NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
32
Cover-glass impressions.—The plate cultivation should be examined
as soon as the colonies appear, by making cover-glass impressions and
staining them, as should also the potato and other cultivations.
Characters of the cultivated bacillus.—Examined in the diverse
stages of their evolution, the cultures show the bacillus under various
forms. The filaments at the commencement contrast by their length
with the relatively short rods, which is the form seen in the blood of the
sick. They are very long, cylindrical, and larger than the rods in the
blood, non-ramified, undulating, tangled into skeins or masses, and
immobile. Examined in their natural condition in a drop of the culture
liquid or in a piece of the culture on solid medium, after the addition of
a drop of sterilized broth, they show the characters already noted; they
appear homogeneous in their whole length, with rare segmentations,
under these conditions; they exhibit a homogeneous protoplasm, trans-
parent and devoid of granulation.
Sometimes the filaments, when
treated by reagents, show segmentation into short segments.
The filamentous bacilli are easily coloured by the various methods
above indicated, by hydro-alcoholic solutions of gentian violet, methyl
violet, fuchsine, rubine, methyl blue, methylene blue, etc., by Loeffler's
blue, by the methods of Gram and Weigert. Staining allows us to
observe the true structure and composition of the filaments. We then
see that they are formed of a hyaline sheath, containing cubical or
elongated protoplasmic masses, separated from each other by transverse
partitions; each of these segments is a vegetable cellule, a microbe.
Examined after 24 hours, and day by day, the cultures show the
bacillus under a new form—the spore form.
Spore formation consists in the appearance of a bright, glisten-
ing, spherical body in the protoplasm of an elementary mass or cell;
these are the seeds of the plant, or, as we term them, spores. This body
gradually enlarges until it reaches its full size, becoming at the same time
oval. The bacilli are thicker at these points than where no spore forma-
tion has set in. Under the most favourable conditions, each cubical or
rod-shaped mass of protoplasm includes one spore, in which case the
bacillar filament contains an almost unbroken row of spores; but in other
cases only an elementary mass here and there contains a spore, the rest
breaking down and becoming absorbed. In the first case, also, the pro-
toplasm of the elements almost entirely disappears, the sheath swelling
up and becoming hyaline, and only the bright spores remain. Their
linear arrangement, however, still indicates that they were formerly con-
tained in one filament. After some days in an incubator, it is observed
that a pulverulent deposit has formed at the bottom of the culture, and
in it there are still some filaments or débris of filaments filled with re-
33
fractile spores; chains of spores still maintained in line by the jelly-like
membrane of the filament; and lastly, masses of spores in a hyaline sub-
stance, which appears to support them, and free spores animated by
brownian movements.
If bacilli grow in the depth of a fluid medium they do not form
spores, and as new bacilli appear, or the old filaments increase in length,
degeneration sets in. This degeneration gradually affects greater and
greater numbers, and when the fluid is exhausted for the formation of
new bacilli it necessarily follows that the whole growth becomes involved
in degeneration, the whole mass becoming smaller, and finally only débris
remaining. Such cultures—namely, those in which degeneration involves
the whole mass of the bacilli—are quite innocuous when inoculated into
animals or into fresh nourishing media. But as long as there are any good
protoplasmic elements of the bacilli left, the culture remains virulent to
rodents with the exception of mice, as will be seen later; and it is capable,
when transferred to a new suitable nutrient medium, of starting new cul-
tures which prove virulent to all rodents and sheep. The same holds good
of the blood and organs of an animal dead of anthrax, provided the animal
be not opened, and its organs, exudations, and urine be not exposed to free
air; for the bacilli not exposed to the free air gradually degenerate, and
the blood and organs of such an animal, although at first deadly poison
to other susceptible animals, become at length quite innocuous. Sys-
tematic observations have shown me that small animals, such as mice
and guinea-pigs, when unopened or buried in earth, become innocuous in
from 5 to 8 days, the bacilli having by this time undergone degeneration
and disappeared from the blood, spleen, and other organs. Pasteur's
statement that in animals, dead of anthrax and buried, the bacilli form
spores, and that these spores are taken up by earth-worms and carried to
the surface of the soil, where they deposit them in their casts, is not
borne out by these experiments (Cornil and Babes). Further, Koch has
proved (Mattheil. a. d. r. Gesundheit samte, 1881), by direct experiment,
that the spores of anthrax bacilli, when mixed with earth, are not taken
up by these creatures.
Spores, as we have seen, form in the cultures; after the first days
they form rapidly in cultures maintained at a temperature of 35° to
37° C. They also form at 30°, 25°, 20°, and 18° C., although more
slowly ; they become more numerous as the culture material gets more
impoverished. Sometimes, under certain conditions, the cultures of the
bacilli may lose the power of forming spores. Cultivated at 42° to 43°
they do not form true spores (Pasteur); brilliant corpuscles, a kind of
false spores, form in the filaments, smaller than true spores and not
endowed with like powers of resisting heat (Chauveau). But these
D
34
bacilli without spores, sown in broth and cultivated at a temperature of
35° to 37° C., produce spores ; they are, therefore, not rendered truly
asporagenous by cultivation at 42° to 43° C. At too low a temperature
the bacillus does not form spores, but it does not lose the power of doing
so when cultivated at a proper temperature; spore formation does not
take place below 15° C. In 1883 MM. Chamberland and Roux ob-
tained asporagenous bacteridia, by causing weak antiseptics to act upon
the cultures, using phenic acid and bichromate of potassium for the
purpose. In this manner the bacillus cultivated for eight days in a
culture medium to which a two-thousandth of bichromate of potash was
added, lost the faculty of producing spores, and remained aspora-
genous in successive cultivations; it killed guinea-pigs in three or four
days, and the cultures sown with the blood of the charbonous guinea-
pig remained asporagenous. Lehmann, in 1887, discovered another
method of producing asporagenous bacilli, which only formed false
spores, and which did not regain the power of forming spores after
having been passed through guinea-pigs and mice, and his method was
repeated cultivation on gelatine. Behring, in 1889, obtained similar
bacilli by cultivation on gelatine, to which rosalic acid or an antiseptic
had been added in a dose insufficient to kill the microbe. M. E. Roux
has pointed out a certain method, and one easy of application, of obtain-
ing cultures of asporagenous bacilli. The following is a résumé of
his method:—Take eleven test tubes and place in them ten cubic
centimetres of veal broth rendered slightly alkaline; leave one as a
control, and add to the others weak doses of phenic acid, such as 2 ten-
thousandths to one, 4 ten-thousandths to a second, and so on, in such a
manner that from the first to the tenth there is a progression of 2 ten-
thousandths per tube; sterilize the tubes at 115° C., after sealing those
containing the acid in order to avoid loss; then sow each of the tubes
with a drop of blood, and put them in an incubator at a temperature of
30° to 33° C. The bacilli develop, if anything, more slowly as the
dose of phenic acid has been larger. The culture is always less abundant
in phenicated broth than in the ordinary broth, and it generally occurs
in flocculi, which remain in the depth of the liquid, if the tubes are
not shaken; at other times the development occurs in the whole mass
of the liquid, and gives it a troubled appearance; it is more especially
in the tubes, which are richer in the antiseptic, that this appearance is
produced. We should, if possible, avoid the cultivation occurring at the
surface of the liquid and forming a crown to it, which adheres to the
side of the tube. The bacilli, which increase in this manner, freely
exposed to the air, outside the culture liquid, quickly form spores,
principally in those tubes poor in phenic acid. When, therefore, floc-
culi appear, we submerge them by agitating the tubes a little. The
35
broth, to which 20 ten-thousandths of phenic acid has been added,
remains sterile, the bacilli sown in it rapidly perishing.
After eight or ten days we take a little of the culture from each of
the tubes and heat it for fifteen minutes to 65° C., and then sow the heat-
ed portion in ordinary veal broth; in a couple of days the sowings made
with material from the tube to which 2 ten-thousandths of phenic acid
have been added show themselves to be fertile. Those made with mate-
rial from the tube containing 4 ten-thousandths and 6 ten-thousandths
often give a culture on the following days, but the others remain for
the most part sterile, and so prove that the bacillus sown was without
spores.
The proportion of the antiseptic necessary to prevent the formation of
spores varies with the composition of the broth, the origin of the bacilli,
and the facility with which air gains admission to the culture. It is
for this reason that we take the precaution of using a number of tubes
with slightly varying quantities of the antiseptic added to them, and
differing in no other respect; unforeseen results often occur in these
experiments. We may find, for instance, a tube containing 6 ten-thou-
sandths of the antiseptic produce no spores, whilst one sown in 10 ten-
thousandths may contain them, although the two tubes have been
sown with the same blood. The bacillus does not suddenly lose the
power of forming spores; if we remove it from the antiseptic medium,
after three or four days of culture only, it will form spores if we sow it
in a non-antiseptic broth. In order that it may become asporagenous it
is necessary that the contact with the antiseptic be prolonged.
Sown in ordinary broth, without the application of heat, the aspora-
genous phenic cultures rapidly develop at 33° C.; and the daughter
cultures have no spores; even after a long time they perish without
having formed them. We may make successive cultivations of these
bacteridia, either in broth, gelatine, nutritive gelatine, coagulated serum,
or liquid serum, without their forming spores. They have not, however,
lost their virulence; inoculated into guinea-pigs they kill them in from 30
to 36 hours and rabbits in from 48 to 60 hours. At the seat of inocula-
tion there is extensive œdema in guinea-pigs, generally greater than
what occurs after the injection of the ordinary bacillus. When we
pass this asporagenous bacillus through a large number of guinea-pigs
and rabbits, inoculating a healthy animal with blood from a dead
charbonous animal, it does not regain its power of producing spores in
cultures, even though its virulence increases. Blood of the rabbit dead
of asporagenous charbon may be placed upon the side of a sterilized
tube closed by a plug of cotton wool, and left for fifteen days without
producing spores in a moist chamber, in contact with the air, and at a
temperature of 30° C. We know that ordinary charbonous blood, placed
D 2
36
under these circumstances, rapidly produces spores. The aqueous humor
of a rabbit's and the sheep's eye is a particularly favourable medium
for the production of spores, and the ordinary bacillus gives, in it,
after twelve hours, beautiful spores, capable of resisting a temperature of
15° C. and 190° C. The asporagenous bacillus does not produce spores
even when cultivated in fresh, aerated aqueous humor. This, then, is
no temporary and passing change of the bacilli of charbon, but a
permanent hereditary change, and at present we are not acquainted with
any method by which we may endow the bacilli so modified with the
power of producing spores.
The appearance of cultures of asporagenous bacilli closely resem-
bles that of ordinary cultures of the bacillus. In broth they give
flocculi which are more easily broken down; the filaments of the
asporagenous bacillus are shorter and slightly thinner; they often
contain in their interior refractile grains, smaller than spores, and not
resistant to heat. At the commencement of the culture the filaments are
transparent and stain well; in proportion as the culture ages, many of
them become granular, swell up and stain badly. Cultivated on gelatine,
the asporagenous bacilli appear to liquefy it more rapidly than do
the ordinary bacilli. Broth in which the sporeless bacilli develop
is less coloured in the incubator, and gives less abundant crystals of
ammonio-magnesium phosphate than that which has nourished the
ordinary bacillus of charbon. The cultures of asporagenous bacilli
finally perish at 33° C. after a longer or shorter time, but generally
within a month. If after some days in the incubator we place them
at the temperature of the air, they remain alive for a much longer
period. Sporeless cultivations have been preserved in this manner sown
in broth for 155 days, at the end of which time they have given cultures;
they did not, however, contain any spores, and after heating at a tempera-
ture of 65° C. for fifteen minutes were sterilized. In other cases, these
asporagenous cultures remained in a cupboard in the laboratory, being
still alive after 81 days; a culture on nutrient jelly was still alive after
47 days. In these old cultures very few bacilli remained alive; to
resuscitate them they should be sown in large quantity, and after several
days we may perceive development taking place; the virulence of these
cultures without spores persists until they perish. A culture taken a
short time before its death or the death of its filaments is active and
destroys guinea-pigs and rabbits. In the mother cultures, on the contrary,
made in the presence of an antiseptic, there is a diminution of virulence,
as MM. Chamberland and Roux have long since pointed out. The
daughter cultures made from phenic cultures about eight days old—that
is, at the time when the loss of virulence is least marked—do not weaken
sensibly in successive cultures in broth. However, it seems that these
37
sporeless bacilli must be in a condition particularly favourable to
attenuation, since they remain exposed in the form of filaments to
the atmosphere at the temperature of the incubator. It is to the
action of air upon the mycelium without spores that MM. Pasteur,
Chamberland, and Roux have attributed the attenuation of the virulence
of the cultivated bacilli at a temperature of 42.5° C. These two con-
ditions—the absence of spores and the continued action of the air on
the filaments—are realized in the case of our asporagenous cultures, but
in spite of this they do not become attenuated. We must consequently
come to the conclusion that the action of air at a temperature of 33° C.
is not sufficient to produce a diminution of virulence ; it must be
increased to a higher temperature, It would be interesting to know at
what temperature it is necessary to make the culture in order to bring
about attenuation of the virulence of asporagenous bacilli. We may
also obtain attenuated asporagenous bacilli either by sowing, as M.
Behring has done, bacilli already attenuated in a medium containing
a small dose of antiseptic material or by prolonging the contact of
virulent bacilli with phenic acid, as we have indicated below. To
obtain virulent asporagenous bacilli we may sow charbonous blood
in a phenicated broth, and after eight or ten days' culture remove a small
quantity of seed and place in sterilized tubes. The bacilli have remained
for too short a time with the antiseptic for their virulence to have
become profoundly altered, and the daughter cultures so obtained are
sporeless and still kill guniea-pigs and rabbits. If we leave the bacilli
for a sufficiently long time in the presence of the antiseptic, its virulence
will diminish, as is well known, and will be incapable of forming spores
or of killing animals.
These asporagenous bacilli give a novel and very interesting
example of the permanent and hereditary modifications which microbes
may be caused to undergo. Since the observations made in M. Pasteur's
laboratory we know that virulence is a quality which microbes may lose
and acquire, and that pathogenic properties are not sufficient to charac-
terize species. The morphological characters are no more certain in
many cases. The manner in which the bacillus forms spores, the
resistance of these to various agents, and the fashion in which they
germinate in suitable media, have been regarded as very characteristic
particulars. It is, however, seen that the power of forming spores may
be modified to such an extent as to cause its permanent loss. The
action of antiseptics is probably not the only method by which this
modification may be brought about. Lehmann had not added any
antiseptic to the cultures in which he found bacilli without spores; he
did not know to what influence to attribute this modification, which was
produced without any intervention on his part. It is then not beyond
38
possibility that in nature conditions may be realized which transform
ordinary bacilli into attenuated and asporagenous ones. The experi-
mentalist, who should meet a similar bacillus, would be very much
puzzled to recognize in it the " Bacillus of charbon." He would not
have the slightest hesitation in regarding it as a species of Saprophyte
without associating it with the bacillus anthracis. It is necessary, in fact,
to have followed step by step, in well-conducted experiments, all the
series of changes of the bacillus to be certain that the inoffensive
and sporeless microbe is the virus of charbon ; microbes are easier to
modify than is generally admitted. It is with great trouble, in fact, that
in our laboratories we preserve them with their characteristic appearance,
in careful conditions of culture. We are not justified either in declaring
that two microbes are not related to each other because their colonies are
not quite the same, and one of them liquefies gelatine more rapidly than
the other. What are these differences when compared with that which
exists between attenuated sporeless and virulent spore-bearing charbonous
virus? Insignificant in truth, and they should not authorize any
experimentalist to regard as issues of different stock, two microscopic
organisms very similar in other respects. The dissimilarity between
microbes of the same origin is due doubtless to the diverse vicissitudes
to which they have been exposed, and to incidents with the nature of
which we are unacquainted. It is sufficient for us to avoid attaching too
much importance to artificial characters and distinctions.
If the work of attenuation of the virus prove to us that virulent
and fatal microbes may become inoffensive, the converse, or the return
to virulence of these attenuated microbes, leads us to suspect that a
saprophyte may become virulent.
The pathogenic organisms with
which we are at the present day acquainted may be ancient saprophytes
which have become progressively adapted to parasitic life. These ideas,
enunciated often enough before, explain the interest which biologists
and pathologists attach to experiments which furnish well-established
examples of these permanent modifications of microbes.
In old cultures we may find abnormal forms, filaments with swellings,
irregular or ovoid, or with a series of granulations varying with their
size. The spores of the bacillus are very refractile, and it is easy to
distinguish them from pseudo-spores, either by staining, or, better, by
heating them for fifteen minutes at a temperature of 65° C., which
destroys the filaments and all material not true spores without destroy-
ing spores which resist a temperature of 90° C. for fifteen minutes.
The spores are difficult to stain, not being affected by ordinary dyes, and
herein they differ from the bacilli. We may, however, stain them in
several ways; firstly, by heating them for nearly an hour at a tempera-
ture of 200° C., or by passing the slide or cover glass, upon which is
39
placed and dried the drop of the culture, through flame, then treating
them with a hydro-alcoholic solution of gentian violet or some other stain.
Secondly, by leaving them for 24 hours in a warm solution of fuchsine
in aniline water (Erlich's solution), decolorizing with nitric acid 30 per
cent. or by absolute alcohol, and exposing them to the action of a hydro-
alcoholic solution of methyline blue. By the first of these methods the
spores alone are stained, the elevated temperature having so altered the
filaments as to render them unable to fix the coloring matter and the
spore able to do so. In the second method a double coloration is
achieved—the spores are stained red and the filaments blue. In conclu-
sion, then, we may repeat that the bacillus of charbon, cultivated in
suitable media, such as broth, produces, after from 24 to 48 hours, exces-
sively long filaments disposed in such a manner as to form flocculi
resembling cotton wool; after the lapse of a further period the filaments
become filled with refractile corpuscles, then lose their contour, and being
destroyed, leave in their place chaplets or spores, which later on become
free, grouped into masses, preserving their lines, and capable of growing
into batonnets or rods or filaments when they are inoculated or sown
afresh. Spore formation occurs when the conditions of the life of the
organism become least favourable, when the medium in which cultiva-
tion is going on becomes poor in nutritive material or oxygen, when the
temperature is low, etc., etc.
The blood of a charbonous animal, or the excretions containing it,
must be exposed to the air in order that spores may be formed. It is
perfectly easy to follow the evolution of the culture and the modifica-
tions of the microbe under the microscope, by sowing it in serum or
aqueous humor placed in a moist chamber and kept at a temperature
of 33° to 35° C. by means of a warm stage. We see the rods double
their length at the end of an hour, elongate to ten times their original
length in two hours, and become transformed into long filaments in
seven or eight hours; then we see small, very refractile, rounded or
ovoid masses appear in the filaments; these are the spores, which are
composed of a very refractile protoplasm and a very resistant envelope.
The spores of charbon form rods or filaments only with difficulty or not
at all in the liquid where they have been produced. However, few of
the cultures are free from filaments, and it is not impossible that the
spores may sometimes germinate in the liquid in which they have been
formed. Be that as it may, the spores inoculated or sown in suitable
media produce rods in the organism or filaments in the culture media.
The cultures of the bacillus produce the disease in animals which receive
them, just as do the charbonous products or blood of the sick.
Antagonism between the " Bacillus " of Charbon and other
Microbes.—The microbes of charbon disappear in the presence of those