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Journal of Experimental Medicine

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ON THEINHIBITORY INFLUENCESPORULATION)
By H I D E Y ONOGUCHI,OF EOSIN UPON

M.D.

(From the Rocke[eller Institute for Medical Research, New York.)

Sporulation requires optimum temperature and suitable nutrient
media. It is greatly influenced by various physical and chemical
agents. Thus, in the case of Bacillus anthracis, the most studied inthis respect of all spore-bearing organisms, no sporulation takes
place at temperatures above 420 C. 2 or below I 4 ° C. a Weil 4 places
the lowest temperature at which sporulation takes place at 7 ° C.
The presence of oxygen seems to be essential to the formation ofanthrax spores. 5 Persistence of conditions unfavorable to sporulation through many successive generations of the organism gives
rise to asporogenous strains in which virulence is often foundgreatly reduced or even totally absent.
Phisalix' succeeded in obtaining an asporogenous stain of B. anthracis bycultivating it at 420 C. for twelve successive generations. Roux ~ induced a similar biological alteration by means of a medium containing certain chemicals.
Potassium bichromate in the ratio of I to 2ooo, or phenol in from 2 to 6 to IO.OOO
added to ordinary bouillon stops sporulation completely. Behring s found thatvarious acids, alkalies, salts and certain antiseptics when used in suitable concentrations prevent sporulation. The antisporulative property of certain dyes wasalso described by Behring,' who states that safranin in I to 30,oo0, and methyleneviolet, cyanid, and malachite green in I to 2oo, ooo to I to 6oo,oco exert a powerful restraining influence upon the growth and sporulation of B. anthracis. After'Received for publication October I, 19o7.
2 Phisalix, Bull. m~d., t892, vi, 533.
* Kitasato, Zeit. f. Hygiene, 189o, viii, I98.
Weil, Zeit. f. Hygiene, 19Ol, xxxvi, 451.
5 Schreiber, Cent. [. Bakt., I896, xx, 353. Well, Arch. [. Hygiene, I9OI, xxxix,
2o5. Klett, Zeit. f. Hygiene, I9oO, xxx, 420. Jacobitz, Cent. [. Bakt., 19Ol, xxx,232. Slupski, Cent. [. Bakt., 19Ol, xxx, 396.
s Phisalix, Bull. m~d., I892, vi, 533.
7 Roux, Annales de l'Inst. Pasteur, 189o, iv, 25.
' B e h r i n g , Zeit. [. Hygiene, 1889, vi, 117.
*Idem, 1889, vii, 17I.
30

Hideyo Noguehi.

31

two months' successive cultivation in the colored agar media, no permanent lossof the sporulating property resulted.
Schreiber TM states that potassium phosphate of a concentration above 3 percent. prevents sporulation of B. anthracis, B. subtilis and B. tumescens. Behring,n Bormans,1~ and Leeleff13 found that B. anthracis does not form spores inblood serum, while Brieger, Kitasato and Wasserman 1. recorded a few instancesin which B. tetani failed to sporulate in an aqueous extract of thymus gland.
While abundant work has been done with various spore-bearing aerobes,
especially with B. anthracis, a similar study with anaerobic organisms has been
so far neglected. In a recent study on the antitetanic property of certain dyes,
Flexner and Noguchi'5 called attention to the fact that eosin in an adequate
strength prevents the sporulation of B. tetani, the experimental details of which
are given in a later paper by Noguchi.leI n the present c o m m u n i c a t i o n I wish to present some o f the results of experiments on the restraining influence of eosin upon
the sporulation of various microbes.
T h e varieties of bacteria
subjected to experiment belonged to the aerobic and to the anaerobic
organisms. O f the first, B. anthracis, B. m e g a t h e r i u m , B. cereus,B. mesentericus, B. subtilis, B. ruminatus, and B. anthracoides, andof the second, B. tetani, B. anthracis symptomaticus, B. botulismus,B. cedema maligni, B. enteritidis sporogenes and B. putrificus werestudied.
Eosin " G e l b " h a v i n g been mixed with a g a r or bouillon inv a r y i n g strengths, the inoculations of bacteria were made as usual.
Stab and slant solid cultures were made. F o r the anaerobic bacteria,
tissue-bouillon and a deep layer of glucose a g a r were employed.
T h e a g a r tubes were incubated in an atmosphere of h y d r o g e n .
T h e results are tabulated.
Table I. indicates that the inhibitory action of eosin is mostintense upon ]3. cereus and B. mesentericus, and least upon B.
anthracoides, while B. subtilis, ruminatus, anthracis and megatherium occupy intermediary positions. All g r o w t h became uncertain
when the concentration of eosin reached one per cent. ; below this~oSchreiber, Cent. f. Bakt., 1896, xx, 353.
nBehring, Zeit. f. Hygiene, I889, vi, 117.
12Bormans, cited in Baumgarten's Jahresberichte, 1895, xi, I38.
~'Lecleff, La Cellule, 1894, x, 349.
1, Brieger, Kitasato and Wassermann, Zeit. f. Hygiene, 1892, xii, 137.
~5Flexner and Noguchi, Jour. of Exper. Med., I9o6, viii, I.
~' Noguchi, Jour. of Exper. Med., I9o7, ix, 281, 291.

32

Influence of Eosin upon Sporulation.
TABLE I.~L'xaminaNon

after zo Days.

dSlant Agar with Eosin" Gelb.'".=z~:dControl (no eosin).
o . o o r per cent.
O.O1~

0.0 5,,

O.I~

0.5~'

9=--;all-+ few+all-4- few

4- all+ many
4- few-4-- all+ all+ all-4-4-4-?

-4-+++?

-4+I+?
!ir-4-i+ ~ spore formation.
- - ~--- n o s p o r e f o r m a t i o n .
-~- ? = s p o r u l a t i o n d o u b t f u l .

strength, multiplication of the bacteria still takes place•
Theseorganisms, when cultivated in bouillon containing varying amountsof eosin " Gelb," appear to be even more sensitive to the action of
the dye than when grown on a slant agar surface. But when thecultures are allowed to stand for many weeks, sporulation takes
place in a medium containing as high concentration of the dye as o.I
per cent. Concentrations above o. 3 per cent. prevent sporulation,
to which effect is added restraint of growth and formation of chainsof bacteria through imperfect multiplication. At times, marked
degrees of involution occur. In no instances was growth discoveredin bouillon containing two per cent. of the eosin.
In deep stab cultures the results were practically identical withthose given in the table. Transplantation of the asporogenous
bacilli into eosin-free media was associated with immediate returnof the spore-bearing power. Many weeks' contact of the sporelessvegetative bacilli with the eosin exerted no enduring effect on thesporogenous property.
The influence of eosin " Gelb " on the sporulation of the anaerobic
species has been shown in Tabie III. Recapitulated, they show that

Hideyo Noguchi.
,.4;433'+~~oooo.~÷÷÷÷÷-t- ÷ ÷÷÷I÷I !

I I

÷++÷÷÷÷÷I÷I II I ~o÷+ioooII÷-~~~-~-H-~I-~I II I~odd6ddd,~

IIInfluence of Eosin upon Sporldation.

34
TABLE

III.--Observalions made afler 2 9 Days on Certain Anaerobes C~ullivaled Aerobicall)' in the Presence of Tissue.

Bouillon Containing [a Small Pieceof Rabbit's
Fresh Liver.

[ B. tetani.

B. anthracis
symptomaticus.

B. botulismus.

B. oedemamaligni.

B enteritid s-~+++?

+++++++-?

+Percentageof 1 , - . . . .
eosin " G e l b " ]in the b o u i l l o no . o o I p e r cent. !
o . o o 3 p e r cent.
o.oI

per cent.

0.0 3

p e r cent.

o. I

p e r cent.

0. 3

per c e n t .

ip e r cent.

-.~~+i+++

Control {no eosin)~sporogenes. I is. putrmcus.

-a_@+

4

No growth

No growth

JiNogrowth

No growth

No growth

No growth+ = spore formation.
-- ~ no spore formation.
= spore formation doubtful.

the eosin in the strength of one per cent. completely inhibits alI
multiplication of the bacteria. The results are only very slightly
different in the case of the cultivation of the anaerobic species freely
in the air, in the presence of tissue (Table III.) and in an atmosphere in hydrogen in deep glucose agar.
The phenomena of restraint are less pronounced in the latter media.
SUMMARY.

Sporulation of B. anthracis, B. subtilis, B. cereus, B. ruminatus,
B. mesentericus, B. anthracoides and B. megatherium does not takeplace in an agar medium containing eosin " Gelb " in a concentration exceeding 0. 5 per cent. In a concentration of o.i per cent.
most of these bacteria fail to produce spores. The greatest sensitiveness is shown by B. cereus and B. mesentericus. In a bouillon
medium sporulation is likewise inhibited by eosin, but after a longer
time--seven weeks or more--sporulation still occurs where theconcentration of the dye equals one-tenth per cent.
Sporulation of B. tetani, B. anthracis symptomaticus, B. botu-Hideyo Noguehi.

35lismus, B. cedema maligni, B. enteritidis sporogenes, and B. putrificus does not take place in a medium containing eosin " Gelb "in concentrations exceeding 0.o 3 per cent. With these organisms,
no difference was noted in the final effect, depending on the medium
employed. No permanent loss of power to produce spores ensues
with the bacteria tested even after long sojourn in the eosinized
media.
It may be stated that on the whole the inhibitory action of eosin
was more pronounced upon the anaerobic than upon the aerobic
species of bacteria employed in these experiments.