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Journal of Experimental Medicine

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THE

D I R E C T C U L T I V A T I O N OF T R E P O N E M A P A L L I D U M P A T H O G E N I C F O R T H E MONKEY.*
BY H 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 I2-I4.

I have described, in a previous communication,1 a method for thecultivation of Treponema pallidum from the testicular lesions ofrabbits inoculated with human syphilitic material containing thatorganism. At that time I had failed to cultivate the pallidum
directly from syphilitic lesions in man, a feat that has now beenaccomplished. In cultivating the organisms from the testicularlesions, I found it advantageous to employ first a fluid medium andlater, after adaptation had taken place, to substitute a solid medium.
This is a satisfactory method for the testicular lesions which arenearly free from contaminating bacteria, but it is not applicable tothe highly contaminated primary lesions in human cases of syphilis.
Two conditions are all-important in the working out of the~method of direct cultivation: first, the maintenance of strict anaerobiosis; and second, the property possessed by the pallida of migrating in the solid medium in which they are multiplying. To meetthese conditions, I employed a high cylindrical layer of a solid
medium consisting of two parts of 2 per cent. slightly alkaline agar
and one part of ascitic or hydrocele fluid, at the bottom of which
has been placed a fragment of sterile tissue, such as rabbit kidney
or testicle. I have concluded that the tissue acts chiefly by removing the traces of oxygen that exist in the lower levels of the medium,and probably also by providing a special form of nutriment. The
solid medium, when inoculated with a fluid containing pallida andbacteria, seems to keep the latter fixed to the inoculation stab while* Received for publication, November 25, 1911.
Noguchi, .four. Exper. Med., 1911, xiv, 99.
90

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91

permitting the pallida to migrate to a distance where they may often
be found in a pure or nearly pure state.
Comparisons have been made between the agar medium described
and the gelatinized horse serum employed by Schereschewsk-y,2in favor of the former. The disadvantage of the gelatinizedserum arises from the fact that the bacteria rapidly liquify it, andacids render it more permeable for bacteria. Hence it is very difficult to separate the spirochmtm from the contaminating bacteria.
DIRECT ISOLATION OF T H E PALLIDUM.

Fifteen cubic centimeters of the medium described are placed
into test-tubes measuring two by twenty centimeters. At the bottom
is placed a fragment of sterile tissue. While rabbit kidney or testicle is preferable, human placenta, sheep testicle, or other sterileorgan will suffice. The medium is prepared in the tubes, after the
addition of the tissue, by mixing two parts of the melted agar at
5°0 C. and one part of ascitic or hydrocele fluid. After solidification, a layer of sterile paraffin oil three centimeters deep is added toprevent evaporation of the medium. A considerable number oftubes should be prepared at a time, and they should be incubated fora few days prior to in6culation, to determine sterility.
The material to be cultivated is obtained from lesions rich in thepallidum, such as chancre, condyloma, or skin papule, from which
are snipped off, after cleaning with sterile salt solution, suitable
fragments. These are immersed immediately into sterile salt solution containing in addition I per cent. sodium citrate. The fragment of tissue, which should be carefully preserved against drying,is then cut into small bits which are used as follows: one bit isemulsified in a mortar with the citrate solution; the others areinserted as such into tubes of culture medium, provided the emulsion
shows, under the dark-field microscope, the presence of the pallidum.
Each of the half dozen bits of pallidum-containing tissue is inserted into a tube of the ascitic fluid tissue agar by being forced tothe bottom by means of a thin blunt glass rod or heavy platinum
loop, and into the same tube several drops of the emulsion are introduced by a capillary pipette also inserted deeply into the medium.
Schereschewsky, Deutsch. reed. Wchnschr., 19o9, xxxv, 835, 126o.

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Direct Cultivation of Treponema PaUidum.

Care is taken to avoid tearing the medium, and about six tubes are
inoculated from each specimen of tissue.
The tubes should be incubated at 37 ° C. constantly for two to
three weeks before examination.
The inoculated tubes usually become diffusely opalescent andshow, as well, a dense opaque growth of bacteria along the stabcanal and about the wall of the tube. When considerable gas isformed, the medium is torn. The cultures are now punctured inseveral places with a capillary pipette which is inserted deeply into
the tube, and the withdrawn contents are examined under the darkfield. The rule is to find large numbers of the pallidum imbeddedin the agar and hence immotile. Should the organisms be present
in a space clear of agar, they show active motility. Bacteria arealways mingled at this stage with the pallidum.
The impure culture is fetid and putrefactive. Purification is
begun by inoculating about twelve tubes with the impure culture bymeans of a charged capillary pipette that is carefully inserted in thecentre of the agar column quite to the bottom of the tube. The
contents of the pipette are expressed by means of compressed air,for which purpose I employ a syringe. The medium must not be
split. The inoculated tubes are again incubated at 37 ° C. for two or
three weeks. The bacteria grow in a dense mass along the stabcanal and on the surface of the medium. The tubes are inspected,
from time to time, and in some a faint hazy zone will be seen toradiate from the stab-canal towards the sides of the tube. This
hazy appearance is caused, usually, by masses of the pallidum thatare growing away from the bacteria. When this growth has spreadsufficiently far away from the stab-canal another transfer is made.
The surface of medium is sterilized with sublimate alcohol that is
poured away. The tube is marked about the middle with a diamond
pencil, and to the cut a pointed red-hot glass rod is applied. The
tube cracks and the upper half is removed. The exposed surface ofagar is sterilized with sublimate alcohol, after which the remaining
moisture is wiped away with sterile absorbent gauze. By gently
bending the agar column, it cracks transversely and exposes a clear
surface on which the hazy growth is readily seen. Now, without
touching the central canal, a capillary pipette is introduced into theHideyo Noguchi.

93

haze and, after confirming, by dark-field examination, that it iscaused by the presence of the pallidum, a series of inoculations are
made in the manner described. It' happens rarely that a pure culture is obtained on the first or second reinoculation; usually several
reinoculations are required.
Not infrequently the growths of the pallidum are almost invisible.
I am inclined to think that this is due to the quality of the culture
medium. In this case, the dark-field microscope is the only reliable
guide as to growth and situation in the culture of the pallidum.
Again, the bacteria in the stab-canal may produce so much acidthat it diffuses into the surrounding layer of medium where it causesa haziness capable of being mistaken for growth.
When ascitic or hydrocele fluid is not available, serum from thehorse or sheep may be used for the purification process; they are,however, less satisfactory than the former fluids. It should bestated that I have found some specimens of ascitic fluid entirely
unsuitable to the cultivation of the pallldum. When a favorable
specimen has been obtained, it should be stored in sterile condition
for future use.
P R O P E R T I E S OF T H E P U R E C U L T U R E S .

The pure cultures of the pallidum that I have studied have shown
certain distinctive properties. First, the colonies of the pallidum
that diffuse into the medium are never sharply, but always faintly
visible, and the degree of visibility tends to be less in the pure thanin the impure state. It. seems to be unusual for the colonies tobecome discrete, although this has been observed when the sampleof ascitic fluid has been very favorable. Second, the growth is mostmarked about the fragment of sterile tissue, I have not thus far
succeeded in obtaining growth without fragments of tissue, although
the medium used was similar in all other respects. In this important
respect', the organisms that I have cultivated differ from thosedescribed by Mfihlens3 and by Hoffmann, 4 who obtained growth ina medium devoid of tissue. I have never observed my growths tocause any coagulation of the serum in a fluid medium, or to produce* M'iihlens, Kiln. ]ahrb., I9iO , xxiii, 339.
H o f f m a n n , Ztschr. f. Hyg. u. Infektionskra•kh.,

1911, lxviii, 27.

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Direct Cultivation of Treponema Pallidum.

putrefactive odors, both of which are noted to occur by Mfihlens
and by Hoffmann.
When the medium is favorable, the first indications of growthappear in about seventy-two hours, and these increase gradually for
two or three weeks, when the growth is at its height. The growth
in a fluid medium takes place very slowly but continues for manyweeks. The medium retains its fluid state indefinitely.
Morphology.--In young cultures, the greater part of the spiroch~et~e are short and actively motile. After growing for about two
weeks, they attain the usual length of eight to twelve curves. Active
motility is continued and the typical cork-screw movement is displayed. When the organisms are imbedded in the agar medium,
they are immotilized. After several weeks, not only is the motility
reduced, but the length has been increased (figures I, 2, 3, and 5).
The ends of the mature pallidum often show fine flagellum-like prolongations which are frequently perfectly curved, although sometimes they are devoid of curves (figure 6). The typical morphological characters are subject Co disturbance by violent handling, as
by the careless introduction into the growing mass of the capillary
pipette or the presence of a trace of oxygen. Then the curves appear irregular, and straight forms occur. When irregular andatypical spiroch~et~e appear in certain tubes, they are due to themedium which is not strictly suitable, or to the presence of a traceof oxygen. To secure perfect and typical cultures, optimum condition.s must be supplied.
Multiplication.--Schaudinn 5 described longitudinal division of
the pallidum which he observed in two specimens. However, later
investigators have described transverse division of the organism.
It is important to ascertain which is the primary, or normal, andwhich the secondary, or abnormal, mode of division. That appearances suggesting both longitudinal splitting and transverse division
are observable is admitted: the question relates to the significanceof each process. Those who regard the longitudinal process as thenormal one for multiplication look upon the transverse process as
a last stage in the series of changes; while those who regard thetransverse process as the normal one look upon the appearance of
Sehaudinn, Arb. a. d. k. Gsndhtsamte., ~9o7, xxvi, H.

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longitudinal splitting as caused by the coiling of one spiral organism,
still united by a thin end filament, about another part of the sameorganism which is undergoing transverse division.
The disputed points relate to unstained and stained preparations
directly obtained from lesions in man or animals in which the possibility of artefacts can hardly be removed. In both cases, the conditions are not distinctly favorable. What is demanded are periodsof development of the pallidum during the stage of active division,and this is provided in the pure cultivation at certain stages ofgrowth. I have now studied about ten strains at various stages ofproliferation and growth and under varying conditions of cultivation, so that I have formed an objective conception of the process.
The mode of division of the pallidum in pure cultures is suchas to admit of ready following of the phenomenon under the darkfield microscope. During the period of active proliferation, numerous dividing individuals may be seen in a single field. SeveraI
classes may be constructed according to the stage of the process(figures 7, 8, 9, and xo) : (a) the body is divided at about two
thirds of its entire length; (b) the body is completely divided andone part is wrapped about the other with regular curves or flat
waves; (c) one half the body is divided and the other not, and thedivided half has separated widely so as to produce a figure Y; (d)the partially divided body diverges at angles to produce V forms ofvarying width. When an acute angle is formed, tenuous protoplasmic bridges may span the interval ; (e) two bodies remain united
by a thin filament, one of which may be of normal or abnormal
form; (f) three or more individuals may be united in a series offine filaments suggesting imperfect transverse segmentation; (g)finally, t w o b l u n t wavy individuals each about double the thicknessof an ordinary pallidum may remain together (figures I I, I2, x3, I4,
and 15)- This last form differs from the others in possessing a few
granules that are definitely arranged and aggregated into bands thatrun across the organism. Whether this condition is degenerative
remains to be determined. The longitddinal form of division is met
with in all the cultures irrespective of the length of the spirochmta~. During the dividing process, the curves may be shallow andmerely wavy; but after the separation is accomplished, the curves

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Direct Cultivation of Treponema Pallidum.

become deeper and regular. Motility is preserved during division,
but is perhaps less active (figure I8, A, B, and C).
The various phases and varieties of the dividing process here
described can best be followed when the spiroch~et~e are embedded
at first in the agar medium that is pressed beneath the cover glass
without disturbing their position. When the specimens are thenliberated into a free space in which a fluid exists, the various phases
can be readily traced as they pass from one to another, and in general in the order given, of the series described. Speaking broadly,
the process is not rapid and requires about two hours for completion.
The final result is two symmetrical halves, although asymmetricaldivision has been observed in rare instances.
P A T H O G E N I C PROPERTIES.

Treponema pallidum is a pathogenic organism. While its natural
host is man, it is capable of implantation upon several higher animals,
notably upon the monkey and the rabbit. I have already reported
the successful inoculation of pure cultures of the pallidum, obtained from the testicular lesions of rabbits, to the testicles of normal
rabbits. 6 Hoffmann7 reported somewhat later the successful production of a specific orchitis by inoculating a pure culture derived
immediately from human tissues. There is no real difference inthese results, since originally all strains of the pallidum are humanin origin. On the other hand, not all strains of the spirochmt~e canbe successfully implanted upon rabbits or other animals, when obtained directly from syphilitic lesions in man. Racial differences
exist naturally, and probably can be produced artificially. The pallidum strains which I cultivated from the testicular lesions of rabbits
and which were pathogenic for these animals, failed to produce skinlesions when inoculated into monkeys. But the strains described inthis paper have been inoculated successfully into the skin of several
monkeys.
In the experimental results to be reported, I employed three cultures of the pallidum derived from as many different human casesof syphilis and two species of lower monkeys, Macacus rhesus and* Noguchi, Jour. Am. Med. Assn., 1911 , lvii, lO2; Jour. Exper. Med., loc. cir.
~ H o f f m a n n , Deutsch. reed. Wchnschr., 1911, x x x v i i , 1546.

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Cercopithecus callitrichus. The inoculations were made by applying ten day old pure cultures in ascitic fluid tissue agar to the freshly
shaven, scarified surface of the skin over the eyebrows, and, also, tothe scarified prepuce, scrotum, or labia. The cultures were smeared
over the prepared surface, and several small injections were madewith a very fine syringe. The final results of the inoculations cannot be stated yet, but the present conditions are given in the following protocols.
PROTOCOLS.

Macacus rhesus, A.--October 2, 1911. A ten day old pure culture of pallidum,strain No. 7 (from a chancre), was inoculated to both eyebrows and the left
labium.
October 9. The traumatic reactions have all disappeared.
October 15. Several small nodules appeared on both eyebrows, and a peasized induration at the site of inoculation on the labium.
October 22. The small nodules still persist, and in some there is an increasein size; some are scaly and congested. No pallida are seen in the expressedjuice. The labium is still indurated.
October 29. Two distinctly indurated nodules on the left, and one small one
on the right eyebrow. On examination of the expressed juice under the darkfield, some active pallida are found. The nodule on the labium shows no pallida.
November 6. The indurated nodules on the eyebrows show superficial ulceration. The expressed juice contains numerous active pallida. The labium is still
indurated, but no pallida are found.
November n . The size of the nodules on both brows is increasing, showing
a moderate degree of congestion. The labium shows only a slight induration.
Photographed. The blood gives a positive Wassermann reaction (figures 16
and 17).
Macacus rhesus, B.--October 2, 1911. Inoculation made as above with thesame culture.
October 9. The traumatic reactions have disappeared.
October 15, Several small indurations on the eyebrows. The skin of thescrotum shows marked induration at the site of inoculation.
October 22. The size of the nodules is increasing.
October 25. Death due to pulmonary tuberculosis. The indurated tissues
were excised and examined for the pallida, but none were found.
Macact4s rhesus, C.--October 2, 1911. Inoculation made as in monkey B.
October 9. The traumatic reactions have disappeared.
October 15. One nodule on the left, and three small nodules on the righteyebrow. The labium is moderately indurated.
October 29. The nodules on the eyebrows have become larger and congested. The labium is indurated.
October 31. Death from pulmonary tuberculosis. Examination of the indurated tissues from the eyebrows shows numerous pallida.
Macacus rhesus, D.--October 2, 1911. A ten day old pure culture of pallidum,strain No. 8 (from a condyloma), was used for the inoculation.

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Direct Cultivation of Treponema Pallidum.

October 9. The traumatic reactions have all disappeared.
October 15. Several small indurations on both eyebrows. The labium shows
marked induration at the site of inoculation.
October 22. No further changes.
October 29. The nodules are somewhat larger and fewer in number. The
induration of the labium is still about the same. The expressed juice of thenodules on the brows shows a small number of active pallida.
November II. The nodules are becoming smaller in size, and the labium ishealing up. No pallida.
Macacus rhesus, E.--October 2, 1911. Inoculation as above.
October 9. All traumatic reactions have disappeared.
October 15. No change.
October 22. No induration.
October 29. No change.
November I1. Still no change.
Macacus rhesus, F.--October 2, 1911. Inoculation with a ten 6ay old pureculture of pallidum, strain No. 9 (from a rectal condyloma).
October 9. The eyebrows are smooth and show no induration.
October 15. Several small indurations on the eyebrows.
October 22. The nodules are disappearing.
October 29. The nodules have disappeared.
November IO. No further change.
November II. Death from pulmonary tuberculosis.
Cercopithecus calIitrichus, G.--October 2, 1911. Inoculations with the same
culture as in monkey F.
October 9. The eyebrows are clean.
October 15. There are several small nodules on both brows.
October 22. The nodules are increasing in size.
October 29. A distinctly raised nodule is seen near the corner of the left
eyebrow. The small nodules are disappearing.
November 6. The nodule on the left brow has become very much congested
and covered with a scaly crust. Under the crust, the nodule is barely covered
with a new epidermis, which by slight scraping caused exudation of a clear
serous fluid. Examination of the exudate showed a few active pallida.
November II. The nodule is still much indurated and congested, but the surface is well covered. By gentle scraping, a serous exudate containing pallida
escapes from the nodule. The blood gives a positive Wassermann reaction.
Photographed.
F r o m t h e s e r e s u l t s it c a n be i n f e r r e d t h a t p u r e c u l t u r e s o f Treponema pallidum a r e c a p a b l e o f c a u s i n g , i n c e r t a i n l o w e r species o f
t h e m o n k e y , l e s i o n s o f a c h a r a c t e r i s t i c f o r m "and h i s t o r y t h a t res e m b l e the l e s i o n s c a u s e d b y i n o c u l a t i o n s o f t h e spiroch~et~e-cont a i n i n g s e r u m f r o m a p r i m a r y s y p h i l i t i c lesion. C o i n c i d e n t a l l y , t h e
d e v e l o p m e n t o f t h e W a s s e r m a n n r e a c t i o n i n the s u c c e s s f u l l y i n o c u lated monkeys has been observed.

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CONCLUSIONS.

A method for the direct cultivation of Treponema pcdlidum fromhuman syphilitic lesions, by the employment of a solid-medium, hasbeen described. By means of it, three of the four strains worked
with were successfully cultivated.
The several pure cultures agree in morphological and culturalcharacters, grow only in the presence of sterile tissue under anaerobic conditions, and do not produce putrefactive odors. The morphology is typical under optimum cultural conditions; it becomes
atypical when the conditions are unfavorable.
In cultures, Treponema pallidum multiplies by longitudinal division. The process is usually symmetrical but occasionally appears tobe asymmetrical.
Inoculation of the pure cultures into the skin of two species oflower monkeys was followed by the production of lesions resembling
the primary syphilitic lesion occurring in man and those caused in
the monkey by inoculation of spirochmtm-eontaining serum fromhuman sources.
During the course of the positive inoculation in the monkey, theblood develops the property of giving a positive Wassermann reaction. Thus the relation of Treponema palligum to this reaction is
supported, and the identity of the cultivated strains with the species
found in human syphilitic lesions established.
EXPLANATIONOF PLATES.

PLATE 12.

FIG. I. Treponemata pallida from a young pure culture in an ascitic agar
tissue medium, four days old at 37° C. Dark-field. X 1,4oo.
Fins. 2 and 3. The same after two weeks.
Fro. 4. Treponematapallida from a chancre. (For comparison.) X 1,4¢o.
Fro. 5. Treponematapallida from a pure culture in an ascitie agar tissue
medium, two weeks at 37° C. The pallida in free space show typicalmorphology.
Fro. 6. A short pallidum with flagella-likeprojections at both ends. X 1,4oo.
Fins. 7, 8, 9, and IO. An ascitic agar tissue culture (pure) showing various
phases of longitudinal division. X 1,4oo.
PLATE I3.
Fro. I I. A mass of dividing pallida from a pure culture in solid media. X 1,4oo.
FIGS. 12 and I3. Peculiar forms of dividing pallida from a pure culture insolid media. X 1,4oo.

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Direct Cultivation of Treponema Pallidum.

Fins. I4 and I5. Dividing forms of pallida in fluid cultures.
Fro. I6. Macacus rhesus, A, with primary indurations on the skin above theeyebrows produced with a pure culture of the pallida. Photographed after
forty days.
Fro. I7. A pallidum in the expressed juice of the lesions of the same monkey.
PLATE 14.
Fro. 18. A schematic reproduction of different phase s of longitudinal divisionof Treponemata pallida in solid culture (pure). The drawings were made fromobservations on living organisms under the dark-field microscope. The description of these forms will be found in the text.
A = symmetrical division ; B ---~asymmetrical division ; C -= irregular forms.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XV.

PLATE 12

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL XV.

~LA~E 1:~.

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

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PLATE 14.

1i1