野口英世著

Journal of Experimental Medicine

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F U R T H E R STUDIES ON THE PROPERTIES OF PUREVACCINE VIRUS CULTIVATED IN VIVO.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, Se~/tember 15, 1917.)

It has been shown that a sample of vaccinia virus, free from bacterial
impurity and at the same time of sufficient strength for practical
purposes, can be propagated in the testes of certain animals, particularly rabbits? There were still several points, however, which
seemed to require further investigation. It was not known whether
or not, through continuous cultivation in vivo in the testicular tissuesof rabbits, the biological properties of the virus would finally undergo
modifications. Since the natural habitat of the virus is the skin,
it is not improbable that'the new medium in which it has been induced
to grow might have a deleterious influence upon it. It was necessary
to determine the viability of such a virus under varying conditions,that is at different temperatures, with the addition of antiseptics,
etc. I t was desirable also to compare the resistance and viabilityof the tes'ticular strain with those of the virus propagated in thedermal tissue. These are questions not merely of purely academic
interest, but of practical importance, inasmuch as the proper utilization of the new vaccine depends to a great extent upon the right understanding of its properties.

Scope and Mode o.[ Experiments.
In tl/e present series of experiments we studied the effect of disinfectants upon the testicular vaccine virus at different temperatures,the influence of diluents under various experimental conditions, theeffects of desiccation upon the virus, and the viability of dried vaccine]
The samples of testicular or of skin virus employed in each instancewere highly active, being capable of producing a confluent eruption
1 Noguchi, H., Pure cultivation in vivo of vaccine virus free from bacteria,
J. Exp. Med., 1915, xxi, 539.
425

426

PURE VACCINE VllZUS C U L T I V A T E D I N VIVO

on the shaved skin of the rabbit in 1:1,000 dilution or higher. The
vaccine preparations were placed in temperatures of 37 ° , 18° , 4 ° , and
0°C. Samples were taken from different test-tubes containing thevirus at intervals in order to determine its strength under various
conditions. During the 1st week the tests were made every 24 hours,
since some of the vaccine samples which were kept at higher temperatures underwent a rapid attenuation of virulence, so that daily
titration or" their strength was imperative for following the course ofdeterioration.
The titration of the vaccine was made by applying a number ofgraduated concentrations of the specimen to a corresponding numberof shaved areas, 10 by 20 cm., of the dorsal skin of rabbits. Special
precautions were taken to have a control virus accompany the seriesof tests made on every animal in order to eliminate the irregular results
due to individual variations in susceptibility to the vaccine virus, forwithout a proper control for each rabbit no accurate estimation of
the vaccine effect may be made. In the present work duplicate
tests were often resorted to. It was found both practical and reliable
to make as many shaved areas as the dorsal and lateral surfaces of
the rabbit's body permits, leaving narrow strips of hair between the
shaved areas to serve as barriers, preventing the accidental overflow of
one dilution to the next area. Readings of the results were made atintervals for about 8 to 10 days after inoculation. The dilutions ofdifferent samples varied according to the rapidity with which theirstrength diminished, but it was customary to prepare 1: 1,000,
1 : 100, 1 : 50, 1 : 25, and 1 : 10 dilutions, and undiluted stock suspension.
The testing of different samples after the preliminary titration was,of course, much simplified, as in a subsequent titration fewer dilutions
were sufficient to estimate the strength of the virus. Strict asepsis
was exercised in handling the vaccine throughout the experiments.
For comparison the regular skin vaccines were employed. These
had to be put up as emulsions containing 40 to 50 per cent glycerol
or 0.5 per cent phenol.

Virulence and A~nities of the Testicular Vaccine Virus.
The first point to determine was whether a strain of vaccine virus
which had been propagated for successive generations during a

HIDE¥O NOGUCItI

427

period of 3 years would acquire an ascending virulence for the testicular tissues while suffering a gradual loss of its affinity for the tegumentary system. In the beginning the strain which was passed on
to the testicular tissue showed a lower virulence for this organ, b u t
it also showed a correspondingly low virulence for the dermal tissue.
Upon attaining a certain degree of virulence .for the testes the virus
manifested a parallel increase of activity for the skin, showing nodisproportion between the titers for the two kinds of tissue. It may
be assumed, then, that by a prolonged passage through the testes, theaffinity for the skin has in no wise been diminished. The maximum
titer obtained by a testicular product in rabbits was that which produced a confluent eruption on the skin of rabbits in a dilution of
1: 10,000. Such specimens were not frequently obtained, and theresult probably depends upon the suitability of the animal used. Thereare considerable individual variations in the susceptibility of the rabbit and the calf, and it is not rare to get a specimen that requires a
1:100 dilution in order to produce a confluent reaction. The titers
with the rabbits averaged about 1:1,000. Of course, a specimen
possessing the activity to produce a confluent reaction in a 1 : 100
dilution is already strong enough to insure 100 per cent of takes
among primates. The individual variations in suitability for producing a testicular virus are paralleled by the susceptibility of the skinof the same animal to the vaccine virus, regardless of the mode ofpropagation.
It has been stated elsewhere l that a highly potential testicular
vaccine can easily set up vaccinia orchitis in the rabbit in dilutions
as high as 1 : 100,000, while oil the skin the same dilution produces a
few eruptions. In this respect the virus seemed to have acquired
an increase in virulence for the testicle but not necessarily to havelost its dermatophilic property. It still remained to be seen whether
this orchitophilic adaptation of the virus was associated also with thegeneral increase of virulence for other internal organs and tissues.
In order to determine this point we tested the emulsions of lungs,
liver, spleen, kidneys, and lymph glands, removed 5 days after
inoculation, of the rabbits which had been successfully inoculated
intratesticularly with the testicular virus. As controls a numberof rabbits were intratesticularly inoculated with unadapted virus,

428

PURE VACCINE VIRUS C U L T I V A T E D I N VIVO

which, however, caused a marked orchitis. In a second series ofanimals the testicular strain was used on the skin, causing the latterto produce a confluent eruption, while several animals were vaccinatedwith the regular skin strain to serve as controls. The results w e r e
uniformly negative, except for a few eruptions in the areas inoculatedwith the lymph nodes in a few instances where the virus, irrespectiveof whether it was of testicular or dermal origin, was given intratesficularly.

Localization of the Vaccine Virus after Subcutaneous and Intravenous
Inoculations.
The introduction of small quantities of the testicular virus, suchas 1 cc. of a 1 : 1,000, 1 : 10,000, etc., dilution, into theblood circulationor under the skin of the rabbit produced no appreciable local or general
symptoms. The injection of 1 cc. of a 1 : 100 dilution, however, sometimes caused a local inflammation and rise in temperature on the 4th
and 5th days. No general eruption was ever observed. The injection of 1 cc. of a 1:10 dilution was sometimes accompanied by a
• high temperature for 3 days, and, in the case of, subcutaneous injection,
local tumefaction and edema, but no generalized eruption. The
injection of 1 cc. or more of the undiluted emulsion produced a grave
illness of 3 or 4 days, with a fever lasting for 3 days. Generalized
eruptions, particularly numerous on the mucous membranes of thenose, lips, mouth, and genitalia, were observed. Camus ~,3,4 noted asimilar phenomenon with the skin vaccine.
The examinations of different tissues removed from the animals
showed that in cases of intravenous inoculation of 1:10 and 1:100
d!lutions, the lymph glands and sometimes, but seldom, one of thetestes contained some virus, but no bilateral orchitis or marked reaction was obtained. In case of a higher dilution than 1:1,000 we
occasionally demonstrated a small quantity of the virus in the lymph
Camus, L., De la vaccine g~n6ralis~e exp~rimentale. Conditionsde sa production, Bull. Acad. m~d., 1916, lxxvi, 342.
Camus, L., R6productlon de la vaccine g6n6ralis6e chez le chien, Bull. Acacl.
m~d., 1916, lxxvl, 376.
4 Camus, L., La vaccine g~n6ralis6e exp6rimentale chez la g~nisse et chez lesinge, Bull. Acad. m3d., 1916, lxxvl, 433.

EmE~ZO s o G v c ~429

nodes, but never in the testes or other organs. Even the injectionof 1 cc. of the undiluted vaccine failed in two experiments to localizein the testes. The other organs, except the lymph glands, were
negative.
The corresponding series of experiments with subcutaneous inoculation did not bring about generalized eruptions even with the strongest
dose used. A small amount of the virusin the adjacent lymph nodes
was occasionally demonstrated, but far less frequently than in theintravenous series.
The rabbits which received the intravenous and subcutaneous
inoculations of the virus were tested for their susceptibility to a subsequent application of a powerful vaccine, both the testicular and thedermal, within several weeks. They were found to be refractory
to the new vaccination, although some of them had originaUy received
only 1 cc. of a 1:10,000 dilution. The testes of these rabbits werealso insusceptible to the inoculation with a highly active virus. The
immunity brought about by the intravenous or subcutaneous inoculaion of the virus was altogether comparable with that conferred by aegular procedure on the skin. A detailed report of this phase of thework will be made later.

Viability and Resistance of the Testicular Vaccine Virus.
Data concerning the viability and resistance of vaccine virus
ought to be abundant, but one does not easily find details of a systematic investigation. From the tixne of Jenner the resistance of thevirus to desiccation has been known, and it was proved nearly 50
years ago 5 that it resists the action of glycerol when the latter is usedin the proper concentration. Later Umeno6 and others found thatphenol in a concentration below 1 per cent does not perceptibly decrease its virulence. Yet much of what was done years ago seems tohave been overlooked, and it may be of sufficient interest to publish
here the experimental data obtained by the writer during the l~ast 3
6 Copeman, S. M., Vaccination. Its natural history atld pathology, London,
1899, 156.
s Personal communicationfrom Professor S. Umeno, Director of the Imperial
Institute for the Preparationof VadcineVirus, Tokyo,Japan.

430

PURE VACCINE VIRUS CULTIVATED I N VIVO

years. N a t u r a l l y , the chief object of the experiments was to s t u d y
the testicular vaccine virus, b u t in some instances, controls with theregular vaccine virus were m a d e as far as it was possible. F r o m then a t u r e of the latter, however, no experiments could h a v e been carriedout to test its viability in a suspension w i t h o u t antiseptics such as
glycerol or phenol, while it was possible to do so w i t h the bacteriafree testicular virus.

Survival of the Vacci~e Virus in Distilled Water and Glycerolated Media,
at Different Temperatures.
April 2, 1915. Three sets of eight test-tubes each were used. After sterilization, 11.6 cc. of distilled water were added to the first tube of each set, and tothe other seven tubes 11.6 cc. of 10, 20, 40, 50, 60, 80 per cent, and pure glycerol
respectively. The contents of each tube were mixed with 0.4 cc. of the stock
emulsion of the testicular strain, No. 948 emulsion, which*had the titer of 1:1,000(confluent). One set was placed in a refrigerator at 4°C., the second set at
18°, and the last at 37°C. 0.5 cc. was taken from each tube and tested
as usual on the skin of rabbits, three or four rabbits being used in order to test thethree sets (24 tubes) simultaneously. At first tests were made daily, or every
other day, but later at semimonthly, monthly, or longer intervals. Tables I,
II, and I I I give the results.
T h e m o s t striking p o i n t d e m o n s t r a t e d in the foregoing experiments
is t h a t the vaccine virus retains its virulence m u c h longer in di~stilled
w a t e r t h a n in a n y of the glycerolated media. P u r e glycerol destroyed
the virus to a considerable extent in a week and completely within am o n t h , even a t a t e m p e r a t u r e o f 4°C., while the virus in w a t e r
remained v e r y active after 1 year. As the concentration of glycerol
diminished its destructive effect was less noticeable, and in 10 to 20
to 40 per cent the virus remained active for a t l e a s t 6 m o n t h s . A t
18°C., the t e m p e r a t u r e of our laboratory, the virus deteriorated
rapidly. T h e virus was killed in 5 days in p u r e glycerol, in 1 m o n t h
in 60 to 80 per cent, in 2 monttls in 10 to 20 per cent glycerol, while
it was still v e r y active in w a t e r after 3 months. These differences
are m u c h m o r e a c c e n t u a t e d at 37°C., where the virus was no longer
alive in p u r e glycerol after 24 hours. I n 80 per cent glycerol it was
a v i r u l e n t in 6 days, in 60 per cent in 7 days, in 50 per cent in 9 days,
and in 10 to 20 to 40 per cent in 28 days. On the other hand, in w a t e r

431

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a trace of the virus was still present for as long as 2 months, when it
still showed a few eruptions on test. The powerful vaccinicidal
property of glycerol is well brought out in this set, and in any experiment bearing upon the viability of the virus this factor shouldbeconsidered. The persistence of the virus in water or in 10 to 20 per centglycerolated water at the temperature of 37°C. is remarkable andbecomes important in interpreting the results in cultivation experiments.

Effect of Diluents upon the Viability of the Vaccine Virus at Different
Tert~peratures.
May 24, 1915. Experiments were made similar to the foregoing but with
distilled water, 0.9 per cent saline solution, Ringer's solution, 50 per cent glycerol,
0.5, 1, and 2 per cent aqueous, and 50 per cent gl¥cerolated solutions of phenol asdiluents. To 19.6 cc. of each of the solutionswas added 0.4 cc. of testieular virus,
No. 992 emulsion. The three sets of tubes were placed at 4°, 18°, and 37°C.
respectively. The results of the tests are given in Table IV.
Table IV confirms the findings of the preceding series of experiments and further shows that at 4°C. the virus was best preservedin Ringer's solution and in 0.5 and 1 per cent phenol water, being still
active at the end of 1 year. In distilled water it was weaker than insaline solution, the latter being almost as good a medium as Ringer's
solution. The deteriorating effect of 50 per cent glycerol was marked
in this instance. The addition of phenol in 0.5 per cent did not affectthe action, although 1 per cent phenol plus 50 per cent glycerol killed
the virus more quickly than either of them separately. Phenol in a
2 per cent solution and agitation of the virus with an excess of chloroform for 3 hours destroyed the virus within 24 hours. The results
at 18°C. and at 37°C. indicate that there is a more complete andrapid destruction of the virus with a rise of temperature. An interesting feature seems to be the longer, if not better survival of thevirus in a carbolized solution (0.5 per ceflt w a t e r ) t h a n in Ringer's
solution, saline solution, or distilled water. In the first 7 days, theactivity of the virus in the latter solutions was greater than in the
carbolized medium, however.

434

PURE VACCINE VIRUS CULTIVATED IN VIVO

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Survival of the Vaccine Virus in Different Atmospheres.

In order to find whether a gradual deterioration of the vaccine virus
could be delayed or prevented by preserving the virus in different
kinds of gases, we placed the vaccine in sealed ampules containing
either hydrogen, nitrogen, oxygen, carbon dioxide, or air. For thispurpose the testicular vaccine, No. 1,035 emulsion (1:1,000 titer),
was diluted 10 times with sterile distilled water, the gases were passed
through the vaccine, and the receptacles hermetically sealed by fusing
the drawn portions with flame. The vaccine was not exposed to heat.
The appearance of the vaccine was not altered by hydrogen, nitrogen,or air, but the passage o f carbon dioxide caused a cleai-ing of the
diffuse turbidity, the precipitates adhering to 'the wall of the container,
while with oxygen, granular precipitates appeared.
Three duplicate sets placed at 4 °, 18°, and 37°C. respectively wereused. The results obtained after a period of 21 days are shown in
Table V.
TABLE

V.

Survival of Vaccine Virus in Various Atmospheres.
Results after 21 days.
Gas.

Hydrogen (sealed) .........................
Nitrogen"..........................
Oxygen".........................
Carbon dioxide (sealed) ....................
Air (sealed) ...............................
" (open tube) .........................

4 ° C.

18 ° C .

37 ° C.

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The virus retained its virulence in all the gases for 3 weeks when
kept at 4°C. but became avirulent at 37°C. From the results
obtained at 18°C., however, it may be concluded that in sealed
ampules containing hydrogen, nitrogen, or ordinary air, the virus retained its virulence somewhat better than in an open receptacle.
Pure oxygen or carbon dioxide gas destroyed the virus completely
at the same temperature.

HIDE¥O

437

NOCUCHI

Effect of ACid, Alkali, and Germicides upon the Vaccine Virus.

Knowledge concerning the resistance of the vaccine virus to various
substances is a prerequisite for handling it properly. In a series ofexperiments hydrochloric acid, sodium hydroxide, tricresol, and phenol
in varying concentrations were mixed with a testicular vaccine emulsion. Tests were made on rabbits at the end of 24 hours, 4 days, and6 days respectively at 4°C. Table VI shows the results.
The table shows that the vaccine virus is completely destroyed bysodium hydroxide added in a" concentration greater than 1: 200,
TABLE VI.

Effect of A cld, Alkali, and Germicides.
Fresh skin vaccine (some cocci).

Testicular vaccine(no bacteria).
Tested for vaccine.

Substance.

Tested forbacteria.

Tested for vaccine.

4 6 days.
24 hrs. days.

4
6 days. hrs.
24 days.
4 d6s.eydays.

24 hrs.

pegceni

H y d r o c h l o r i c acid.

Sodium hydroxide.

Tricresol.

Phenol.

Control.

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438

P U R E VACCINE VIRUS CULTIVATED I N VIVO

while hydrochloric acid destroyed it almost completely in a corresponding concentration. On the other hand, the contaminating micrococcio f the fresh skin pulp showed a different relation, resisting theaction of even 1 per cent sodium hydroxide solution for at least 24
hours, while they were completely sterilized by a 0.5 per cent hydrochloric acid solution. The action of tricresol and phenol is similar,t h e difference being quantitative rather than qualitative. The virus
resisted 0.2 per cent tricresol or 0.5 per cent phenol for many days,
as did also the contaminating bacteria. In the case of phenol, andin a lesser degree tricresol, the destructive effect was comparatively
more severe upon the micrococci than upon the vaccine virus. The
margin was, however, narrow. A bacterial spore cannot be sterilizedb y a concentration which will not destroy the vaccine virus completely.
The action of iodine was next studied in various ways, because of
its effectiveness as a germicide. It was employed as a local antiseptic in the form of an alcoholic solution or as Lugol's solution.
In a series of experiments we made a number of dilutions of tinctureof iodine by using 10 per cent ethyl alcohol water as diluent. To
0.9 cc. of each dilution, 0.1 cc. of a 1 : 10 dilution of a strong testicular
vaccine virus was added, the mixture incubated for 1 hour at 37°C.,
and then tested on rabbits. It was found that the diluent alone, thatis a 10 per cent alcohol water, exerted no effect. On the other hand,
the mixtures containing a dilution of the tincture of iodine above
1 : 10,000 became avirulent.
The mixture which contained a dilutionof i : 100,000 gave several eruptions instead of the confluent reaction
which took place when controls without iodine were used. The experiment demonstrates how destructive this element is for the vaccine
virus. Lugol's solution (iodine 1 part, potassium iodide 2 parts, andwater 300 parts) destroyed the virus in a 1:100 dilution but had noeffect in a 1:1,000 dilution. Attempts were made to influence thecourse of the vaccine reaction by administering a considerable amountof Lugol's solution or sodium or potassium salts by intravenous or
subcutaneous injections for several days before and after the vaccination. No effect was perceptible. The iodide salts failed to reducethe virulence of the vaccine virus even when mixed in vitro in a 30
per cent solution and kept 1 hour at 37°C.
Attempts were made to sterilize the vaccine virus simultaneously

HIDE¥O NO6UCHI

439

with its application to the skin or at various intervals afterwards.
I t was found that tincture of iodine in a concentration stronger than
1: 10 inhibits the development of the eruption; in 1 : 400 dilution, it
prevented the process from being confluent. Lugol's solution usedin full strength reduced but failed to check the infection. The
application after 24 hours of tincture of iodine in concentrated forms
did not noticeably influence the vaccine infection.
So far no visible organism has been found as the causative agent ofvaccinia. From the viewpoint of classification it seemed important
to study the effects of certain protoplasmic poisons on the virus.
For this purpose 0.1 cc. of testicular vaccine, No. 1,062 emulsion, wasmixed with 0.5 cc. of sodium oleate, sodium taurocholate, sodium
glycocholate, and sodium cholate, in varying concentrations. After
1 hour at 37°C. the mixtures were tested on rabbits. It was foundthat all the salts destroyed the vaccine virus in a 1: 10 dilution. In a
1 : 100 dilution a small portion of the virus still survived, while thevirus in control tubes was caPable of producing a confluent reaction.

Effect of Desiccation upon the Vaccine Virus.
Vaccine virus is known to withstand desiccation for a long time,
but more exact knowledge is desired as to its reaction to dryness,the length of time it will survive in the dry state, and how the driedvaccine virus compares with moist emulsions at different temperatures. Many microorganisms, especially those which pass the filter,
resist desiccation and remain viable for a long time. Most enzymes
retain their activity better in the dry than in the moist state, especiallyat the higher temperatures. The question becomes one of considerable importance in the case of the vaccine virus, because of the rapid
deterioration which attends the moist preparation of the virus intropical countries. If the dried vaccine proved to resist the conditionsof moisture and temperature similar to those of a tropical climate
better than the liquid emulsion, it would be a great advantage topreserve the vaccine virus in the dried form.
On several occasions we have dried quantities of the organ pasteof testicular vaccine virus by means of a vacuum pump. Afterdesiccation quantities were weighed, powdered, and preserved in440

PURE VACCINE VIRUS CULTIVATED IN VIVO

hermetically sealed ampules or left in an open receptacle. One setof specimens was placed at 4°C., some at 18°, and others at 37 °.
The controls consisted of aqueous emulsions of the undried portionof the same tissue paste.
The reduction in weight through desiccation was not uniform,
and no constancy could be expected on account of the variation indegree of edema of the organs (Table VII).
TABLE VII.
Reduction of the Weight of the Vaccine Paste through Desiccation.
Emulsion No.

Organ.

Original weight.
gm.

gft~.

866
876

Testis.

2.0
2.5
2.0
2.0

0.38
0.38
0.3
0.32
0.32
0.25

877

878
1,045
1,046

tt
st

1.85

1.6

Resld~e.

The vaccine virus did not in any instance show its original titerafter desiccation. The loss of virulence, as determined by employing
corresponding quantities of the dried and moist materials, amounts
to half or even more of its original strength. This was unexpected,
but was true with all the dried materials. The insolubility which
attends the desiccation of the vaccine paste may play a part in theloss of virulence. To resuspend and dissolve the powdered material isdifficult. The results obtained during a period of 18 months indicatethat the dried material remains still viable, although reduced to
one one-hundredth or less of the original titers, for about 12 to 18
months at 4°C. and 18°C., but becomes inert within 30 to 60
days at 3 7°C. The control specimens in the moist state showed similar
relations.
From the above findings it is evident that the process of desiccationas carried out is considerably destructive to the vaccine virus, andthat it does not protect it from the gradual deterioration_due to age
which takes place at different temperatures.

HIDEYO NOGUCHI441

SUMMARY.

1. The virulence of vaccine virus for the testicular tissues increases
until its maximum is finally reached. The selective increase is not
associated with any loss, reduction, or modification in its virulence
for the skin. A highly potent testicular vaccine is also highly active
for the skin.
2. The testicular strain of vaccine virus has no more tendency tolocalize in various organs than the ordinary skin strain. Both may
localize in adjacent lymph nodes when introduced intravenously,
subcutaneously, or intratesticularly in sufficiently large quantities,
but other organs are not involved.
3. Intravenous inoculation of an excessive amount of a powerful
vaccine virus (1 to 2 cc. of undiluted stock emulsion), irrespective ofwhether it is from the testis or the skin, will result in a generalized
eruption over the entire body surface of rabbits. The eruption may
be confluent on mucous membranes of the moutl~, nostrils, genitalia,
etc. Intratesticular or subcutaneous inoculations of the same virus
fail to produce this effect.
4. Subcutaneous or intravenous introduction of much smaller quantities of the virus does not cause an appreciable local or general reactionin the rabbit. But the animals which have once received these injections become refractory to a subsequent vaccination as applied tothe skin. It seems probable that an active immunity has been conferred.
5. Experiments on the viability and resistance of the testicularstrain of vaccine virus indicate that the virus is best preserved whenemulsified with Ringer's solution or 0.9 per cent saline solution. Distilled water, while apparently one of the best diluents, fails to keep
the virus active as long as Ringer's or saline solutions. As would be
expected, the lower the temperature is, the longer the virus retains
its viability. At 18° or 37°C., the deterioration of the virus proceeds rapidly. However, a small part of the virus survives after
many weeks' standing at 37°C.
6. Of the two most commonly employed chemical agents for the
ripening (eliminating bacteria) process of the green vaccine pulp,
glycerol and phenol, the latter is the less injurious. Phenol in con-

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centration above 2 per cent destroys the virus within 24 hours at any
temperature, but it has almost no injurious effect when used in 0.5
to 1 per cent. On the other hand, glycerol is a powerful vaccinicide.
When used in full strength it destroys the virus within 24hours, evenat 4°C. In a concentration of 40 per cent, that ordinarily recommended for the ripening, the virus retains some of its virulence forabout half a year at 4°C., while at higher temperatures the same concentration kills the virus within 1 to 2 months. The virus preservedin distilled water or Ringer's solution under similar temperature conditions remains more active during this period. From this it may beconcluded that glycerol is not an indifferent agent, as is assumed b y
many, but a powerful vaccinicide when used in high concentrations.
The injurious effect is markedly accelerated at 18° or 37°C.
7, The vaccine virus retains its virulence better in a sealed tube
containing either hydrogen, nitrogen, or air than in an open receptacle.
The virus deteriorates when placed in a sealed tube with oxygen or
carbon dioxide.
8. Desiccation decreases to a considerable degree the virulence ofthe vaccine virus. In the dried state the virus retains its viability
about as long as does the emulsion, b u t it is not protected from thedeterioration caused b y age under various conditions.
9. Iodine is a powerful disinfectant for the vaccine virus, b u t its
sodium and potassium salts have no effect. Various bile salts destroy
the vaccine virus when employed in sufficient concentration.