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Journal of Experimental Medicine

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T H E DRUG-FASTNESS OF SPIROCHETES TO ARSENIC,MERCURIAL, AND IODIDE COMPOUNDS IN VITRO.
BY SEINAI AKATSU, M.D., AND HIDEYO NOGUCHI, M.D.
(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, November 22, 1916.)

It has been known for some time that in trypanosomiasis the try,
panosomes which have survived the first effect of an arsenic germi~cide, such as atoxyl or arsacetin, offer a greater resistance to a subsequent dose of the same drug. By subjecting the organisms torepeated injections of an arsenic medicament, one tends to create,both in animals and in man, a strain or race which resists the arsenic
chemotherapy to such an extent that the term arsenic-fastness hascome into existence as a brief designation of the modified strain, xA similar phenomenon has been observed in spirochetosis in fowls
and mammals. Even with a typical bacterium, there seems to exist
a possibility of raising the original resistance against a certain arsenic
compound to a considerable degree, as was shown by Marks ~ in his
experiment with the paratyphoid bacillus, which he was finally able
to cuitivate in a medium containing eight times the amount of arsenious acid which the organism was able to withstand at the beginning of his experiment. Marks accomplished this within a periodof 3 years by successive transplantations from a weaker to a graduallystronger concentration of the acid.
In syphilis, even before the discovery of Treponema pallidum,
it had long been suspected t h a t the causative agent of this diseaseacquires a gradual tolerance to the action of mercurial and iodide
preparations. The intermittent form of treatment generally adopted
by clinicians, with gradually ascending doses of the medicaments
for each course, bears sufficient evidence of this assumption. That
1 Ehrlich, P., and Hata, S., Die experimentelle Chemotherapie der Spirillosen,
Berlin, 1910.
2 Marks, L. H., Ueber einen arsenfesten Bakterienstamm, Z. Immunitatsforsch., Orig., 1910, vl, 293.
349

350

DRUG-FASTNESSOFSPIROCHETES

unicellular organisms, under certain environmental influences, gradually acquire tolerance to certain toxic substances is not inconceivable, inasmuch as instances are not wanting where a group of cells,
or an entire organization of much higher multicellular organisms,
becomes adjusted to new surroundings when the change is brought
about by degrees. In short, the state of drug-fastness is well known
among the metazoan organisms.
• To establish definitely whether Treponema pallidum acquires anincreased resistance to various antisyphilitic therapeutic agents isof great practical importance, for the regulation of dosage of medicaments must necessarily be guided by the changes which occur atthe same time in the resistance of the parasites. It may be recalledthat Ehrlich assumed from analogy with trypanosomiasis and fowl
spirochetosis that Treponema pallidum becomes readily fast or insensitive to the action of arsenic compounds, and with this idea heevolved his therapia sterilisans magna, that is, a total sterilizationof Treponema pallidum in the system of the infected individual bya single administration of salvarsan, in which he combined a maximum parasitotropic and a minimum organotropic activity. The benefit derived from his efforts to achieve the aim of therapia sterilisans
magna has been great, but in its practical application salvarsan has
fulfilled only a part of Ehrlich's expectation. The consensus ofopinion is that salvarsan has to be used in many successive doses,
like other antisyphilltic medicaments, in order to obtain the full
benefit of the drug. The introduction of salvarsan and neosalvarsan
as routine medicaments in the treatment of syphilis raises once more
the question whether or not the repeated injections of these preparations tend to produce an arsenic-fast strain of Treponema pallidum.
This vital point, as well as those with regard to the alleged fastnessof the pallidum against mercurial and iodide compounds, has neverbeen proved experimentally, probably because of the fastidious character of Treponema pallidum in its behavior in the experimental animals available for studying these points.
For ascertaining experimentally that the syphilitic organism becomes gradually more tolerant to the sterilizing effect of various arsenic, mercurial, and iodide preparations, four different methods
suggest themselves: (1) to determine in ~vo the resistance of Trep-

SEINAI AKATSU AND HIDEYO I~'OGUCHI

351

onema pallidum to the germicidal action of the drug in question,
both before and after the administration of the drug; (2) to subject in vitro the pallidum derived from normal and treated animals
to the action of various concentrations of the drug and then to inoculate them into a series of suitable animals; (3) to cultivate theorganism instead of inoculating it into animals as in (2); (4) todetermine the degrees of tolerance of pure cultures of Treponema
pallidum to a given medicament by testing the organisms in vitro
against gradually increasing doses of the drug.
The first two methods are practically unavailable for the present
problem, since there is no animal in which an accurate estimationof the relation between the number of the parasites and the severity
and character of the lesions produced could be made with uniformity,and no method is yet known which will permit the cultivation of thepallidum directly from the animal tissue emulsions, as would be required if we were to adopt the third procedure. We have, therefore,
left these three methods out of consideration and followed the fourth,the only procedure which may be carried out with the various factors
under ready control and with a fair degree of constancy in results.
In the present work, we have chosen not only the syphilitic spirochete, but also some allied non-pathogenic varieties, and tested themagainst some of the representatives of antisyphilitic medicaments.
We do not consider that the phenomena observed in vitro on purecultures of spirochetes can be directly transferred to the processes
occurring in the animal or human body, but we believe that theobservations reported in this paper warrant sufficient interest as such. 3
EXPER~ENTAL.

Method.

As the test objects, three strains of pure cultures of Treponema
paUidum, one of Treponema microdentium, and one of Spirochttta
refringens were employed. Against each of the above organisms,
salvarsan, neosalvarsan, bichloride of mercury, and Lugol's solutions Akatsu, S., The Resistance of Spirochetes to the Action of Hexamethylenetetramine Derivatives and Mercurial and Arsenic Compounds, Y. Exp. red.,
1917, xxv, 363.

352

DRUG-I~ASTN~SS 0 ~ SPIROCHETES

were used. The solutions of salvarsan and neosalvarsan in sterile
distilled water were each time freshly prepared, the proportion being
1 : 10,000. The solution of bichloride of mercury was made up to
1 : 10,000 during the early part of the experiment and became 1 : 1,000
as the concentration was gradually increased in order to keep thevolume of the solution of the sublimate to be added to the media
within a certain limit. Lugol's solution was prepared by dissolving
1 gm. of iodine and 2 gm. of potassium iodide in 300 cc. of distilledwater. In the experiment a 1 : 10 dilution was first used, then an
undiluted solution, (1 part of iodine, 2 parts of potassium iodide,and 300 parts of distilled water), and finally one three times as concentrated as the usual undiluted solution (1 part of iodine, 2 partsof potassium iodide, and 100 parts of distilled water).
Because of certain technical difficulties, the use of solid media
was restricted to a small series. The majority of the experiments
were carried out by means of a fluid medium consisting of equalparts of neutral bouillon and ascitic fluid with a piece of fresh kidney of a normal rabbit. To maintain the relative volumes of the
drug solutions and the culture media, none of the former was addedin a volume greater than 1 cc. or less than 0.05 cc. When the requisite amount of the drug solution exceeded 1 cc., a corresponding quantity of a tenfold stronger concentration was employed in order tokeep the volume within the specified limit. The total volume of
the culture medium in each tube was made uniformly 5 cc., including the quantity of drug solution added.
A series of test-tubes was set up, each containing a piece of fresh
rabbit kidney, and inoculated with the pure culture of the spirochete. To the tubes were added first the ascitic bouillon (equalparts), and then the drug solution in the quantity indicated for eachtube. The whole content of the tube was thoroughly mixed bygently inverting the tube a few times, then covered with a layer ofsterile paraffin oil, and finally incubated at 36°C. in an anaerobic
apparatus. At the end of 14 days, the cultures were examined for
growth under the dark-field microscope. From the tubes showing
varying degrees of growth, transfers were made into a new seriesof media containing different doses of the drug. Again, after afortnight's incubation, transfers were similarly made, the process
being repeated every 2 weeks.

S E I N A I A K A T S U AND HIDEYO I~OGUCHI353

Results of Experiments.
It was possible to find certain quantities of each drug, the additionof which to the culture media completely inhibited the growth ofthe organisms. As the quantities of the drug were gradually reduced,
the organisms grew in corresponding abundance. With a very small
quantity of the drug, no appreciable effect upon the growth was seen.
But between the two extremes there were doses which distinctly
showed the inhibitory influence of the drug as indicated by the retardation and meagerness of growth. In the border-line tubes theorganisms were fewer in number, less active, and often granular inappearance. Numerous degenerated forms were seen in these tubes.
To conform with the purpose of the present work, transfers weremade both from the border-line tubes and from those containing
smaller amounts of the drug into a number of new media, to some ofwhich ascending doses of the drug solution had been added. After
14 days in the incubator at 36°C., the cultures were examined for
growth in the manner already described. From repeated observations it was discovered that no growth or very scant growth was
obtainable when a transfer was made from one of the border-line
tubes to a tube containing an equal quantity of the drug, and thateven if there was any growth in the second transfer, it was not furtiler transferable. The organisms seemed to succumb to the continued effect of the drug in such a concentration. Under these conditions there was no evidence that the organisms had in any way
acquired an increased resistance to the drug.
On the other hand, the organisms growing fairly well in the tubes
with somewhat smaller amounts of the drug were found capable oftransplantation into new media, in the presence of not only thesame amount, but also of somewhat larger doses. In other words,
the minimal inhibitory doses became gradually larger with eachtransfer. In the accompanying tables we present a general aspectof the effect of drug-containing media upon the gradual developmentof tolerance by the spirochetes.
The point of interest here lies in the fact that the organisms under
the influence of moderate quantities of an adverse drug acquire agreater resistance to it and are capable of doing so progressively up
to a certain point. As will be described, the various organisms era-

354

DRUG-FASTNESS OF SPIROCHETES

ployed in this experiment behaved differently towards different
agents, and our results permit the tentative generalization that thegroup of treponemata gains, under certain conditions, a definite,
though rather slight degree of resistance to salvarsan and neosalvarsan, while its resistance to the bichloride of mercury ~and iodide?)
preparation is strikingly augmented under similar circumstances.
In Tables I and I I the reaction of different spirochetes towards
the influence of each of the chemical preparations used in the present
work is shown.
TABLE I.
Transferred to the tubes containing.
First transplantation trom drug media to drug media.

Dose.
3

1

From tubes conmining
the
doses
and
showing the
growth indicated.

4

No
drug.

Dose 1, causing total inhibition

(-).
.4-

Dose 2, causing slight growth( < <.4-).
Dose 3, causing moderate
growth (<.4-).
Dose 4, causing good growth

.4-

+

.4-

+

.4-

.4-+

:.4-

<.4-

.4-

+.4-

<+

<.4-

.4-

.4-+

:.4-

(+).
Dose 5, causing no inhibition(++).
Control tube without any drug,
showing luxuriant growth(++).
TABLE II.
Transferred to the tubes containing.

Third transplantation from drug media to drug media.

Dose.

No

drug.
2

From the second
transplants
containing the
doses
and
showing the
growth indicated.

Dose 2, showing moderate ~ < ÷growth (<.4-).
Dose 3, showing good growth
<.4-

.4+

<.4-

.4-

T

.4-.4-•4-+.4-".4-.4-.4-.4-.4-.4-.4-< <.4- <.4--4-.4-.4-(+),

Dose 4, showing good growth(+).
Control tube without any drug,
showing luxuriant growth

(++).

3

SEINA1AKATSU

AND~tlDEYO

355

NOGUCltI

Table I I I represents the results of experiments with salvarsan andshows how various pallidum strains, and microdentium and refringens,withstood the action of the drug.
TABLE

III.

Salvarsan.
Maximum Doses in Which Abundant Growth Still Occurred on Successive
Transplantations.
Generation in drug media.

T. pallidum.

1

2

~ng.

fng.

0.05
•0.02
0.03
~
R ...........
"
Z. A .........
0.02
0.04
T. microdentium.. ......
0.01 0.02
0,02 0.02
S. refringens ..........
Strain McD .........

0.03

4

mg.

0.06
0.04
o,o4
0.04
O.04

!

tttg.

0.07
0.04
0.05
0.06
Accident.

5

6

mg.

mg.

rag.

0.07 0.08
0.05 0.1

0.1

0.12

0.12
Accident

0.07 0.07
0.04 0.04

0.05

0.08

The figures denote the amount in milligrams of salvarsan containedin 5 cc. of the culture medium. The first column shows the doses ofsalvarsan in which various strains of the spirochetes still thrived;
the second those in which the organisms grew well on the first transplantation; the third those for the second transplantation, etc. I t
will be seen that the initial tolerance of the pallidum and refrlngens
was slightly greater than that of the microdentium, while the ratewith which tolerance increased, up to the end of the fifth and sixthtransplantation, was greatest with the mivrodentium and pallidum,
these having attained about six to seven times their original tolerance within 3½ months. The refringens still remained sensitive
to the drug during this time.
Table IV shows the results with neosalvarsan, and they agree
in general with those obtained with salvarsan, except that larger
quantities were tolerated on account of its weaker action. The
pallidum and microdentium reached four to five times their initial
tolerance, while the tolerance of refringens was least affected.
In general, the tolerance of the pallidum and microdentium tosalvarsan and neosalvarsan was definitely raised, while that of therefringens seems to have been least increased. With the pallidum

356

DRUG-FASTNESSSPIROCHETES

OF

TABLE

IV.

Neosalvarsan.
Maximum Doses in Which Abundant Growth Still Occurred on Successive
Transplantations.
Generation in drug media.
1

T. pallicIum.

Strain McD. . . . . . . . . . . . . . . . . . . .
" Z. A . . . . . . . . . . . . . . . . . . . .
T. microdentium . . . . . . . . . . . . . . . . . .
S. re#ingens . . . . . . . . . . . . . . . . . . . . . .

2

3

4

$

6

mg.

mg,

m.g,

mg.

mg.

mg.

mg.

0.03
0.02
0.03
0.02
0.03

0.07
0.05
0.04
?
0.06

0.08
0.06
0.06
0.03
0.08

0.1
0.06
0.06
0.07
0.08

0.12 0.15
0.07 0.08
0.07 0.08
0.1 0.1
0.I 0.1

0.15
0.08

0.1
0.1
0.1

strains, the rise in tolerance was gradual and the probable limit
was not reached until the fifth or sixth transfer, which was madeat the end of the 12th or 14th week. With the m i c r o d e n t i u m andrefringens, especially the latter, the limit of tolerance was reachedmuch sooner, and it could not be raised further after the fourthtransfer. In salvarsanized media, the refringens reached the limiton the second transfer and remained unchanged for the rest of theexperimental period.
The results obtained with bichloride of mercury, compared withthose with salvarsan and neosalvarsan were striking. Table V showsthat the initial tolerance of all the spirochetes was almost the same,
being 0.01 mg. per 5 cc. of the medium. One of the p a l l i d u m strains
tolerated 0.02 mg. The increase in tolerance rapidly rose from thefirst transfer and progressed gradually for the following generations.
In T r e p o n e m a p a l l i d u m the limit was not reached until the 16th
week (seventh transfer), while that of the m i c r o d e n t i u m had alreadybeen reached on the 8th week (third transfer), and that of the ref r i n g e n s on the 12th week (fifth transfer). Even the p a l l i d u m strains
could not tolerate much higher concentrations, the absolute limit
being 1 nag. in 5 cc. of the medium, when the color of the tissue, as
well as of the ascitic fluid, becomes gray within a few days at 36 ° C.,and black streaks appear in the tissue.
The striking feature of the experiment is the high rate of increase
in tolerance through the organisms' becoming accustomed to the357

SEIN'AI AKATSU AND HID:EYO NOGUCHITABLE V.
B i d d o r i d e of M e r c u r y .
M a x i m u m Doses i n W h i c h A b u n d a n t Growth S t i l l Occurred on Successine
Transplantations.
Generationin drug media.
1

T. pallidum.
Strain McD . . . . . . . . . . . . . .
"" Z. A . . . . . . . . . . . . . . .
T. ralcrodentium . . . . . . . . . . . . .
S. refringen¢ . . . . . . . . . . . . . . . .

2

3

4

5

7

6

8

rag.

rag.

rag.

rag.

rag.

rag.

mg.

rag.

0.02
0.01
0.01
0.01
0.01

0.07
0.03
0.02
0.02
0.015

0.13
0.07
0.07
0.07
0.03

0.17
0.1
0.07
0.1
0.07

0.2
0.2
0.15
0.1
0.1

0.23
0.3
0.3
0.1
0.3

0.5
0.7
0.5
0.1
0.3

0.7
0.5
0.1
0.3

mercurial salt. With the pallidum, it was between 35 and 70 times
the initial tolerance; with the refringens, about 30 times; and withthe microdentium, 10 times. We did not believe that these delicate
organisms could be made resistant to the action of so simple andpowerful an inorganic disinfectant as bichloride of mercury. The
above observations seem to point to the possibility that bichlorideof mercury forms, with certain constituents (proteins and lipoids)of the culture medium, a compound which induces tolerance on thepart of the spirochetes.
In Table VI the results obtained with Lugol's solution are given.
The figures in the first and second columns do not indicate themaximum doses in which the spirochetes could grow, but those which
TABLE

VI.

Lugol's Solution.
Amounts Contained in Successive Transplantations.
Gene~tion in drug media.
1

T. pallidum.
Strain McD . . . . . . . . . . . . . . . . . . . . . . . . . . .
" Z. A . . . . . . . . . . . . . . . . . . . . . . . . . . . .
T. microdentium . . . . . . . . . . . . . . . . . . . . . . . . .
S. refrlngens . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

3

4

5

¢~.

ifC:.

¢,4~°

t,6.

at:.

0.03
0.03
0.02
0.01
0.02

0.05
0.04
0.05
0.06
0.03

0.5
0.5
0.5
0.3
0.3

1.5
0.7
1.0
0.8
0.5

2.5
1.5
2.0
0.8
1.0

6

2.5
2.5
2.0
2.0
2.0

358

DRUG-FASTNESSOFSPIROCIIETES

had been arbitrarily added to the media at the beginning of the experiments, with the purpose of gradually accustoming the organisms
to the action of iodine and iodide. The doses given in the third to
the sixth columns indicate the quantities added to the media where
the spirochetes could still grow well. It may be stated here thatthese organisms, before passing through the iodinized media for some
generations, could not grow in the presence of 0.7 cc. of Lugol'ssolution in 5 cc. of the culture media.
The color of Lugol's solution soon disappears from the media
when the mixture is placed at 36°C. Even the addition of 2.5 cc. did
not preserve the brown color over 48 hours. The most importantfactor affecting the antispirochetal action of the iodine solution was
found to be the size of the fresh tissue added. The larger the tissue,
the safer the spirochetes were from the inhibitory or sterilizing influence of the preparation. For this reason our experiments were carriedout with as uniform a size of the tissue as practicable for each tube,
and the results presented in Table VI may be considered approximately correct. It is true also with salvarsan, neosalvarsan, andbichloride of mercury, but the interference from this source wasmuch less disturbing than with the iodine solution. Thus our figuresin all the experiments should be taken as expressing merely theapproximate values.
At the end of the experiments, a comparative study of the drugfast and the ordinary stock cultures of the spirochetes in relation
to their resistance to salvarsan, neosalvarsan, bichloride of mercury,and Lugol's solution was undertaken. The results are shown in
Tables VII, VIII, IX, and X.
Attempts to carry out parallel experiments in solid media met
with certain technical difficulties, in that the drugs had to be added
while the mixture of the ascitic fluid and agar was still in fluid condition; that is, at a temperature of about 45°C. When salvarsan,
neosalvarsan, and bichloride of mercury were mixed with the media,
a distinct turbidity resulted, and solidification of the media at once
followed, sometimes rendering a uniform distribution of the drug
impossible. It was for this reason probably that we were unable toobtain results of definite constancy to warrant the assumption of
the existence of an increased drug tolerance in the spirochetes when

SEINAIAKATSUANDHIDEYO

TABLE

359

NOGUCHIVII.

Salvarsan.

In 5 cc. of media containing.

Strain McD., which grew in the
tube containing 0.06 rag.

Strain McD., never in contact
with salvarsan.

Growth.

Growth.

<+
+
++
++
++

+
++
++

mg.

0.2
0.1
0.05
0.01
0.005
No drug.

TABLE

N

VIII.

Neosalvarsan.
I n 5 cc. of m e d i a containing.

Strain R., which grew in the
tube containing 0.1 mg.

Strain R., never in contact with

Growth.

Growth.

<+
<+
+
++
++

<+
<+
++

neosalvarsan.

mg.

0.2
0.1
0.05
0.01
0.005
No drug.

TABLE

IX.

Bichloride of Mercury.

In 5 cc. of media containing.

Strain Z. A., which grew in the
tube containing 0.5 rag.

Strain Z. A., from original solid
culture, never in mercuric
chloride media.

Growth.

Growth.

mg.

1.0
0.5
0.1
0.05
0.01
Nodmg.

<+
+
++
++
++

N
m

++

360

DRUG-FASTNESS OF SPIROCHETESTABLE X.

Lugol's Solution.
In 5 cc. of media containing.

2.0
1.0
0.5
0.1
0.05
No drug.

Strain McD., which grew in the Strain McD., never in contact withtube contaimng 2 ¢c.
Lugol's solution or iodide.
Growth.

Growth.

<+
+
++
++
++
++

+
+
++

-4-

passed through the drug-containing media for several generations.
Nor was it possible to demonstrate the acquired drug tolerance bymeans of solid media, which was easily seen in the fluid media. We
are not prepared to offer an explanation for this phenomenon. Perhaps in fluid media the drugs enter into combination with certain
tissue ingredients of the media and in this form induce a gradualtolerance on the part of the spirochetes, whereas with solid media
the drugs are held in the agar in such a manner as not to become modified enough to produce increased resistance in the spirochetes.
With regard to the duration of the acquired drug tolerance of the
spirochetes, a series of experiments was carried out in which the drugged
strains were returned to the ordinary fluid media without any drug.
From each successive generation, tolerance tests were made withvarying concentrations. The results are summarized in TableXI.
As may be seen from Table XI, the acquired tolerance to various
drugs decreases fairly rapidly. Within a period of 8 weeks, during
which four transfers through ordinary media were made, the acquired
tolerance of the pallidum and refringens to salvarsan and neosalvarsan
entirely disappeared, and that of microdentium disappeared evensooner. The disappearance of the acquired tolerance to bichlorideof mercury was more gradual, requiring about 10 weeks for complete
restoration of the normal sensitiveness to this salt. In this respect,
the paratyphoid strain used by Marks 2 differs greatly from the spirochetes, as the former passed through forty-six passages before returning to the initial tolerance.

361

SEINAI AKATSU AND HIDEYO NOGUCHITA~BLE XI.

Maximum Doses of Each Drug in Which Various Drug-Fast Strains of the Spirod, etes Still Grew Well after Being Returned to Drug-FreeMedia for Successive Generations.
Sslvarssn.

me.

rag.

rag.

Neo~lvarsan.

,*

~

rag,

rag, rag°

Bichloride of
mercury.

-~

me,

me.

me.

kfter one generation in drug-free media .... 0.1 3.05 0.05 ioA5 o.1 o.1 9.5 0.1 0.1" two generations in "0.05 3.03 0.03 0.1 0.050.08 3.3 0.1 0.1
"
three
"
"
"
"
0.05 9.02 0.02 0.05 0.02 0.04 3.05 0.07 0.05
0.03 0.010.02 0.03 0.02 0.03 3.04 0.04 0.0~
. . . . 10.02 3.01 0.02 ~.03 0.02 0.03 9.02 0.01 0.01
"
five
,
u
,
,
tt
SIX
tc
tt
tc
tt
0.02 0.01 0.02 3.03 0.02 0.031 9.02 0.01 0.01....

* Pallidum, Strain M c D .
S ~ Y .

In the foregoing experiments we attempted to determine whether
or not, by subjecting several varieties of spirochetes to increasing
doses of certain chemotherapeutic agents, a gradual increase of resistance to the latter could be shown. For this purpose, pure cultures of Treponema pallidum, Treponema microdentium, and Spirochceta refringens were used against the action of salvarsan, neosalvarsan, bichloride of mercury, and iodine-iodide potassium solution
in vitro. For culture media, the usual ascites-broth-tissue medium
as well as solid ascites-agar-tissue medium was used. After permitring the spirochetes to grow for a fortnight in media containing
certain quantities of each drug, transfers were made from tubes
showing various degrees of growth to the next series of tubes containing the same drug in still higher concentrations, and similartransfers repeated every 2 weeks. The results of the experiments
may be briefly summarized as follows:
1. Treponema pallidum and Treponema microdentium have, within
3 to 4 months, increased their tolerance to salvarsan and neosalvarsan to five and one-half times their original mark. With Spirochceta
refringens the increase was about three times.

362

DRUG-FASTNESSOF SPIROCHETES

2. Against the action of bichloride of mercury, the amount of
increased tolerance of Treponema pallidum was about 35 to 70 times
the original, while that of Treponema microdentium was about 10
times as much and was reached within 10 weeks. Spirochceta refringens resisted 30 times the original dose.
3. There was an unmistakable increase of resistance of thesespirochetes to the action of the iodine-iodide solution (Lugol's solution) when they were grown for several generations in fluid media
containing the iodine solution, but the rate of increase between theinitial and the acquired tolerance was slight. In general, the addition of Lugol's solution to fluid media has a weak inhibitory influence
upon the growth of the spirochetes, requiring for the total suppression of growth a quantity of over 0.7 cc. to 5 cc. of the culture media.
The tolerance reached was for about three times that amount.
4. A similar tolerance phenomenon has not been established whenemploying a solid instead of a fluid medium containing the drugs.
No explanation is offered except a suggestion that the drugs heldin the agar do not enter into combination with certain tissue constituents of the medium as they are able to do with tissue elements
in fluid media. This may be a factor necessary for inducing drug
tolerance in these organisms in vitro.
5. The increased drug-fastness in vitro has a limit beyond which
no further advance can be made. This limit varies with different
species of spirochetes.
6. The acquired drug-fastness in vitro gradually disappears when
the spirochetes are cultivated again in the drug-free media for several
generations.