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Journal of Experimental Medicine

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THE:NATUREOF

THE ANTITETANICEOSIN.

BY H I D E Y ONOGUCHI,ACTION

OF

M.D.

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

In a previous publication I Dr. Flexner and I gave the results
which we obtained from a study of experimental tetanus in rats
and guinea pigs subjected to the influence of certain photodynamic
anilines. To recall briefly the main facts brought out there, it may
be stated that a solution of eosin, of a certain concentration, destroys in vitro the haemolytic and tetanospastic principles of tetanus
toxin, and when applied to the site of inoculation of tetanus sporeson threads, or tetanus-toxin, either entirely prevents the appearanceof the tetanic symptoms, or delays the appearance and diminishes
the effect, and, in rats, sometimes prevents the fatal issue. In a
great number of instances in which the eosin is applied directly tothe spore-infected area in rats, the lives of the animals can besaved. The next step in this investigation was the determinationof the mode of action of the eosin through which the effects alreadydescribed are produced.
The present paper deals with the manner of action of eosin upon
Bacillus tetani without and within the body of animals. The subject falls naturally, therefore, into the two main divisions of the~ction of eosin upon the tetanus bacillus in vitro and in vivo.
THE

ACTIONOF E O S I N

UPON

THE

TETANUSB A C I L L U S IN

VITRO.

Relating to this topic, the following points were considered: ( I )the influence of eosin upon the germination, growth, sporulation,
morphology, and toxin production of B. tetani; (2) the bactericidal and sporicidal effects of eosin upon B. tetani; (3) the viabilityof B. tetani in eosinized media; (4) the toxin-producing power ofB. tetani exposed to the action of eosin.
' F l e x n e r a n d N o g u c h i , ]our. of Exper. Med., I9O6, viii, I.

281

282

Antitetanie Action of Eosin.

Cultivation of Bacilhts tetani i~z Eosini.:ed Culture Media. (a)Eosinized Ghtcose BouiIlon.--Two kinds of e o s i n - - " rein " and" g e l b " - - w e r e employed in strength of o . o o i per cent., o . o i percent., o . I per cent., and I per cent. A v i g o r o u s culture of thetetanus bacillus was inoculated and incubated at 37 ° C. in an atm o s p h e r e of h y d r o g e n for IO days.
The control cultures were abundant; they contained typical bacilli forming
few threads and showing few spores. The growth in o.ooi per cent. eosin was
equal to the control, but microscopically the thread forms predominated and no
spores whatever could be found. The growth in o.oI per cent. eosin was equal
to the control, but upon microscopic examination only asporogenous thread forms
were seen. The growth in o.I per cent. eosin was slight and showed single,
asporogenous bacilli. No growth whatever took place in the I per cent. eosin
medium.
T h e first effect which the eosin produces, apparently, is to suppress sporulation of the bacilli, and the n e x t is to increase thread
formation. T h e limit of concentration of eosin which permits
restricted g e r m i n a t i o n is between o . I and I per cent., and is probably not far f r o m the f o r m e r concentration. A later d e t e r m i n a tion placed this limit at o.2 per cent. T h e cultures p r e p a r e d as
described were filtered t h r o u g h porcelain and tested f o r toxicity
upon rats. T h e n u m b e r of 111. 1. d. per cubic centimeter of filtrate
was as follows:
Control = iooo m.l.d.
o.oo~ per cent. eosin ~ 7oo m.l.d.
o.ot per cent. eosin ~ IOO m.l.d.
o.I per cent. eosin ~ I m.l.d.
I.o per cent. eosin ~ o m.l.d.
N o r e m a r k a b l e difference in the properties of the two kinds of
dye w a s noticed. T h e reduction in toxicity is g r e a t e r than the reduction in g r o w t h caused by the eosin. T h e diminished toxicity
l~_as, doubtless, a two-fold o r i g i n : it arises f r o m the diminishedmultiplication of the bacillus, and f r o m the i n j u r y exerted by theeosin upon the toxin originally produced. T h e degree of this destruction can be deduced f r o m the relative s t r e n g t h s of the filtrates
f r o m the control and the o . o o I per cent. eosin cultures, and ita m o u n t s to a b o u t 3 ° per cent. As the eosin strength g r o w s , theeffects upon the nmltiplication and toxicity increase, but not in
equal ratio. A c o r r e s p o n d i n g c o m p a r i s o n of the h,'emolysin (tetan-Hideyo Noguchi.
olysin) present in the several filtrates was also made.
centimeter contained the following units:

283
Each cubic

Control filtrate contained *C.H.D. 20 *M.H.D. 333
o.ooi per cent. " "rein" "
"
I2
"
242
0.00I
"
" "gelb " "
"
IO
"
2oo
o.oi
"
"
"rein " "
"
4
"
66
o.oi
. . . . . .
gelb . . . . . .
3
"
50
o.I
"
"
"rein " "
"
o
"
o
o.I
"
"
"gelb """o"o
* C.H.D. = complete h~emolytie dose; M.H.D. = minimal (trace) hmmolytic
dose.
Eosin destroys or suppresses the tetanolysin in the cultures as
it does the tetanospasmin, a fact which could be predicted on thebasis of the observations of F l e x n e r and N o g u c h i 3 Note should
be taken of the g r e a t e r activity in this respect of eosin " g e l b " ascompared with eosin " rein."(b)
Eosinized
GlucoseAgar.--The
advantage for the study ofB.
tetaniwhich glucose a g a r has over glucose bouillon is derivedf r o m the fact that in the f o r m e r medium sporulation is much moreabundant, and is already easily noticeable a f t e r t w e n t y - f o u r hours
growth. A t the end of seven days spores are numerous, and at
the expiration of about t h i r t y days, no vegetative bacilli remain.
Hence, to glucose a g a r the following strengths of both eosins wereadded, o . o i per cent., 0.02 per cent., 0.0 5 per cent., o.I per cent.,
0.2 per cent., 0. 5 per cent., and I per cent. Stab inoculations werem a d e ; cultivation at 37 ° C.
T h e g r o w t h s were well-marked in the tubes below 0.0 5 per cent.
s t r e n g t h ; above this limit the g r o w t h s diminished progressively
with increase in concentration. On microscopical examination, it
was found that o.I per cent. of eosin was the limit of concentration
for spore formation. Below this concentration, sporulation took
place; at and above it, no sporulation occurred even a f t e r threemonths. T h e n u m b e r of spores in eosinized media is below then u m b e r in o r d i n a r y media; the situation of the spores in the f o r m e r
media is the centre of the body. Transplantation of the atypicalsporulating bacilli to simple glucose a g a r brings about immediate
"Op.

cit.

284

Antitetanic Action of Eosin.

return to typical mode of sporulation. The limit of germination
is for eosin " rein " about o. 5 per cent., while for complete germination, it is about o.2 per cent. Strengths above o. 5 per cent. inhibit
vegetation of spores, but they do not kill the spores. Eosin " g e l b "is somewhat more inhibitory. Concentrations above o.o2 per cent.
prevent sporulation, but permit vegetation; above o.5 per cent. novegetation occurs.
(c) Eosinized Tissue B o u i l l o n . 3 - - T h e tetanus bacillus grown
aerobically at 37 ° C. in tissue bouillon produces abundant sporesii~ a few days. The sediment which forms in the cultures in a
few days, is composed largely of spores. The effect of eosin" g e l b " on this process was studied by adding strengths of o.ooi
per cent., 0.003 per cent., o.oi per cent., 0.03 per cent., o.I per
cent., 0. 3 per cent., I per cent., and 2 per cent. of the dye. The
tissue absorbs a part of the dye; the cultures were incubated at
37 ° C. for twenty hours when first examined, and subsequently fortwenty-nine days longer.
After 2 o H o u r s .

Control.
Eosin " g e l b " o.ooI %Good growth ; gas formation.
Idem.

.

.

0.003

Idem.

~

~

O.OI

Less growth.

.

~,

0.0

,c

~

O.I

,,

,,

0 3

3

Idem.
Slight growth.
Doubtful growth.
No growth.
Idem.

C.S. ~ single bacilli ; no spores.
C.S. chiefly single, few threads,
no spores.
C.S. bacilli chiefly in chains, nospores.
C.S. long threads only showing
vacuoles and irregular
contours.
Idem.
C.S. very few single bacilli, small
number of spores.
C.S. few bacilli and spores.
C.S. very few spores.
Idem.

After 3° D a y s .

Control.
Eosin " g e l b " o.ooi
,,
,,
0.003
~

~

0.01

,,

,,

o.o 3

~

~

O.I

,,

,,

0. 3

Fluid clear ; deposit heavy.
Idem.
Idem.
Idem.
Idem.
Slight deposit.

C.S. all bacilli bear spores.
C.S. numerous threads and spores.
C.S. nearly exclusively in threads ;very few spores.
CIS. threads ; no spores.
Idem.
C.S. a few short chains ; many single bacilli ; few spores.

No change.
No change.
No change.

* C.S. indicates cover slip p r e p a r a t i o n s s t a i n e d in the u s u a l way.
t T h e o b a l d S m i t h , J o u r . o f t h e B o s t o n S o c . o f t h e M e d . S c i e n c e s , 19oi, iii, 34o.
Tarozzi, C e n t r a l b l . f. B a k t . , etc., Orig., I9o5, x x x v i i i , 619.

Hideyo Noguchi.

285

The foregoing observations are in conformity with those alreadyrecorded. In spite of the vigorous growth of the bacilli in thismedium containing small quantities of eosin, the biology of theorganisms is influenced directly as regards their power to segment
and to form spores. With increasing concentrations of the eosinthese effects become more pronounced, until in the concentrationof o.I per cent. only part of the transplanted spores are enabled
to germinate, and the vegetative bacilli produced by this imperfectgermination are restrained from free multiplication. This processof imperfect vegetation comes, I believe, to play a very interesting
part in developing immunity in inoculated rats treated with eosin,of which phenomenon I will have occasion to speak hereafter.

(d) Bactericidal and Sporocidal Properties of Eosin upon B.
tctani.--Glucose bouillon cultures of the tetanus bacillus do notform spores within forty-eight hours, hence such cultures can be
employed to test the bactericidal effect of strong solutions of eosin.
It was found that such spore-free cultures when mixed with eosinto the concentration of 2 per cent., and kept for fifteen minutes indiffuse light, fail to grow upon replantation. If the eosin strength
falls below i per cent., not all the bacilli are killed. Contact fortwenty-four hours of vegetative sporeless bacilli with eosin in solutions above o.I per cent., causes their death. The bactericidal
effect of eosin is increased by exposure to the sun: eosin in astrength of 0.02 per cent. can cause in eight hours the death ofvegetating bacilli, when exposed directly to the sun's rays. If theexperiments are made under anaerobic conditions the results are
not essentially different.
The spores of the tetanus bacillus~display far greater resistance.
Solutions of eosin of 5 per cent. and 0.05 per cent. failed to bringabout their destruction after exposure under aerobic and anaerobic
conditions to the direct rays of the sun for thirty hours.
( e) Viability of B. tetani in Eosinized Media.--Cultures eightyeight days old of the tetanus bacillus in glucose agar containingo.I per cent. eosin " gelb" showed no spores. On transplanting
from these cultures into glucose agar, a feeble growth containing
few spores after seven days was secured. Transplantations into
esoin-free media from this slight growth gave cultures which be-286

Antitetanie Action o/ Eosin.

haved, in all respects, in a normal manner. From this experiment
the conclusion can be drawn thai concentrations of eosin which are
not quickly fatal 'to sporeless tetanus bacilli reduce in the first generation their power of reproduction but do not suffice to kill themoutright even after long periods of contact.
([) Can B. tetanus be Rendered Durably Asporogenous byt~'osin?--Eosin in the strength of o.oi per cent. reduces sporeformation in B. tetani and in the strength of o.I per cent. prevents
it entirely. A strain of B. tetanus was cultivated in o.oI per cent.
and o. I per cent. successively through many generations. At eachtransfer to eosinized medium a control culture was made in glucoseagar to observe the point of final disappearance of the spore-bearing
faculty. In the case of eosin " rein," the bacillus withstood fairly
well the successive implantations into the eosinized medium, but thefaculty to produce spores in ordinary media was not lost after
eight generations, covering a period of three months, in eosinizedglucose agar. A reduction of the spore-formation was noted in
the first generations in the plain glucose medium. In the case of
eosin " gelb," the bacillus survived for three generations only ino.I per cent. eosin-glucose-agar, and for the eight generations in
the O.Ol per cent. medium. No permanent alteration of spore-bearing capacity was effected in the latter cultures.
(g) Is the To.tin Producing Power of B, tetani Affected by its
Growth in Eosinized Media?--Two cultures of B. tetani were kept
in o.~ per cent. eosin " g e l b " glucose-agar for eighty-eight days,
after which they were renewed by transplantation to glucose
bouillon. After forty-eight hours growth at 37 ° C., these culturesand suitable control cultures were filtered through porcelain. The
toxicity of the filtrates was approximately equal. The deduction
from this experiment is obvious: no permanent influence upon thetoxin-producing faculty of B. tetani is exerted by long contact ofeosin in concentrations below the bactericidal limit.
THE

ACTIONOF EOSIN

UPON

THE

TETANUSBACILLUS IN

VIVO.

The next subject of the study taken up related to the manner inwhich eosin acts in preventing tetanus in rats inoculated withtetanus bacilli or their spores.4 The effects to be explained are,' F l e x n e r a n d N o g u c h i , op. cit.

Hideyo Noguchi.

287

briefly, these: rats, beneath the skin of which tetanus spore-threads
are placed, regularly develop tetanus and die. If, however, the inoculated rats are treated by injections of eosin about the sporethread, m a n y recover, and some even fail to develop any symptomsof tetanus. Injection of the eosin in other parts of the body m a ydelay the appearance of tetanus and the fatal issue, but does not
suffice wholly to prevent them. T h e effect of the eosin on the local
reaction to the tetanus bacilli in the inoculated rats is to be explained.

Fate of Spores Introduced into the Body on Threads.--Eighteen
rats ( w e i g h i n g about 9 ° grams each) were inoculated beneath theskin of the thigh with spore-threads of B. tetani, free f r o m toxin,
on J u n e 8, I9o6. T h e six of these left untreated (controls) developed tetanus in two to six days time (average, three d a y s ) .
Death usually resulted on the third day a f t e r the appearance of thetetanus. Cultures were made f r o m the threads and the liver. 5
T e t a n u s bacilli were recovered f r o m the threads in all six animals
and f r o m the liver in three animals. T h e r e m a i n i n g twelve rats
were treated with eosin " rein." Doses of o.2 to o.4 c.c. of a 2
per cent. solution were injected successively on three days, beginning immediately a f t e r the inoculation, about the thread, and intermittently ( e v e r y other d a y ) for t h r e e more injections. T h etreated animals did not develop tetanus, and a part o f them werealive and healthy four months a f t e r the inoculation.
T h e first question which I asked myself is, Do the tetanus spores

germinate and multiply under the influence of the eosin, and what
becomes of the germinated bacilli?
Experiment. Rat No. 7. A part of the spore-thread was removed forty-eight
hours after inoculation and during the eosin treatment. Cover slips preparedfrom it showed many spores, a small number of bacilli and a few leucocytes. A
second portion of the thread was removed on the ninth day (day of last eosin
injection). Cover slips showed very few bacilli and many spores and leucocytes.
Cultures gave pure growths of B. tetani. On the thirteenth day, the remainderof the thread was removed. Now only spores could be found, and leucocytes
were no longer present. The rat remained well.
Rat No. 8. Same treatment; examination on fifth day; among many spores asmall number of bacilli--vegetative forms--were seen.
Tarozzi, Cenlralbl. f. Bakt., etc., I9o6, xl, 3o5, 45I.

288

Antitetanic Action of Eostn.

T h e study of the spore threads in the two inoculated rats show
conclusively that the injections of eosin do not wholly prevent thegermination of the spores, but that the germination is largely suppressed; and it also renders the view v e r y probable that of theg e r m i n a t e d spores few or none multiply in the eosinized tissues,
while a f t e r a short time ( f e w days) the vegetated bacilli themselves
disappear. Attention is called to the observation that the tetanus
spores remain in an intact state for m a n y days in the inoculated
region o f the body.
T h e next question which I asked myself is, What becomes event-ually of the ungerminated spores, and, supposing they remain locallyin the tissue, do they suffer any alteration in toxin-producing power?
Experiment. Rat No. 9. Inoculated June 8. Eosin-treated. No symptoms,
June 24. Removed the spore-thread which was found to be surrounded by
fibrous tissue and adherent to the fascia. Eosin had disappeared. Cover slips
showed many spores and no bacilli. A pure culture of B. tetani obtained in tissuebouillon.
Rat No. zo. The thread was removed nineteen days after inoculation. The
result was in all respects like that of rat No. 9. Toxin of the usual strength
was yielded by the cultures.
Rat No. H. A part of the spore-thread was removed twenty-three days after
the inoculation. Cover slips showed many spores which yielded a pure cultureof B. tetani containing a strong toxin. The second wound healed without giving
rise to tetanus. Animal remained well.
B y these observations it is shown that the healing of the local
wound containing the eosinized threads, proceeds in a m a n n e rsimilar to that of a sterile w o u n d ; and it is also shown that, by
virtue of the eosin treatment, the tetanus spores are reduced to thevalue o f inocuous foreign bodies. 111 the course o f this process,the tetanus spores, already quiescent, remain in the healed tissues for
an indefinite time. T h e y are not devitalized by the tissues, or,apparently, altered in any essential way, at least, their toxin-producing power is not impaired by the new conditions u n d e r which
they survive.
If the results obtained in vitro and in vivo are comPared certain
correspondences, as well as certain differences, can be noted. Perhaps the most important difference consists in the disappearanceof the vegetative bacilli in the body, and their persistence in thetest tubes, in the tests with eosin; and the germination of the re-Hideyo Noguehi.

289

strained spores in the culture once the eosin is no longer present,
while, in the body, the tissues already advanced in healing restrain
the vegetative propensities of the spores, although the toxin has
been entirely eliminated.
The healing of the wound of the second operation in rat No. I I,
without giving rise to tetanus, all the eosin having meanwhile disappeared, is an interesting fact, and one that deserves further consideration. The cultivation experiment proved that living tetanus
spores, capable of toxin-production remained in the thread; andyet the remainder of the thread, in the tissues, gave rise to nosymptoms. It might be thought indeed, that the spore threads,
after this long sojourn in the body, are not capable of directly infecting a second animal. A test of this possibility was easily made.
E.vperiment. Rat No. ~2. A n o r m a l rat w a s inoculated on J u l y 9 b e n e a t h
t h e s k i n of t h e t h i g h with a s p o r e - t h r e a d r e m o v e d on the t h i r t y - s e c o n d day f r o m
a n e o s i n - t r e a t e d rat. O n J u l y II the a n i m a l w a s tetanic, a n d on J u l y 13 it w a s
dead. A p u r e c u l t u r e of B. tetani was recovered f r o m the thread.

The apparent innocuousness of the second operation for theremoval of spore-threads in animals treated long before with eosin,
and the great susceptibility of the normal rat to the threads after
this long residence in the body, brought up the question of thepossible existence of a local immunity to tetanus in the eosintreated animals. This idea was, of course, capable of experimental
verification. The experiments relating to it form the subject ofa separate publication.
SUMMARY.

Eosin, if present in cultures containing tetanus spores, prevents
the germination of these spores when its concentration (in glucose
bouillon) reaches 0.2 per cent. When the concentration of theeosin sinks to o.oi per cent., germination of the spores is no longer
inhibited, but the vegetative bacilli developed from the spores execute a highly restrained form of multiplication. When the eosin
concentration sinks to o.ooi per cent., vegetation and multiplication of the bacilli become more active, but no new spores are formed
even after long periods of time. With glucose agar it is not until
the concentration of the eosin in the cultures falls to o.o 5 per cent.
that sporulation again appears. At this concentration of the eosin,

290

Antitetanic Action of Eosin.

very few spores are formed ; but as the eosin sinks lower and lower,
sporulation becomes more active, until with o.ooi per cent. it isessentially of normal degree. In concentrations of o.oo 3 per cent.,
eosin prevents perfect segmentation of the multiplying bacilli, withthe result that, finally, long and convoluted threads of bacilli areproduced. The spores which are formed in a medium containing
o.oi per cent. of eosin are often situated at the centre and not at
one pole of the bacilli.
Eosin in a strength of 2 per cent. is capable of destroying thevegetative bacilli, if the contact is prolonged to fifteen minutes,
and in strength of o.I per cent., in twenty-four hours. Placing
this latter mixture of bacilli and eosin in the sunlight greatly hastens
the bactericidal effect, and the bacilli are found to be incapable ofgrowth at the end of several hours. Eosin in high concentrations
is not capable of killing the tetanus spores, even after long exposure to sunlight (thirty hours).
The toxin production of tetanus bacilli grown in eosinized culture media diminishes as the concentration of the eosin increases.
This effect is brought about partly by the restraining action of thedye on vegetation, and partly by its detoxicating action upon the
poison.,The toxin-producing power and the virulence of tetanus bacilli
are not permanently modified by contact with eosin for a long
period, or by successive cultivations in eosinized media.
Eosin is likewise capable of restraining the vegetation of tetanus
spores in the animal body. In spore threads inserted beneath theskin of rats, and surrounded with eosin in solution, a very restricted
vegetation takes place. If the injections of eosin are repeated,vegetation soon ceases and the vegetated bacilli degenerate and disappear.
The ungenninated tetanus spores remain alive in a latent condition indefinitely in the healed wound beneath the skin. These
spores do not lose power to grow outside the body, or inside thebody of animals under favorable conditions, or to produce toxin
in a characteristic manner.