野口英世著

Journal of Experimental Medicine

所収論文

こ の PDF は Journal of Experimental Medicine に 掲 載 さ れ た 論 文 を Rockefeller
University Press の許可（2020 年 3 月 18 日付）を得てアップロードしています。

C U L T U R A L STUDIES ON M O U T H SPIROCH.,GTzG( T R E P O N E M A M I C R O D E N T I U M AND
MACRODENTIUM).*
BY HIDEYO

NOGUCHI,

M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)
PLATES 7--1I.

In the normal oral cavity of man and animals, three distinct
types, at least, of spiral organisms have long been recognized. The
first type is much larger and thicker than the other two and has
only a few flat curves. It is known as Spirochceta buccalis (Cohn,
1875 ) . The second and third types are more delicate in structure
and possess a thin flexible body and quite regular, closely set shallow
curves, the difference between the two being that one is smaller thanthe other. The smaller type is called Spiroch(eta dentium (Koch,
I877 ), while the medium form bears no particular name, but has
been designated the "medium f o r m " by Hoffmann and vonProwazek. 1
The relative number of the three types of spiroch~et~e varies
greatly according to the conditions of the mouth and to the localities from which the material is obtained. Thus, in my experience,the smallest type is more abundant in the deposit at the juncture
between the teeth and the gum, and in the cavity of a carious tooth;while the medium and buccalis types are more frequently found in
the mucus about the tonsils and pharynx, and in large numbers inulcerative stomatitis.
As regards the pure cultivation of these organisms, our knowledge has been very meager. The orfly investigator who has succeeded in isolating them from associated bacteria in a culture is* Received for publication, Nov.ember 6, 1911.
1 Hoffmann, E., and vor~ Prowazek, S., Untersuchungen fiber die Balanitis undMundspirochaten, Centralbl. f. Bakteriol., zte Abt., 19o5, xli, 741, 817.
81

82

Cultural Studies on Mouth Spirochcetw.

Miihlens (19o6). ~ The descriptions of his cultivated spirochmt~eagree, in part, with the morphological features of Spirochceta
dentium (Koch) and, in part, with those of the medium type.
From this fact, Mfihlens suggested that the medium type is nothing
more than forms that, under certain conditions, spring out of thesmaller type. However, he left this point unsettled. As the work
to be presented in this paper shows, I was able to obtain at least
two distinct types of mouth spiroch~et~e in pure cultures. One ofthem is the smaller type and has affinities with Spirochceta dentium(Koch), and the other belongs to the medium type. The morphological and cultural characteristics of these two organisms are so
distinct that no confusion of the two should be made. It is not
improbable that the culture of Miihlens contained both the smaller
and the medium types. In fact, on two occasions, I have obtainedsuch a mixed culture.
Hitherto, on account of the lack of pure cultures, a reliable meansof differentiating various spiroch~et~e was impossible, and a loose
terminology, such as has been in use for the spiroch~et~e of themouth, was all that could be employed without risk. But, now thatsuccessful separa.tion in pure cultures of morphologically distinct
forms has been accomplished, it is possible to define two distinct
species of mouth spirocha~t~e. I therefore propose the name of
Treponema microdentium for the smaller, and Treponerna macrodentium for the medium type. My reason for including these two
types under 'the general specific name of " d e n t i u m " with the prefixes of " m i c r o " and " m a c r o " is that they belong unquestionably
to the same genus, while the buccalis type belongs rather to anothergenus. In general characteristics, the micro- and macrodentium
agree with the genus Treponema (Schaudinn) to which also thepallidum belongs.
CULTIVATION OF TREPONEMA MICRODENTIUM.

A small amount of tooth deposit, preferably from a young child,is put in a tube containing a large quantity of sheep serum water*Miihlens, P., Ueber Ziichtung von Zahnspiroch~iten und fusiformen Bacillen
auf kfins~lichen (festen) Nfihrb6den, Deutsch. reed. Wchnschr., 19o6, xxxii, 797;
Mfihlens, P., and Hartmann, M., Ueber Baciltus fusiforrnis und Spiroeh~et~dentium, Ztschr. f. Hyg. u. Infectionskrankh., t9o6-o7, Iv, 81.

Hideyo Noguchi.

83

and a piece of sterile tissue (testicle or kidney of rabbit or sheep). ~The culture medium thus inoculated is now covered with a layer ofsterile paraffin oil. It is best to inoculate about three tubes which
are incubated for about ten days at 37 ° C. During this time, themedium may become firmly or loosely coagulated through the actionof bacteria simultaneously growing in the culture; a small amountof the culture is taken with a capillary pipette from the bottom of
the tube and examined under the dark-field microscope. Numerous
active small spiroch~et~e and other bacteria are usually seen. The
next step is to purify the spirocha~tm from the accompanying
bacteria• The same principle is employed as for the purification of
the pallida. For this purpose, a series of wide and tall tubes containing serum agar, in the ratio of I to 3, and a piece of sterile
tissue (preferably sheep or rabbit) are inoculated with a capillary
pipette with the impure fluid culture by inserting it carefully into
the agar along the middle line until the point reaches the bottomof the tube. Then the contents o f the pipette are gently forced inby means of a rubber bulb or a syringe, care being taken not to
split the agar column by means of the compressed air. It is best not
to empty the pipette wi,thin the agar, but to withdraw it with Someof the contents still present. After withdrawal, the agar is covered
with a layer of paraffin oil and the tubes are put in a ,thermostat.
In ten days or two weeks, the tubes begin to show a hazy colonyof spiroch~et~e spreading out from the stab canal that is now filled
up with a whitish mass of bacterial colonies. When the size of the
hazy colony becomes large enough to allow fishing, without risk o ftouching the stab canal, the tube is cut, the agar column split acrossvery gently, and a sterile capillary pipette carefully inserted into the
hazy zone from the clean newly cut surface of the medium• The
material removed shows, under the dark-field, the presence of innumerable active spiroch~et~e. Pure cultures can then be obtainedby inoculating a new medium with this colony by means of a capillary pipette. It may, of course, happen that purification may not
at first be successful, so that it is necessary to repeat the same procedure umil success is obtained•
8Noguchi, ]our. Am. Med. Assn., 1911, lvii, lO2; lour. Exper. Med., 1911,
xlv, 99.

84

Cultural Studies on Mouth Spirochcet~.
C U L T I V A T I O N OF T R E P O N E M A M A C R O D E N T I U M .

The same technique is used for isolating the microdentium.
Special precautions must be taken ( I ) to obtain material in which
the larger dentium organisms are predominating, as from a caseof Vincent's angina or ulcerative stomatitis; (2) to enrich thelarger type by several transplantations of the impure culture in
fluid media; and (3) to select carefully the colonies that are characterized by fainter visibility and slower growth. The pure cultivation of the macrodentium is more tedious than that of the microdentium.
Sometimes it happens that the impure cultures show no, or only aslight coagulation of the serum constituents. In this instance, thepurification is comparatively easy because the solid media do not
become turbid and the delicate colonies of the spiroch~et~e radiatingfrom the central stab canal can easily be recognized. But when thebacteria growing in the impure culture, as is the rule, produce acidand perhaps gas, the purification is rendered very difficult because
the solid media become so turbid or torn up with gas that thecolonies of the spiroch~et~e are not easily seen, or, when seen are
difficult to isolate. Under such circumstances, it is advisable either
to select better material for inoculation or to reduce the acid-producing bacteria by leaving the impure cultures for four or five
weeks, during which time the spiroch~etm remain alive and stilltransplantable while the bacteria often die. It may, however, benecessary to repeat this procedure several times before successful
purification is attained.
Treponema Microdentium.--(Figures I to IO.) When cultivatedin a serum agar tissue medium, a faint haze becomes perceptible
within seventy-two hours at 37 ° C. along the stab canal. The density and dimensions of the hazy growth increase slowly for manydays until the agar column two to three centimeters below the surface becomes diffusely hazy and opalescent. Along the stab canal,
the growth becomes so dense that it appears as an irregularly thickened, whitish streak without definite contour. During the first andsecond weeks, numerous small 'spots of separate colonies scattered
here and there appear around the stab canal, which are often seen
to grow in the space between the agar column and the wall of theHideyo Noguchi.

85

test tube. Young cultures have only a faint odor, but after about
three weeks, the odor becomes characteristically fetid.
This spirocha~ta measures less than 0.2 5 of a micron at the
middle of the body and gradually tapers towards both extremities,
which are sharply pointed. The length varies according to the ageof the culture: young cultures show many short forms, and oldones may reach eight microns in length and show, on the average,
fourteen curves. The number of shallow and rectangular curves
varies also according to the length of the specimen; but the angle
and size of each curve are strikingly constant, although diminishing
gradually ,towards the extremities. The pointed ends are usuallydrawn out straight along the hypothetical axis. The motion of the
spiroch~et~e is quite active and rotating, and the body is flexible.
Under certain conditions, a long thin flagella-like projection is observed at one or both ends. The general appearance of the organism
is a straight line. W h e n such a culture is disturbed by a capillary
pipette or platinum loop, the regular straight forms become less evident and peculiarly ~wisted specimens more numerous. In these disturbed specimens, the curves, nevertheless, resemble the original.
Singularly enough, these contracted individuals swim swiftly by aserpentine or vibratory motion. When the flagellum of one end iscaught by a fixed object, the spiroch~eta swings actively as if attempting to liberate itself. Longitudinal division has frequently
been observed in pure cultures.
As long as oxygen is completely excluded from a fluid medium,straight typical forms are produced; but when the medium is imperfectly anaerobic, the spiroch~etm take on irregular and nontypical shapes.
The microdentium causes a loose coagulation of the serum constituents that renders litmus colorless after about two weeks' growth
and the cultures exhale a strong fetid odor.
The injection of a culture of this organism into the testicles ofrabbits and the subcutaneous tissue over the eye of a Macacus
rhesus monkey, causes, within twenty-four hours, a marked induration that persists for about a week; however, no spirocha~t~e weredetected in the inflamed tissue.
Treponema Macrodentium.--(Figures II to 16.) In the serum

86

Cultural Studies on Mouth Spirochcet~.

agar tissue medium, after about four or five days at 37 ° C., it grows
as a faint hazy colony which is almost transparent and without
demarcation towards the outside. Isolated colonies are rarely produced, and strict anaerobiosis is required for any growth to takeplace.
According to the condition of the culture, the morphological features of the macrodentium are subject to wider variations thanthose of the microdentium. In young cultures the organisms areplump and short with rather irregular shallow curves. The extremities taper off abruptly. U n d e r the dark-field, the body of theorganism shows double refraction; namely, a feeble line of refraction along the axis, bordered on both sides with stronger zones ofrefraction. The body is flexible and capable of creeping with greatease through the agar in all directions. When in a clear space, theorganism swims freely with swinging or vigorously vibrating movements• Certain specimens have been observed in which the bodyshows a longitudinal cleft or division, and long delicate flagellalike projections, with minute curves, are attached to one or both ends.
Specimens from an older culture are somewhat longer and thinner
and taper more gradually towards the ends; the curves are almost
rectangular, shallow, and quite regular; the body shows no longer
double refraction, except near the middle. Motility seems to be lostin certain individuals, except for a few curves at the extremities that
may still exhibit a swinging motion• Young organisms showing
double refraction and having two to eight curves may measure 0.7
to I.O micron in width and 3 to 8 microns in length. Older specimens having fourteen or more curves measure usually o. 3 of a
micron in width and I2 microns or more in length. The spiroch~et~e grown in a fluid medium show about the same dimensions,but many long specimens, corresponding to the length of several
ordinary ones, may appear. Besides these, other individuals occur,
the bodies of which are much heavier in the middle and show moreclosely set, regular, rectangular curves, and of which one fourth to
one third of the body toward the extremities gradually tapers off tosharply drawn out points with comparatively few shallow curves.

Hideyo Noguchi.

87

I N V O L U T E D FORMS.

I have already stated that when the exclusion of the oxygen is
not complete, the microdentium produces many irregularly waved
forms in a solid or fluid medium (figures I8 to 23). These individuals swim freely and are actively motile. By transplanting theminto a new medium, under strict anaerobic conditions, they reproduce the normal forms. In fact, the forms which are commonly
observed in a smear from tooth deposit consist of these irregularindividuals; and it is only in an artificial medium, under strictly
anaerobic conditions, that the regular straight forms showing aseries of uniform curves appear. This fact is also true of themacrodentium.
On one occasion, I observed in an impure culture, in a fluid
medium growing under imperfect anaerobic conditions, numerous
spiral organisms with extremely regular, almost rectangular curves.
Their length varied from four to sixty microns or more, and, withthe corresponding number of curves, each averaged 1.8 microns.
They were usually straight, finely tapered towards the ends, andapparently showed no real motility. Their bodies were flexible,
but no modification of the form of the curves could be made out.
The shorter ones, with few curves, are very thin (0.3 of a micron),while the longer specimens may be twice, three times, or four times
as thick as the main portions of the body of the smaller varieties.
Even with the longest forms, no cross septa, such as are easily recognized in certain other large spirocha~ta~, were seen. On the other
hand, many larger forms showed a central cleft suggesting longitudinal division, which may run along paff or the whole of the axis.
Transplantation of such cultures into a new fluid medium, kept
under similar conditions of cultivation, causes these peculiar immobile spiral forms to reproduce indefinitely; while when cultivatedin a solid medium under strictly anaerobic conditions they fail togrow at all.
Although I am inclined to view this finding as an example ofinvolution of the macrodentium, I have not yet concluded whether
we may not have before us rather a new spirocha~ta than merely an
involuted form: The culture in which these spirocha~t~e appearedcame from scrapings of an ulcer of the mouth of a child of three88

Cultural Studies on Mouth Spirochcetoe.

years of age, in whom ulceration, attended by fever, has been occurring regularly every three weeks since infancy. I am indebted to
Dr. La Fetra for access to this case.
CONCLUSIONS.

I have described the successful cultivation of two distinct speciesof Treponema from the oral cavity of man. The species differ
from one another morphologically and in respect to their culturalproperties. As the two species have now been clearly separated andcultivated for the first time, I propose for them the names Treponema microdentium and macrodentium, respectively.
Division along the longitudinal axis has been demonstrated forboth Treponema macrodentium and microdentium. When the
micro- and macrodentium are cultivated under imperfect anaerobic
conditions, they undergo involution, but still continue to vegetate tosome extent. Finally, a spiral organism has been cultivated anddescribed that may be merely an involuted form of the macrodentium, or it may be a new species altogether.
E X P L A N A T I O N OF PLATES.
PLATE 7.
FIGS. I, 2, 5, and 6. Treponema microdentium in a pure fluid culture, twenty
days at 37 ° C. X 1,4oo.
FIG. 3. Treponema microdentium in a pure solid culture, ten days at 37 ° C.;a few forms show longitudinal division.
FIG. 4. A preparation of the microdentium from a pure culture in a solid
medium, five days at 37 ° C. X 1,4oo.
FIG. 5. An exceptionally long chain of the microdentium from a pure culture
in a fluid medium.
FIGS. 7 and 8. Treponema microdentium from a pure culture in a solid
medium, twelve days at 37 ° C. Giemsa stain. X 1,4oo.
PLATE 8.
FIG. 9. A schematic reproduction of a dark-field view of the microdenfium. A
seven day old pure culture in a solid medium. X 1,4oo.
FIG. io. The same as figure 8, but the culture has been disturbed by repeatedpunctures, and in this case the m~crodentium shows m a n y involuted forms,
X 1,400.

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XV.

PLATE 7.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XV.

PLATE 8.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XV.

PLATE g.

THE JOURNAL OF EXPI~RIMENTAL MEDICINE VOL. XV.

PLATE 10.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XV.

PLATE 11.

HideyoNoguchi.

89

PLATE 9.

FIGS. II, 12, and 13. Treponema macrodentium from a young pure culture ina solid medium, seven days at 37 ° C. X 1,4oo.
FIGS. 14 and 15. The same from an older culture, many showing longitudinal
division.
PLATE IO.

FIG. 16. A schematic reproduction of a dark-field view of a young solid
culture (pure) of the macrodentium, ten days old at 37 ° C. X 1,4oo.
FIG. 17. Treponema pallidum from a pure solid me6ium, fourteen days old
at 37 ° C. ( F o r comparison.) X 1,4oo.
PLATE I I.

FIGS. 18, 19, 20, 21, 22, and 23. Involuted forms ( ? ) of the macrodentium in
a n impure fluid culture. They show undeniable longitudinal division.