Diffuse
daylight was beneficial, darkness sometimes proving injurious,
(Staphyl. pyog. aureus and B. fluorescens). Direct sunlight
(3 days) produced colorless cultures.

The results of Beck and Schultz do not seem very conclusive;
the non-effect of the colored light may have been due to the slight
intensity of the rays which passed through their light filters. In a
recent paper Oliver (17) used colored glass plates.

A simple comparative study as to the effect of direct sunlight
upon a series of my organisms gave the following results. One loop-
ful-inoculations were made upon slant agar from 18 hour bouillon
cultures which had been grown in the dark ; these were used as
controls. The cloudy bouillon cultures were then exposed to the
March sunlight (according toDieudonnd as effective as that of
July), and similar agar slant cultures inoculated at intervals. These
agar cultures were then grown in the dark and examined after 24 hours,
48 hours, and ten days. The results are appended.

Table VIII.

24 hours' growth after exposure to sun of

min.

5 min.

15 min.

30 min.

Gr.

Pigm.

Gr.

Pigm.

Gr.

Pigm.

Gr.

Pigm.

fe. prod. I
B. niberbalt.
3. ruber miq.

+ +

dotted

5. rutilus (n. sp.)
5. amylor. (n. sp.)
B. rosac. metali.

si.

+ -f

prod. I
5. niberbalt.
S. ruber miq.
J. rutilus (n. sp.)
5. amylor. (n. sp.)
5. rosac. metali.

+ +
+ 4-
+ +
4- +

48 hours' growth after exposure to sun

- red

_l_

pink

_l_

pink

- red

4- +

pink

-1-4-

4- pink

--red
- red

+ +

orange red
4- slight red

+ +

orange red
pink

- red

mge

+ + +

+ +red

*J+

+ 4-+ red

pink
pink
pink
pink
f red

10 days' growth after exposure to sun

J. prod. I

4- +

+ 4- red

thin

pink

thin

piuk

thin

pink

5. ruber bait.
3. ruber miq.

+ +

+ + red
+ + red

thin

pink

+ 4- red

thin

pink

+ red

thin

pink
+ dull red

5. rutilus (n. sp.)

_l__l_

+ + red

thin

pink

thin

pink

thin

pink

$. amylor. (n. sp.)

4.+

+ + dark red

-j--j-

4- + dark red

4--|-

+ 4- dark red

-1-4-

4- + dark rec

5. rosac. metali.

4-4-

+ + orange

+ +

+ 4- orange

-f

thin

pale

prod. I
ruber bait,
ruber miq.
rutilus (n. sp.)
amylor. (n. sp.)
rosac. metali.

48 hours after 2 hours exposure to sun.
Growth normal, pigment faint pink

thin, faint pink

very slight, slight

normal, faint pink

thinner normal

no development

From the tables it will be seen that the 24 hour cultures pre-
sented a curious developmental result. The cultures of B. ruber
balticus which had been made after 30 minutes' exposure to the
sun showed more development than the control, while the other
exposures exhibited a comparative decrease of effect, a 5 minutes'
exposure giving less growth than one of 15 minutes. Greater
development of a 15 minutes' exposure as compared with one of
5 minutes was also shown by B. prodigiosus, B. ruber miquel,
and B. amyloruber, so that the possibility of an accidental
mechanical error, e. g., the transference of a loopful of germs crowded
at one side of the tube by heliotaxis, was excluded from consideration.
The experiment was repeated for B. ruber balticus, B.
rutilus, and B. amyloruber with the same results.

A possible explanation of the phenomenon may be suggested.
Gotschlich (5) has remarked that brief exposure of a culture to
an injurious influence may react beneficially to the culture as a whole,
by cutting out the weaker organisms, and leaving only the u Aus-
nahmezellen" ; that is, that there may be selective death-rate. On this

supposition the first effect of the sunlight was the destruction of a
great number of the less resistant organisms, which accounts for the
slighter mass-development of the cultures after the 5 minute exposure.
For the remaining and more resistant cells we must then assume that
the actinic effect of a 15 minute exposure was stimulating, promo-
ting cell division, somewhat as exposure to increased osmotic pres-
sure 1 ) or to lack of oxygen, to heat, etc., induces it in unfertilized
parthogenetic eggs. As the eggs in these experiments must be
exposed only briefly and then returned to their normal environment
if maximal results are to be obtained, so with the bacterial cells.
The accelerating effect of the sunlight on growth does not seem to
be an enduring one, for later observations upon the same cultures
show that development is in reality permanently hindered; as much,
in the case of B. prodigiosus, B. ruber balticus, and B.
rutilus, by 5 minutes' exposure as by one of 30 minutes, and
nearly as much by 5 minutes as by one of 2 hours.

The pigmentation of B. amyloruber and B. ruber balticus
also showed, in the 48 hour agar culture, a distinctly better color
after the 15 minute exposure. The chromogenesis of the others of
the series was markedly decreased by a 5 minute exposure, and only
B. ruber miquel showed any recovery after ten days. The greater
resistance of B. amyloruber, which was as brilliantly pigmented
after two hours' exposure to the sun as at first, was probably due to
its recent isolation from river water.

No attempt was made to determine the further history of these
cultures.

4. Summary.

The experimental and comparative work done on these cul-
tures of pigment bacteria may be roughly summarized as follows:

1) Notwithstanding the occasional loss of power of pigment
production by a previously chromogenic organism, the character of
the pigment is markedly constant among red chromogenic bacteria.
By constancy is here understood the appearance of pigment of definite
color upon nutritive media of known composition and under defined
environmental conditions.

2) A collection of about forty red cultures selected at random
fell readily into four main groups, according to slight but constant
differences in the character of the pigment as determined by a
standard color scale.

3) Sports, or discontinuous variations, such as white or light
colored colonies on a plate, viscosity of growth etc. sometimes oc-
cur. In general however the socalled important biological characters
are not subject to discontinuous variation.

1) Loeb, J., Further Experiments on Artificial Parthenogenesis and the
Nature of the Process of Fertilization. (Amer. Jour, of Physiol. IV. 1900. p. 178.)
Matthews, A. P,, Some Ways of Causing Mitotic Division in Unfertilized
Arbacia Eggs. (Amer. Jour, of Physiol. IV. 1900. p. 343.)

4) Considerable nonnal variation in biological characters is
seen among the members of the Pro digios us group on ordinary
media. In many cases this does not exceed the variations shown
by a series of inoculations made from the same culture. It was
noticeable that although gas production varied for most of these
organisms, the ratio of C0 8 to the total gas remained comparatively
constant.

5) Within comparatively narrow limits the pigment color of the
Prodigiosus group may be altered by changing the composition
of the nutritive medium.

6) Also by variation in the composition of the nutritive medium,
cultures usually distinct from one another in pigment character can
be made to approximate, e. g. to change from orange to violet
red; or to assume pigment qualities previously foreign to them,
e. g. metallic luster. The former characters are regained upon
transference to the original medium.

7) There is a tendency to violet red pigment in more acid, to
orange red in more alkaline media.

8) Dextrose and saccharose in peptone agar medium favor
pigment formation much more than does lactose.

9) The ability or non-ability to produce pigment in non-proteid
media is a particularly constant character for each of the different
members of the Prodigiosus group; but strains which are
otherwise approximate in biological characters may differ in this
ability.

10) Two strains in the Prodigiosus group, viz. B. ruber
in die us I and II, differed from all the others in the ability to
produce pigment in pure asparagin solution, without MgS0 4 or
K 2 HP0 4 .

11) The effect upon pigment production of adding dextrose and
saccharose to a standard asparagin solution is similar to that of
concentrating the solution by increase of asparagin content. Here
again, lactose is shown to be without effect upon pigment.

12) There is probably little or no correlation between luxuriance
or vigor and the power of pigment formation. Hence pigment
production does not appear to be essential to the life-processes of
an organism.

I). Notes on Groups of Bed Chromosome Bacilli.

The division into groups of the pigment bacteria by means of
the color scale falls in closely with the division according to other
biological characters as shown by the species description table.
Aside from the differentiation made in the discussion of pigment
production above, the inter-relationship of the members of some
groups may be briefly described as follows:

I. The Prodigiosus group.

A. Gelatin liquefied.

1. No gas in dextrose, lactose, or saccharose.

B. prod. VIII, B. amyloruber, B. fuchsinus.

2. Gas in dextrose only.
B. prod. I, II, III.

3. Gas in dextrose and saccharose only.

B. prod. IV, VI, VII, B. ruber indicus I, II,
B. rutilus.

4. Gas in dextrose, lactose and saccharose.

B. prod. V, B. plymouth ensis, B. miniaceus, B.
kilien sis.

B. Gelatin not liquefied.

1. Gas in dextrose only.
B. ruber miquel.

n. The Lactis erythrogenes group.

The members of this group are characterized by the production
of soluble red pigment. The appended table shows also some inter-
mediary forms described by Dyar (loc. cit.).

Sol. red. pig.

Insol. pig.

Gel. liq.

Agar gr.

Milk

Gr. at

37V,

1)B. lactis ery. I

agar, gel. and milk

yellow

lux. soft

coag. alk.

2) II

n j>

pale yel.

3) B. ery. rugat.

Dyar

(Dyar)

yellow

folded

4) B. helvolus

Zimm.

+ slowly

lux. soft

5) B. granul.

Dyar g
6) B. rubefaciens

gel. and agar

pale yel.
yellowish

granular
smooth

coag. acid.

in gel.

7) B. lactorubef.

+ milk

white

8) B. rutilescens

gel. and agar

lux.

smooth

A culture from Krdl of B. ros e oflu or esc ens Tataroff,
which is said by Migula to be identical with B. lactis ery-
throgenes, was evidently atypical, showing thin white growth
and no pigment. It was non-motile, non-liquefying, and had no
effect upon milk.

III. The Rubricus group.

These cultures were of interest because they are typical forms
of a group of red chromogenic organisms quite different from the
Prodigiosus group. I have no doubt that the whole series of
small and non-motile, non-liquefying, slow growing red forms, i. e.

distinct from B. ruber miquel, are much more closely related
than the members of the P r o d i g i o s u s group , if they are not
all identical. This includes forms isolated and described by
Dyar, B. zeta, B. delta, B. ferruginous, B. salmoneus,
B. rhodocrous Overbeck, B. finitimus ruber, B. haema-
t o i d e s Wright etc. Some of these are recorded as liquefying
gelatin slowly or very slowly. The pigment ranged from salmon
pink and orange to red. Milk is either unchanged or alkaline.

I desire to express my grateful thanks to -Professor Edwin
O.Jordan, of the University of Chicago, under whose advice and
direction the work embodied in "this paper was carried out
(See Table IX p. 4647, Table X p. 4851.)

Bibliography 1 ).
General.

1) Chester, F. D., Manual of determinative bacteriology. New York 1901.

2) E is en berg, J., Bakteriologische Diagnostik. Hamburg und Leipzig 1891.

3) Fliigge, C., Die Mikroorganismen. Leipzig 1896.

4) Fraenkel, C., Grundrifi der Bakterien kunde. Berlin 1894.

5) Kolle, W. and Wassermann, A., Handbuch der pathogenen Mikro-
organismen. Jena 1902.

6) La far, F., Technical Mycology, transl. Salter. Vol. I. Lond9u 1898.

7) Lehmann, K. B. and Neumann, E. 0., Atlas and principles of bac-
teriology, ed. from the second German ed. by G. H. Weaver, Phila. and
London 1901.

8) Lustig, A., Diagnostik der Bakterien des Wassers. Jena 1893.

9) Mac, E., Traite pratique de bacte'riologie. Paris 1891.

10) Migula, W., System der Bakterien. Bd. II. Jena 1900.

11) Steinberg, G. M., Manual of bacteriology. New York 1892.

12) Zimmermann, O. E. E. , Die Bakterien unserer Trink- und Nutzwasser.
Chemnitz. I und II. 1890. III. 1900.

13) Dieudonne*, A., Beitrage zur Beurteilung der Einwirkung des Lichtes auf
Bakterien. (Arb. aus dem Kaiserl. Gesundheitsamte. Bd. IX. Wien 1894.
p. 405. (cf. full bibliography there given).

13a) , Beitrage zur Kenntnis der Anpassungsfahigkeit der Bakterien an ur-
spriinglich ungiinstige Temperaturverhaltnisse. Ib. 1894. p. 492. (with biblio-
graphy).

14) Beck, M. und Schultz, P., Ueber die Einwirkung sogenannten mono-
chromatischen Lichtes auf die Bakterienentwickelung. (Zeitschr. f. Hyg.
Bd. XXIII. 1896. p. 490.)

15) Galeotti, G. , Eicherche biologiche sopra alcuni batteri cromogeni. (Lo
Sperimentale. VoL XLVI. 1892. Fasc. 3. p. 261.)

16) Luckhardt, A. E., Ueber Variabilitat und Bedingungen der Farbstoff-
bildung bei Spaltpilzen. Diss. Freiburg 1901.

17) Oliver, C. A., An experimental study of the effects of change of color
upon pigment-bacteria, (Amer. Journ. Med. Sci. Vol. CXXIII. 1902. p. 647.)

18) Noesske, H., Versuche iiber die Bedingungen der Farbstoffbildung des
Bacillus pyocyaneus. (Beitr. z. klin. Chirurg. Bd. XVIII. Tubingen 1897.
p. 103.)

19) Schneider, P., Die Bedeutung der Bakterienfarbstoffe fur die Unter-
scheidung der Arten. Diss. Basel 1894. (Arb. d. bakt. Inst. Karlsruhe. 1895.
Abdr. im Centralbl. f. Bakt. Bd. XVI. p. 633.)

20) Sullivan, M. X., The chemistry of bacterial pigments. (Abstr. im CentralbL
f. Bakt, Bd. X. 1903. p. 386.)

1) Articles not seen by the writer are bracketed [ ], and the place from
which the reference was obtained is subjoined.

Bio

Morphology

Cultural Features

Name of Organism

Source

. .

Nutrient
broth tube

Nutrient
agar tube

Gelatine
plate

Gelatine
stab

Potato tube

Fermentatioa
tube

, &,*

2 a

gjM

i o3 o

i-s

J'-| | 5

-c

O QQ

3"^

1 >

o-l

B. prodigiosus I, II,

III

-f-

-(-

.:-

_(-

B. prodigiosus IV,
VI, Vft

B. prodigiosus V

-r

B. VIII

_j-

B. ruber indicus I, II

_l_

B. ruber plymouth-
ensis I, ft, III
B. kiliensis, B. ruber

balticus

B. miniaceus I, II, III

-1-

B. rutilus (n. sp.)

B. amyloruber (n. sp.)

B. fucnsinus

-r

B. ruber miquel

-r

B. rubricus (n. sp.)

B. rufus (n. sp.)

-t-

_l_

-r

B. ruber zimmermann

_l_

, __

B. havaniensis

B. lactis erythrogenes

I, II

B. rubefaciens

B. lactorubefaciens

;

B. rutilescens (n. sp.)

B. mycoides roseus
B. mycoides coral-

;

' r

linus (n. sp.)
B. latericeus (?)

4-
4-

-r

4-
4-

B. rubro pertinctus
B.rosaceus metalloides

-i-

-t-

4-
4-

B. mesentericus ru-

ber, 1-IV

-r

- 47 -

IX.

logy

Biochemical Features

Pathogenesis

SL : 3 : 1

> ~ is

= r c8

Liquefaction Gas production

Milk

Nutrient agar tubes

Mice

precip

lus

Lus rare
hts

lus
lus

+
+

sweet

red
red

- 48

Table

Bio

Morphology

Cultural Features

Nutrient
broth tube

Nutrient
agar tube

Gelatine
plate

Gelatine
stab

Potato tube

Fermentation
tube

Name of Organism

Source

"i :L

-g

-S
IS

-j~

13
Q

S *

B. rubidus (Eisenberg)
B. ruber aquatilis

--.

(Lustig)

water

-j-

_i_

_;_

B. sardinae (Du Bois

St. Se"vrin)

-r

B. carneus (Tils)

_l_

B. ruber Berolinensis

(Fraenkel)

water

_l_

_j_

_ L

_j.

B. sulfureus (Holz-

schewnikoff,

-f

B. subfulvus (Matzu-

schita). B. tuberi-

genus 4, (Gonner-

mann)

_j-

B. pneumonicus agi-
lis (Schon)

B. viguali (Matzu-

schita) (B. Vigual)

-f

_l_

_j_

_J_

_;_

_l_

red

B. liquefaciens com-

munis (Stern berg)

-f

B. pyocinnabareus

wrinkl.
red

(Ferchmin)

pus

-r

_i_

red

B. The've'nm

pus

B. ruber lactis (Conn)
B. rubiginosus (Keru)
B. tuberosus (Kern)

milk

B. rubescens (Zim-

mermann)

-\-

_i_

_j_

dry

B. velatus (Matzu-

schita)

red

B. colere laterum

water

B. exiguus (Wright)
B. haematoides

.^_

(Wright)

B. epsilon (Dyar)

air

B. zeta (Dyar)

air

B. delta (Dyar)

water

B. ferruginous (Dyar)
B. finii inm-i ruber

air

-i

(Dyar;

_l_

B. salmoneus (Dyar)

- 49 -

logy

Biochemical Features

Pathogenesis

Liquefaction

Gas prodaction

Milk

Nutrient agar tube

! Mice

a o

1
1

li
1

1 Gelatine

-I-

brick

-f-

red

_i_

_j_

red

pink
yellow

red

si.

;

pink

red

pink

brick

reddish-

pink

_i_

salmon

Sl.

50 -

Table X

Bio

Morphology

Cultural Features

Name of Organism

Source

Nutrient
hroth tube

Nutrient
agar tube

Gelatine
plate

Gelatine
stab

Potato tube

Fermentation
tube

Q
1

1
1

1 Wrinkled

Charac-
teristic app
ranee

5
p.

1
00

Luxurian

B. rhodochrous Over-

beck (Dyar)

B. rubefaciens pyo-
genes (Matzuschita)
Bact. rubrum (Mi-

gula)

_;_

_j_

_^_

Bact. erythromyxa
Zopf (Migula)
B. nibescens (Jordan)

Bact. carnosum

(Kern)

_l_

_I_

_j_

Bact. roseum (Losski)

-j-

_|-

-f

rose

B. nitrogenes (Mat-

zuschita) B. deni-

trificans II, Burri

and Stutzer)

-r

_j_

_^_

_>_

_|_

_{_

B. rubescens (Matzu-

schita) (B. oogenes
hydrosulfureus j.

Zorkendorfer)

-f

_^_

_l_

_^_

B. subrubiginosus

(Maschek)

>|.

_j_

B. lupini (Matzu-

schita)

_j_

B. pseudomycoides

(Migula)
B. coccineus (Pansini)

B. rubellus (Okada)

B. thermophilus

liquef. aerobiuB

(Oprescu)

_|_

_l_

_^.

_j_

B. thermophilus

liquef. tyrogenes
(Oprescu)

1 +

B. mycoides ruber

(Matzuschita)

_|_

-f

B.Dan teci (LeDantec)

-f

_l_

B. apicum (Cane-

strini)

_l_

-1-

B. rubiginosus (Ca-

tiano)

_j_

_l_

_j_

-|-

_j_

B. subcoccineus (Ca-

tiano)

-;-

-f

_l_

_j_

B. kermesinus (Ta-

taroff)

-f

-i-

-f

_j_

-|-

-f

B. rosaceus marga-

rineua (Jolles and

Winkler)

_|_

_l_

_(_

_ _

_l_

-f

B. subrubeum (Kern)

-f

continued.

- 51

logy

Biochemical Features

I Pathogenesis

Liquefaction

Gas production

Milk

Nutrient agar tubes

Mice

1 Grows at bod?
temperature

Facultative
anaerobe

| Gelatine

Blood serum

Saccharose
broth

1 Nitrate reduced

'o
c

Alkaline

.2
1

Fluorescence

Intra-peritoneal
inoculation

pink

-r

red

pink

flesh

red

rose

red

reddish

red

-}-

red

+55

red

path, for bees

red

orange

red

rose

- 52

21) Woolley, P. G., Experiments made to determine the effect of sugar upon
the pigment-formation of some chromogenic bacteria. (Johns Hopkins Bull.

Vol. X. 1899. p. 130.)

22) Fermi, GL, Die Leim- und Fibrin-losenden und die diastatischen Fermente
der Mikroorganismen. (Archiv f. Hygiene. Bd. X. p. 154.)

B. prodigiosus.

23) 1819. Bizio, B. [Gazzetta privilegiata di Venezia. Aug. 24.] Spica No. 44
below.

1823. [Bibliot. Ital. o sia Giorn. di letterat., scienze ed arti. Vol. XXX-
p. 275, Milano.l Spica No. 44 below.

24) 1844. See also id., Comptes rendus, Paris. Vol. XVIII. p. 951.

25) 1824. Sette [Memoria storico-naturale sull' arrossamento straordinario di
alcune sostanze ah'mentose. Venice]. Spica, ib.

26) 1839. Ehrenberg, Chr. G. [Monatsber. iiber die zur Bekanntmachung ge-
eigneten Verhandl. d. Kgl. preufl. Akad. der Wissenschaften, Berlin.] Migula.

27) 1850. Fresenius, G. [Beitrage zur Mykologie. Frankfurt a. M. 1850 bis
1863].

28) 1866. Erdmann, O. Bildung von Anilinfarben aus Proteinkorpern. (Journ.
f. prakt. Chemie. Bd. III. p. 385.)

29) 1872. Schroeter, J., Ueber einige durch Bakterien gebildete Pigmente.
(Cohns Beitr. z. Biol. der Pflanzen. Bd. I. Heft 2. p. 109.)

30) 1872. Cohn, F., Untersuchungen iiber Bakterien. (Beitr. z. Biol. d. Pflanzen.
Bd. I. Heft 2. p. 127. See p. 151 ff., Ueber Pigmentbakterien.)

31) 1875. - [I. Heft 3. p. 142 ff.?J

32) 1875. Helm, O., Ueoer Monas prodigiosa und den von ihr erzeugten Farb-
stoff. (Archiv f. Pharmacie. Bd. III. p. 19.)

33) 1879. Wernich, A., Versuche iiber die Infektion mit Micr. prodigiosus.
(Cohns Beitr. z. Biol. d. Pflanzen. Bd. III. p. 105 (pubd. 1883).

34) 1886. Liborius, P., Beitrage zur Kenntnis des Sauerstoffbedurfnisses der
Bakterien. (Zeitschr. f. Hygiene. Bd. I. p. 115.)

35) 1887. Schottelius, M., Biologische Untersuchungen iiber den Micrococcus
prodigiosus. (Printed from Festschr. fur Koelliker. Leipzig. Abstr. in
Centralbl. f. Bakt. Bd. II. p. 439 (1887).

36) 1888. Strazza, G. Beitrage zur Lehre iiber die Biologic der Mikroorga-
nismen. (Wiener med. Jahrb. Heft 1.)

37) 1888. Wasserzug, E., Variations de forme chez les bacteries. (Ann. dePInst.
Past T. II. p. 75, 153.)

38) 1889. Kuebler, P., Ueber das Verhalten des Micr. prodigiosus in saurer
Fleischbriihe. (Centralbl. f. Bakt. Bd. V. p. 333.)

39) 1892. Griffiths, A. B. Sur la matiere colorante du Monas prodigiosa.
(Comptes rendus de 1' Academic de Science. T. CXV. p. 321.)

40) 1892. Gorini, C. [Studi experimentali sul latte. Roma.] (Abstr. in Centralbl.
f. Bakt. Bd. XII. p. 666.)

41) 1893. Das Prodigiosus-Labferment. (Hygien. Rundschau. Bd. III. p. 381.)

42) Bordoni-Uffreduzzi, G., Fall von fuchsinahnlicher Bakterienfarbung
des Fleisches. (Hygien. Rundschau. Bd. IV. p. 12.)

43) 1896. Scheurlen, E., Geschichtliche und experimentelle Studien iiber den
Prodigiosus. (Archiv f. Hygiene. Bd. XXVI. p. 1.)

44) 1899/1900. Spica, P., Sulla materia colorante prodotta dal Micrococcus
prodigiosus. (Kivendicazione di prioritd, per Bartolomeo Bizio.) (Atti del Reale
Istituto Veneto di scienze, lettere, ed arti. Vol. LIX. Parte seconda, dis-
perse 10. p. 10251031.)

45) 1899. Rosenberg, W. W., Beitrage zur Kenntnis der Bakterien farbstoffe,
insbes. der Gruppe des Bact. prodigiosum. Diss. Wiirzburg.

46) 1900. Kuntze, W., Ein Beitrag zur Kenntnis der Bedingungen der Farbstoff-
bildung des B. prodigiosus. (Zeitschr. f. Hygiene. Bd. XXXIV. p. 169.)

47) 1900. Ritter, G. Zur Physiologic des B. prodigiosus. (Centralbl. f. Bakt.
Bd. VI. p. 206.)

48) 1900. Marx, H., Die Pathogenitat des B. prodigiosus. Eine Bemerkung zur
Farbs toff prod uktion der Bakterien. (Archiv f. lain. Chirurgie. Bd. LXII.
p. 347.)

49) 1902. Kraf t,E., Beitrage zur Biologic des B. prodigiosus und zum chemischen
Verhalten seines Pigments. Diss. Wiirzburg.

49a) 1903. Bertarelli, E., Untersuchungen und Beobachtungen iiber die Bio-
logie u. Pathogenitat des Bacillus prodigiosus. (Centralbl. f. Bakt. Bd. XXXI V.
p. 193.)

B. ruber indicus (Koch).

50) K o c h , R. f Berichte iiber die Reise zur Erforschung der Cholera. 1884.] M i gu 1 a.

51) Fraenkel, C., Grundrifl der Bakterienkunde. 1891. p. 229.

52) Fliigge, op. cit. as 3), p. 302.

53) Migula, op. cit. as 10), p. 306.

B. plymouthensis (Fischer).

54) Fischer, B. Bakteriologische Untersuohungen auf einer Reise nach West-
indien. (Zeitschr. f. Hygiene. Bd. II. 1887. p. 74.)

55) Voges, O. , Ueber einige im Wasser vorkommende Pigmentbakterien.
(Centralbl. f. Bakt. Bd. XIV. 1893. p. 314.)

B. ruber balti CUB \ (Breunig)>
B. ruber kiliensis /

56) Breunig, J. [Bakteriologische Untersuchung des Trinkwassers der Stadt
Kiel. Diss. Kiel, 1888.] Migula.

57) Laurent, E., Etude sur la variability' du bacille rouge de Kiel. (Ann. de
1'Inst. Pasteur. T. IV. 1890. p. 465.)

58) Petrow, N., Ueber einen neuen roten Farbstoff bildenden Bacillus. (B. sub-
kiliensis). (Arbeit des Bakt. Inst. der Grossh. Hochschule zu Karlsruhe, 1902.
p. 273.)

B. miniaceus (Zimmermann).

59) Zimmermann, op. cit. as 12), Reihe I. p. 46.

B. fuchsinus (Boekhout and de Vries).

60) Boekhout, F. W. J., und de Vries, J. J. Ott, Ueber einen neuen
chromogenen Bacillus. (Centralbl. f. Bakt. Bd. IV. 1898. p. 497.)

B. ruber (Zimmermann).

61) Zimmermann, O. E. R., op. cit. as 12), Reihe I. p. 24.

B. havaniensis (Sternberg).

62) IS tern berg, G. M., Manual of bacteriology. 1892. p. 718.

B. lactis erythrogenes (Hueppe).

63) Grotenfeldt, G., Studien iiber die Zersetzungen der Milch. (Fortschr. der
Med. Bd. VII. 1889. p. 41.)

64) Tatarof f [Die Dorpater Wasserbakterien. Diss. Dorpat, 1891. p. 21, p. 60?]
Migula.

65) Dyar, H., On certain bacteria from the air of New York City. (Ann. N. Y.
Acad. Sci. Vol. VIII. 1895. p. 324.)

66) Galeotti, G., see No. 15, ante.

B. rubefaciens (Zimmermann).

67) Zimmermann, op. cit. as 12), Reihe I, p. 26.

B. lactorubef aciens (Gruber).

68) Gruber, Th., Ueber einen die Milch rosa farbenden Bacillus. (Centralbl. f.
Bakt, Bd. VIII. 1902. p. 457.)

B. mycoides roseus (Scholl).

69) Grotenfeldt, loc. cit, p. 46.

70) Ei sen berg, op. cit. as 2), p. 84.

71; Migula, op. cit, p. 482 (B. mycoides)!

B. latericeus (Adametz).

72) Adametz, L., [Die Bakterien der Trink- und Nutzwasser. Mitteil. der osterr.
Versuchsst f. Brauerei u. Malzerei in Wien. Heft 1. 1888. p. 50.] Chester,
op. cit as 1), p. 173.

73) Dyar, loc. cit. as 65), p. 361.

B. rubropertinc tu s.

74) Grass berger, R., Ueber die nacn intraperitonealer Injektion von Markt-
butter bei Meerschweinchen entstehenden Veranderungen. (Miinch. med. Woch.
1899. p. 343.)

B. mesentericus ruber (Globig).

75) Globig, Ueber einen Kartoffelbacillus mit ungewb'hnlich widerstandsfahigeu
Sporen. (Zeitschr. f. Hygiene. Bd. III. 1888. p. 323.)

B. rosaceus metalloid es (Tataroff).

76) Tataroff [Die Dorpater Wasserbakterien. Diss. Dorpat, 1891.] Migula.

77) Hefferan, Mary, An unusual bacterial grouping. (Centralbl. f. Bakt.
Bd. III. 1902. p. 689.)

Reference list of red chromogenic Bacteria.

(cf. Table X.)

B. rubidus Eisenberg. Bakt. Diagnostik, p. 88. 1891

B. ruber lactis (Conn). Rep. Conn. Agric. Sta. 1899

Bact. pyocinnabareum (Ferchmin. "red pus") Centralbl. f. Bakt. Bd. XIII.

p. 103. 1894

Bact. rubiginosum (Kern) Arb. d. techn. Hochschule zu Karlsruhe. Bd. I.

Heft 4. 1896

B. rubiginosus (Catiano). Cohns Beitrage. Bd. VII. p. 538. 1896

B. subcoccineus 539. 1896

B. coccineus (Pansini). Virchows Archiv. Bd. CXXI1.

p. 437 1890

B. sardinae (Du Bois St. Se'vrin). Ann. Past. T. VIII. p. 152. 1894

B. rubellus (Okada). Centralbl. f. Bakt. Bd. XI. p. 1. 1892

B. carneus (Tils). ' Zeitschr. f. Hyg. Bd. IX. p. 294. 1890

Bact. rubrum (Migula). Migula. p. 488. 1900

Bact. erythromyxa (Zopf). p. 487.

(Zopf himself (Ber. d. deutsch. botan. Gesellsch. 1891. p. 22) , calls his

form a micrococcus).
Bact. pseudomycoides (Migula).

Bact. carnosum (Kern) cf. Kern above also Migula. p. 485.
Bact. tuberosum (Kern) p. 490.
B. rubescens (Jordan). Rep. Mass. State Bd. of Health, p. 835. 1890

B. roseus (Fischer) Die Bakterien des Meeres. p. 22 1894

B. rubrofuscum (Fischer) p. 36

B. mesentericus roseus (Zimmermann). Zimmermann. II. p. 26 1890

See Lustig. p. 72. Name (Kruse) see Fliigge p. 303.
Bact. roseum (Losski). Die Mikroorganismen des Bodens. Diss. Dorpat. See

Migula, p. 484.
B. kermesinus (Tataroff). Die Dorpater Wasserbakterien. Diss. Dorpat. 1891

See Migula p. 858.

B. exiguum (Wright). Mem. Nat. Acad. Sci. Vol. VII. p. 447. 1895

B. haematoides (Wright). p. 448.

B. epsilon (Dyar). Ann. N. Y. Acad. Sci. Vol. VIII. p. 369. 1895

B. zeta - p. 369.

B. delta p. 368.

B. ferrugineus (Dyar) p. 361.

B. fimt iiniis ruber (Dyar) ,, p. 361.

B. salmoneus (Dyar)
B. rhodochrous (Overbeck. Dyar)

(Overbeck himself called the organism a micrococcus)

See his art., Nov. Act. Leop. Carol. Acad. Vol. LV. D. 16. 1891

B. ruber berolinensis (Fraenkel, p. 252) Fliigge. Die Mikroorganismen.

p. 303. 1896

B. sulfureus (Holzschewnikoff). Fortschr. d. Med. Bd. VII. p. 201. 1889

B. subfulvus (Matzuschita) = "B. tuberigenus 4", Gonnermann, Landw.

Jahrb. Bd. XXIII. p. 656. 1894

B. velatus (Matzuschita) "B. tuberigenus 5". Gonnermann, Landw.

Jahrb. Bd.XXIII. p. 657.

B. Lupini (Matzuschita) "B. tuberigenus 7", Gonnermann, Landw. Jalirb.

Bd. XXIII. p. 657.
B. pneumonicus agilis (Schou). Fortschr. d. Med. Bd. III. p. 483. 1885

See Neumann, Zeitschr. f. klin. Med. Bd. XIII. p. 73. 1888

B. viguali (Matzuschita) = Bac. [i Vigual, Fliigge. Bd. II. p. 283.
B. liquefaciens communis (Sternberg). Fliigge. Bd. II. p. 315.
B. rosaceus margarinicus (Jolles and Winkler). Zschr. f. Hyg. Bd. XX. p. 105. 1895
B. nitrogenes (Matz.) B. denitrificans II. Burri und Stutzer, Centralbl. f.

Bakt. Abt. II. Bd. I. p. 362. 1895

B. erubescens (Matzuschita) = B. oogenes hydrosulfureus Zorkendorfer, Arch.

f. Hvg. Bd. XVI. p. 391. 1893

B. subruoiginosus Maschek.

B. rubefaciens pyogenes (Matzuschita). Centralbl. f. Bakt. Bd. XXIX. p. 378. 1901
B. subrubeus (Bact. subrubeum Kern). [Arb. aus d. Hochsch. zu Karlsruhe. Bd. I.

p. 450.1

B. thermophilus liquef. aerobius (Oprescu). Arch. f. Hyg. Bd. XXXIII. p. 166.
B. tyrogenus Ebenda, p. 171. 1898

B. subthermophilus (Matzuschita) = B. thermophilus IV Rabinowitsch. Zeitschr.

f. Hyg. Bd. XX. p. 161. 1895

B. sacchanphilus (Laxa). Centrabl. f. Bakt. Abt. II Bd. IV. p. 362. 1898

B. mycoides ruber (B. mycoides roseus?) Matzuschita. Archiv f. Hyg. Bd. XLI.

p. 251. 1901

B. Danteci (Le Dan tec). Ann. Past. Bd. V. 1891. p. 656. Named by K ruse,

Fliigge. Bd. II. p. 270.

B. apicum (Canestrini) Fliigge, Bd. II. p. 233.
B. mesentericus rubiginosus (Senger). Centralbl. f. Bakt. Bd. III. p. 603.

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