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Journal of Experimental Medicine

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PURE CULTIVATION OF SPIROCHiI~TA PHAGEDENIS(NEW SPECIES), A SPIRAL ORGANISM FOUNDI N P H A G E D E N I C L E S I O N S ON H U M A N
EXTERNAL GENITALIA.*BY H I D E Y O NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research,N e w York.)
PLATES 32 AND 33.

In the ulcerative processes situated around the genital region ofman one encounters, besides various forms of bacteria, a numberof spiral micro6rganisms belonging to the genus Spiroch~eta orTreponema. Thus Schaudinn and Hoffmann 1 described Spirochceta
refringens; von Prowazek and Hoffmann, 2 Spirochwta balanitidis;
R6na, 3 Spirochceta gangrcenosa nosocomiali.¢; Corbus and Harris, 4forms resembling Vincent's spirillmn; Mulzer, 5 Spirochwta pseudopallida; and Pollandfl a large spiroch~eta with five or six bluntcurves.
While Spirochceta refringens is now known to be non-pathogenic,
the etiological relation of Spirochceta balanitidis to balani,tis erosiva
circinata or that of Spirochceta gangrwnosa nosocomialis to ulcus
gangramosum genitalicum has not been definitely established. Several difficulties must be overcome in order to settle this question.
First of all, it is not easy by morphology alone satisfactorily toidentify a spiral organism found in certain lesions with the organisms previously described by these authorities. For example, there* Received for publication, May I5, I912.
1Schaudinn, F., and Hoffmann, E., Arb. a. d. k. Gsndhtsamte, I9o5, xxii, 527;
Berl. klin. Iq/chnschr., I9o5, xlii, 673.
2 Hoffmann, E., and yon Prowazek, S., Centralbl. f. Bakteriol., Orig., I9O6,xli, 74I, 817.
s R6na, S., Verhandl. d. deutsch, dermat. Gesellsch., I9o7, ix, 47I.
* Corbus, B. C., and Harris, F. G., Jour. Am. Med. Assn., I9o9, lii, I474.
5Mulzer, P., Berl. klin. Vr'chnschr., I9O5, xlii, II44.
e Polland, R., I/Vien. klin. Wchnschr., x9o5, xviii, I236.
261

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Pure Cultivation of Spirocha~ta phagedenis.

are certain investigators 7 who fail to recognize any essential
morphological differences between Spirochceta refringens and Spirochceta balanitidis, while others s hold that there are at least four
distinct varieties which were indiscriminately described under thename of Spirochceta refringens. From this it is easily seen how
difficult it is to establish the etiological relation of any one of thoseorganisms to a given definite pathological condition with which it
is associated.
The second difficulty is that up to the present we have been unable to isolate these organisms in pure culture and have, therefore,been unable to determine their pathogenicity. The same lack ofclearness surrounds the organism of Rdna and those of the others.
It appears that only a systematic cultural study of all these organisms can clear up the situation and it is for this reason that I
describe in this article a spiral organism that has been isolated andgrown in pure culture from a rather slowly progressing phagedenic
lesion on the external genitalia of a woman.
Material and Cultivation.--The entire left labium showed anenormous swelling with induration, redness, and slight edema. The
surface was moist with a seropurulent discharge from the vagina.
In the center of the indurated region was an ulcer two by four centimeters and this was covered along its thickened edge with whitish
serofibrinous matter. W h e n touched, the ulcer was painful and it
bled readily. The lesion had been present for about ten days andwas increasing slowly in area. Under local anesthesia a portion of
the ulcerated tissue was removed and used for the cultivation
experiments.
The tissue thus obtained was rinsed thoroughly in sodium citratesaline solution and then ground in a sterile mortar by adding a
fresh lot of citrate solution. In the resulting emulsion, the darkfield microscope revealed numerous irregularly wavy organisms
which had a motility of their own that was slow but unmistakable.
The length of the spirochmt~e varied between four and thirty
Rille, Miinchen. reed. Wchnschr., 19o5, lii, 1377; Kraus, A., Arch. f. Dermat.
u. Syph., 19o6, lxxx, 255.
8Eitner, E., Mi~nchen. reed. Wchnschr., 19o7, liv, 770; Richards, G. IV[.O., andHunt, L., Lancet, 19o6, i, 667.

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263

microns, but was usually about fifteen microns ; the width was abouto.75 of a micron. The number of waves varied greatly, somehaving as many as eight and others only one or two, and some werealmost straight. The wave length also varied greatly in different
spiroch~et~e, Very frequently the organism took the form of theinterrogation point. The ends were definitely pointed although not
sharply drawn out. The short spirocha~t~e bent their bodies very
slowly almost to a semicircle and then stretched out suddenly witha whipping motion. Some forms moved the terminal portion of thebody to the left and right very much as an earth worm moves its
head. The organisms were stained by Giemsa's stain, but did not
retain the Gram stain.
The cultivation experiments with the material described above
were carried out by means of a special medium and procedure described by me 9 for obtaining pure cultures of Treponema paUidum,
Treponema mucosum, and Spirochceta refringens directly from contaminated human materials. Briefly stated, a number of ascitic
agar tissue tubes were inoculated with the emulsion by means of asterile capillary pipette. A f t e r inoculation the ~ubes were covered• with sterile paraffin oil and incubated at 37 ° C. After a few days
the stab canals of ~these tubes were filled up with the dense whitishcolonies of the bacteria (chiefly staphylococci), but the rest of theagar column remained perfectly clear. At the end of one weeksome tubes commenced ~o show a very faint haze at various
points along the stab canal, suggesting a colony of the pallidum.
The intensity and size of the haze gradually increased, until, after
about two weeks, ,there was an unmistakable growth of someorganism. I left the tubes un6isturbed for four weeks andthen examined them to determine the cause of this hazy appearance. When e~he hazy portion of the media was taken out
with a capillary pipette and examined under the dark-field microscope it showed a mass of rather heavy spiral organisms and alarge number of round refractive bodies. Some of the organisms
still showed distinct, apparently normal outlines, while others weregranular and partly disintegrated. The round bodies were doubtless derived from the breaking up of these partly disintegrated
DNoguchi, It., orour.Exper. Med., I912, xv, 9o, 466.

264

Pure Cultivation of Spirocho~ta pl~agedenis.

forms. Transplants were made at once to a series of fresh culture
tubes (ascitic agar tissue medium) in order to purify the culture.
After several successive transplants a pure culture of the organism
was finally obtained, and during the ten months that have elapsed
since its purification the culture has passed through twenty subcultures.
Properties of Pure Cultures.--In the ascitic agar tissue medium
a faint haze appears around the tissue within forty-eight hours
and gradually extends upwards. The density of the growth increases, and within ten days it can ,be recognized withom difficulty(figure I). The organisms grow at 37 ° C., but not at 15 ° C. The
strain is still transplantable after having been cultivated for threemonths at 37 ° C.
The length of different organisms varies considerably. In young
cultures the majority of the spiroch~etm measure ten to fifteen
microns and show only one or two waves (figures 2, 3, 4, and 5).
In old cultures many organisms a,ttain a length of twenty to thirty
microns, and the waves are more distinct and numerous (figure 6).
The curvature and alternation of the waves are very irregular, andthe changes in form are very gradual (figures 7, 8, and 9). The.
width of the body measures about 0. 7 to 0.8 of a micron and is notalways uniform throughout the entire length. In a long form there
may be seen a nodular swelling and constriction at the middle orat each third of the body. The organisms usually have ends thatare fairly pointed, although they may end obtusely. Young forms
are frequently straight, but they may bend their bodies into parabolic
curves, and then suddenly straighten out again. They also have asluggish forward movement like that of a creeping worm. The
terminal portion of the body is quite flexible and is moved like afeeler. Longer individuals are less motile. There is no flagellum,termin,al projection, or undulating membrane. In a very old culture the majority of the organisms become granular and finallydisintegrate into small fragments. At the same time there appear
a large number of spherical bodies measuring 1. 5 microns in diameter. These round bodies are often attached to the ends or the sidesof short forms that are still well preserved (figures IO and I I ) .
When stained with Giemsa's solution these bodies show a small dot

H i d e y o Noguchi.

265

of chromatic material at one pole, but do not take the ordinary
bacterial spore stains. Another interesting feature of the organisms
is that, under certain undetermined cultural conditions, they produce
during disintegration numerous round or ovoid bodies of varying
sizes, some as large as three microns in diameter, scattered singly, inpairs, or in clusters (figure 12). At one side of the protoplasmic
mass of these bodies a careful examination reveals the presence ofa highly refractive spot. In some of the larger forms there are twoo f these spots. In certain cultures there were transitions between
the spherical bodies and those that contain one or two highly refractive spots in their more solid appearing protoplasm. The highly
refractive points of the latter take a deep red stain with Giemsa'ssolution. Forms were also observed which showed more or lessirregular refraction in different parts of the spiral body. Thiswas possibly due to cross-bar concentration of the chromatin before
the granular segmentation of the organism. In a stained preparation these refractive portions take a deep nuclear stain.
The organism is difficult to stain with most anilin dyes and does
not retain the Gram stain. With Giemsa's solution it stains red.
It is a strict anaerobe and requires for its growth the presence of:fresh sterile tissue in the ascitic fluid agar (the serum .of rabbit orsheep is unsuitable). The protein constituents of the tissue andascitic fluid are not visibly altered through its growth, but a distinct
putrefactive sour odor somewh'at resembling butyric acid develops
in the culture.
P a t h o g e n i c i t y . - - W h e n introduced intradermically into a M a c a c u sr h e s u s monkey and into rabbits an acute inflammation follows
within twenty-four hours, but the reaction subsides in about threedays without causing an ulcerative process. In the testicles ofrabbits it causes a temporary induration which disappears completely within forty-eight hours.
I d e n t i f i c a t i o n o f the O r g a n i s m . - - S i n c e the recent investigationso f Gonder, 1° Gross, n Schellack, 12 Novy, 13 yon Prowazek, 14 Keys~oGonder, R., Centralbl. f. Bakteriol., Orig., 19o9, Mix, 19o.
~1Gross, J., Mitt. a. d. zo61. Station z. Napel, 191o, xx, 41.
~*Schellack, C., Arb. a. d. k. Gsndhtsamte, 19o8, xxvii, 364; 19o9, xxx, 351.
~sNovy, F. G., and Knapp, R. E., lour. Infect. Dis., 19o6, ill, 291.
•4yon Prowazek, S., Centralbl. f. Bakteriol., Orig. , 19o8, xlvi, 229.

266

Pure Cultivation of Spirocha~ta phagedenis.

selitz, 15 Swellengrebel, a6 a n d others, the classification of a spiralorganism has become considerably more difficult. These authorities have shown that typical spiroch~et~e may fail to possess all thecharacteristics formerly regarded as differentiating the spiroch~et~efrom the spirilla; e. 9'., longitudinal fission, presence of an undulating membrane, absence of flagellum, etc.
In differentiating spiroch~et~e from spirilla, the importance once
attributed to the mode of division has been still further decreasedby the discovery of Sehmeidlechner;1~ that a group of bacilli multiplies by longitudinal division.
The flexibility of the body was also once considered to be characteristic of spiroch~et~e, but the organisms of the relapsing fevers(hitherto called spiroch~et~e or spirilla indiscriminately) seem to haveas much flexibility as any of the spiroch~et~e, and yet on account of
the fact that they are supposed to divide transversely, they havebeen called spirilla by certain authorities (Novy, is Fraenkel, 19
Borrel, ~° Lev,aditi, 21 etc.).
It seems that spiroch~etm, and some varieties of spirilla, havecertain features that are possessed by the protozoa, and others thatare characteristic of the bacteria. They occupy, therefore, an intermediary position between the protozoa and bacteria.
All the various spiroch~et~e that I have studied have shown features which are more highly differentiated than those seen inbacteria. For example, in most of the spiroch~et~e we observe
during certain periods of their life the secretion of a small roundbody that stains like chromatin. The organisms often concentrate
the chromatin material at one part of the body and then undergo apeculiar segmentation. The granules thus liberated seem to remain
alive and at certain periods develop into spiral forms. Again, atlo Keysselitz, G., Arb. a. d. k. Gsndhtsamte, 19o6, xxiii, 566.
x~Swellengrebel, N. H., Compt. rend. Soc. de biol., 2907, Ixii, 213; Ann. del'Inst. Pasteur, 19o7, xxi, 448, 562; Centralbl. f. Bakterio'l., Orig., 19o9, Mix, 529.
1, Schmeidlechner, K., Ztschr. ~. Geburtsh. u. Gyniik., 19o5, lvi, 291.
~8Novy, F. G., and Knapp, R. E., loc. clt.
~gFraenkel, C., Berl. klin. Wchnschr., 19o7, xliv, 682; Centralbl. ~. Bakter{ol.,Orig., 19o8, xlvii, 471; Miinchen. reed. Wchnschr., 19o7, liv, 2Ol.
20Borrel, A., Compt. rend. Soc. de biol., 19o6, lviii, I38.
22Levaditi, C., Ann. de l'Inst. Pasteur, 19o6, xx, 593, 924.

Hideyo Noguchi.

267

certain stages of their life some of the spirocha~t~e become bandedor dotted with the chromatin substance.
These primitive nuclear phenomena and also the distinct
differentiation of the cytoplasm of some species into ectoplasm
and entoplasm appear to me sufficiently characteristic to holdthese organisrns together under the group of spirochaetae without classifying them either with the bacteria or with the protozoa.
Upon the basis of the conception just outlined I place my organism
among the spirochaetae, but should further investigation make itpossible to distinguish between spiroehaetae and spirilla, and should
it be evident that the organism is wrongly classified I should like tobe permitted to correct the error.
Returning now to the question of whether the organism here
described has been previously found by other investigators, it appears that the spiroch~etae reported by R6na and Polland bear acertain resemblance to my organism and may possibly be identicalwith it, but I am not justified in concluding that this is the case, andto do so might be misleading; for if my spirochaeta is different, Ishould be giving wrong cultural and biological properties to theirorganism. In order to avoid such confusion I propose to call myorganism Spirochceta phagedenis, a conventional name merely indicating its source, but not necessarily showing its etiological relation
to the lesion. In the event that this particular organism is foundconstantly in the phagedenic ulcers on the external genitalia andnot in other conditions, its etiological significance would be established. Through lack of material I have up to the present been
unable to pursue this subject further.
CONCLUSIONS.

I. A hitherto undescribed spiral organism has been isolated inpure culture from a case of mild phagedenic ulcer on the external
genitalia of a woman.
For this organism the name Spirochreta
phagedenis is proposed.
2. Spirochwta phagedenis is a strict anaerobe and grows in thepresence of fresh tissue in ascitic agar. It produces no apparentchange in the media, but a somewhat offensive odor develops in theculture tube.

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Pure Cultivation of Spirochceta phagedenis.

3. Spirochceta phagedenis incites a slight i n f l a m m a t o r y r e a c t i o ni n the s k i n o f a Macacus rhesus m o n k e y a n d in the s k i n a n d testicles
o f rabbits.
4- I t s etiological r e l a t i o n to the p h a g e d e n i c lesions o n the e x t e r n a lg e n i t a l i a has n o t y e t b e e n d e t e r m i n e d .
EXPLANATIONOFPLATES.

PLATE 32.
FIC. I. A stab culture of Spirochwta phagedenis in ascitic agar tissue
medium, three weeks old, at 37° C.
Fins. 2 and 3. Smears from a pure culture, three weeks old, stained with
Giemsa's solution.
PLATE 33.
The dark-field appearance of Spirochceta phagedenis from pure cultures.
FIGS. 4 and 5. Young forms with few waves.
FIG. 6. An average form with shallow, irregular curves.
Fins. 7, 8, and 9. An entangled mass of organisms, with occasional sporelike spherical bodies.
Fins. IO and II. Formation of spherical bodies which are seen still attachedto the organisms or already in the free state.
FIG. 12. Plasma granules in an old culture.
FIo. I3. Spirochcta refringens from a pure culture (for comparison).

THE JOURNAL OF EXPERIMENTAL'MEDICINE VOL. XVl.

PLATE 32.

T H E J O U R N A L OF E X P E R I M E N T A L MEDICINE VOL. XVl.

PLATE 8:3.