野口英世著

Journal of Experimental Medicine

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PURE CULTIVATION OF SPIROCHTETA REFRINGENS.*
BY HIDEYO NOGUCHI, iVf.D.
(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)
PLATE69.
In 19o5, in the article in which the discovery of Treponema pallidum was announced, Schaudinn and Hoffmann described a coarse
spirochaeta found in superficial lesions o n t h e genitals. This organism was observed not only in syphilitic, but also in non-syphilitic
lesions. It is differentiated from the pallidum by its larger dimensions, its plump shape, its comparatively few, irregular curves, andby its readiness to take various anilin dyes. The Giemsa solution
stains it violet red. Schaudinn and Hoffmann named this organism
Spirochceta refringens, and considered it non-pathogenic.
While the organism frequently comes under observation in examining scrapings for the diagnosis of syphilis, yet our exact knowledge of its biology has been fragmentary for the reason thatno one had succeeded in isolating it in pure culture. Unquestionably, certain investigators have obtained a growth of this spirochaeta in highly impure cultures, but a study based upon such contaminated material aided little in advancing our knowledge.
In my systematic attempt to secure pure cultures of various spirochaetae, I have succeeded in growing this organism in pure culture.
The strain of Spirochcta refringens that I cultivated was derived
from the scrapings of a moist condyl0ma around the anus of afemale patient. Under the dark-field microscope, the scrapings
showed a moderate number of the refringens. The material wasground in a sterile mortar with sodium citrate solution, and theemulsion thus prepared was inoculated into several tubes of solid
culture medium consisting of one part of ascitic fluid, two parts ofslightly alkaline agar, and a piece of fresh rabbit kidney. The*Received for publication, March 4, 1912.
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Hideyo Noguchi.

467

technique employed was the same as that for the cultivationof the pallidum 1 from a human being. A f t e r two weeks' incubation
at 37 ° C. an impure culture was obtained. By the same process thatwas used for the pallidum and for the mouth spiroch~et~e, therefringens was finally purified.

The Properties of the Pure Culture (Figures ~ to 5) .--Spirochceta
refringens grows luxuriantly in the deeper part of a tube of ascitic
tissue agar, and forms hazy colonies that gradually extend from thedeeper parts of the tube to the upper. It is an anaerobic organismand no growth takes place within two centimeters of the surface ofthe medium. At 37 ° C., the growth becomes visible on about thefourth day after the inoculation, and proceeds for some weeks. It
does not produce any visible changes in the medium, and the culture
is free from any odor. Unlike the pallidum, a growth is obtainable
in the medium without the addition of fresh tissue, although, likethe microdentium, the growth is poorer when the tissue is absent.
In pure culture, Spiroch(eta refringens measures, on the average,
about 0. 5 to 0.75 o f a micron in width, and about 6 to 24 microns inlength. Chains of two, three, and four or more individuals may
be seen. They are connected with one another by means of a thinfilament. The number of curves varies with different individuals,
but, as a rule, the middle part of the body is merely wavy, while thetwo extremities are more regularly and deeply curved. Not infrequently, however, the majority of the spirocha~t~e in certain cultures
are fairly regularly curved throughout their entire length, the distance between two curves measuring about 2 microns. The endsof the organism are pointed, and to one or both of them is usuallyattached a fine, long, more or less stiff, but elastic projection, which
has regular minute curves.
In a culture growing under optimum conditions, the spiroch~et2e
are very motile. U n d e r these conditions the curved flagella-likeprojections rotate with extraordinary rapidity around their point ofattachment to the spiroch~et~e. While the flagella-like projection isrotating rapidly, the body of the spiroch~eta may remain motionless, or may be traversed by waves, convex curves becoming concave, and concave curves becoming convex. The waves begin at* N o g u c h i , ]our. Exper. Med., I912~ xv, 9o.

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Pure Cultivation of Spirocha3ta refringens.

one end and may pass along the entire body, or may stop after
having passed over only a few curves. Whenever motility is observed in the body of the spiroch~eta, the movements are repeatedat frequent intervals. In a dead specimen, the curves are much
more irregular and are often almost straightened out.
The body of the refringens was thought by Schaudinn to beribbon-like, but in pure culture I find it to be cylindrical. In therefringens cultivated by me, there is no undulating membrane.
Regarding the mode of multiplication, I am still unable to decide
whether or not a longitudinal division occurs before the spiroch0eta~
are found attached end to end in pairs or in chains of several individuals. The ultimate division of these chain forms is transverse.
Pathogenicity.--Several attempts were made to produce lesions
in the testicles of rabbits, or in the skin of Macacus rhesus monkeys,
but thus far without a positive result.
G E N E R A L CONSIDERATION.

That Spirochwta refringens constitutes an independent species,and not a form representing merely a certain stage of Treponema
pallidum, is proved by the present study and by my published observation on different strains of the pallidum in pure culture. Neither
the refringens nor the pallidum lose their characteristics at any
stage of cultivation. As a consequence, they may always be distinguished even in cultures.
A few investigators in questioning the evidence for the specificity of Treponema pallidum for syphilis have argued that, if therefringens is a form of the pallidum, the latter cannot be specificfor syphilis, because the former is found in so many non-syphilitic
lesions. As the two organisms are now shown to be different, thisargument can no longer be employed.
Spiroch(eta refringens belongs, doubtless, to the genus Treponema, but, like the other members of this genus, it is entirely distinct
from the pallidum.
There are still some important problems that await solution bymeans of pure cultures. Balanitis erosiva circinata is a clinicallywell defined disease, and in the lesion a large spiroch~eta resembling
the refringens is usually found. Von Prowazek and HoffmannT H E J O U R N A L OF E X P E R I M E N T A L

M E D I C I N E VOL. XV.

P L A T E 69.

Hideyo Noguchi.

469

considered this organism to be its causative agent. Mindful of thefact that the refringens has a wide distribution in the genital region,
one naturally seeks a means by which the two morphologically alliedforms, the refringens and balanitidis, can be definitely differentiated.
This is possible only through the growth of both organisms in pureculture. Unfortunately, I have seen but few cases of this type ofbalanitis and for this reason I have been unable to attempt seriously
its cultivation.
From the point of view of cultivation, another problem of interestis the relation between Spirochceta buccalis and the refringens.
There is at present much controversy in regard to the identity ofthese spiroch~et~e. Some consider them identical, while others regard them as independent varieties. My numerous attempts toisolate the buccalis in pure culture have thus far been fruitless.
There seems to be no doubt but that the buccalis is much more difficult to grow on artificial medium than the microdentium or macrodentium. In an impure culture of the buccalis, I was much impressed with the morphological similarity that it bears to therefringens, but the question of the identity of these two spiroch~et~erequires further study.
S U M MARY.

A strain of Spiroch6eta refringens has been obtained in pure culture, and its morphological and cultural characteristics have beenstudied. The strain possessed no pathogenicity for rabbits ormonkeys. It probably belongs to the genus Treponema, but it iseasily differentiated from Treponema pallidum, Treponema microdentium, and Treponema macrodentium. Its relation to Spiroch6eta
balanitidis and Spirochceta buccalis is still undetermined.
EXPLANATION

OF

PLATE

69.

FIG. I. A schematic drawing of Spirochcetarefrlngens from pure cultures(dark-field).
FIG. 2. Spirochcetarefringens from a three weeks' old pure culture inascific tissue agar, at 37° C. (dark-field). >( i,IOO.
FIG. 3. Spirochwtarefringens from a pure culture seven days old (darkfield). X I,IOO.
FIGs. 4 and 5. Spirochctarefrlngens from a pure culture two weeks old(dark-field). X i,IOO.