NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
40
of putrefaction when they are sown in the same medium. Their develop-
ment may be retarded or stopped in culture media when, either intention-
ally or unintentionally, they are sown with other ærobic microbes as some
common bacteria are; in such cases it may happen that the microbe of
charbon develops little or not at all, or that it perishes after a certain
time. Inoculated alone into the organism they generally kill, although
they have to battle against the cells; but inoculated in company with
common bacteria they may perish without causing any disturbance.
(Pasteur.) The antagonism is not always so marked; and the microbe
of charbon may develop at the same time as others in the same medium;
as, for instance, in the milk of the cow attacked with specific mammitis
sown with the cultivated microbes of the two diseases. (Nocard.)
Nevertheless, the antagonism of the microbe to other microbes is worthy
of mention. We know that the bacillus anthracis develops badly
in a medium wherein the microbe of fowl cholera already exists, that
it is possible to render inoffensive the inoculation of the microbe in that
rabbit by preceding it with, accompanying it by, or following it by the
inoculation of the microbe of erysipelas (Emmerich, Watson, Cheyne,
Pawlowsky); that the inoculation of certain inoffensive or pathogenic
microbes (bacillus prodigiosus, staphylococcus pyogenes aureus, pneumo-
coccus) may also exercise a certain protective influence against the
microbe (Pawlowsky) ; that the microbe of " Rouget " may hinder the
effects of the microbe of charbon on the guinea-pig (Zagari); that a
saprophyte bacterium closely resembling the bacillus anthracis, growing
and giving spores like it, but without its pathogenic action on small
animals, and capable of giving them a certain immunity against the
bacillus anthracis, has been found in air and water (Hueppe and
Wood).
Again, new observations have been made in these later days relative
to the antagonism of the bacillus to other microbes. Bouchard, Frenden-
rich, Guignard and Charrin have remarked that the pyocyanic bacillus
lessens the infection of anthrax in the rabbit and guinea-pig; it has also
been found that sterilized pyocyanic cultures exercise an antagonistic
action to the bacillus of charbon (Woodhead and Cartwright Wood).
Boin and Rœser have found that yeast, which is not harmful to the
organism, lessens the effects of the virus of charbon; and Buchner has
observed that sterilized cultures of the bacillus of Friedlander may arrest
the development of charbon.
By what mechanism is this effect of one microbe upon another pro-
duced ? is it the direct influence of one microbe or its products against
the other, or is the system not excited to intervene more energetically ?
The reply to this question is in part decided by the researches made by
41
Blagavettehensky. This investigator has found (1) that the simul-
taneous inoculation into the eye of pyoeyanic bacilli and the bacillus
of charbon generally opposes the development of the latter without, at
all events in the immense majority of cases, rendering the animal re-
fractory to a future charbonous infection; (2) that pyoeyanic bacilli in-
oculated at some little distance from charbon bacilli exercise a much
slighter effect upon them; (3) that the pyoeyanic bacilli exercise a direct
action upon the bacilli, hindering their development; (4) that suc-
cessive injections of the two microbes into a being do not prevent the
development of charbon; (5) that sterilized cultures of the pyoeyanic
bacilli can only influence the development of the bacilli when used
in very large doses; (6) that outside the body development is arrested;
(7) that the proximity of the products furnished by the pyoeyanic
bacillus exercises a harmful influence on the development of the bac-
teridia; (8) that the products of the bacillus exercise the harmful
influence.
Resistance of the "bacillus" to Physical and Chemical
Agents.—The microbe of charbon, as we have seen, presents itself under
two principal forms—under the form of rods or filaments and under the
form of spores. Its resistance to the various agents, which have the effect
of sterilization, is very variable according as it is found under one or the
other of these forms. The rods preserve their virulence and their lives
for a short time only, when they are deprived of air or oxygen; they are
destroyed without forming spores and are resolved into inoffensive granu-
lations when they are placed in a situation deprived of air or oxygen, or
in an atmosphere of carbonic acid gas; they are in this case asphyxiated
more quickly when the temperature is elevated to near 38º to 40º C.
Charbonous blood taken with due antiseptic precautions from a living
animal or from one recently dead, does not contain putrefactive microbes;
it only contains charbonous rods; it loses its virulence after a certain
time, which varies according to the surrounding temperature, when
placed in a vessel containing no air or in an atmosphere of carbonic acid;
to vegetate it requires the presence of air.
We have seen that the microbe only vegetates at certain temperatures—
from 12º to 43º or 44º C.,—and that it does only form spores between
15º and 43º C.; above 45º multiplication by segmentation ceases. Low
temperatures have no great influence on the microbe; the rods resist 45º
C., and the spores are not killed by a temperature of 70º C. or even 130º
C. Rods and filaments are killed by a temperature of 100º C., by cook-
ing the media in which they are found, and even by a temperature of 60º
C. or even 55º, 50º, and 45º C., and they are exposed for a sufficient length
of time. The spores have a greater power of resistance and bear heat
much better; they resist a temperature of 95º for ten minutes in a
42
moist atmosphere, and can bear 80º C. for some time; they are capable
of resisting temperatures of even 100º, 110º, 115º, 120º and 123º, when
they are ancient, dried, or heated in dry air (Koch). Sometimes, however,
contrary to the observations of Koch and Wolffhugel, who have seen
spores not destroyed by a heat of 140º until after three hours' exposure
M. Massol has found that they are always destroyed after an exposure
of 57 minutes in dry air at a temperature of 100º C.
To resume, spores resist a temperature of 95º C. for ten minutes in a
moist atmosphere, whilst they are destroyed in less than five minutes
when the temperature is carried to 100º C. Below 80º C. they preserve
their vitality for a considerable time; we may heat them for a number
of hours at 70º without destroying them (Roux). All the spores of a
culture do not present the same degree of resistance to heat.
At a temperature of 70º C. the spore of charbon does not perish for
a very considerable time when heated without the presence of air; in
the liquid in which it was formed this long heating does not sensibly
modify its virulence; it is necessary that the heating be prolonged to
nearly the limit of the resistance of the spores for the virulence to be
lessened; and, on the other hand, even though by this means the virulence
is lessened, its lessening does not appear to be of the nature of a true
attenuation, for bacilli born of spores which have been heated in
this manner for 165 hours at a temperature of 70º C., kill guinea-pigs and
rabbits. At the same temperature the spores, heated in the presence of
the air, die more quickly; and, before dying, lose their virulence, or it
becomes sensibly diminished. Inoculated into animals they remain in-
active from 24 to 36 hours, but their culture is still virulent. Some-
times, when we inoculate cultures of spores heated in the air for a longer
period (64 hours), a real attenuation of their virulence may be observed
which may be preserved in successive cultures. Oxygen, on account of
its oxidysing properties, is then the agent which interferes most wich
it. Spores already attenuated by Pasteur's method are more easily acted
upon by heat than are spores which have not been attenuated. Spores
heated in the presence of oxygen undergo the same modifications, with
little less rapidity than those heated in the presence of air.
The filaments from spores heated in air or without air rapidl y form
spores in cultures (Roux).
Rapid desiccation kills the rods and filaments; slow desiecation does
not prevent the formation of spores, which explains how it was that
Davaine found charbonous blood virulent a long time after desiccations
The spores once formed bear desiccation, preserve their properties for a
very long time, resist putrefaction, alternate lowering and raising of
temperature, humidity, desiccation, etc. Dried spores may preserve their
virulence for years.
43
The action of light upon the microbe is important. M. Arloing's ex-
periments led to the following results: Istly, that gaslight modifies the
vegetation of the microbe but little, that it retards the evolution of the
spores sown in broth, without modifying their pathogenic properties;
2ndly, that sunlight is more active, hinders the evolution of the spores
and the development of the bacillus, and gradually attenuates cultures;
3rdly, that exposure to the sun for two or three hours in July, between
35º and 39º C., destroys the power of vegetating in broth culture media
freshly sown with spores; 4thly, that it requires 27 to 30 hours' exposure
to the sun, in the same conditions, to sterilize the mycelium of the same
bacillus in a state of full development, and that before death takes
place the microbe undergoes a series of progressive alterations which
transform it into a vaccine of less and less activity.
It had been considered that the least resistance of the spores, shown
by these results, was due to the fact that those which were sown com-
menced to germinate despite the action of the rays of the sun, and
that the young mycelium in course of formation rapidly undergoes
the sterilizing influence of the sun's rays. And this explanation of
the phenomenon appeared to be demonstrated by the results obtained
by M. Straus, who observed, with regard to the resistance of the
spores, that when they are immersed in pure water germination does
not occur for four hours, whilst in broth it may commence rapidly (in
two hours). Nevertheless, M. Arloing obtained death of the spores after
an hour's exposure to the rays of the electric light acting on cultures
kept in ice at a temperature at which germination is impossible. In
addition to this, putting aside the intimate mechanism which presides
over sterilization, it remains an established fact that exposure to light
more or less rapidly destroys the spore of charbon, that it may kill
it very rapidly, or not till 29 or 54 hours have elapsed (Roux), and
that its action is more or less rapid in proportion to the intensity of
the light and according to the surrounding medium which, being
modified, when culture has already commenced, renders the steriliza-
tion slower. On the other hand, it is averred that the action of
light is due to the presence of oxygen, and that the spores of bacillus
autbracis live longer when they are exposed to the action of light
without air. Similarly, M. Roux, experimenting on the spores of
charbon, believed that the action of light is due to that of oxygen,
and that the action of light favoured the action of the air, and that in
the absence of air the spores are modified much more slowly. Under
the combined action of light and air there is produced in a culture
medium a chemical change which arrests the evolution of the germs;
generally, flasks containing broth and the germs do not cultivate when
placed in a stove, after two hours' exposure to light; whilst those
44
containing only broth after two hours of exposure to the action of
light, and to which spores are afterwards added, cultivate for the most
part when placed in the incubator; nevertheless, when the broth has
been exposed for three or four hours the spores shown in it do not
germinate. Yet the spores are not killed neither after two hours'
nor after seven hours' exposure to the action of light; and when shaken
in a medium which has been exposed to the action of light, and in
which they will not germinate, then sown in a medium which has not
been so exposed, they vegetate; germination is, it is true, if anything
the more retarded the longer the spores have been exposed, or the
longer they have been kept in the medium which has been exposed to
the action of rays of light. Exposed broth, in which the spore does
not develop, is suitable for the cultivation of the filaments; germina-
tion is arrested by a modification of the medium, which is insufficient
to arrest the multiplication of the rods. Oxygen plays an important
part in the modification of the medium, for, after exposure, the
spores exposed in a medium deprived of air germinate in the same
medium when it is aerated, whilst those which have been exposed to
the action of both light and air do not germinate when so treated.
In addition to this, broth deprived of air or placed in an atmosphere
of carbonic acid may remain exposed to the action of light for many
hours and afterwards may serve for the cultivation of the rods or
spores; whilst a few hours' exposure to light and air renders a broth
unsuitable for the development of the spores. The action of light
and air is rapid when the receiver is freely open and contains
only a small quantity of broth. We may increase the effect of light
and air by adding to the broth substances which oxydise easily; in
such manner, veal broth to which glucose has been added, becomes,
after exposure, more unfavourable to the germination of spores than
broth to which sugar has not been added. A medium which has
become unsuitable to germination after the exposure may regain its
original properties when kept in diffused light or partial darkness.
Cultures of spores which have been exposed to the action of light
without air, preserve their germinating power for a long time, becoming
slower and slower in performing the process as they remain for a longer
period in the medium. Cultures of spores which have been exposed,
without air, for 83 hours, have not sensibly lost their virulence. It is
the same with cultures of spores submitted for 54 hours to the action
of light and air. The filaments, as M. Arloing has demonstrated, exposed
to the sun and air in broth for a sufficient length of time, become by
degrees attenuated, and their cultures follow the same course (Roux).
Under the double influence of oxygen and light, oxydation, which is
hurtful to the life of the microbe, occurs. It is averred lastly by
45
Duclaux that spores exposed in a dry state may preserve their power of
vegetating for a month or more. The oxygen, which is necessary for
the development of the bacillus anthracis, may, under such circumstances,
contribute to its attenuation, or may even destroy it. Its action is more
particularly marked in adult bacilli, the spores bearing its presence
much better. Compressed oxygen also acts more energetically on the
rods or filaments than on the spores. When chemical germicides are
caused to act upon the microbes we still observe great differences
between the resistance of the rod and that of the spore; the adult bacilli
are killed more rapidly and with much smaller doses than are the spores.
Bromine, iodine, and chlorine may be used as germicides when we are
dealing with the baciilus anthracis even in the spore form. According
to Davaine 7 milligrammes of iodine sterilizes a litre of fluid in which
has been placed 1 centimetre cube of charbonous blood; it arrests the
development of charbonous microbes in culture media in which it has
been dissolved, and kills the spores after 24 hours' contact (Koch). Char-
bonous spores perish after an hour's exposure to chlorine water and the
rods are destroyed more quickly (Perroneito). Chlorine gas, 25 to 50 per
cent., kills the spores of charbon in a minute (Galtier), but in practice
these agents are little used. As to sulphurous acid, we know that it is
unable to sterilize the spores of charbon; and after the experiments of M.
Thionot we cannot count on sulphurous acid in large quantities used for
24 hours to destroy the microbe of charbon. Essence of turpentine
which has been considered as capable of destroying the bacillus and its
spores (Pasteur), may be inactive against the latter (Koch). Solution of
nitric acid must be used in high percentage to kill the spores, whilst a 2 per
cent. solution of hydrochloric acid should be sufficient to kill the bacilli
and their spores (Koch). A 5 per cent. solution of cresyl rapidly destroys
the adult bacillus; but the spores resist its action for a considerable time —
24 hours according to some, and more than 20 days according to
others (Esrnarch). With regard to the action of carbolic acid the follow-
ing remarks by Koch may be studied with benefit:—"Anthrax spores,
dried upon silk threads, were placed in carbolic solutions of various
strengths and kept in corked bottles. A thread was removed from time to
time, and planted on nutrient jelly. The following shows the results:—
Carbolic Solution.
2
5
1
2
3
4
5
per cent.
„
„
„
„
„
„
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Days of Exposure.
1, 3, 5*
1, 3
1, 2, 3, 4, 5, 7, 15
1, 2, 3*, 4*, 5*, 7*
1, 2*, 3*, 4*, 5*, 7
1*, 2*, 3, 4
1*, 2, 3, 4
46
"The plain figures in the second column signify that the spores
removed from the disinfectant solution on those days developed on the
nutrient jelly as actively as in the control experiments, and had there
fore not been affected by the action of the carbolic solution. The
figures bearing an asterisk signify that the spores transferred to the
jelly on those days yielded scanty or retarded growths, less active than
those of the control experiments. The doubly underlined figures in
dicate complete sterilization—that is, that the spores removed from the
carbolic solution on the corresponding days yielded no growth whatever
upon the jelly.
"Thus 1 and 2 per cent. failed to destroy anthrax spores within a
week; 3 per cent. took seven days; 4 per cent. took three days; and 5 per
cent. required more than one day.
"The action of carbolic solutions upon the bacilli destitute of spores
were of a very different character. Bacilli from the spleen of a mouse,
dead of anthrax, in which spores were not formed, were dried upon silk
threads and subjected to the same experiment. Two minutes' exposure
to a one per cent. solution killed the bacilli. It was also found that
blood from an animal dead of anthrax, mixed with an equal volume of 1
per cent. carbolic solution and allowed to stand for a short time, did not
infect an animal into which it was injected; but if the strength of the
carbolic solution were only 0.5 per cent., anthrax ensued.
"With regard to the inhibition of the growth of bacillus anthracis,
threads charged with anthrax spores were placed in a series of seven
vessels, each containing 10 cc. of fresh clear blood serum; and to these
were added respectively 1, 2, 4, 6, 8, 10, and 15 drops of a 2 per cent.
carbolic solution. They were protected from evaporation and pollution by
dust by means of a cover. In the first four the growth was as active as in
the control experiments; in the fifth it was delayed, but ultimately equally
vigorous; in the sixth there was only a scanty growth of short and
crooked threads; in the seventh there was no growth whatever, but the
spores still grew vigorously, after 72 hours' immersion, when transferred
to fresh nutrient jelly.
"These experiments were repeated with similar quantities of fluid, the
blood serum, however, being replaced by a solution containing 1 per
cent. of peptone, and ½ per cent. of meat extract. Five drops of 2 per
cent. carbolic solution had no effect, ten drops caused marked retardation,
and twenty drops entirely prevented the growth.
"Hence one part of pure carbolic acid in 850 parts of the cultivating
fluid completely prevents the growth of anthrax bacilli; whilst one part
in 1,250 causes marked diminution of activity."
Perroncito states "that 2, 3, 4, 5 per cent. of solution of carbolic
47
acid rapidly destroys the rods and filaments of bacillus anthracis, but
the spores resist a 5 per cent. solution for 24 days."
Koch also remarks of several compounds of carbolic acid which
were tested as to their power of destroying anthrax spores, but all
proved to be less powerful than the pure acid, and he decided that car-
bolate of zinc stands next to carbolic acid as a disinfectant.
Anthrax spores were found to be absolutely unaffected by lying for
110 days in a 5 per cent. solution of carbolic acid in oil, or 70 days in a
solution in alcohol.
Even the sensitive anthrax bacilli were not more affected by 5 per
cent. solution of caroblic acid in oil than by pure oil, as will be seen
from the following table:—
Carbolic acid, 5 per cent. in olive oil
„ „ 1
Pure olive oil .
„
.
„
.
.
.
.
.
Days of Exposure.
.
.
.
1, 2, 3*, 4*, 6
1, 2, 3*, 4*, 6
1, 2, 3, 4*, 6
The same holds good as regards thymol and salicylic acid. It might
be supposed that the enveloping membrane of the spores is impervious
to carbolic acid unless softened by the action of water upon it, but the
action of carbolic and other vapours on the dry spores tells against
this view. If carbolic oil comes into contact with substances containing
water,—the tissues of the body, for instance,—doubtless part of it is taken
up and exerts some antiseptic action. It appears that sulphuric acid
must be used pretty strong to destroy the spores of charbon; absolute
alcohol kills the adult rod, but not the spore. Weak solutions of potash
and soda destroy the rods, whilst the spores resist solutions of from 7
to 20 per cent. (Perroncito). A 5 per cent. solution of permangauate of
potassium sterilizes the spores of charbon. Chloride of lime in strong
solution instantly destroys the adult bacillus, and can kill the spore in five
minutes (Franz Nissen). Lastly, bichloride of mercury is with hydro-
chloric acid and chloride of lime an agent to be recommended. The
solution at 1 per mille, and more particularly stronger solutions varying
between 2 and 5 per mille, may be advantageously used in practice even
against the spores. Davaine observed that a solution of one in a hundred
and fifty thousand could destroy the virulence of charbonous blood. Koch
found that even in the enormous dilution of 3 parts per million, it
entirely arrests the development of the bacilli. Further experiments
showed that 1 per 1,000 watery solution of mercuric chloride, mercuric
nitrate, or mercuric sulphate destroyed anthrax spores in ten minutes.
Anthrax spores, which had been exposed for ten minutes to a 1 in 20,000
solution of mercuric chloride, and then washed in alcohol, gave no growth