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Journal of Experimental Medicine

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THE SURVIVAL OF LEPTOSPIRA (SPIROCH~ETA) ICTEROH2EMORRHAGI2E IN NATURE; OBSERVATIONSCONCERNING MICROCHEMICAL REACTIONSAND I N T E R M E D I A R Y HOSTS.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, February 27, 1918.)

A solution of the question of the survival of Leptospira icteroheemorrhagice in nature may be brought about (1) by following up directly the actual conditions to which the spirochetes cast off by thehosts or artificially mixed with urine or feces will have to submit, or(2) by mixing the spirochetes with each in turn of the various bacteria
commonly encountered in feces, sewage, or soil, and then determining
the results of their simultaneous existence in the same. media,
As I shall show, the spirochete of infectious jaundice is a very delicate organism and is rapidly overwhelmed by most of the bacteria
from intestinal contents, sewage, or soil.

Urine in Rdation to'Leptospira icterohcemorrhaglce.
The effect of urine upon the viability of Leptospira icterohcemorrhagiee is of practical importance, since it has been found by previous
investigators,. 2. 8 that the urine of about 77 per cent of the patients recovering from infectious jaundice still contain~ the spirochete after
a period of 2 to 5 weeks. They made the interesting observation that
two-thirds of the positive urines, some containing numerous spirochetes, failed to produce the infection in guinea pigs. Since the urines
came from cases of 15 days' standing or longer, the fact may be ex1 Ido, Y., Hold, R., Ito, H., and Wani, H., J. Exp. Med., 1917, xxvi, 341.
Gamier, M., and Reilly, J., Compt. rend. Soc. biol., 1917, lxxx, 38.
8 CappeUani, S., and Frugoni, C., Sperimentale, !917, lxxi, 335.
609

610

SURVIVAL

OF LEPTOSPIRAICTEROHZE~OP.RHAGI2E

plained b y an attenuation in virulence of the organism during thecourse of the disease. I have seen one case in which the urine wasstill infective for guinea pigs 1 m o n t h after the onset of the disease.
I n one series of experiments a sample of urine freshly collected froma healthy individual, who had no history of ever having had jaundice,
was tested for its action upon the spirochetes. 10 cc. a m o u n t s of
TABLE

I.
culrain.nto

Changes inappearance andLeptospira icterohvemorrhagi~eintroduced into.

reaction.

~ted
tab1.5
rate

3
~ays.
NormalUrine

Clear, stronglyacid.

urine ..........................
10 ec. +0.1 N sodium hydroxide

cc .............................

<+

0.1
Clear, neutral.

~......

+

Urine 10 cc. + 0.1 N sodium hydroxide 0.2
CC. . . . . . . .

". . . . . . . . . . . . . . . . . . . . . . . . . . .

Precipitate +,slightly alkaline.

+

Precipitate + +moderately
alkaline.

<+

Urine 10 cc. + 0.1 N sodium hydroxide 0 4
ec ....................................

Urine 10 cc. + 0.1 N sodium hydroxide 0.8
cc ...................................

•

Precipitate+++,
markedly alkaline.
the urine, which w a s strongly acid and had a titer such tt{at 10 cc. ofit required 7 cc. of 0.1 N sodium hydroxide to become moderately
alkaline, were measured into a n u m b e r of test-tubes, to each of which
was added normal sodium hydroxide solution, the quantities added
varying in each case in order to obtain a series of reactions from theoriginal acidity of t h e spec/men to a markedly alkaline reaction.

I~IIDEYO NOGUC]KI

611

Cultures were set up in two parallel series, using in each series theoriginal and partially neutralized portions of the urine, but adding to
one series suitable amounts of rabbit serum and citrate plasma. Table
I summarizes the results.
As the table shows, the jaundice spirochetes survived at least 24
hours in the portion of urine to which a quantity of from 0.1 to 0.4
cc. of normal sodium hydroxide solu~tion had been added, but notrace of them could be found in the original urine or in that receiving
0.8 cc. of the alkali. In plain Ringer's solution alone the organism
lived 24 hours under similar conditions. After 48 hours there wereno spirochetes in any of the tubes of the first series.
The results obtained with the urine containing rabbit serumand citrate plasma were different from those of the other series.
There was a good growth in all the tubes containing the unalkalized
urine, and also in those to which had been added from 0.1 to 0.4 cc.
of normal sodium hydroxide solution. The growth was better andlasted longer in the tubes in which the urine showed a neutral orslightly alkaline reaction than in the unmodified or more strongly
alkalized urines. There was no growth in the tube to which 0.8 cc.
of the normal sodium hydroxide had been added. While there wasunmistakable growth in the urine media with rabbit serum and citrateplasma, the organisms were viable for only 1 week at the longest.
The presence of the urine apparently reduces very much the nutrient
value of the rabbit serum and citrate plasma, as is shown by the factthat the use of Ringer's solution instead of urine enables the spirochetes to multiply progressively for at least 3 weeks. Not only is
the urine devoid of cultural value for the organism, but its presencein an otherwise suitable medium renders the latter less suitable forthe growth of the organism.

Feces in Relation to Leptospira icteroheemorrhagice.
The escape in feces of living Leptospira icterohcemorrhagice fromexperimentally infected guinea pigs seems to be rather frequent.
Ido and his coworkers, 1 for example, succeeded in producing typicalspirochetosis in seven out of eleven animals tested with a corresponding number of specimens of feces; yet in spite of this high percentage of612

SURVIVAL OF LEPTOSPIRA ICTEROH.~MORRHAGI~E=;~~-~ i ° ~'" ~'~- ~

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614

SIIIIVIVAL O F L E P T O S P I R A

ICTEIIOH.~MORI~HAGI2E

positive results, it was by no means easy to demonstrate the presenceof the spirochetes in feces with the dark-field microscope, the organism being found only once in the stools from 60 guinea pigs having
spirochetosis. The specimens of feces which were infective usuallycontained erythrocytes. In human cases one specimen of feces outof seven was found to be infective.
There is apparently a possibility, then, that the spirochetes are
excreted from patients in the feces. To determine the viability ofthe spirochetes under these conditions, a series of experiments was
performed in which they were added in large quantities to feces,both with and without a simultaneous addition of blood ingredients..
Two specimens of feces from normal individuals were used, and two
from cases of jaundice in children, with the characteristic clay color.
Each specimen was used in three ways: (1) as a moderately thickemulsion (1:10) in Ringer's solution, without sterilization; (2) thesame sterilized by autoclave; (3) a Berkefeld V filtrate of a dilute
fecal emulsion (1 : 100). Two sets of tubes were set up, each containing 5 cc. of one of the variously prepared suspensions. To each of
the tubes of one set were added a few drops of defibrinated blood and1 cc. of serum from a normal rabbit, and to all the tubes were added
2 cc. of a richly growing culture of either the American or the European strain of the organism. Both sets of tubes were then placed
at a temperature of 26°C.
The fate of the spirochetes under these conditions was followed
daily b y direct microscopic examination and indirectly by inoculation tests on guinea pigs. Table I I summarizes the results obtainedwith the European strain. The results with the American strain
were practically identical and are consequently not recorded here.
As is apparent from the table, the spirochete cannot survive in a
fecal emulsion, even when there are present sufficient nutrient elements, longer than 2~ hours at a temperature of 26°C. That thisfact is due to the simultaneous presence of various bacteria, which
rapidly overgrow the delicate spirochetes and deprive them of thenecessary nutrient substances, is inferred from the much longer survival of the spirochetes in the tubes containing the sterilized emulsion,
particularly in those to which were added the blood and serum. In
the latter tubes, in fact, there was a temporary multiplication of the~EYO~roctrCHI

615

organisms lasting several days. In the tubes containing a dilute,
sterile, fecal filtrate, the spirochetes survived at least 4 days, and the
addition of blood and serum caused the filtrate to become a suitableculture medium, if not equally as good as Ringer's or saline solution.
Inoculations made with the mixtures of the non-sterilized fecal emulsions, with and without the blood and serum and the spirqchetes,
applied to the scarified skin of guinea pigs, were all negative. Where
there was an actual multiplication or survival of the organism, as in
the case of sterile suspensions or filtrates, with or without the addition of blood, the animal inoculations were positive.
Judging from the foregoing experiments, it seems highly improbable that, under natural conditions, the causative agent of icterohemorrhagic spirochetosis survives for any length of time after it
has left the human body in the feces. It is probably rapidly destroyed by the common bacterial flora of the intestinal tract.

Polluted Water and Soil in Relation to Leptospira icterohcemorrhagice.
Samples of water were collected from the East River (a tidal river),from sewage, and from a stagnant cesspool in New York City. I t
is needless to say that such water is highly contaminated with various
bacteria. In one series of experiments the water was used as it
was, in another it was autoclaved in order to destroy contaminating
bacteria, and in another it was filtered. An emulsion of freshly excreted horse stool was used in one series. The experimental data are
given in Table I I I .
The results show that the spirochetes are not capable of multiplying or even of surviving for any length of time in these contaminatedwaters. They invariably disappeared in 48 hours. Even when thecontaminating bacteria were removed by autoclaving or filtration
and rabbit serum was added, only indifferent media resulted, andwithout the addition of an adequate amount of a suitable nutrient
medium (rabbit serum in this experiment) no culture couM beobtained:
The question of how long a rich culture of the spirochetes willremain viable when mixed with distilled water and left unprotectedfrom dust in a room was next determined. A Flanders strain, havinggrown luxuriantly in rabbit serum, Ringer's solution, and agar mix-

616

SURVIVAL

OF LEPTOSPI~A
TABLE

Ltplos#ira

ia~rolwemorrhagig

ICTEI~OS_.~MORI~tAGI2~

III.

introduced into.

Growth Growthofofbacspiroteria, chotes.

R i n g e r ' s s o l u t i o n 4.5 cc. + r a b b i t s e r u m 1.5 cc. ( c o n t r o l ) . .
-East Riyer water 6 cc...
+++. . . . .
'4 . 5 cc. + r a b b i t s e r u m 1.5 ec . . . . .
+++
"
"
"
autoclaved, 6 cc...
-""""4 . 5 cc. + r a b b i t s e r u m 1.5
CC..

+ + +
--' ----+

S e w e r w a t e r 6 cc . . . .
+++""4 . 5 cc. + rabl~it s e r u m 1.5 cc . . . . .
+++""autoclaved, 6 cc ....
-"""
4 . 5 ec. + r a b b i t s e r u m 1.5 c c . . .
-----+++

S t a g n a n t w a t e r 6 cc.
+ + +
-"
"
4 . 5 cc. + r a b b i t s e r u m 1.5 c c+++-""
a u t o c l a v e d , 6 cc.
--"""
4 . 5 cc. + r a b b i t s e r u m 1.5 cc.
-+ + +
Horse stool emulsion 6 cc..
"""4 . 5 cc. + r a b b i t s e r u m 1.5 cc . . . . . .
"""autoclaved, 6 cc.
""""
4 . 5 co. + r a b b i t s e r u m

+ + +
+ + +
--

1.5 c c . .

Sewer filtrate 6 cc...
""4 . 5 cc. + r a b b i t s e r u m 1.5 c c . . . . .

Survivalo!
spirochetes.

Many wks.

2 wks.

Many wks.

Many wks.

----

(?)

-

+

---

-++

Accidentallycontaminated.

More than 3wks.

t u r e for 22 days, was placed in distilled water _(ten times the volumeof the culture) and then allowed to stand in the laboratory w i t h o u t
being covered. The distilled water was not sterile, but contained
a few large motile bacilli. The results were as follows:
24 hrs: Spirochetes +++;
opalescent.
48 hrs.: Spirochetes+++;++;
3 days:"•4 - ;

5

g~

gg

,6
7

~¢
t*

g~
~¢

+;
<@;

active and long; numerous motile bacilli; fluid slightly
active; more bacilli.
"
,,
,,
"
probably more bacilli.
many immobile;""nearly all dead.

617

HIDEYO NOGUCHIThe spirochetes remained active and numerous for 48 hours, but
all of them gradually disappeared within a week. A drinking water,
therefore, richly contaminated with spirochetes, will not be infectious
longer than a week.
Samples of soil were collected from several localities in and about
New York City for use in an experiment performed to ascertain how
long soil will harbor spirochetes under experimental conditions. The
samples were rich in organic matter and some came directly fromfertilized ground. They were all neutral in reaction. One specimenof soil was obtained from a deeper stratum than the others and was
yellowish gray in color. All were purposely contaminated with the
spirochetes and determinations of their continued presence in it madedaily. No spirochetes could be detected after 72 hours, while there
was always an abundance of bacteria. The spirochetes seem to be
rapidly overgrown by the contaminating bacteria.
¢

Various Bacteria in Relation to Leptospira icterohcemorrhagice.
When the spirochetes are excreted from the infected host, either inthe feces or in the urine, their immediate fate will depend upon thepresence of various putrefactive bacteria which are always found in
the soll in which the feces or urine is deposited. Today we know allof the more common varieties of bacteria that inhabit the intestinal
tract or that may be found in unclean objects or soil. There are, ofcourse, a great number of anaerobes as well as aerobes, but since thespirochete in question is an obligatory aerobe, 4 the study of the relation of the bacteria to it becomes much simpler. We have, therefore, to direct our attention only to the part played by aerobic bacteria under natural conditions.
There are many ways of conducting such a study, but I have chosen
an indirect one; namely, that of observing the effect of the simultaneous presence of the spirochete in question and each in turn of thosebacteria which are likely to coexist with it at the moment when theinfected feces, urine, or dead rodent becomes subject to the decomposing forces of the organic world.
A number of culture tubes containing media suitable for the growth
4 Noguchi, H., ]. Exp. Med., 1918, xxvii, 593.

618

SURVIVALOF

LEPTOSPLRA ICTEROH~EMORRHAGIrE

of spirochetes was prepared, and all were inoculated with the organism.
T h e tubes were then inoculated with various bacteria and placed in a
TABLE IV.

Bacteria.

Control without bacteria ..................
B. fcecalis

alkaligenes.....................

of
bac-

of
spiro-

Survival of
spirochetes.

Remarks as tohemolysis in

M a n y wks.
12 d a y s .

m

B. aerogenes .............................

48 hrs.

B. doacce ...............................

24 "B. coli ..................................

24
48
48
,48

B. clysenterice Shiga . . . . . . . . . . . . . . . . . . . . . .
B.

Flexner-Harris ...............

"

B. typhosus.............................

B. paratyphos~sB.

"

B ........................

"

24 "
24 "
24 "

vulgaris .......................

24 "
24 "
24 "

B. pyoeyaneus...........................

B. suipestifer............................

B. subtilis ...............................

24 "
24 "

B. mesenterlcus

24 "B. suicidus.............................

..........................

+
+
+
m

<+
<+

B. xerosis ...............................

24 "B . s p . ? large, m o t i l e , c h r o m o g e n o u s . . . . . . . .

4 days.
5"
24 hrs.

Streptococcus Pr .........................
"
B r o w n F 17 . . . . . . . . . . . . . . . .
""A 1 .................

""

C 2 .................

""

W 18 . . . . . . . . . . . . . . . .

""K 4 ..................

5 days.
8
"
3
"
5
"

"

"

S 6 ..................

6

"

"

H 6 ..................

24 hrs.

Pneumococcus Type I ....................
""II ...................
"" III ...................
""IV ..................
Streptococcus"

aureus.....................

aZbus ........................

+
<+
<+

"
"
"

...........................

B. prodlgiosus
B. proteus

A ........................

media.

+++"

3 days.
24 hrs.
3 days.
24 hrs.
48 "
48 "

p

<+

slowly.

t h e r m o s t a t at the temperature of 26°C. The culture media consistedof 1.5 cc. of rabl~it serum, 4.5 cc. of Ringer's solution, 1 cc. of citrateplasma, and 1 drop of defibrinated rabbit blood. Observations werem~)~¥o NOOtTCm

619

made of the growth, survival, or disappearance of the spirochetes, thegrowth of the bacteria, and the presence or absence of hemolysis in
the cultures. The results obtained during a period of 2 weeks arerecorded in Table IV.
It is apparent from the recorded observations that the more vigorous the growth of a bacterium, the less is the possibility that the spirochetes in the same medium will multiply. The longest period of survival of the spirochetes, except in the control tubes, was observed in
the media simultaneously inoculated with Bacillus fiecalis alkaligenes.
Certain strains of streptococci, notably the n~)n-hemolytic types,
seem not to have interfered for a certain period, after which, however,the spirochetes rapidly disappeared from the culture. In the presence of most of the intestinal bacteria, such as Bacillus coli, Bacillus
aerogenes, Bacillus cloacce, etc., the spirochetes were not only unable tomultiply but were rapidly destroyed within 24 hours. It m a y be
added that no growth of the spirochetes took place in ordinary bouillon, either with or without the simultaneous inoculation of the bacteria just enumerated. The bacteria grew vigorously in the bouillon.

M icrochemical Reactions.
The resistance of various spiral organisms to the solvent action ofbile, bile salts, saponin, and sodium oleate has been a subject ofstudy for many years, and it was once thought t o differentiate theprotozoa from the bacteria. Although this view is no longer valid,because some bacteria have been found to act like protozoa and viceversa, the fact is of sufficient interest to make worth while a determination of the resistance of the present organism to these reagents (TableV).
The jaundice spirochetes appear to be highly sensitive to the destructive action of the bile 5 and bile salts when employed in concentrations of 1:30 or more, while saponin exhibited no injurious effectupon them, even when used in as high a concentration as 10 per
cent. The action of sodium oleate was stronger than that of thebile or bile salts and produced a granular disintegration of the organism in a dilution of 1: 10,000. Among the organisms which under5 Gamier, M., and ReiUy,J., Compt. rend. Soc. biol., 1917, lxxx, 41.

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621

NOGUC~IIwent this disintegration, however, was a number of actively motile,
apparently intact organisms.
The destructive action of the rabbit bile as well as of the bilesalts and sodium oleate was considerably reduced by the addition ofserum, as shown in Table VI.
Ido and his coworkers1 observed that in spite of the difficultyof finding spirochetes in the bile when it was examined under
the dark-field microscope, two out of three specimens of the bile ofguinea pigs dying of experimental spiroch~etosis icteroh~emorrhagica
were capable of producing typical infection in the guinea pig. ThisT A B L E VI.

Leptosplra
iaerohmr~orrhagi~
introduced into.

--i

I0 p e r c e n t r a b b i t
b i l e 1 cc. + c u l t u r e

1 cc.

10 p e r c e n t s o d i u mt a u r o c h o l a t e 1 cc.

+ culture Icc.

R a b b i t serum 0.5 ! N o a p p a r e n t ef- Nearly all a c fect. All acCC,
tive.
tive.
60 per cent rabbit M a n y gone,
M a n y dead a n dserum 0.5 cc.
some motile.
distorted. A
few motile.
20 per c e n t rabbit Nearly all gone. Nearly all gone.
serum 0.5 cc.
6 per cent rabbit All gone.
All gone.
serum 0.5 cc.
2 per cent rabbits e r u m 0.5 cc.
Ringer's solution
0.5 cc.

10 per cent s o d i u mo l e a t e 1 cc. +culture 1 cc.

0 + c u l t u r e 1 cc.

N e a r l y all ac- All motile.
five°

M a n y active.

~¢

g¢

$g

Nearly all gone.

"$g

All gone.
g~

~g

gg

~g

gg

may be ascribed to the fact that in these specimens of bile there was
mixed a certain amount of the b l o o d a n d also the serous exudate
from the affected liver, which, by virtue of their w~ll known inhibitory effect upon the solvent action of the bile salts, must have protected some spirochetes from destruction in the bile. Guinea pig
bile was affected by the serum in the same way.
A parallel series of experiments with a specimen of ox bile obtained
from an abattoir gave somewhat contradictory results. In this instance the addition of the rabbit or horse serum failed to check thedestruction of the organism by this bile, which had a much stronger

622

SURVIVAL OF L E P T O S P I R A I C T E R O H / E M O I ( R H A G I . ~solvent power than that of the rabbit or guinea pig. At all events,
the amount of the serum necessary to nullify the destructive actionof the bile is so large that the escape of the spirochetes in the bile
seems less probable than would appear from the observations of theinvestigators just quoted. Perhaps the impairment of hepatic function through the spirochetal infection of the organ m a y lead to adecrease of the bile salts in such a specimen.

Leptospira icterohcemorrhagice and Intermediary Hosts.
It has been shown b y previous investigators that the spirochetes
may remain in the organs of certain rodents without producing serious
illness, and that they may be excreted in the urine. From the experiments already described, it seems improbable that the spirochete cansurvive very long after leaving the infected hosts. The infection ofman, therefore, must result from contact with the spirochete before
its destruction under natural conditions; that is, the carrier rodents
must be present in places frequented by man. But while this sourceof infection may explain many cases of infection, there are a few inwhich the infective agent cannot be traced in this way.
The question of insect carriers has been taken up b y Reiter, 6 who
obtained only negative results with certain biting flies, fleas, and bedbugs. In the present study opportunities were afforded the writer toascertain whether or not the larvae of certain varieties of flies or mosquitoes could become infected with spirochetes when fed on infectedguinea pig liver or raised in a stagnant water tank into which anabundance of the culture had been put.
The larvae of the common house-fly were allowed to feed for 2 days
on infected material consisting of several pieces of the liver and kidneyof a guinea pig killed in the last stage of experimental Well's disease.
They were then transferred to a clean receptacle and fed for 5 days on
a non-infected mass of horse manure, and at the end of that timethey were crushed into an emulsion and smeared over depilated areasof the skin of guinea pigs. The emulsion was also examined for spirochetes under the dark-field microscope. The examination revealed
no spirochetes, and the guinea pig remained normal.
Reiter, H., Deutsch. reed. Woch., 1916, xlii, 1282.

H I D E Y O NOGUCttI

623

A similar experiment with the larvae of bluebottle flies (Calliphora

vomitoria) gave only negative results.
In another series of experiments, about 50 cc. of a rich culture of
spirochetes (Japanese strain) were added to 150 cc. of stagnant waterin which twenty-five mosquito larvae had been living for some time.
The water was neutral in reaction and was quite clear and transparent
at the time when the culture was introduced. The larvae swam about
actively in the usual manner after the addition of the culture. A
drop of the contaminated water examined under the dark-field microscope contained numerous active spirochetes. There were a few bacteria. After 24 hours at room temperature, the water became somewhat turbid. Most of the larvae were still active, but the number ofthe spirochetes was diminished and that of the bacteria increased.
At the end of 48 hours there was a scum of bacteria over the surface of
the water and no spirochetes could be found. All but six of the largest
larvm had died. The water was full of bacteria and infusoria. It is
possible that the death of the mosquito larvae and of the spirochetes
was the result of overcrowding by the bacteria and infusoria , increased
suddenly by the addition of the culture media to the water. The surviving larvae were kept in the same water for 5 days and then crushed
into an emulsion to be used for an infection experiment on a guinea
pig and also for examination under the dark-field microscope. The
results were entirely negative.
Another series of experiments was performed with adult mosquitoes(Culex pipiens) by first allowing them to feed on an infected guinea
pig, in the blood of which had been found spirochetes, and then, after
6 days, causing them to bite normal guinea pigs. No infection resulted from their bites.
Wood ticks (Dermacentor andersoni) failed in several experiments totransmit the infection from guinea pig to guinea pig. Leeches(ttirudo medicinalis) were allowed to suck blood from an infectedguinea pig until their bodies were engorged. In the blood escaping
from the wound inflicted by the leeches a few spirochetes could befound under the dark-field microscope. These "infected" leeches were
kept at room tetnperature for 7 days and afterwards in a cool roomat 15°C., being taken out "at the end of intervals of 2, 3, 4, 6, and 8months'and made to suck normal guinea pigs, b u t so far no infection

624

SURVIVAL OF LEPTOSPIRA I C T E R O H _ ~ M O ~ G I / E

has been produced. Some of the leeches died in the meantime, but
those which still survived at the end of the 3 month interval were examined for the presence of spirochetes. The viscid, dark reddish,decomposed (?) blood showed no spirochetes under the dark-fieldmicroscope, nor did it cause infection when tested on guinea pigs.
Some of the tissues were examined by the silver impregnation method,
but with negative results. Apparently there is no multiplication of
the spirochetes after their ingestion by leeches, and no infection canbe induced by the bite of the latter after a period of 1 week.
S U M M A R y AND C O N C L U S I O N S .

1. Leptospira icteroh6emorrhagice is unable to grow in the urine,
either with or without the addition of suitable culture ingredients,
the acidity of the urine being detrimental to the growth. It survives
less than 24 hours, unless the urine is neutralized or slightly alkalized,
when the period of survival is somewhat longer. If suitable nutrient
ingredients are added to the neutralized or slightly alkalized urine,
the organism is able to grow for about 10 d a y s , after which multiplication ceases.
2. Feces from normal or jaundiced persons destroy Leptospira icteroh¢morrhagi6e within 24 hours when a rich culture is added and the
mixture allowed to stand at 26°C. The addition of blood serum andcorpuscles does not prevent the destruction of the organism. Autoclaved specimens and filtrates of unheated feces do not constitutea suitable medium in which to keep the organism alive for any lengthof time, but the addition of blood corpuscles and serum in adequate
quantities renders them fairly satisfactory as media. Under natural
conditions Leptospira icterohcemorrhagide cast off in the feces cannot
survive more than 24 hours.
3. Polluted water, sewage, and soil will not serve to keep Leptospira icterohcemorrhagi~ alive for more than 3 days at the most.
When deprived by filtration or autoclaving of their bacteria they become indifferent diluents and may be used to make up a culture medium when mixed with sermn and citrate plasma of a.suitable animal
Sterilized soil with a neutral reaction, when added to a culture, has
an unfavorable effect upon the growth of the organism.

HIDE¥O NOGUCHI

625

4. Most of the aerobic bacteria found in feces, sewage, soil, and tap
water inhibit the growth of Leptespira icteroh6emorrhagicewhen inoculated into the same medium. Bacillusftecalis alkaligenes and manystrains of non-hemolytic streptococci caused the least interference,
although growth was never so vigorous or lasting in the media i nwhich they were present as in the control media. Certain pathogenic
bacteria (Bacillus typhosus, Bacillus paratyphosus, Bacillus dysenterice,pneumococcus) are antagonistic to the growth of the spirochete.
5. Leptospira icterohcemorrhagice is highly sensitive to the destructive action of bile, bile salts, and sodium oleate, but resists the actionof saponin. In this last respect it differs from many so called spirochetes. The destructive action of these agents is counteracted byblood serum.
6. The larvae and adults of the Culex mosquito, the larvae of thehouse-fly and bluebottle fly, wood ticks (Dermacentor andersoni), andleeches failed to become carriers of the spirochetes when fed on infected guinea pigs or their organs; that is, they cannot play thepart of an intermediary host of Leptospira icteroheemorrhag#e.