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Journal of Experimental Medicine

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CULTIVATION OF TREPONEMA CALLIGYRUM (NEWS P E C I E S ) F R O M C O N D Y L O M A T A O F MAN.*
BY t t I D E Y O N O G U C H I , M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)
PLATES 6 A N D 7.

While engaged in the cultivation of various species of spiroch~etm
found in the oral cavity or about the lesions on the external genitals
and the anus of human beings, I encountered on two occasions a
spiroch~eta which morphologically occupies an intermediary position
between Treponema pallidum and Spirochceta refringens. The material was taken in one instance from a condyloma latum in which
both the pallidum and this organism were present. In another instance, it was found in a condyloma situated around the anus of aboy, nine years of age, in whom the existence of syphilis was firlally
ruled out by clinical and serological examinations. 1 In this case,the condytomatous tissue was excised and inoculated into the testicles of three rabbits by the usual method, but no lesion resulted
during a period of four months. An emulsion of the tissue examined under the dark-field microscope showed some contaminating
bacteria, but no other spiral organisms save the one under consideration. In the sections stained by the Levaditi silver method, thespiroch~et~e were found on the surface, but not within the tissue.
No pallidum was detected. The spirochmta in question could hardly
be mistaken for Treponema pallidum as it appeared decidedly
thicker than the latter, ialthough some specimens had quite deep andregular cur~es. The extremities w e r e obtuse or sharply tapered,and some possessed delicate, finely curved projections at o n e o r bothends. The movements were chiefly those 0f rotation around theaxis, but there was also a change in the form of the curves. Neither* Received for publication, October I, 1912.
XA medico-legal case.

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Cultivation of Treponema calligyrum.

a vibratory nor lateral movement such as is common with Treponema
rnacrodentium occurred. The spirochmt~e measured about 0.35 to
0. 4 of a micron in width, and ranged from a few microns to twenty
microns in length. The number of curves was determined by thelength of the organism, and in typical specimens the distance between the curves was usually about01.6 microns. The curves were
not as closely set as in the pallidum. Compared with Spirochcta
refringens it was somewhat thinner, more regular, and possessedmore numerous curves and showed less motility. Stained with
Giemsa's solution, it was red with a faint violet tint.
Cultivation.--Emulsions of the condylomatous tissue were usedin both cases. The method employed consisted in inoculating aseries of tubes containing a mixture of ascitiC fluid (one part) andslightly alkaline agar (two parts) containing a piece of fresh sterile
tissue at the bottom of each tube. The introduction of the emulsion was made with the aid of a capillary pipette connected with asyringe. After inoculation the tubes were covered with a layer ofsterile paraffin oil and placed in a thermostat at 37 ° C. At the endof ten days, a hazy zone radiating from the central stab canal,
which was now densely filled with the growth of bacteria, appearedalong the canal up to three centimeters of the surface.
Purification.---After two weeks' growth some of the best tubes
were selected and the organisms in the hazy zone were examined
under the dark-field microscope. The colonies from the first case(syphilitic) revealed the presence of two distinct varieties of spiroch~etm; namely, a thin, regularly and deeply curved organism, anda thick but also regularly curved one. The thin type was undoubtedly the pallidum and the thick one the spiroch~eta in question. The
colonies developing in the tubes inoculated with the material fromthe second case (non-syphilitic) showed the presence of only one
type of spiroch~eta which was in all respects identical with the form
found in the original material.
I was able to separate these organisms from the bacteria. 2 Whilecolonies of the syphilitic material contained both the pallidum andthe second organism in the first generation, the pallidum type finallydisappeared in subsequent subculture, thus leaving the thicker type2Noguchi, H., Jour: Exper. Med., 1912, xv, 9o.

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in pure condition. The purified culture of the second or non-syphilitic material yielded solely colonies of this organism.
Properties of Pure Culture.--~Vhen transplanted into the ascitic
agar tissue medium, it develops s.o far in three or four days as toshow hazy, somewhat opalescent colonies about the tissue. During
the following several days the colonies gradually spread towards theupper part of the medium, thus rendering the latter unevenly filledwith hazy opalescent colonies. At the end of four weeks the growth
usually reaches the greatest height that it ever attains,--about two
centimeters from the surface. The colonies are diffuse and usuallysomewhat more dense than those of Treponema pallidum. The
organism grows well in fluid medium containing ascitic fluid whencultivated by the special method described for Treponema pcdlidum. 3
The growth of this spiroch~eta does not bring about any visible
change in the medium and there is no perceptible odor produced.
Like Spirochceta refringens it is capable of growing in ascitic agar
without the addition of fresh sterile tissue, although the growth isfar bettei" in the presence of the tissue. The organism is a strict
anaerobe. There is no growth at room temperature.
Morphology.--(Figures I, 2, 6, 12, 13, and 14.) In solid cultures three or four days old the majority of the organisms are shortand vigorously motile, while in cultures which have been growing
for about two weeks they are longer and less active. In fluid
medium long specimens as well as short ones are present from the
beginning. The length as well as the width of the organisms ismore variable in the flu~id cultures.
The spirochmta in pure culture measures about 0.35 to 0.4 of a
micron in width and 6 to 14 microns in length. Forms as short as 4
microns or as long as 3 ° microns, according to the age and nature ofcultures, may occur. In old fluid cultures, individuals measuring
in width as little as 0. 3 or as much as 0.45 of a micron are apt toappear. The curves are very regular and quite deep. The distance
between the curves is about 1.64 microns and the depth of a curveis about I to 1. 5 microns. The apex of curves is not angular, but
Noguchi, Jour. Exper. Med., 1912, xvi, 211.
4The wave length of Treponema pallidum measures about 1.2 microns andthat of Treponema microdentium about I micron.

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Cultivation of Treponema calligyrum.

more or less rounded. In the short specimens the curves are lessconstant a n d often obliterated through movements of the body.
While the curves of less motile specimens remain unmodified during movement, those of vigorously active specimens change theirform in an undulatory manner. A small number of individuals
have shallower curves or become almost straight, especially near the
middle portion of the body; but I regard these as the irregularities
due to the abnormal effect of artificial cultivation. The body has
a uniform width until toward the terminal portion which ends ina sharp point. In this respect it resembles gpirochceta refringens inpure culture, and not Treponema macrodentium, whose body gradually thins out towards t'he ends (figure 8).
The spiroch~eta shows often a delicate, regularly curved, rather
stiff projection attaclled to one or both ends. The length of thisappendage varies in different specimens, but usually measures about
six microns. The presence or absence of this projection seems
tO depend greatly upon cultural Conditions and the age of the cultures. It has appeared oftenest in fluid cultures about two weeks o,ld.
The varieties of movements are chiefly rotatory, sometimes undulating, and seldom lashing. I have never observed lateral vibration such as is commonly seen in active specimens of Treponema
macrodentium, 5 Spirocha~ta obermeieri, 6 and Spirochceta gallinarum, ~ in puke cultures.
The organism stxins reddish violet by Giemsa's solution,- faintly
red by earbol fuchsin, but not by Gram's method.
Division.--Longitudinal division has been observed, but transverse division may also occur.
Pathogenicity.--Repeatedattempts to produce lesions with purecultures :of this organism in the skin of monkeys (Macacus rhesus,
Cercopithec~is callitrichus, Papio babuan), arid in the testicle orcornea of rabbits resulted negatively.
Differentiation from Allied Species.--There is no difficulty indifferentia~cing this organism from Treponema microdentium s andNoguchi, Your. Exper. Med., 1912, xv, 81.
6 Noguchi, idem, 1912, xvi, 199.
Noguchi, idem, 1912, xvi, 62o.
s Noguclai, idem, 1912, xv, 81.

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93

Treponema mucosum, 9 as both the latter are m~lch smaller and produce anunpleasant odor in culture (figures I0, II, and 18.) Differentiati.on from Treponema pallidum in culture offers no di~cultywhen one is dealing with the average and thinner types of the pallidum, 1° because this ,organism is altogether too thick to be confused
vcith the average specimens of pallidum. On the other hand, a certain precaution is required when one is dealing with a strain of pallidum belonging to the thicker type. 11 In this instan,ce, especiallywhen one I~cks means of compar, ir~g both species in pure culture underexact and parallel cultural conditions, the two are apt to be confused, for it is not impossible that, by providing one species with
one condition and the other with another, the morphological features
will approach each other so nearly that considerable resemblance
m a y result. In my cultivation work this difficulty did not arise, because most of my strains of the cultivated pallidum belonged to theaverage type2 2 Besides, in cases when the heavier pallidum strains
were obtained from the testicular lesions of rabbits, there was no
possibility of obtaining the above species, and when a dubious strain
w a s isolated directly from human chancres or condylom'ata, onlythat which produced characteristic lesions in animals (monkeys)
was designated as the pallidum. 1.
Since obtaining two strains of this organism in pure cultures I
have compared it with the thicker type of the pallidum under parallelcultural conditions. The study showed that under the same conditions even the thick type of pallidum is still decidedly thinner than
the specimens here described. U n d e r unfavorable conditions, especially in fluid medium, the width of the pa.llidum (thick type) may
reach in some Specimens that of the thinnest of the spiroch~et~e inquestion, but the majority of pallida h'ave a width of about 0. 3 ofa micron, while in the latter 0.4 to 0.45 is the average. In suitable
solid medium, the thick pallidum never exceeds 0.3 of a micron,while the other specimens measure usually 0.35 to 0.4 of a micron,with few exceptions. Moreover, the curves of the pallidum are
9Noguchi, dour. Exper. Med., 1912, xvi, 194.
~oNoguchi, idem, 1912, xv, 2Ol.
Noguchi, idem, 1912, xv, 2Ol.
Noguchi, idem, 1912, xv, 2oi.
Noguchi, idem, 1912, xv, 90.

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Cultivation of Treponema calligyrum.

more closely set and m o r e rigid than those o f the present organism.
A n o t h e r point o f difference between the two species is that in ascitic
a g a r the p allidum never grows without fresh sterile tissue, while
the other can g r o w without tissue. 14
T h e next species entering into consideration are Spirochwta~ balanitidis 1~ and refringens and Treponema macrodentium. Spirochcetabalan.itidis is described as having a wiSth o f 0. 5 to 7-5 microns
and a length o f IO to 12 microns. Its curves are r a t h e r regular andquite deep. Besides, the balanitidis is described as having a bandlike body, an undulating membrane, and a periblastic projection.
T h e present species is decidedly thinner, has a cylindrical body, andno undulating membrane. Spirochwta refringens 16 is much coarser
and has wavy, irregular curves, although in pure cultures the curves
m a y be quite regular in some specimens (figure 9)- T h e appearance o f the g r o w t h and the cultural conditions required aresimilar f o r the refringens and the present species. Treponema
macrodentium 17 resembles the present organism in length, but differs f r o m it because o f slightly less width, tapering body, i r r e g u l a r
curves, creeping and lateral v i b r a t o r y movements (figure 8 ) .
W h e n viewed under the dark-field microscope there is no difficultyin distinguishing them. F u r t h e r , the m a c r o d e n t i u m in pure culture
never grows without the tissue.
Identification.--In the foregoing pages I have distinguished thisorganism f r o m Treponemata pallidum, microdentium, mucosum,
and n~acrodentium, and f r o m Spirochceta refringens by means ofmorphological and biological properties present in pure cultures. It
14Whether or not a strain of pallidum which has suffered a marked morphological and biological deviation from the origin M culture, such as appears to be
the case with the strain I sent to Paris, will grow in a medium without tissue, I
cannot say. This strain in my possession still remains unmodified since its isolation from a condyloma latum a little over a year ago. The preservation of morphological and biological characteristics in my hands is undoubtedly due to themore strictly parasitic condition provided in my culture medium, while the deviation of the same strain from the original type in the hands of Levaditi andDanulesco (Compt. rend. Soc. de biol., 1912, lxxiii, 256) can easily be explainedby the modification'of cultural methods and media adopted by these authors.
~ Hoffmann, E., and yon Prowazek, S., Centralbl. ~. Bakteriol., Ite Abt., Orig.,
19o6, xli, 741.
~*Noguchi, Jour. Exper. Med., 1912, xv, 466.
a' Nognchi, idem, 1912, xv, 81.

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95

was further differentiated from Spirochceta balanitidis from the description and photographs given by Hoffmann and yon Prowazek.
The question now arises whether or not the present species corresponds with some oflaer organisms described by previous investigators.
Mulzer in 19o 5 described a small spiroch~eta res,embling thepallidum occurring in cases of ulcerating carcinoma of the uterus orbreast and proposed the name of Spirochceta pseudopallida, is This
organism m a y have been identical wi~h the variety found in casesof carcinoma by Hoffmann, 19 Krienitz, ~° Loewenthal, 21 Kiolemenoglou and yon C u b e y Sch,oltz, ~3 Loewy, 24 and Schmofl. 25 Kr~enitz'sorganism is called oCpirochceta microgyrata~ while that of Hoffmann
is called Spirochceta gangrvence carcinomatosce. The lack of thedescriptions regarding their behavior in the living state and ofother biological properties renders it impossible to idemify t~hepresent organism with any of these. In 19o 7 Veszpr6mi ~6 proposed
the name Spirochceta gracilis for a small organism found in an
ulcerating neoplasm of the j a w which communicated with the buccal
cavity. The description of the morphology and the figures of theorganism make it probable that the organism described by him
belongs to the dentium varieties and is especially allied to Treponema
macrodentium. At all events, Spirochceta gracilis cannot be identified with the present organism. In 19o 9 Levaditi and Stanesco ~obtained in mixed cultur,es a spiroch~eta from syphilitic as well .as
r~on-syphilitic lesions and pus occurring on the human genitalia.
Mulzer, P., Berl. kiln. Wchnschr., 19o5, xlii, 1144.
19Hoffmann, E., Berl. klin. Wchnschr., 19o5, xlii, 880; Deutsch. reed. Wchnschr.,
19o6, xxxii, 967; Atlas der fitiologischen und experimentetlen Syphilisforschung,
Berlin, 19o8.
,oKrienitz, W., Centralbl. f. Bakteriol., zte Abt., Orig, 19o6, xlii, 43.
Loewenthal, W., Berl. klin. Wchnschr., 19o6, xHii, 283.
22Kiolemenoglou, B., and yon Cube, F., Mi~nchen. reed. Wchnschr., 19o5, lii,
1275.
Scholtz, W,, Deutsch. reed. VJchnschr., 19o5, xxxi, 1487; 191o, xxxvl, 215.
Loewy, K., ~trch. ~. Dermat. u. Syph., 19o6, lxxxi, lO7.
25Schmorl, G., Mi~nchen. reed. Wchnschr., 19o7, liv, 188; Deutsch. reed.
Wchnschr., 19o7, xxxiii, 876.
~*Veszpr6mi, D., Centralbl. ~. Bakterlol., ~te Abt., Orig., 19o7, xliv, 332, 408,
515, 648.
,TLevaditi, C., and Stanesco, V., Compt. rend. Soc. de blol., 19o9, Ixvii, 188.

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Cuttivation of Treponema ccdligyrum~

This .organism was described as having a width of o.33 to o. 5 of a
micron, a length of 3-5 Co 15. 5 micron.s, as presenting fairly regularcurves, and as being more actively mo.tile than the pallidum and.staining bluis'h red with Giemsa's solution. As regards pathogenicity no
conclusion could be drawn, as they used mixed cultures to produce
an acute in.flammation of the prepuce of a chimpanzee, and did not
rule out the part played by contaminating bacteria in the process.
They ascribe the inflammatory process to their spirochmta, which
they call Spirochceta gracilis. They did not identify their organismwith Veszprdmi's, previously so named, or make any reference tohis previous work. Thus if Levaditi and Stanesco applied thisname to their organism without knowing that it was already used
by Veszpr6mi, and if their organism was not identical with that
described by this author, they must seek a new appellation. Oll theother hand, if they had identified their organism with that of
Veszpr~mi, ~he species we are describing is clearly different fromtheirs. Judging from the description and also the source fromwhich Levaditi and Stanesco obtained their mixed cultures, I am,
however, inclined to think that their organism was probably identicalwith that described by me in the present article. 2s Even if theirorganism were the same as mine, it would be impossible to accept
the name gpirochceta gracilis for this species as, in doing so, itwould be erroneously identified with that of Veszpr6mi. On thisaccount, and since I have obtained the new species in pure culture
and studied its properties systematically, I feel justified in proposing
as its name Treponema calligyrum. 29
CONCLUSIONS.

I. On the surface of genital or anal lesions, either syphilitic or
non-syphilitic, there may be found occasionally a spiroch~eta resemE. Hoffmann, in his latest publication (Finger, E., H a n d b u c h der Geschlechtskrankheiten, Vienna, 1912, 828), remarks that while Spirochwta balanitidis and£pirochceta refringens are much coarser organisms than Treponema pallidum,
yet there may be found some specimens which are quite thin and may cause difficulty in being distinguished f r o m the pallidum. H e does not state, however,whether the thin forms are to be considered as an independent variety or only
atypical forms of the refringens or balanitidis.
~9I take pleasure in acknowledging my indebtedness to P r o f e s s o r Gonzalez
Lodge of Columbia University for selecting a suitable adjective.

Hideyo Noguchi.

97

bling Treponema pallidum, but somewhat thicker than the latter.
In general characteristics, this organism occupies an intermediary
position between Treponemc~ pallidum and Spirochceta. refringens.
2. The treponema was obtained in pure culture from two cases.
For the organism the name of Treponema calligyrum is proposed.
3. Treponema: calligyrum is non-pathogenic for monkeys andrabbits.
4. Treponema calligyrum can be distinguished from Treponema

pallidum, Treponema rnicrodentiu,m, Treponema. macrodentium,
Treponema n,ucosum, and Spirochceta refringens either by themorphology, cultural and biological properties, or all these conditions in combination.
5. Under certain cultural conditions Treponema calligyrum becomes thinner and may cause some diffictdty of differentiation from astrain of the thick type of Treponema, pallidum, especially when thelatter is grown under conditions which tend to render it thicker.
There is no such .difficulty when the average and thin types ofTreponema pallidum are concerned. On the other hand, by providing the calligyrum with conditions which lead to the appearanceof thicker specimens and by supplying Spirochceta refringens withconditions of growth that favor the development of individuals thatare thinner and more regularly curved, the refringens may be madeto resemble the calligyrum in general appearance; but under iderrticalcultural conditions they can be readily distinguished.
EXPLANATIONOF

PLATES.

PLATE 6.
FIGS. I and 2. Treponema calligyrum in pure culture, ten days old, in solid
medium. Giemsa stain. X I, IOO.
FIGS. 3 and 4. Treponema pallidum in pure culture, four weeks old, in fluid
medium. Giemsa stain. X 1,1oo.
FIG. 5. Treponema pallidum f r o m orehitis of the rabbit. Giemsa stain.
X 1,100.
PLATE 7.
FIGS. 6 to II. Schematic reproduction of dark-field views of various speciesof T r e p o n e m a in pure culture. FIG. 6. Treponema calligyrura; FIG. 7. Treponerna pallidum; FIG. 8. Treponema macrodentium; FIG. 9. Spirochceta refringens;
FIG. IO. Treponema microdentium; Fig. 11. Treponema mucosum.

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Cultivation of Treponema calligyrum.

FI~S. 12 to 14. Treponema calligyrum in pure culture, ten days old, in solid
medium. Dark-field view. X I,IOO.
Ft~s. 15 to 17. Treponemapallidum in pure culture, four weeks old, showing
morphological variations among different strains. Dark-field view. X I,IOO.
FI~. 18. Treponema microdentium in pure culture, ten days old, in solid
medium. Dark-field view. X I,IOO.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL, XVII,PLATE 6.

(NOGUCHI: Cultivation of Tre#onema calligyrum.)THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVll.

PLATE 7.

(NoGucaI: Cultivation of Treponema calligyrum.)