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Journal of Experimental Medicine

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A M E T H O D F O R T H E P U R E C U L T I V A T I O N OF
PATHOGENIC TREPONEMA PALLIDUM (SPIROCH.tETA P A L L I D A ) . *BY H I D E Y O NOGUCHI, M.D.

(Fron~ the Laboratories of the Rockefeller Institute for Medical Research,

New York.)
PLATES 8 TO 12.

Schereschewsky ( I ) , in 19o9, was the first to show that Treponema pallidum (Spirochceta pallida) might be cultivated. His
method consisted in inserting a piece of human tissue containing
pallida deep down into a high layer of gelatinized horse serum.
In this medium, the gelatinized serum commences to liquefy about
the tissue and the spiroch~et~e grow together with bacteria whichwere introduced with the inoculated material. Schereschewsky,
however, never succeeded in obtaining a pure culture of the pallidum.
Miihlens (2), in 191o, and Hoffmann (3), in 1911 , both utilized
Schereschewsky's method for obtaining first generations of the pallida and later succeeded in purifying their strains by means of
M/ihlens's horse serum agar. While the spiroch~et~e cultivated bySchereschewsky, Mfihlens, and Hoffmann exhibited the typical mor. hological features of the pallida, these investigators, nevertheless,were unable to reproduce syphilitic lesions in animals by means ofeither pure or impure cultures. Subsequently, Schereschewsky
stated that he had never been able to cultivate a virulent strain of
the pallida, while Mfihlens employed the term "pallida type" todesignate his strain because it possessed no pathogenicity whatever.
On the other hand, Bruckner and Galasesco (4), in 191o , andSowade (5), in 1911, reported that they succeeded once in reproducing syphilitic lesions in rabbits by the injection of rather large
quantities of " y o u n g impure cultures" derived from gelatinizedascitic fluid or horse serum media which still contained the originalsyphilitic tissues. Neither investigator, however, was able to grow
* Received for publication, June 8, 1911.
99

100

Pure Cultivation of Pathogenic Treponema paUidum.

a second generation of these so-called cultures in any medium;
hence the question must remain open as to whether the successful
production of the syphilitic lesions was due to actually cultivatedspiroch~eta~ or simply to the original spiroch~et~e that had survived.
While there are few today who would deny that Treponema
pallidum is the causative agent of syphilis, yet the final proof canonly be brought forth through the reproduction of syphilitic lesions
by means of pure cultures of the micro6rganisms. This had been
done by no one up to the time of the present report.
In the present communication, I desire to report the positive
reproduction of syphilitic lesions in rabbits by means of pure cultures of some of the several strains of Treponema pallidum which
I obtained during I9IO and I9II.
MATERIALS FOR OBTAINING THE CULTURES.
Unlike all previous investigators, who used syphilitic tissues directly from human cases, I have employed the spirochmta~-containing testicular tissue of rabbits which had been inoculated withhuman syphilitic material for the purpose of cultivating the pallidum.
The rabbit testicle, when inoculated with human material rich inpallida, swells gradually after a fortnight's incubation period andreaches its maximum, as a rule, in from four to six weeks. The
testicle not only swells but its consistence becomes considerably increased through infiltration of cellular elements due to the presenceof the pallida, which, at this period, reach an enormous number.
From this first generation in rabbits any number of generations
can subsequently be derived by transmitting the strain, at appropriate intervals, from rabbit to rabbit. The use of rabbit material
offers at least two important advantages over human material;
namely, ( I ) the ease with which large quantities of spirocha~ta-containing material may be obtained at any time, and (2) the fact thatthe spiroch~et~e are almost free from banal bacteria, especially after
several passages through rabbits.
In my present work, I used ten different strains of pallida whichwere passed into rabbits through many generations. One of thesestrains was transmitted from man to the rabbit by Captain Henry

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101

J. Nichols, of the United States Army, while working at the Rockefeller Institute ; the remaining nine strains were obtained by me.
CULTUREMEDIA ANDMETHODSOF C U L T I V A T I O N .

Various culture media were tried, but without success. These
media were pure rabbit plasma, oxalate plasma of rabbit and horse,with and without the addition of calcium chlorid (for removal ofoxalate), a plasma and neutral agar mixture, a plasma and gelatin
mixture, serum water, serum agar, serum bouillon to which various
protein cleavage products were added (tryptophan, asparagin,
glycil-glycin, alanin, etc.), and finally the gelatinized horse serum
prepared according to Schereschewsky.
The only suitable medium in my hands is a serum water (sheep,
horse, rabbit) to which a piece of sterile rabbit tissue is added.
The rabbit tissues best adapted to this purpose are the kidney andtesticle. The heart muscle is also good, but liver is unsuitable,
since it is not only difficult to obtain in a sterile condition, but evenwhen sterile, supplies enough carbohydrates to render the medium
strongly acid in cases where certain bacteria are simultaneously
cultivated. I usually use tubes 20 centimeters high and 1. 5 centimeters wide and fill them with 16 cubic centimeters of serum water(I part serum and 3 parts distilled water). After the completionof the usual fractional sterilization at IOO° C. for three days (fifteen minutes each day), a small piece of freshly removed sterile
tissue is placed in each tube. The tubes are incubated at 37 ° C.
for two days and then examined for their sterility. To each tube
a layer of sterile paraffin oil is now added in order to shield the
medium from contact with the air and to prevent evaporation.
With regard to the method of cultivation, it should be stated thatstrict anaerobic conditions are very important in obtaining the first
generation of pallida. For this reason, I employ a combination ofhydrogen gas, vacuum and pyrogallic acid in an anaerobic apparatus.
With the fresh tissue at the bottom of the culture tube (Theobald
Smith's principles for anaerobiosis) and the above apparatus, acomplete anaerobic condition is obtained. There may be certain(denaturalized type) strains of pallida which grow under less

vae,

[lie

TEXT-FmtlR~ I shows the apparatus in which three conditions can be combined. The inoculated test tubes are placed inside of a jar which already contains a solution of pyrogallic acid at the bottom. Hydrogen gas is first passed
from the tube indicated through the pyrogallic acid solution, for about ten
minutes. A f t e r the air is driven off through the other tube (with stop-cock),the jar is sealed at both ends. The jar is then connected with a vacuum forabout thirty minutes, in order to exhaust the air. When exhaustion has reached
its maximum a concentrated solution of potassium hydrate is allowed to flow infrom the tube indicated. Before the use of the potassium hydrate, by passinghydrogen the solution is freed from oxygen as much as possible. The
potassium hydrate solution is now mixed with the pyrogallic acid solution in thejar. If there is any trace of oxygen inside the jar the mixture will quickly turnbrownish or even dark brown, but if there is no oxygen it will remain almost
colorless for at least many hours, when a light brown color may appear. The
jar is now closed, and once more hydrogen gas is passed through the mixture.
Before putting the jar in the thermostat it is best to exhaust the air once morebecause this collapses the rubber tubing at one end, and a leak in the jar caneasily be detected by the disappearance of the collapse (negative pressure) of
this end. I have found it advisable to pass hydrogen gas through the inoculated
culture media by means of a long, sterile capillary pipette for about five minutes
before they are put in the anaerobic jar.

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103

strictly anaerobic conditions (such as are furnished by Schereschewsky's and Miihlen's media), but these stains seem to be nonpathogenic, at least so far as is indicated by the cultures of Schereschewsky, Mfihlens, and Hoffmann.
To obtain the first generation of the Treponema pallidum invirulent form, the following conditions are essential: ( I ) the presence of suitable fresh sterile tissue in serum water, (2) strict
anaerobiosis, (3) a slightly alkaline reaction as furnished by theserum and tissue, and (4) a temperature of about 35 ° to 37 ° C.
The presence of agar or gelatin seems to interfere with the successful growth of the first generation of the spirochmt~e. On theother hand, when once adapted to the artificial serum water tissue
medium, the pallidum grows well under less strictly anaerobic conditions. In fact, certain strains even grow well in a medium containing agar, provided that suitable fresh tissue is placed low down
in a high column of medium (serum water or serum agar), and that
it is covered with paraffin oil. Certain pallidum strains refuse to
grow in a solid medium for several generations, and none of mypallidum strains grew in any medium without the presence of tissue.
Thus all the pure pallidum strains are being cultivated in a tissuecontaining medium. In this respect they differ greatly from thosedescribed by Miihlens and Hoffmann, since the spiroch~et~e of theseinvestigators grew without difficulty in various media without tissue and even without serum.
T H E RESULTS OF CULTIVATION EXPERIMENTS.

Out of ten strains of Treponema pcdlidum, six have been cultivated in the serum water tissue medium. The first strain was
obtained in October, I9IO, and it has just reached its twenty-fifth
generation, while the remaining five strains were cultivated during
the last six months. All the strains are now going on very well.
It must be emphasized that to obtain these six cultures almost innumerable series of unsuccessful cultivations were carried out
with each strain before one suddenly started to grow for the firsttime. I am still unable to explain why it is so difficult to obtaina first' generation of culture with any pallidum strain; but probably
the strictly parasitic nature of this organism is accountable.

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Pure Cultivation of Pathogenic Treponema pallidum.

Purification.--Except in one instance, the pallidum has grown
together with other bacteria. At first the pallida would .not growin any solid medium and I was unable to purify them from a fluid
culture. After numerous fruitless efforts, I finally succeeded, however, in isolating the pallidum by permitting it' to grow through a
Berkefeld filter. O f course, only certain filters were suitable toprevent the bacteria from growing through at the same time. The
spiroch~et~e were found to have passed through the filter after about
the fifth day. Later it was found that certain strains of pallida
would grow together with the bacteria along the stab canal in aserum agar tissue medium. But while the bacteria did not growout into the surrounding medium, the pallida grew out gradually,
as was indicated by a light, almost transparent zone of haziness.
Thus the spiroch~et~e grew away from the stab canal in a purestate. From this hazy colony, I was able to obtain pure culturesfrom four different impure strains. The purified pallidum grows
well in a serum agar tissue, but even better in a serum watertissue medium. No marked change in the culture medium is observed under anaerobic conditions. The growth does not extend
very far from the bottom, where the tissue lies. I was unable,
furthermore, to cultivate the spiroch~et~e in any medium without
the addition of tissue. The mere addition of glucose to the serum
agar (without tissue) does not enable the pallida to grow. When
compared with the characteristics of the spirocha~t~e cultivated by
Miihlens and Hoffmann, a striking difference is observed, since
their spiroch~et~e grew in plain serum agar, in serum bouillon, andeven in plain agar. Moreover, their strains produced a peculiar
penetrating odor in serum-containing media, while my strains areodorless.
CHARACTERISTICS OF T H E CULTIVATED TREPONEMA PALLIDUM. 1

In the serum water tissue, the pallidum commences to multiply
after forty-eight hours and continues to grow slowly for at least
four or five weeks (probably longer). In the young cultures many
1The descriptions of the flagella-like projections and spore-like bodies frequently observed in the cultivated pallida are given in the explanations of theplates.

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105

short and rather heavy specimens are seen, some with only a few
curves. In a culture ten or twelve days old, the spirocha~t~e are of
the usual length and have typical curves. As they grow older, thepallida may become very long, and often a mass of entangled
pallida is formed. The cultures are quite active though probably
less uniformly motile than the younger cultures. On the other
hand, when cultivated in the serum agar tissue medium, the hazyzone of growth around and above the tissue becomes perceptible
in about three days (sometimes two, sometimes four days). It
becomes more distinct in a week or ten days. Isolated colonies are
seldom formed apart from the tissue. When the inoculation ismade while the medium is still in a melted state (at 45 ° C.), thepallida grow only around the tissue, but no individual colonies
develop. It is in a stab culture only that isolated colonies appear,
owing, undoubtedly, to the communication established by the stabcanal between the colonies and the tissue. There is no coagulation of the serum or tissue constituents through the growth ofthe pallida. The morphology of the pallida cultivated in solid
media is quite typical, and it is difficult to distinguish it from
specimens just taken from human or animal lesions. Their motility
is quite characteristic and there is no difficulty in differentiatingthem from Spirochceta dentium. In young cultures, the length ofthe pallidum is generally less than that of the specimens in the tissue
or in an older culture, and motility is almost undiminished evenafter growth for some weeks. In this point they differ from thestrains of Miihlens and Hoffmann, which became immobile after
about ten days.
Pathogenicity.--So far only two strains of pure cultures havebeen carefully tested for their virulence. Both strains have produced typical lesions in the testicle of the rabbit. The lesion became noticeable about two weeks after inoculation, and the testicles
were excised, under anesthesia, at the end of about four weeks for
examination (dark field, and Giemsa, Levaditi, and the usual
methylene blue and eosin stains). The first strain (cultivated in
October, I9Io, in the mixed state, and purified through a Berkefeld
filter in January, I 9 I I ) was inoculated into eight rabbits on February 2, when a ten day old culture in a serum water tissue medium

106

Pure Cultivation of Pathogenic Treponema pallidum.

was used. Two rabbits showed lesions in the course of two to five
weeks. The testicles were excised under anesthesia on March 3
and showed typical syphilitic orchitis with an abundance of thepallida. The second strain (cultivated impurely in M a r c h - - f r o m
the 6th to 26th--and purified in a serum agar tissue medium on
April 16, I9I I ) was inoculated into four rabbits on April 26, whena seven day old culture in a serum water tissue medium was used.
Three of the four rabbits showed typical lesions in three to five
weeks. The aspirated tissue juice from the swollen testicles contained an enormous number of actively motile pallida. One ofthe positive animals was castrated on May 24, I 9 I I , and thehistological examination revealed the appearance shown in theillustrations.
CONCLUSION.
In conclusion, it may be pointed out that this is the first timethat Treponema pallidum of Schaudinn has been proven beyond all
doubt t'o have been obtained in pure culture. The method of cultivation described would appear also to be suitable for obtaining indefinite generations of the microSrganism. Doubtless slight modifications will adapt it to a larger number of strains and possibly
to the cultivation of all strains and to still other species of treponema. Finally, it may now be accepted as established that thetesticular lesions produced in rabbits by means of syphilitic materials are the result of the multiplication of the pallida and notof some associated indefinite parasite.
BIBLIOGRAPHY.
I. Schereschewsky, J., Ziichtung der Spirochaete pallida, Deutsch. reed.
14/chnschr., 19o9, xxxv, 835; Weitere Mitteilung fiber die Ziichtung der
Spirochaete pallida, ibid., 125o; Bisherige Erfahrungen mit der geziichteten
Spirochaete pallida, ibid., 1552.
2. Mfihlens, P., Reinziichtung einer Spirochiite (Splrochaeta pallida?) aus elnersyphilitischen Driise, Deutsch. reed. Wchnschr., 19o9, xxxv, 1251; ?3bet
Zfichtungsversuche der Spirochiite pallida und Spirochiite refringens sowie
Tierversuche mit den kultivierten Spirochiiten, Klin. Jahrb., I9IO, xxiii,
339.
3. Hoffmann, Beitriige zur Reinziichtung der Spirochaete pallida, Ztschr. f. Hyg.
u. Infectionskrankh., 1911, lxviii, 27.

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4. Bruckner, I., and Galasesco, P., Orchite syphilitique chez le lapin par cultures
impures de spiroch&tes, Compt rend. Soc. de biol., I9IO, lxviii, 684,
5. Sowade, H., Syphilitische Allgemeinerkrankung beim Kaninchen durch intrakardiale Kulturimpfung, Deutsch. reed. I¥chnschr., 1911, xxxvii, 682.
EXPLANATIONOF PLATES.

PLATE 8.

FIG. I. A two weeks old colony of the pallida grown out from an impure
stab culture in a serum agar tissue medium. (Translucent view.)
FIG. 2. A three weeks old pallidum colony grown out from an impure stab
culture in an ascitic fluid agar medium. (A view in reflected light.)
FIG. 3. Two weeks old pallidum colonies in a pure culture grown in an ascitic
fluid agar tissue medium. The colonies are situated along two stab canals.
FIGS. 4, 5, 6, and 7. Treponema pallidum from a ten day old pure culture in. ahorse serum agar tissue medium. Giemsa stain. X 1,28o.
Fins. 8 and 9- Long forms of pallidum from a pure culture. Giemsa stain,

X 1,28o.
FIG. IO. A section of a pallidum colony irL an ascitic fluid agar tissue medium,
Giemsa stain. X 1,28o.
FIG. ii: A characteristic appearance of pallida under the dark-field microscope. From a ten day old pure culture grown in an ascitic fluid agar tissue
medium. The peculiar radiating arrangement of the pallida is due to the effort
of each pallidum to liberate itself from the entangled mass and it can, be observed within about five minutes after the slide is prepared. The mass breaks up
gradually and the constituent motile pallida scatter singly or in smaller groups.
(Semi-schematic.)
PLATE 9.

FIGS. 12, 13, 14, and 15. A three weeks old impure culture of pallida in a sheep
serum water tissue medium. The figures show the extraordinary morphologicalconditions of the organism. Figure 13 shows a mass of entangled pallida.
Giemsa stain. X 1,28o.
FIGS. 16 and 17. A ten day old impure culture in a similar medium. Giemsa
stain. X 1,28o.
FIG. 18. A seven day old impure culture, showing many short forms. Giemsa
stain. X 1,28o.
FIG. 19. A delicate, typical form from a two weeks old impure fluid culture.
Giemsa stain. X 1,28o.
FIGS. 2o and 21. Pure cultures of pallida in a sheep serum water tissue
medium (ten~ days old). Giemsa stain. X 1,28o.
PLATE IO.

FIGS. 22 to 34. From impure pallida cultures in a sheep serum water tissue
medium (twenty-five days old). The figures show that in spite of the enormous
length of the pallida, the curves are typical. Of special interest may be the ringformation shown in figure 23. Figure 34 shows a peculiar short, but acute,curvature. Burri's Indian ink preparation. X 1,28o.

108Pure Cultivation of Pathogenic Treponema pallidum.
PLA~ IL

FIGS. 35 to 39. Specimens of pallida from experimental syphilitic orchitis of
the rabbit. For comparison with the cultivated pallidum. Giemsa stain. X 1,28o.
Fie. 40. The same stained by Burri's method. )< 1,28o.
Fie. 41. A mass of entangled pallida derived from the syphilitic testicle of
the rabbit. The preparation is made from an emulsion kept for 24 hours at 37 ° C.
Giemsa stain. X 1,28o.
Fit. 42. A section of liver of a congenitally syphilitic fetus. Levaditi
method. X 1,28o. ( F o r comparison.)
FIG. 43. A section of testicle of a rabbit inoculated with the pallida fromhuman chamcre. Levaditi method. X 1,28o. ( F o r comparison.)
FiGs. 44 and 45. Sections of testicles of rabbits inoculated with a seven day
old pure pallida culture grown in a sheep serum water tissue medium. Levaditi
method. X 1,2go.
PLATE X2.
Fro. 46. This figure shows several paltida with delicate flagella-like projections at one or both ends. The projections have very regular curves along their
entire length, and are often seen to assume an angular position with relation to
the pallida. Sometimes more than one projection may be observed at one end.
It is not uncommon to see the projection attached laterally to the body of a shortrod-like form of pallidum. The projections are not seen in most cultures.
Another interesting feature shown in this figure is the presence of peculiar
spore-like round bodies in some pallida. Sometimes several round bodies areseen in a group, and these are entangled in a mass of short, irregular forms ofpallida. It is not rare to find a round body connected with one or two young
pallida as though the latter were just sprouting from the former. The pallida
with these round bodies are motile. The size of this spore-like round body isvariable. (Schematic; dark field.)
FIGS. 47 to 48. The round bodies mentioned above are shown here (Giemsa
stain). They are difficult to stain, but when stained they take the chromatin
stain. The larger ones are more or less irregular in shape and not strictly round;these seem to stain more easily than the small ones. In the right lower side of
figure 47, there is one large body which shows a certain significant structure of
the chromatin substance. The nature of these bodies is still unknown, but there
is a striking similarity between these and a certain stage of development of
Spirochceta balbianii studied by Perrin.

T H E JOURNAL OF E X P E R I M E N T A L MEDICINE VOL. XlV.

PLATE 8.

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. XlV.

PLATE 9.

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XIV

PLATE 10.

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XlV.

PLATE 11.

T H E J O U R N A L OF EXPERIMENTAL MEDICINE VOL. XIV.

PLATE 12.