野口英世著

Journal of Experimental Medicine

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A METHOD FOR CULTIVATING TREPONEMAPALLIDUM IN FLUID MEDIA.
BY HIDEYO

NOGUCHI,M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)
Treponema pallidum is an obligatory anaerobe which together
with certain other spiroch~et~e forms a special class of organisms
whose growth requires the presence of a fresh, sterile tissue in culture media. 1 The growth of Treponema paIlidum in any suitablemedium is very slow, and continues for a considerable length o ftime. In a solid medium, for which the pallidum shows a particularpreference, the growth continues for about two months after transplantation. On the other hand, the cultivation of the pallidum in afluid medium, with the addition of fresh tissue, by means of ananaerobic jar in which oxygen is removed through the combinationof vacuum, displacement with hydrogen and absorption by pyrogallol, has given rather inconstant results. In one series of cultivations the growth may be quite favorable, while in others it may failaltogether. The loss of time and material from unsuccessful fluid
cultivations is very great and the entire procedure is therefore inneed of improvement.
The method which I am about to describe in detail has alreadyproved to be entirely satisfactory and possesses certain special
features that are lacking in the usual anaerobic cultural methods
now in general use. In the case of the usual method, an ordinary
test-tube is filled with the fluid in which the cultivation of an organism is intended, the fluid is then inoculated, and cultivated in asealed j a r under anaerobic conditions. The process is quite satisfactory for the cultivation of most anaerobic bacteria, but, as has
already been mentioned, it is unreliable for obtaining growth of theIt may be stated that the addition of sterile fresh tissue to a fluid medium
alone is not only inadequate to make the pallidum grow, but is unable to keep it
alive for any length of time. In this respect the pallidum is very different fromthe obligatory anaerobic bacteria, as the latter grow abundantly in such a medium.
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212

Method for Cultivating Treponema pallidum.

pallidum in a fluid medium even with the addition of fresh tissue.
The failure to effect cultivation of the pallidum by this method is
doubtless due to the fact that with this process the removal ofoxygen is often too incomplete to enable the inoculated pallidum togrow or even to survive the time required by the fresh tissue toabsorb the remaining oxygen and render the fluid perfectly anaerobic. In other words, Treponema pallidum is too delicate an organism to survive the unfavorable conditions which last for sometime within the fluid in which it is freshly transplanted. The sourceof this difficulty can, however, be removed by providing the organism with a more suitable medium in which it can at once begin toadjust itself. A solid medium consisting of a mixture of ascitic
fluid and agar with the addition of a fresh, sterile tissue fulfills thisrequirement. Thus the new method which is designed to exclude
this difficulty is composed of two distinct parts; one for the solid
and the other for the fluid cultivation. By combining both the solidand the fluid culture media for simultaneous cultivation, the shortcoming of the latter is easily supplemented by the former which ishighly suitable for the growth of ,the pallidum. By this method
the pallidum grows at first in the solid medium as usual, and then,when the cultural .conditions in the fluid portion become favorable,
it migrates from the solid portion into the fluid and multiplies thereabundantly.
A solid medium in this method not only serves as a reservoir of
the growing spiroch~et~e for the fluid culture, but it is also a good
indicator for determining the purity of the culture; for should anycontamination occur, it is infallibly indicated by the development of
the colonies of bacteria in the agar column, while it is quite difficult
to detect a contamination with certain bacteria in a fluid medium
alone. As will be seen later, the entire process of cultivation iscarried on without the aid of a sealed jar, vacuum, or pyrogallol.
The state of growth, or any changes that may take place in themedium, can easily be observed at any time without disturbing thecultivation, and, if desired, the content of the culture may, underaseptic precautions, be drawn out and examined. These are thefeatures which may be regarded as special advantages of the newprocess in comparison with the older procedures.

Hideyo Noguchi.

213

METI-IOD.

Construction of the Culture Tube.--This consists of a test-tube,
i. 7 centimeters wide and 2o centimeters long, whose bottom ends
in a hollow projection made by fusing a short piece of a strong
glass tubing, 0. 7 of a centimeter bore, to the perforated bottom(text-figure, I ) ; a large test tuSe, 2. 5 centimeters wide and 15
centimeters long (text-figure, 2 ) ; and a perforated rubber c o r k ,size 5 (text-figure, 3). These parts are combined by connecting
the two tubes by means of the rubber cork (text-figure, 4). TheTEx¢-FIG.

F o r description see text, p. 213.

upper tube is intended for the solid, and the lower for the fluid
culture medium.
Preparations for Cultivation.--The double tube just described isthoroughly cleaned and dried; the upper tube is then plugged with
non-absorbent cotton. Just before use the whole is sterilized in anMethod for Cultivating Treponema pallidum.
autoclave. In the meantime pieces of sterile, fresh rabbit kidney
are prepared. A slightly alkaline agar (2 per cent.) is also freshly
prepared, autoclaved, and kept in a fluid state. A sufficient quantityof sterile paraffin oil should be kept on hand.
Process of Cultivation.--I. After the double tube cools, one or
two pieces of the sterile, fresh tissue are put into the lower tube.
This is done by removing the rubber cork and the upper tube together, which are quickly replaced and fitted as tightly as possible.
These parts must not be loosened again. Another piece of tissue isnow put into the upper tube. The tissue goes down to the perforated bottom of the tube, bm it should be large enough not to passthrough the connecting hole into the lower tube:
2. A f t e r tissues have been placed in both tubes, the lower tube isfilled with ascitic fluid or a mixture of ascitic fluid and bouillon.
This is done by means of a large, sterile bulb pipette whose tubular
portion is so drawn out as to pass through the connecting tube witha margin of space that permits the air that is displaced by the fluid
to escape.
The lower tube must be filled up until there is no air
bubble inside the tube.
3. The inoculation of the pallidum is next made. By means of along capillary pipette, made by drawing a sterile glass tubing, asufficient quantity of a well growing culture is aspirated and used
for inoculation of the double tube. F o r this purpose a syringe witha connecting pressure rubber tubing is recommended. First thecapillary pipette is inserted into the fluid of the lower tube into
which some of the contents are forced; then the remaining portionof the culture in the pipette is emptied into the upper tube just about
the tissue.
4- When the inoculation has been performed, the upper tube isfilled with a solid medium. The medium is prepared by mixing
o n e part of ascitic fluid with two parts of slightly alkaline agar ina sterile flask. The mixing is done when the temperature of themelted agar is about 420 C. Before solidification of the ascitic
agar sets in, the medium is quickly distributed into the upper tube.
The quantity of the medimn is about fifteen cubic centimeters.
5. The last step is to add-an arbitrary amount of sterile paraffinHideyo Noguchi.

215

oil in order to cover the surface of the solid medium. About three
cubic centimeters suffice (text-figure, 5).
6. The culture is then incubated at 37 ° C.
For the sake of clearness I have described the procedure as if I
were dealing with a single set of double tubes, but in practice it is
my custom to employ twelve sets at a time. The advantages ofusing several sets at a time are twofold. The first is an economic
advantage: when one kidney is removed from a normal rabbit itusually furnishes enough tissue to fill at least six sets. As I usuallysacrifice one rabbit, I have enough tissue for a dozen sets. The
second advantage in making several sets is that, owing to thenumerous steps that must be taken in making one series of cultivation, the possibilities o f contamination are very great and it is best
to provide against this accident so as to insure success.
As regards the capacity of the culture tubes, it is understood thatit depends altogether upon the purpose of the work and whether or
not a large quantity of the culture is desired. As the nature of mywork requires as large a quamity of the material as I can obtain, I
use comparatively large tubes. 2 But, according to my experience,
it is not advisable to employ still larger tubes, as the result may be
an enormous waste of material when accidental contamination takes
place.
The method described in this article is especially adapted for thepure cultivation of Treponema pallidum, Treponema microdentium,Treponema macrodentium; Treponema refringens, Treponema mucosum, and Treponema pertenue in fluid media, when these organisms have been growing already in solid media. It is unsuitablefor obtaining the growth of these spiroch~etm when they are associated with other bacteria.
SUMMARY.

I. A method is described for obtaining pure cultures o f Trepo,nema pallidum and allied species of spirochmtm in fluid media.
2. The principle of the method is based upon the superpositionof a favorable culture medium upon a less favorable one.
3. The mettiod here described is not suited to the growth of
spirochmtm when they are a d m i x e d w i t h contaminating bacteria.
2 The fluid culture of Treponema pallidum is used for the preparation of luetin
as well as for immunity studies.