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Journal of Experimental Medicine

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ETIOLOGY OF YELLOW FEVER.
VI.

CULTIVATION, MORPHOLOGY, VIRULENCE, AND BIOLOGICALPROPERTIES OF LEPTOSPIRA ICTEROIDES.
BY H I D E Y O NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
PLATES i TO 3.

(Received for publication, May 2, 1919.)
Cultivation.
As the nature of the causative agent of yellow fever was unknown,
it was necessary at the beginning of these experiments to formulate
a spedal method of cultivation. The methods employed have been
similar to those recommended1 for the cultivation of Leptospiraicterohcemorrhagice (Inada and Ido). Instead of the serum and citrateplasma of the rabbit or other animal, serum and plasma from nonimmune persons were used during the early stage of the cultivation"
experiments. The principal medium consisted of a mixture of 1
part of the serum and 3 parts of Ringer solution, used in a combination of the liquid form and a form made semisolid by adding melted
neutral agar (0.3 per cent), the liquid half (8 cc.) of the medium being
superimposed on the semisolid half (8 cc.) in a tall culture tube suchas that used in the cultivation of spirochetes. *The first step in the inoculation of the medium was to mix about
0.5 to 1 cc. of the citrate blood, drawn from the median basilic veinof the patient, with the lower or semisolid portion of the medium,
while the latter was still in the fluid state (42°C.), and allow the
mixture to solidify by cooling. The serum-Ringer dilution was t h e npoured on the semisolid portion and about 0.5 to 1 cc. more of thesame blood introduced. A layer of paraffin oil was finally added to
1Noguchi, H., J. Exp. Med., 1918, xxvii, 575.
Noguchi, H., J. Exp. Med., 1912, xvi, 199.
13

14

ETIOLOGY O F YELLOW F E V E R .

VI

cover the surface of the medium. When making a culture with thecitrate blood from a patient no human citrate plasma was added to theliquid portion of the medium, as the.plasma contained in the blood was
sufficient to form a 19ose fibrin throughout that portion. When subsequent subcultures were set up, however, 0.5 to 1 cc. of citratehuman or rabbit plasma was introduced into the liquid portion after
inoculation. The presence of a loose cobweb fibrin in the culture
medium seems to favor the growth of certain organisms. I The conditions provided for in this form of culture medium would allow thegrowth of various microorganisms requiring different degrees ofoxygen tension. In a later period 2 to 3 cc. of the citrate blood wereused for each of two or three large flasks (50 cc.) with correspondingly
larger quantities of culture medium (25 cc.).
JDirect Cultivation from Yellow Fever Patients.
Cultures were made from eleven cases of yellow fever, with only
three successful isolations of the leptospira. In the first instance(Case 1) I failed to detect the organisms under the dark-field microscope, but in a culture 3 days old (kept at 26°C.) a few active leptospiras were seen. This was inoculated into four guinea pigs, all ofwhom died later of the typical experimental infection. One of theguinea pigs had epistaxis and melena in addition to intense jaundice,advanced degeneration of the liver, acute parenchymatous nephritis,
ecchymoses in the lungs, stomach, and intestines. 8 This culture waspresently lost through a secondary fungus contamination, which wasdifficult to avoid under the conditions in which the work had to be
carried on.
The second positive growth was obtained with the blood derived
from Case 4 on the 3rd day of the disease. The organisms werereadily detected in the culture after 5 days at 30°C. A few leptospiras were present in the blood when carefully examined in thestained preparations, and the guinea pigs inoculated with this specimen came down with the typical symptoms in 8 days. The culture
proved to be pathogenic for guinea pigs.
3 Noguchi, H., J. Exp. Med., 1919, xxix, 565.

I~IDEYO NOGIIClaI1S

The third successful direct cultivation of the organism from yellow fever patients was obtained with the blood from a fatal case
(Case 6). The blood was drawn on the 5th day of the disease andput immediately into six tubes containing the culture medium (October 19, 1918). On October 26 one of the inoculated tubes showed thepresence of the leptospira under the dark-field microscope. The
culture proved to be capable of producing the typical symptoms andlesions in guinea pigs, pups, and marmosets.*

Cultivation from Experimental Animals.
The method employed for obtaining a culture of Leptospira icteroides was the same as that outlined for direct cultivation from humanblood, except that normal rabbit serum and citrate plasma were usedinstead of human. The blood was obtained from the heart before
the death of the animal. The method was not always successful
in the first generation, but it was nevertheless the most reliable ofthe various combinations tried, In a later subculture the additionof the citrate plasma becomes unessential, although a better growthis had when it is added.
Six strains (Cases I to 6) of yellow fever leptospira have been
maintained to date by passage in guinea pigs. No direct culture wasobtained with the blood from Cases 2, 3, and 5, although cultures
were finally obtained from the blood of guinea pigs or marmosets
inoculated with the passage strain. These cultures, whether obtained directly from the blood of yellow fever patients or indirectly by
way of animal inoculation, were found to be uniform in their characteristics and could be maintained in culture for many months. The
leptospira isolated from yellow fever cases is extremely sensitive to
any alien microbic intrusion, not surviving the slightest contaminationin culture, and the failure to obtain a culture directly from yellow
fever cases can in every instance be explained through the occurrenceof secondary contamination.
When the guinea pigs reach the stage of collapse, with intense
jaundice, it is seldom possible to detect the leptospira in the circulation, and a positive transfer to normal guinea pigs becomes uncertain.
4Noguchi. H., J. Exp. Med., 1919, xxix, 585.

16

ETIOLOGY OF Y E L L O W F E V E R .

VI

Cultures set up with this blood usually remain sterile, and the leptospira is not found in the liver and kidneys. In experimental infectious
jaundice, on the contrary, the leptospira was almost always found in
the later stage of the infection. The extreme lability of Leptospiraicteroides may account for the negative animal inoculation and microscopic findings in so many cases of yellow fever.

Morphology.
The organism which occurs in the blood and tissues in yellow fever
patients in Guayaquil, as well as in those of animals experimentally
infected with the blood or tissue of yellow fever patients, is an extremely delicate filament measuring about 4 to 9 microns in lengthand 0.2 of a micron in width along the middle portion. It tapers
gradually toward the extremities, which end in immeasurably thin
sharp points. The entire filament is not smooth but is minutely
wound at short and regular intervals, the length of each section measuring about 0.25 of a micron. The windings are so placed as to form azigzag line by the alternate change of direction of each consecutive
portion at an angle of 90 ° .
The organism is unrecognizable by translucent light but becomesquite visible under a properly adjusted dark-field illumination. It
possesses an active motility, consisting in vibration, rotation, rapid
bipolar progression, and sometimes twisting of parts of the filament.
When it encounters a semisolid substance it penetrates the latter by aboring motion, and while passing through it the body assumes a
serpentine aspect with few undulations, the elementary windings
undergoing no modification.
The organism manifests remarkable flexibility to almost any angle
while changing its course of progression in a semisolid medium. In a
fluid medium it has fewer and quite characteristic movements. One
end is usually bent in the form of a graceful hook, and, while rapidly
rotating, the organism proceeds in the direction of the straight end,
the hooked end apparently serving as a sort of rear propeller. When
extricating itself from an entanglement, however, the same hooked
end seems to act like the front propeller of an airplane. Many
specimens are seen with both ends hooked, the organism then ro-

I-IJ.OEYO NOGUCHI

17

tatlng in a stationary position unless one hook is larger and more
powerful as a propeller than the other. The rapid rotation makes
the organism appear like a chain of minute dots. From the dynamic
point of view the portions which include the several windings fromthe extremities represent the motor apparatus of the organism. I
have never seen a specimen that doubled at the middle portion of the
body while lying in a free liquid medium. The motor or terminal
portions may be regarded as comparable with the flagella or terminal
filaments seen in a spironema or treponema.
The organism is difficult to stain with ordinary aniline dyes, but
can be made distinct by osmic acid fixation and one of the Romanowsky stains (Giemsa, Wright, Leishman). When stained with Fontana or carbolized gentian violet solution after mordanting with 5
per cent tannin plus 1 per cent phenol the organism appears as amoderately heavy, slightly undulated filament without a clear elementary indentation. The peculiar forms resembling the letters C
and S are quite characteristic. Specimens fixed with methyl alcohol
seldom retain the elementary spirals. The beauty of the organism
as it appears by dark-field illumination is never well retained in astained preparation, even in the best specimens. In the latter it
appears almost as a totally different organism.
From the findings described it is evident that the present organism falls in the general order of so called spirochetes, but in thestrict sense of the term it is neither a bacterium, a spirochete, a spironema, nor a treponema, but belongs to the genus Leptospira, of which
Leptospira icterohcemorrhagice, Leptospira hebdomadis,5 and Leptospirabiflexa have already been described. 1
The study of the strains of Leptospira icteroides obtained fromyellow fever cases in Guayaquil showed the organisms to be of somewhat smaller dimensions than the various strains of Leptospira icteroImmorrhagice in m y possession (six strains), as is readily seen from thephotographs of the organisms shown in Figs. 1 to 9. The difference isstriking when the pictures of the two organisms are compared, particularly in the case of Strain 6 of Leptospira icteroides, which is considerably smaller than any of the other strains.
Ido, Y., Ito, H., and Wani, H., J. Exp. Med., 1918, xxviii, 435.

18~,TIOLOGY OF "gELLOW FEVER.

VI

The strains of Leptospira icterotu~morrhagice isolated from wildrats caught in Guayaquil are also shown (Figs. 10 to 13). These
are seen to be similar to the other strains of Leptospira icterohaemorrhagice, shown in Figs. 5 to 9, and are coarser than the organism
obtained from yellow fever cases.
The photographs shown in the three plates were taken for the purpose of comparison at the same time, under similar conditions, andwith the same magnification.

Cultural Propertges.
Leptospira icteroides does not multiply in a medium in which
there is no access to oxygen. In a dense solid medium it grows wellwithin the zone or layer to which a trace of oxygen can still penetrate,
but no deeper. I t grows best when the supply of oxygen is not excessive, as when a thin layer of liquid paraffin is poured over the surface of the culture medium. A certain amount (above 10 per cent) ofa suitable blood serum is essential for its growth. Various bacterial
culture substances such as peptone, meat extract, various carbohydrates in different forms, or combinations, are unsuitable; their presence in the serum-containing media apparently neither favors nor
impedes development. The percentage of sodium chloride (tried ashigh as 2 per cent) in the medium seems to have but little influence,and either isotonic saline, Ringer solution, or distilled water may be
used as diluent. The organism is highly sensitive to the reaction of
the medium, the optimum growth being obtained with a reactionslightly alkaline to litmus paper, not stronger than 0.025 N. It
grows well in a neutral medium, but not in one with an acid reaction
to litmus paper.
The addition of phenol red to culture media in a ratio of 1 cc. of a
0.0025 per cent solution to 10 cc. of medium has no perceptible disturbing effect upon the growth of Leptospira icteroides. Growth takes
place in culture tubes in which phenol red indicates the values ranging from pH 6 to pH 7.4. In the case of cultures containing rabbit
serum phenol red is gradually decolorized to a trace of pink.
Growth is much more rapid at a temperature of 37°C. than at
25-26°C., but the organisms remain viable much longer at the latter

HIDEYONOGUCIII19

temperature. No growth is obtainable at a temperature above 42°C.
or below 10°C.
Erythrocytes present in the culture do not undergo any special
aIteration that can be ascribed to the growth of this organism, nordoes the hemoglobin. The serum proteins seem in no wise modified
and remain transparent. No external changes of the culture media,
except a light layer of grayish haze over the surface of a solid orsemisolid medium observed in richly growing old cultures, takeplace, and for this reason the growth of the organism can be ascertained microscopically only.
It has been noticed that Leptospira icteroides shows a particularpreference for a semisolid medium such as is provided by the presence of agar (0.3 per cent) or by loose fibrin. They entangle themselves in the substance in large numbers and move about in it very
actively. Continuous multiplication goes on in thi.~ type of medium.
After a few weeks the growth may become so dense as to render theuppermost layer of the medium faintly grayish. This peculiarity ofthe organism may partly account for its predilection for the parenchymatous organs such as the liver and kidney.
Leptospira icteroides multiplies through transverse fission.

Virulence.
While the pathogenic properties of Leptospira icteroides for different species of animals Ihave not been exhaustively studied, it has
been shown that most of the domestic an~raals, such as the donkey,
horse, sheep, pig, and cat are completely refractory to the injectionof the organism. Very young dogs, not older than 6 or 7 weeks,
are found to succumb to experimental infection. None of the birds
so far employed for experiment has been found to be susceptible.3
Among the mammals the guinea pig appears to be most susceptible andthe marmoset somewhat less so. For this reason the guinea pig has
been chosen for determining the degree of virulence of several strainsof Leptospira icteroides.
The mode of inoculation consisted in intraperitoneal injection indescending doses of a culture 2 to 3 weeks old. The guinea pigs
used varied from 300 to 350 gm. A 2 to 3 weeks old culture (26°C.)of this organism, grown in a semisolid rabbit serum medium with

20

ETIOLOGY OF YELLOW FEVER.

VI

0.15 per cent agar, m a y contain 50 to 100 leptospiras per field (Leitz~ oil immersion and ocular 4). I n order to arrive at an accurate det e r m i n a t i o n of virulence different strains would h a v e to be used incorrespondingly c o m p a r a b l e concentrations. B u t this is extremely
difficult in the case of an organism which forms entangled masses" ofm a n y individuals or shows a t e n d e n c y to congregate in varying n u m bers a b o u t the particles of culture medium. I n the present series ofexperiments suspensions of cultures of different strains were so prep a r e d as to m a k e each contain a p p r o x i m a t e l y an equal n u m b e r of organisms in the suspension from which higher dilutions were prepared.
E a c h strain was used in successive tenfold dilutions, and one or two
guinea pigs were inoculated with 1 cc. each of each dilution. Because
TABLE I.
Determination of Virulence of Leptospira icteroides, Strain 1.
Guinea pig No.

Q u a n t i t y of culture,

Incubation ort me after inoculation
to onset of fever.

cc.

days

1

0.1
0.01
0.001
0.00010.00001
0.000001

3
4
5
3½
4
5
No fever.

Result.

Died in 7 days.
Survived (!). No jaundice.
Died in 9 days.
" " 10 "Survived.

of individual variations in resistance a m o n g the guinea pigs in alllater experiments two animals were used for each dilution.
F o u r strains of Leptospira icteroides were studied in this way.
T h e results are recorded in the folloMng protocols.
Experiment /.--Aug. 10, 1918 (at the Guayaquil Yellow Fever Hospital)
Strain 1. 18 day culture of the second generation, grown on semisolid humanserum agar at 30°C. (Table I.)
Experiment 2.--I)ec. 2, 1918. Strain 3. 20 day culture of the third generation, grown on semisolid rabbit serum agar medium at 26°C. (Table II.)
Experiment &--Jan. 2, 1919. Strain 5. 3 week culture of the third generation, isolated from a marmoset experimentally infected with a visceralemulsion from a guinea pig which died of the typical infection after inoculation
with blood from a fatal yellow fever case. The culture was grown on the same
medium as that used for Strain 6, at 26°C. (Table III.)21

I:IIDE¥O NOGIICitITABLE II.

Determination of Virulence of Leptospira icteroides, Strain 3.
Guinea pig No.

Quantity of culture.

Incubation, or
time after inoculation
to onsetoffever.

C6.

days

1

1

2
3
4
5
6
7

1

8

9
10
11
12
13

0.1
0.1
0.01
0.01
0.001
0.001
0.00010.00010.00001
0.00001
O. 000001

Result.

Died in 8 days.
Survived (!).
Died in 9 days.

5
5

s½
4

5
5

....

8½ "

Survived (!).
Died in 11 days.
- "
" 9 "
Survived.
tg

No fever.
7
No fever.

t~

TABLE III.

Determination of Virulence of Leptospira ieteroides, Strain 5,
Guinea pig No.

Quantity of culture,i
Incubation, ort i m e after inoculation
to onset of fever.

CO.

days

Result.

3
3
21
3½

Died in 7 days.

0.00010.00001

5
3~
5
4
6

0.00001

No fever.

1

1

2
3
4
5
6
7
8
9
10
11
12
13

0.I
0.1
0.01
0.01,
0.001
0.001
0.00010.000001
O. 000001

"

"

6

"

"

"

7

"

"

" 10

"

....
10
" " 9
Survived.

"
"

~t
~t
~t

Experiment 4 . - - J a n . 2, 1919. Strain 6. 20 day culture of the third generation, directly derived from h u m a n blood (not passed through guinea pigs),
grown on semisolid rabbit serum agar at 26°C. There were about 25 organisms
per field in the original suspension. (Table IV.)22

ETIOLOGY

0]? YELLOW FEVER.

VI

The foregoing experiments show t h a t the strains of L e p t o s p l r a
icteroides possess, on the whole, a strong virulence for guinea pigs.

In two cases (Nos. 1 and 6) the minimal lethal dose was 0.00001 cc.,in one (No. 5) 0.0001 cc., and in another (No. 3) 0.001 cc. B u t
in the experiments with Strains 1 and 3 some of the guinea pigs
receiving as large a q u a n t i t y as 1 cc. or 0.1 cc., showed only a transient
febrile reaction and speedily returned to normal, notwithstanding
the fact t h a t 0.0001 p a r t of these doses killed other guinea pigs in
the same series of experiments. This is not altogether exceptional,
TABLE IV.
Determination of Virulence of Leptospira icteroides, Strain 6.
Guinea pig No.

Quantity of culture,

Incubation, or
time after inoculation
to onset of fever.

#G.

days

1

1

4

2
3
4
5
6
7

0.1
0.1
0.01
0.01
0.001
0.001
0.00010.00010.00001

3

8
9

10
11
12

13

0.00001

0.000001
0.0OO001

Result.

Died in 7 days.
" " 10 ~c

3½
4
s (~)

" "
" "
....

3

"

5

4
Doubtful.
3
6
4
No fever.

6
8
7

" 9½
" 10
" " 10Survived.
Died in 8 days.
Survived.
"C~because in the higher dilutions there were instances in which a smaller
dose induced a fatal infection while a larger one failed to do so.
Again, in fatal instances the severity of the infection did not parallel
the a m o u n t of culture injected. In other words, the susceptibilityof guinea pigs varies considerably among different individuals. In
another series of experiments, not y e t reported, it was noticed t h a tcertain guinea pigs possess an almost complete natural i m m u n i t y to
Le'ptospira icteroides.
This becomes an important factor in a consideration of the percentage of successful transmissions of this organism from h u m a n cases to guinea pigs.

HIDEYO NOGUCHI

23

Gradual Loss o/Virulence through Cultivation.
All the strains of Leptospira icteroides were brought to New York
from Guayaquil on semisolid rabbit serum agar. The cultures were
kept at ordinary temperature during the journey (about 28°C. inthe tropics and 15°C. after reaching the United States). They had
been renewed in Guayaquil on October 26, 1918, and were tested fortheir pathogenicity for guinea pigs in New York on December 2,
1918; that is, 37 days after the transfer into new media. Intraperitoileal inoculations were made into guinea pigs of 1 cc. of Strains 1,
3, 4, 5, and 6, and t h e animals developed the usual symptoms andlesions characteristic of the infection produced by these strains,
showing that under the circumstances described the organism remained
virulent for 37 days.
On December 9, 1918, some of the older cultures of Strains 1 and 3,
which had stood over 4 months since cultivation, were also tested,
with.varying success. Strain 1 was still quite virulent, but Strain 3
failed to produce a fatal infection. In a subsequent experiment,
however, by using six guinea pigs, each being inoculated with 2 cc.
of the culture intraperitoneally, it was possible to obtain a fatal infection in one of the animals. Through this guinea pig the virulenceof the culture was again raised to its original height; that is, it again
became capable of causing typical infection in guinea pigs in smaller
quantities.
It should be noted that examination of the viscera, especially thelungs, of the guinea pigs which escaped death or severe infection
from the inoculation of an attenuated strain, by killing them at theend of about 14 clays from the time of inoculation, usually revealed
the presence of old hemorrhages of greater or less extent in the lungs.
Perhaps it may prove a useful procedure for ascertaining the outcome of transmission to inoculate several guinea pigs with the bloodof a yellow fever patient and examine the lungs within a period of
from 10 to 14 days. In this way, notwithstanding the absence of
striking external manifestations, the results of inoculation can be
more accurately followed. Unfortunately this fact was not known at
the time of the experiments, reliance being placed upon the development of a fatal infection.

24

ETIOLOGY OF YELLOW F E V E R .

VI

For the past 4 months horse serum has been used for culture
media, since it was easily obtainable at much less expense and inlarger quantities than rabbit or sheep serum. It is far less sarisfactory, however, for the cultivation of Leptospira icteroides, thansheep serum, which in turn is much inferior to rabbit serum. In
testing out the virulence of different strains of the organism recently, rather rapid loss of virulence has been encountered. The
reason was not at first clear, the period of time being comparatively
short, but it soon became evident that cultivation of Leptospira icteroides on sheep or horse serum media leads to rapid diminution of virulence, since the cultures grown on rabbit serum media remained pathogenic. By following the course of development of these strains inmedia containing rabbit serum and phenol red it was found that theoriginal pinkish color of the indicator gradually becomes paler until
it fades to a trace. The color may be brought back to a deeper hue
by the addition of disodium phosphates, b u t it never reaches the original grade. Apparently a change takes place in the media as well "as in
the indicator through the growth of the organism. Such a change has
not been observed in the cultures grown on media containing sheep orhorse serum. Whether the persistence of virulence of the organism inthe media containing the rabbit serum has any relation to the phenomenon just described or whether they are two unrelated coincidental phenomena has not been further studied.

Resistance and Viability.
Leptospira icteroides is a non-spore-bearing organism and offers
little resistance to the action of heat, desiccation, putrefaction, or
disinfectants.
Heating to 55°C. for 10 minutes or freezing and thawing kill theorganism, and complete desiccation promptly destroys its vitality.
In the presence of various bacteria, such as Bacillus coli, Bacillus

aerogenes, Bacillus subtilis, Bacillus mesentericus, Bacillus pyocyaneus,
pneumococcus, staphylococcus, Streptococcus hcemolyticus, etc., Leptospira icteroides is destroyed within a short time. The more numerous
the bacteria the quicker the disappearance of the leptospira; hence indecomposing excreta or urine, sewer or stagnant water, of in con-HIDEYO NOGUCH1

25

taminated foodstuffs, no leptospira can be found 24 hours after being
introduced into them.
On the other hand, certain contaminating fungi or non-putrefactive and non-acid-producing bacteria, sometimes bacilli and sometimes cocci, have been found growing in the cultures of Leptospiraicteroides without seriously interfering with the viability of the latter,
which are actively motile among the intruding fungi or bacteria. In
such a contaminated culture these intruders do not cause any perceptible modification of the culture medium, except that their discrete colonies may be found imbedded here and there in the medium.
When a pure culture of the organism was poured into a cup ofsterile distilled water and left unprotected from the air or dust the
leptospiras survived several days, but finally disappeared, partly because of the lack of nutrition and partly because of bacterial growth.
The leptospira intentionally added in large quantity to fecal matterkept at room temperature disappeared within a few hours.
On several occasions attempts were made to infect the larva~ of
Stegomyia calopus by introducing emulsions of liver or kidney containing a large number of the organisms into the receptacle with thelarva~, but no leptospira could be found in such a mixture after 2 hours.
In this respect the virus of yellow fever is one of the least resistant of
all pathogenic organisms which have been obtained in culture. In
m y experience there has seldom been an impure culture of thisorganism.
Another interesting phenomenon in connection with the organism
in question is that it soon dies out; it may degenerate within 12 hours
in a piece of liver or kidney removed from an infected guinea pig andkept at a temperature of about 10°C. In hundreds of instances a
leptospira was found only rarely in the liver, kidney, or blood fromguinea pigs which had died of typical experimental yellow fever several hours before autopsy. In this respect Leptospira icteroides
differs considerably from Leptospira icterohcemorrhagiceisolated fromthe cases of infectious jaundice in Japan or Europe, the latter being
still easily recoverable from animals kept over night after death.
With regard to the resistance of the organism to the action of various ordinary disinfectants the work is still incomplete. It has beenfound, however, that it is readily killed within 5 minutes by 2 per

26

ETIOLOGYOFY ELLO W F E V E R .

VIcent phenol or 0.1 per cent bichloride of mercury. In a 10 per centsolution of sodium taurocholate, sodium glycocholate, or sodium
cholate the organism promptly disintegrates, but saponin has no injurious effect upon it. Human or animal bile dissolves the organism
rapidly when used in concentrations stronger than 30 per cent.

Filterability.
A noteworthy characteristic of Leptospira icteroides is its ability• to pass through the pores of filters. Some experiments to determine
this point were carried out as early in the investigation as the transmission of Strain 1 from the human case to guinea pigs. By theuse of Berkefeld filters V and N with suction by means of a water pump
it was possible to filter an emulsion of the liver and kidney of a guinea
pig experimentally infected with the passage strain 7 days previously.
The clear filtrates, which were bacteriologically sterile, were inoculated
intraperitoneally into normal guinea pigs in doses of 10 cc. each on
August 8, 1918. Both animals came down with typical symptoms
after 7½ and 8 days respectively. In the blood of these animals asmall number of leptospiras were demonstrated 24 hours before death.
In the emulsions of the liver and kidney the organisms were alsopresent, and upon further passage to normal guinea pigs the emulsion
proved to be infectious.

Possibility of the Existence of a Granular Phase in the Life of Leptospiraicteroides.
That there m a y exist a granular phase of life in various membersof the family of spirochetes has been repeatedly suggested by investigators. Balfour, Fantham, Leishman, and Todd 6 advanced the ideathat the spirochetes of relapsing fevers in man and fowls pass througha granular stage at some time in their life. The following observation
seems strongly to suggest the possibility that this phenomenon also
occurs in the life of Leptospira icteroides.
6Balfour, A., Internat. Congr. Med., 1913, xxi, 275. Fantham, H. B., Ann.
Trop. Med. and Parasit., 1914, viii, 471. Leishman, W. B., Internat. Congr. Med.,
1913, xxi, 282. Todd, J. L., personal communication.

mD~.YO NOGUCra27

The various cultures of Leptospira icteroides made at Guayaqnil
on October 26, 1918, were brought back to New York on November
24 without special accident. But on examination no leptospiracould be found in any of the tubes containing cultures of Strain 5,
although the other cultures were growing well. A thorough examination of the eleven tubes of Strain 5 was continued for several days
without success.
Six culture tubes which were made on October 18, 1918, with theblood from Marmoset 4, severely infected with Strain 5, and which
had been showing a fairly good growth on October 26, were also
examined. These tubes showed no spiral organisms. There werelarge numbers of refringent granules imbedded in the culture medium
in which the leptospiras had been abundantly present a month previously. These granules appeared to be the degenerated remains of
the leptospiras. The hope of recovering the strain from these cultures was almost abandoned, but as a last resort a dozen guinea pigs
were inoculated with 1 cc. of the contents of each of these tubes.
Some of these animals in due time came down with typical symptoms.
The spiral forms of the leptospiras were found in varying numbersin the blood, liver, and kidneys of these animals, and a culture of thestrain was regained.
It is of course possible that these old culture tubes contained thespiral leptospira in such small numbers that they escaped microscopic detection, but it is also possible that they existed in a granularphase under certain conditions.
SU'M-~ARY.

By the employment of methods designed to promote the growthboth of aerobic and anaerobic organisms, particularly those belonging
to the class of spirochetes, it was possible to obtain a pure cultureof a delicate organism, the morphological features of which place itin the genus Leptospira. On three occasions, that is, from three outof eleven cases of yellow fever, the organism was directly cultivated.
These three strains were found to induce the characteristic symptoms and lesions when tested on guinea pigs. The organism wasdesignated Leptospira icteroides.

28

ETIOLOGY OF YELLOW FEVER.

VI

Leptospira icteroides was also obtained in pure culture from theblood of guinea pigs which succumbed to infection after being inoculated with the blood or organ emulsions from patients sufferingfrom yellow fever. These cultures also proved to be virulent whentested on susceptible animals.
The morphological characteristics and certain biological propertiesof the organism were considered in detail. It is invisible undertranslucent illumination and is difficult to stain by most aniline
dyes. It is highly sensitive to the presence of bacteria and is
rapidly destroyed in a medium in which certain other organisms arepresent. The presence of blood serum (man, sheep, horse, rabbit,etc.) seems to be essential for its growth. It grows well at a temperature of about 25-26°C. and more quickly at 37°C., though at thelatter temperature it dies out within a few weeks. At 25°C. underfavorable conditions and in suitable culture media it remains viable
for several months without losing its virulence. Leptospira icteroides
multiplies by transverse division.
The virulence attained by some strains was such that 0.00001 cc.
of a culture could induce typical fatal infection in guinea pigs. Thereexists a considerable variation among guinea pigs in their susceptibility to Leptospira icteroides.
The organism is killed within 10 minutes at a temperature of
55°C. and is also destroyed by complete desiccation or freezing andthawing. Bile and bile salts dissolve it in certain concentrations,but not saponin.
Leptospira icteroides passes through the pores of Berkefeld filters~? and N, and there is a possibility of its having a granular phase oflife under certain conditions.
E X P L A N A T I O N OF PLATES.
PLATE 1.

FIG. 1. Dark-field view of a 2 week culture on semisolid rabbit serum agar
medium of Leptospira icteroides. Strain 6 (Case 6). X 3,000.
FIO. 2. The same. Strain 4 (Case 4).
FIO. 3. The same. Strain 5 (Case 5).
FIo. 4. The same. Strain 3 (Case 3).-

HIDE¥O

NOGUCHI

29

PLATE 2.
FIG. 5. Dark-field view of a 2 week culture on semisolid rabbit serum agar

medium of Leptospira icterol~emorrhagice. Japanese s t r a i n . ~ × 3,000.
FIG. 6. The Same. British strain.
FIG. 7. The same. French strain.
FIG. 8. The same. American Strain 1.
FIG. 9. The same. American Strain 2.
PLATE 3.

FIG. 10. Dark-field view of a 2 week culture on semisolid rabbit serum agar
medium of Leptospira icterohcemorrhagice. Group 8 strain obtained from wildrats in Guayaquil. × 3,000.
FIG. 11. The same. Group 11 strain.
FIG. 12. The same. Group 30 strain.
FIG. 13. The same. Group 30 strain.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXX.

PLATE1.

(Noguchi: Etiology of yellow fever. VI.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. X X X .

PLATE 2.

(igoguchi: Etiology of yellow fever. VI.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. X X X .

PLATE 3.

(Noguchi: Etiology of yellow fever. VI.)