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Journal of Experimental Medicine

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T H E P U R E C U L T I V A T I O N OF SPIROCH2ETA DUTTONI,SPIROCHTETA KOCHI, SPIROCHA~TA OBERM E I E R I , A N D S P I R O C H 2 E T A NOVYI.*BY HIDEYO NOGUCHI, M.D.
(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)
PLATES 19 A N D 20.

There are at least four distinct species of spirochaetae that a r e
responsible for the diseases known as relapsing fever. The organisms in the blood of patients suffering from the relapsing fever ofEurope were first discovered by Obermeier i in 1873 , and since thenthis species has been known as £pirochceta obermeieri. The second
variety is found in the Mood of those who have contracted the disease known as African tick fever; it was discovered in 190 4 simultaneously by Ross and Milne, 2 and by Dutton and Todd. a Thisvariety bears the name of Spirochcetc~ duttoni. In I9O5 the third
species was discovered in Africa 'by Koch 4 who assumed that it was
identical with other varieties already known at that time; but the
recent investigations of Novy and FraenkeP indicate that Koch's
organism is distinct from the obermeieri or the duttoni. F o r Koch's
organism Novy proposed the name £pirochceta kochi, The fourth ~species, £pirochceta novyi, was found by Norris 6 in 1906 in New
York in the blood of a patient with relapsing fever, and was afterwards transmitted to rats and mice. Although very similar inmorphology to Spirochceta obermeieri, Fraenkel, 7 Uhlenhuth and* Received for publication, June I, 1912.
10bermeier, O., Berl. klin. Wchnschr., 1873, x, 152, 378, 391, 455.
2Ross, P. H., and Milne, A. D., Brit. Med. ?our., 19o4, ii, 1453.
3Dutton, J, E., and Todd, J. L., Brit. Med. ?our., 19o5, ii, 1259.
4Koch, R., Deutsch. reed. l/Kchnschr., 19o5, xxxi, 1865; Berl. klin. Wchnschr.,
19o6, xtiii, 185.
5Fraenkel, C., Med. Klln., 19o7, iii, 928; Mi~nchen. med. Wchnschr., 19o7, liv,
2Ol; Berl. kiln. Vdchnschr., 19o7, xliv, 681; Hyg. Rundschau, 19o7, xvii, 263.
6Norris, C., Pappenheimer, A. M., and Flournoy, T., #our. Infect. Dis., 19o6,
iii, 266.
*Fraenkel, C., Berl. klin. Wchnschr., loc. clt.
199

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Pure Cultivation of Spirochcetce.

Haendel, s Schellack,9 Strong, 1° Manteufel, 11 al~d others, consider
Spirochwta novyi to be a distinct species, chiefly on account of theimmunity phenomena. 12
There is another variety which was described by Carter ~a in India,
but it is not yet definitely determined whether or not this is identicalwith Spirochceta novyi or Spirochceta obermeieri, although Novy
considers it a distinct species on account of its being somewhat
thinner than any of the other organisms.
Schellack gives the following comparison of various species:
Splrochceta duttoni: length, 24 to 3o microns; width, 0.45 of a micron.
Splrochceta obermderi: length, 19 to 2o microns; width, o.39 of a micron.
Splrochceta novyi: length, 17 to 2o microns; width, o.31 of a micron.

My success in causing other spirocha~tae to grow in culture media
made it seem advisable for me to apply similar principles to theblood spiroch~etae, Spfroch~eta duttoni, Spirochceta kochi, Spirochceta obermeieri, and Spirocha~ta nouyi, especially as no one had
succeeded in obtaining in vitro a culture of any of these spiroch~et~e
in which growth was kept up for many generations by ~transplanting
the organisms from culture to culture? 4
There are some investigators who observed a decided increase in
the number of the organisms when citrated blood was kept at room
temperature or in the thermostat, but they failed t o obtain asecond generation. According to Novy and Knapp, blood containing the spiroch~etae may remain infectious for many days, but notrue cultivation is effected.
s Uhlenhuth, P., and Haendel, Arb, a. d. k. Gsndhtsamte., 19o7, xxvi, i.
' Schellack, C., Arb. a. d. k. Gsndhtsamte., 19o8, xxvii, 364.
10Strong, R. P., Philippine Your. Sc., 19o8, iii, 231.
=1Manteufel, Arb. a. d. k, Gsndhtsamte., 19o8, xxvii, 327.
==Novy, F. G., and Knapp, R. E., .[our. Infect. Dis., 19o6, iii, 291. Breinl, A.,
and K~nghorn, A., Lancet, 19o6, i, 668; Mere. Liverpool School Trop. Med.,
19o6, xx, 61.
aaCarter, H. V., Deutsch. reed. VFchnschr., 1879, v, 189, 351, 386.
1~Williams, L. A. and R. S., Mere. Liverpool School Trop. Med., 19o6, xxi, IOI.
Dural, C. M., and Todd, J. L., Lancet, 19o9, i, 834. Levaditi, C. (CompL rend,
Acad. d. sc, 19o6, cxlii, IO99), and Novy, F. G., and Knapp, R. E. (Your. An*.
Med. Assn., 19o6, xlvii, 2152), succeeded in keeping up the growth of Spirochceta
kochi and Spirochceta novyl (formerly thought to be the obermeieri by Novy),
respectively, for many generations by means of collodion sacs placed in the peritoneal cavity of rabbits; but their work does not enter into consideration in thediscussion of cultivation in vitro.

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201

As will be described in detail later, I have found t h e conditions
that are necessary for obtaining cultures in vitro of the organismsof the" relapsing fevers. These conditions are somewhat different
from those required by the other spiroch~etm I have cultivated.
Thus, for the pallidum, microdentium, maerodentium, mucosum,
refringens, and phagedenis, a strict anaerobiosis is necessary;while
for the relapsing fever spirochaetae the presence of oxygen seems tobe essential. M y cultivation experiments will be reported in thefollowing pages.
Material for Cultivation25--Four different strains of spirochaet~ewere employed for the experiments here described: £pirochceta
duttoni, Spirochceta kochi, Spirochceta obermeieri, and £pirochwta
novyi. All of these have been preserved for many years in ratsor mice.
Method of Cultivation.--After trying various culture media andmethods t found the following to be the most suitable and reliable
for the growth of all of the blood spiroch~et~e. Into each of a number of sterile test-tubes (I use tubes 2 by 20 centimeters) is placed apiece (not too small) of sterile fresh tissue, usually rabbit kidney,
and then are added a few drops of citrated blood from the heart ofthe infected mouse or rat. Following this, about fifteen cubic centimeters of sterile ascitie or hydrocele fluid are quickly PoUred into
the tubes, and the con~tents of some of the tubes are covered with alayer of sterile paraffin oil, while the rest are left without the oil.
The tubes are now transferred to a thermostat at 37 ° C.
I t is essential to obtain the blood from the infected animal absolutely aseptically. In my experiments, under ether anesthesia, theblood was drawn directly from the heart and was then mixed immediately with citrate solution (1. 5 per cent. sodium ,citrate inphysiological saline solution). It is best to obtain the blood between
the forty-eighth and the seventy-second hours after the inoculationof the animal. The blood suspension thus prepared was examined
for the spiroch~et~e under the dark-field microscope and was thenused for the inoculation of the culture tubes.
ayI am under great obligation to Professor F. G. Novy for placing at. mydisposal his strains of Spirochceta_duttoni,Spirochcetaobermeieri,and Spirochceta
novyl. The Spirochwta kochi employed by me was obtained from Dr. B. T.
Terry.

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Pure Cultivation of Spirochwta~.

It is very important to employ samples of ascitic fluids whichcontain no bile, but which form a loose fibrin in the culture tube,
for many specimens are unsuitable just because they contain too
much bile or do not cause the formation of fibrin when mixed withthe fresh tissue in the culture tube. Fluids which have been heated
to 56 ° to 6o ° C. for thirty minutes, or which have been filteredthrough ,the Berkefeld filter are unsuitable. The addition o fbouillon or sugar to the fluids also diminishes their cultural value.
Sterile tissue kept a day or two on ice is less suitable for cultivation
purposes than fresh tissue, and may prove not at all suitable.
It is not necessary to introduce a large number of spirochaet~e
into the culture tubes. An examination of the culture made immediately after my inoculations usually showed one organism in every
twenty or thirty fields, but sometimes it was almost impossible tofind a spiroch~eta even after a long search.
Results of Cultivation.--By the method described above I havesucceeded in obtaining pure cultures of the four varieties with which
I experimented; i. e., Spirochwta duttoni, Spirochcetct kochi, Spirochceta obermeieri, and Spirochieta novyi. Spirochwta kochi was thefirst to be cultivated and has already gone through twenty-nine
generations within six months. Spirochceta n ovyi was the last to be
cultivated and is now in i~ts fourth passage. It ma.y be stated that
subcultures can be obtained in the tissue ascitic fluid media by inoculating into it about 0. 5 of a cubic centimeter of the original culture.
For growth in these subcultures t h e addition of a small amount of
normal rat or human blood is advantageous but not essential.
P U R E C U L T I V A T I O N OF S P I R O C H 2 E T A D U T T O N I .

Rate of Multiplication.--When a small number of organisms
is inoculated, the original culture tube shows almost no sign ofmultiplication within twenty-fore- hours. On microscopical examination, after forty-eight hours one perceives a slight increase in thenumber of organisms, but at the seventy-second hour the number ofspiroch~et~e has increased decidedly. Four t o s i x days after inoculation the organisms have increased so much that one may be seen
in each field. On the eighth or ninth day the maximum in multipli-

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203

cation is reached, and in every field many actively motile organisms
may be seen singly, in chains, or in masses. On the tenth day there
comes a sudden change. Not only have the spirocha~t~e ceased toincrease, but a beginning diminution in their number may be observed. The active organisms are very few and the other spiroch~et~e seem to be on the verge of disintegration. The spiroch~et~e
are seen to lose their protoplasm, and the spiral framework of the
body is left bare. Many.spherical bodies and irregular protoplasmic
masses make their appearance. In cultures which are not covered
with oil and which are more than fifteen days old it is exceptional tofind an active spiroch~eta, 'but I should mention that 'the spiroch~et~emultiply somewhat more slowly and disappear also somewhat laterin the tubes in which the contents are covered with paraffin oil thanin the tubes Containing no oil.
At 15 ° C. no multiplication takes place and the organisms undergo
degeneration very promptly. No growth is obtainable in an atmosphere of hydrogen or in vacuo.
Effect upon the Culture Media.--Apparently there is no visible
alteration in the culture media in which the spiroch~et~e are rapidly
multiplying. The tissue may look somewhat paler, but no coagulation of the fluid (ascitic or hydrocele) takes place, and no odor is
produced by the growth.
Tran~plantations.--Subcultures can be made at any time between
the fourth and ninth days. It is not advisable to make the subinoculations earlier than the fourth day or after the culture has passed
its period of maximum growth. By using about 0. 5 to I cubic
centimeter of the culture any number of successive transfers (socalled passages) may be obtained. It is extremely important thatno bacterial contamination should occur, because the Simultaneous
growth of certain cocci or bacilli causes the spirocha~t~e to disappear
from the culture.
Morphology.--(Figures I to 5-) In young cultures short forms
with only two or three curves are very numerous. In the culturesapproaching the maximum growth (eighth to ninth day) the lengthof the organisms is about that seen in normal infections. The curves
are very regular and deep. The spiroch~et~e may be single, paired,or in chains. They are vigorously motile. T h e y usually show a204

Pure Cultivation of Spirochc~tw.

long, regularly curved, delicate projection at each end of the body.
In old cultures, after ten days, the spirochaetae lose their motility
and the protoplasmic masses concentrate at varying points along
the spiral skeleton; There are many organisms which show one ormore spore-like spherical bodies attached laterally at irregular intervals (figure 2). In such old cultures there are usually many spiralskeletons entangled or embedded in granular protoplasmic remains(figure 5), But even in these old cultures a few active spirochaetae
are occasionally met with. Old cultures may be temporarily rejuvenated, i. e., for three or four days, by introducing into them fresh
ascitic fluid and a piece of sterile fresh tissue, but degeneration thatis almost complete soon follows.
It is interesting to notice that the spirochaet~e grown under a layerof paraffin oil are somewhat thinner than those grown in-the absenceof oil.
Mode of Division.--(Figure 4-) In luxuriantly growing cul,tures it is difficult to decide whether the spiroch~etae undergo longitudinal or transverse division, as they are usually seen in pairs or•chains, united to each other 'by a thin filament. Their general appearance suggests transverse fission, but no undoubted instance oftransverse division has thus far been observed. At the same time
there are always a few instances of unmistakable longitudinal division. Moreover, I have repeatedly found cultures in which, probably due to certain unfavorable conditions, almost every organism
showed partial longitudinal division. When these organisms werewatched for some time under the dark-field microscope, the process.of longitudinal division could be observed.
Pathogenicity.--gpirochceta duttoni cultivated in vitro remains
infectious for rats and mice. I have been able to infect rats andmice with cultures that were in their ninth passage, and as far as
the severity of the infection is concerned no difference has been
seen between that caused by cultures and that caused by inoculating
blood obtained from infected animals. But, as will be pointed out
when Spirochwta kochi is discussed, it is possible that a diminution
in the virulence may yet be observed when the number of passages
has become sufficiently large.

Hideyo Noguchi.
PURE

CULTIVATION

OF

205

SPIROCItFETA

KOCtII.

Spirochceta kochi is the first variety of the spiroch~eta~ of relapsing
fever that was successfully cultivated by me. In most respects it isvery similar to Spirochceta duttoni. Spirochxta kochi has been
growing in vitro since February, 1912 , and by renewing the culturesr~gularly every week no difficulty has thus far been experienced in
keeping the culture growing. _As a rule, the maximum growth isreached on the ninth day. As in the case of Spirochceta duttoni, novisible alteration of the media results from the growth and there isno perceptible odor at any period of its cultivation. Morphologically (figures 6 to I I) it is almost indistinguishable from Spirochceta duttoni ex.cept that it is possibly a trifle thinner than ,the latter.
It divides longitudinally and possibly also transversely.
Pathogenicity.--Spirochceta kochi cultivated in vitro retained forseveral passages all its usual virulence and killed rats and micefairly constantly, but when tested after the tenth passage in vitro it
no longer killed these animals. In the rats and mice that had been
inoculated, spiroch~et~e developed in large numbers, but their appearance was delayed to the third or fourth day. The sixteenth passage
also caused an infection in these animMs, but was apparently lessvirulent than the tenth passage. F r o m this it appears that thevirulence of these organisms is being gradually attenuated by prolonged cultivation in vitro. The fourth passage of the culture failed
to infect rabbits, guinea pigs, cats, dogs, a chicken, or a Macacus
rhesus monkey.
P U R E C U L T I V A T I O N OF S P I R O C H ~ T A

0 B E R M E I E R I . 16

Spirochceta obermeieri multiplies more rapidly in cultures in vitro
than Spirochceta duttoni or Spirochceta kochi. Within twenty-fourhours there is a decided increase in the number of organisms in theculture and the maximum growth is usually reached on the seventhday, when every field shows several spirocha~t~e. The decline commences after the seventh day, and within the next two days degeneration sets in. Under the cover of paraffin oil the organisms growmore slowly and remain active for a day or two longer than. in the
tubes containing no oil.
~ Synonymous with

Spirochceta recurrentis.

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Pure Cultivation of Spirochc~t~.

Transplantations.--Successful subcultures are best obtained byinoculating new media with about one cubi,c centimeter of a culturethat has shown active growth for three to four days, although aculture at the height of its growth (seventh day) can also be used. 17
I have found it always advantageous to provide two series of tubes,- - o n e with and the other without the paraffin oil layer, because notinfrequently the growth is more abundant and vigorous in one thanin the other of these two series. I have been unable to determine thecause of this irregularity, but there is no difficulty in continuing the.cultivation if both conditions are provided. No growth takes placeat room temperature.
Morphology.--( Figures 12 to 17. ) In young cultures (not older
than three or four days) there are, besides the active specimens of
average length and width, numerous very motile short organisms
which show only two or three curves. Among these forms one
frequently encounters the skeletons of degenerated organisms,
which were no doubt introduced into the culture with ,the inoculation material and died afterwards. In regard to their generalappearance there is a striking contrast between the spirochmtae foundin the blood of an infected mouse or rat and those seen in young
cultures. The former are quite regular in their curves and length.
The cultivated organisms are usually irregularly wavy and shorter.
The movements are similar in both; at one time there is a serpenfine forward movement and at other times there are violent lateral
vibrations which involve the middle portion of the body and bothends. In older cultures (six or seven days) the lengths of theorganisms are more uniform and approach the standard observed in
specimens from the blood, but the curves are still shallower andmore irregular. The Organisms may be entangled in large masses
or may be found in pairs or in chains (figure 14). Some specimens
have blunt ends to which a mass of minute refractive granules areattached (figure 16). In this stage every organism is actively
motile.
The cultures which have just passed their maximum growth entera period of decline o r degeneration. These cultures contain num1~The addition of several drops of fresh, undefibrinated, normal rat bloodfavors the growth decidedly.

Hideyo Noguchi.

207

erous motionless forms which have irregular concentrations of protoplasm at various portions of the spiral skeleton. To many organisms are now attached one or more spherical bodies measuring
about o.7 of a micron in diameter and resembling spores. Thesebodies are, however, not within the protoplasm, but are attached tothe sides of the spiroch~et~e. Free spherical bodies may also befound. There are many skeletons of spirals which show no longer
any masses of protoplasm. Sometimes these degenerated forms areclosely bound together by a mass of granular protoplasm that varies
in size and form. Occasionally quite normal active spiroch~et~e areseen.
If left alone the degenerative changes just described proceed
rapidly and within three or four days almost no spiroch~et~e with
protoplasm can be found. On the other hand, if fresh tissue andascitic fluid are added, one can rejuvenate these cultures for Cwo to
three days, but under these conditions the vigor of the multiplication is limited and feeble.
Mode of Division.--( Figure 15. ) In pure cultures Spirochceta~)bermeieri mul,tiplies by longitudinal division, and possibly also bytransverse fission. The longitudinal division occurs in almost everyorganism whether it be single, paired, or in a chain, and irrespectiveo f the length of the spiroch~eta. As stated already, the spiroch~et~e.cultivated in vitro are less curved than those found in the blood.
They are usually somewhat broader, especially when showing longitudinal division. The organisms which are about to divide aresomewhat less vigorously motile. The first sign of division is the.appearance at any part of the organism of a short narrow cleft
parallel to the axis of the spiroch~eta. Within a few seconds anothershort cleft comes into view at some distance from the first one;then, after short intervals, a third and fourth cleft are seen. The.organism now appears as if its two halves were connected with eachother by several protoplasmic bridges of varying width. At thesame time each of the halves begins to jerk in a peculiar way, themovements of the halves being alternately in opposite directions,T h e positions of the several clefts change, one taking the place ofanother. The fluid character of the protoplasmic bridges which~connect the two halves is thus suggested. Finally two clefts fuse

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Pure Cultivation of Spiroch~tce.

together into a longer cleft, and into this a third and fourth cleft aregradually merged. The organism is now split into two parts, except
for the extremities, which in time divide completely. Thus two
spirocha~tm are formed from one, and, as a rule, one of them dashes
away immediately. In some instances the newly formed daughter
cells are held together by a thin filament. On the other hand, atransverse division seems also to occur. This mode of division m a y
be indicated by the presence of two or more organisms joined
together by a thin filament. It frequently happens that the lengthof one member of the pair of spirocha~t~e thus connected is muchgreater than the other. This appearance suggests that the pair wasformed by the transverse division of a longer individual. Nevertheless, the frequency of the occurrence of the transverse fission in
culture must be less than that of longitudinal division, because thelatter can be seen in any multiplying culture, while an absolutely
convincing case of the former has not been observed by me.
Pathogenicity.--The culture of Spirochceta obermeieri retains its
virulence for rats and mice as was proved by the results of inoculating these animals with tl{e seventh passage in vitro. Whether or
not the virulence may gradually become attenuated after the cultivation has been continued for a long time will have to be determinedlater.
PURE CULTIVATION OF SPIROCI-ITETA NOVYI.

This variety is now recognized as a distinct species, chiefly
through the studies of Fraenkel, Schellacl~, U hlenhuth and HaendeI,
and others. Their conclusions are based upon immunity phenomena, although certain morphological peculiarities are also pointed
out. It was first noticed by Schel!ack that Spirochceta novyi resembles Spirochwta obermeieri more closely than it does the Africart
varieties, but it is somewhat thinner than the Spiroch6eta obermeieri.
Norris, Pappenheimer, and Flournoy, as well as Novy andKnapp, TM observed a certain multiplication of this organism incitrated blood kept a,t body temperature, but a second transfer
In their early articles this variety was dealt with as Splrochceta obermeieri.
as there was still no differentiation established between these two species. Schellack in 19o8 was the first to give this organism the name of Spirochceta novy~
and separate it from the obermeieri.

Hideyo NoguchL

209:showed no further growth. Briefly stated, no culture in the strict,sense of the term was obtained.
In my experiment I obtained a growth of this organism in thesame kind of medium as that in which the other three strains were
cultivated. I have, however, experienced more difficulties with thisvariety than with the rest, but have at last succeeded in getting it tog r o w in vitro. It is now in its fourth passage. 19
The general characteristics (figures 18 to 21) of the culture arealmost identical with those of Spiroch6eta obermeieri except thatthe novyi is somewhat ,thinner than the latter. It shows longitudinal
division as well as forms suggestive of transverse division. U p to
the present the virulence of ,the organism growing in vitro is com-.
parable to that of the spirochmt~e contained in the blood of infected
rats and mice.
SUM IV[ARY.

I. A method for the pure cultivation of Spirochceta duttoni,
Spirochcta kochi, Spirochveta obermeieri, and Spirochceta novyi is
described in this paper. In vitro these strains reach their maximum
growth after seven, eight, or nine days at 37 ° C. For their multiplication they require the presence of a piece of fresh sterile tissue
and a body fluid capable of forming a loose fibrin with the tissue.
T h e presence of some oxygen seems indispensable for their growth,:since they fail to grow in an atmosphere of hydrogen or in vacuo.
N o growth was obtained at room temperature.
2. From cultures that show a good growth subcultures can bemade and the growth can be kept up in this way for many passages.
3- The pathogenicity of these organisms is not lost ,by cultivation,although there is a tendency for the virulence to become attenuated,after growth in vitro has continued for a long time.
4. Longitudinal division has been observed and was followed
amder the dark-field microscope in fresh preparations from cultures.
It occurs in all the species irrespective of the length of the organism.
'Transverse division seems also to occur, but I have not yet observed
the entire process.
19A more vigorous growth in subcultures can be obtained by adding several<trops of fresh, undefibrinated, normal rat blood to the media.

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Pure Cultivation of Spirochc~t~.
EXPLANATIONOF

PLATES.

PLATE 19.

Series A.--Schematic reproductions of the spiroehaetae under the dark-fieldmicroscope.
FIGS. I to 5. Different stages of growth of Spirochceta duttoni in pure culture. Fig. I, young forms; Fig. 2, average forms with spore-like bodies in oneof the spirochmtae; Fig. 3, a mass of spirochaetae; Fig. 4, longitudinal division;
Fig. 5, degeneration forms.
FIGS. 6 to II. Different stages of growth of Spirochwta kochi in pure culture. Fig. 6, young forms; Fig. 7, average forms ; Fig. 8, spirochmtre with sporelike bodies; Fig. 9, an entangled mass of the spirochretae; Fig. IO, longitudinal
and transverse division; Fig. II, degeneration phase.
FIGS. 12 to 17. Spirochceta obermeieri in pure culture. Fig. 12, young
forms; Fig. 13, usual forms; Fig. 14, a mass of growing spirochaet~e; Fig. 15,
longitudinal and transverse division; Fig. 16, blunt forms with granulation;
Fig. 17, degeneration phase.
FIGS. 18 to 21. Spirochcta novyi in pure culture. Fig. 18, young forms;
Fig. 19, average forms; Fig. 20, longitudinal and transverse division; also a few
spore-like bodies; Fig. 21, degeneration stage.
PLATE 20.

Series B.--Microphotographs of the spirochaetae from the preparations fixedin sublimate alcohol and stained for twelve hours with the Giemsa solution.
X I,I00.

FIGS. 22, 23, and 24. Spirochceta duttoni from pure cultures (fourth generation). Fig. 22, average forms; Fig. 23, one long and two short forms; Fig. 24,
an entangled mass.
FIGS.25 and 26. Spiroehceta kochi from pure cultures (twenty-fourth.generation). Fig. 25, young forms; Fig. 26, near the time of decline (tenth day).
FIGS. 27, 28, 29, and 30. Spirochceta oberrneieri from pure cultures (fourfl
generation). Fig. 27, an average and a small f o r m ; Fig. 28, a doubled-up f o r m ;Fig. 29, entangled spirochaet~e near the time of decline (eighth day) ; Fig. 30, a
peculiar form consisting of partially fused spirochaetoe, probably a result of imperfect longitudinal division in succession. This often occurs in a less favorable
culture medium. It is not an artifact produced during the fixation, because it
can be seen in a fresh preparation under the dark-field microscope; each individual of the mass is actively motile.
FIGS. 31 and 32. Spirochceta novyi from pure cultures (fourth generation).
Fig. 31 shows the average forms, and Fig. 32 shows a doubled-up specimen.

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVl,

PLATE 19.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVI.

F'LATE 20.