NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
24
virulent. This proved at least that the virulence did not reside in the
liquid part.
Pasteur recognized the formation of spores in a pathogenic microbe
connected with his studies on the causes of the diseases of silk-worms,
and he found that these spores were very resistant bodies and were
capable of germination several years after their formation; but this
fact not having been verified in the case of the bacillus of Anthrax,
showed Davaine's observations to be incomplete and allowed the
discussion to continue.
P. Bert in 1876 discovered that oxygen at a pressure of several
atmospheres killed anatomical elements and living beings, whilst
it was inactive upon diastases, soluble ferments, and venoms; he
submitted the bacillus of charbon to its action; he also submitted
this virus to the action of absolute alcohol; and he observed in both
cases that inoculation gave a rapidly fatal malady, without the appear-
ance of the bacillus ; in these cases the charbonous blood had become
septic, contained spores, and produced septicæmia, the disease from
which the inoculated animals died.
Koch in 1876 first observed the multiplication of the bacillus
of charbon by the formation of spores; he cultivated it in blood plasma,
aqueous humor, in the presence of air; he placed a minute quantity of
charbonous blood in a drop of serum or aqueous humor; he saw the
bacillus lengthen and form an inextricable felting of mycelium.
He saw it not only form segments, but also very refractile
spores, very close together and regularly spaced in the interior of
the filaments. He saw the filaments become destroyed, leaving in their
place spores laid out in a line and maintained in position by a uniting
substance formed of the last vestiges of the filaments; he lastly saw
the spores isolate themselves and become free. He inoculated the spores,
causing charbon; he cultivated them in blood plasma, aqueous humor
in the presence of air; he saw them stretch, lengthen, and form rods.
He observed that they were more resistant and preserved their virulence
for a longer period than the rods.
Briefly, Koch, by cultivating the bacilli in the aqueous humor or
serum, in the presence of a moist atmosphere at a temperature of 35° to
37° C., saw the rods lengthen considerably, double, tenfold, one hundred
fold, their length, turning about and forming an inextricable network
or felting, lose in some hours their uniform structure and their trans-
parency, their contents become granular; very shortly granulations
or very refringent spores were formed more or less close together and
at regular intervals, and resembling in their form and distribution inter-
laced chaplets of pearls. These dissociate and leave in their place lines of
25
spores which were maintained in position by a mucous uniting substance,
but soon separated, becoming free. He observed that at 35° C. the
bacilli rapidly developed and presented spores at the end of 24 hours,
whilst at 30° C. they did not form them for three hours, at 18° to 20° C.
at the end of two or three days. Below 12° and above 45° C. the
bacilli did not develop. He recognized that the development of the
bacilli required the presence of air, and that if cultivated in a medium
poor in oxygen, they became muddy, broke up, and were destroyed.
Pasteur in 1877 devoted himself to the study of contagious disease;
he completed the demonstration of the parasitic nature of charbon; he
demonstrated, by employing the method of successive cultures inaugur-
ated by him, that the bacillus was the morbific agent of charbon, and
that, if the virus resisted the action of compressed oxygen, this was due to
the great tenacity of the spores which it contained, and which, contrary
to what is the case with the rods, are not influenced by it.
Koch had observed that the microbe of charbon might reproduce
itself by scission or by the endogenous method. During the life of a
sick animal, the batonnet multiplies by scission, elongates, is compressed,
bends in one or several places, separates into several segments or baton-
nets, which in their turn comport themselves in a similar manner after
death. The batonnets cease to multiply by scission, oxygen being want-
ing and septic bacteria get the upper hand. The bacilli, however, under
certain conditions, produce spores before their destruction, which, once
formed, may preserve and perpetuate the virulence for a long time.
Pasteur, as we have said, submitted charbonous virus to successive
cultures outside the organism, and repeated the experiment a great
number of times, and, at last, isolated the bacillus from all the ele-
ments which accompanied it in the drop of blood employed to sow the
first culture; these elements did not multiply and finally disappeared;
the bacillus alone multiplied and became purer in each successive cultiva-
tion; it was obtained pure under the form of filaments; and by inoculat-
ing the liquid which contained it, charbon was induced with multipli-
cation of the bacillus. If the culture liquid or charbonous blood
was filtered through porcelain, it was deprived of the bacilli and
ceased to be virulent. The method of successive cultures, repeated
a number of times, permitted us to get rid of all the elements which
accompanied the bacillus, such as red and white globules, granular
matter which were introduced in sowing the first culture, and trans-
mitted in less and less numbers to some of the subsequent ones. The
cause of charbon then is the bacillus and no other element, since the
bacillus alone was preserved in the whole of the cultures, and neither
a dissolved substance nor any poison whatever produced by it and
26
regenerating itself at the same time in the successive cultures, since
once the bacteridium is removed from the blood or culture liquid,
as is the case when it is filtered through plaster or porcelain, the
malady cannot be produced by the inoculation of the liquid thus cleared.
If it be supposed that the plaster filter causes any change in the plasma
which traverses it so as to destroy in it any virulence which it might
be endowed with, the contrary may be proved by using numerous
double paper filters, or by leaving charbonous blood in a place free
from germs. It separates into two parts; the plasma, obtained by
decantation, is deprived of bacilli which have become caught and
imprisoned in the coagulated part, and it does not produce charbon on
inoculation. In addition to this the blood of the fœtus which does not
contain bacilli is not virulent, which would not be the case were the
virulence due to a liquid substance or poison. Toussaint, as Davaine had
recognized before him, observed that the bacteria had all their virulence
at the time of death before putrefaction had attacked the body, and
that they were destroyed on the development of putrefaction; that
they died, when they were deprived of air, if anything more rapidly
at an elevated temperature. Having filtered charbonous blood through
4 to 8 thicknesses of paper, he found that the filter composed of four
leaves allowed rods to pass and the liquid obtained cause a charbon
when inoculated; whilst the filter of 8 thicknesses did not allow them
to pass, and the liquid did not consequently induce charbon on inoculation.
He found that it was possible to abridge the period of incubation by
injecting a large number of batonnets into the blood. He cultivated
charbonous blood in the aqueous humor and studied the transformations
of the bacillus. It is then clearly demonstrated that charbon owes
its appearance and development to the introduction into the organ-
ism of a microbe in the form of rods or spores. The bacillus is then
the factor and the only factor of charbon, for in cultures we obtain it
pure and free from any formed or amorphous particle.
Seat of the Virus—Characters of the Bacillus in the living ani-
mal and fresh cadavers.—The contagium of charbon is a microbe; and
this microbe, as we have already seen, is met with in the blood of the
sick, in their various organs, in their excretions, more particularly when
the disease has arrived at its termination. It is notably abundant
in the engorged spleen, in the diseased ganglia, in the vessels of con-
gested organs, etc.; it may be found in the fæces, urine, in the
discharge from the nose, and more particularly is this the case when the
excretions are sanguinolent. Feser, Chambrelent, and Mossous, Koubas-
saf and Nocard have found it in the milk of the cow and of the guinea-
pig.
27
The bacteridium of Davaine, still called Bacillus anthracis, presents
itself in the blood of a sick animal, or shortly after death, under the form
of batonnets or short rods only. These rods are either simple, or they
may be formed of two, three, or more segments, straight, or sometimes
crosier-shaped or bent and cylindrical. They are often articulated by an
enlarged extremity presenting somewhat similar appearance to that of
the articulatory surfaces of the phalanges. In the median line of
these articulations is a clear, transverse line altogether characteristic.
They vary in length according to the subjects from which they are
obtained, and to their seat; they are longer in the guinea-pig than in the
mouse, and in both than in the ox; longer in the exuded material, in
the œdema which accompanies inoculation, than in the blood, where
they are more rapidly divided into their individual segments on account
of the movement to which they are constantly exposed; they also vary
in thickness in different species and individuals; they are generally
thinner in the ox than in the rabbit, etc. Their length may vary from
3 to 20 milimetres, and their breadth is between 1 and 1.5 m.
The common appearance presented by charbonous blood in a fresh
condition magnified 1,500 diameters is that the red corpuscles are
agglutinated, deformed, and mixed with a profusion of small straight
batonnets articulated or simple, but never presenting themselves under
the form of long filaments. In the blood of guinea-pigs the bacilli
are often, after 24 hours' inoculation, more numerous than the cor-
puscles which they smother, so to speak, by taking up the oxygen
necessary for their life, and to that of the anatomical elements which
receive from the blood the material for their nutrition. The rods never
contain spores when taken from a living animal or from a fresh cadaver.
They are composed of a very refractile protoplasm and of a less refractile
envelop, which, however, is very resistant; around the rods may be ob-
served a thin clear hyaline and gelatinous zone. There are as many
unicellular microbes as there are segments: these are vegetable cells,
always multiplying by segmentation or scission in the bodies of the sick.
Segmentation is announced by the appearance of a transverse line, some-
times very thin, and which may only be evident on the application of
reagents or heat. Seen in the blood, in the pulp of organs, the spleen,
ganglia, etc., in the gelatinous exudation into the subcutaneous con-
nective tissue, which contains less when less coloured and containing less
hæmorrhagic infracts, they are easily recognized, even when examined
unstained, on account of their elongated form, their refractility, immo-
bility, numbers and arrangement, etc. In a drop of blood set up on a
slide and examined with ordinary illumination, magnified 350 to 500
diameters, we ordinarily observe agglutinated red globules forming irre-
gular masses and leaving spaces in which the rods may be clearly seen.
28
The bacillus resists the action of potash, ammonia, sulphuric acid,
etc., preserving its form after having been submitted to the action
of these substances. When we treat a ninfinitesimal portion of blood
or dried pulp of an organ with a drop of acetic, or, better, formic acid,
we see all the anatomical elements pale and disappear, whilst the bacilli
persist perfectly clearly. The bacillus stains well with all aniline
dyes, gentian violet, methyl blue and violet, fuchsine, rubine, etc.
We may obtain beautiful preparations with the ordinary methods of a
simple coloration, and more especially by double staining by Gram or
Weigert's method. These preparations may be made with blood, exudate
from diseased points, pulp of an organ, as the spleen, ganglia, liver, lung,
marrow of bone, etc., epiploon villosities of the intestine, etc., and sections
of organs previously hardened in alcohol.
Staining the rods.—Nothing is easier than to make stained prepara-
tions of the blood, fluids, and pulp of various organs. These take the form
of cover glass preparations and are made as follows:—A small quantity
of the material is placed between two cover glasses, which are then pressed
gently and carefully together so as to expel any excess of the substance,
leaving only a very thin layer between them; they are then gently drawn
apart, sliding one over the other, and allowed to dry in the air, care being
taken to exclude dust, etc. The thin layer which exists upon each cover
glass is now fixed by passing it quickly several times over a flame, pre-
ferably that of a spirit lamp or a Bunsen burner, care being taken to
keep the coated surface of the glass uppermost. They are now allowed
to cool and are afterwards placed in a hydro-alcoholic solution of one of
the aniline dyes for a few minutes; they are then washed with distilled
water, then with spirit to dehydrate them, and cleared with clove oil or
not, then mounted in Canada balsam and examined, preferably with an
oil immersion and illuminated by means of an Abbe's condenser, when it
is desired to make a minute examination. In simple staining the fol-
lowing method gives excellent results. Place the specimens in solution
of Lœffler's blue, which is composed of one part of an alcoholic solution
of methylene blue and three parts of a one in ten thousand solution of
potassium, and allow them to remain in it for from a few minutes
to half an hour; they are then rapidly passed through distilled water
slightly acidulated with acetic acid and then treated as ordinary
specimens.
Double-staining may be obtained by the method of Gram. The speci-
mens are stained for five to ten minutes with Gram's solution composed
as follows :—
Aniline water
.
.
.
.
.
.
.
Absolute alcohol
.
.
.
.
.
.
.
Saturated solution of gentian violet
.
.
.
.
10 parts
1 part
1 part ;
29
they are then washed and placed for three minutes in the following :—
Iodine . . . . . . . . .
Iodide of potassium
.
.
.
.
.
.
.
Distilled water
.
.
.
.
.
.
.
1 part.
2 parts.
300 parts.
They are again washed, decolorized by absolute alcohol ; washed again
and stained with picrocarmine, or weak alcoholic solution of Eosin or
Vesuvin; they are finally washed, dried, and mounted. Double staining
may also be accomplished by Weigert's method, which is as follows :—
Place for two to five minutes in a 1 per cent. watery solution of gentian
violet, wash in alcohol, rinse in water, and transfer to picrocarmine
solution (Weigert) for from half to an hour. Treat with alcohol until
the colour is almost washed out.
Galtier recommends the following application of Weigert's method:—
Commence by staining the preparation in picrocarmine for some minutes;
wash it in distilled water; then place it for fifteen minutes in Weigert's
solution, made as follows:—
Cold saturated solution of methyl violet
.
.
.
.
Absolute alcohol
.
.
.
.
.
.
.
.
Aniline oil
.
.
.
.
.
.
.
.
.
6
11
3
wash and dry, then clear with a mixture of
White aniline oil
.
.
.
.
.
.
.
Xylol . . . . . . . . .
2 parts.
1 part.
Wash in pure xylol so that no tint of picrocarmine remains, and then
mount in balsam; the ground is rose-coloured and the microbes appear
violet.
Preparations made with the epiploon or villi of the intestines may
be coloured directly upon the slide or cover glass, in the same manner as
preparations of the pulp of organs. We can place on the slide a small
portion of the epiploon which contains only very fine vessels, or we may
equally place a villus; it is left to dry and is fixed by gently heating
either in a stove or by passing through a flame, we then proceed as above
directed. Sometimes with the epiploon we may adopt a different pro-
ceeding : we may stretch a piece across an excavated cork, pass it through
a mixture of equal parts of alcohol and ether, then plunge it into Gram's
violet; afterwards treat with iodine solution, clear with absolute alcohol
stain in eosine and mount like a section. Fine sections may be stained
in the same manner.
Characters of the cultivated Ballcius.—The microbe of charbon
may live, develop, and multiply outside the body, when the conditions
necessary for its nutrition and respiration are combined with a tempera-
ture and degree of humidity suitable to it. It requires for its well-being
a medium composed of nitrogenous and mineral material, such as serum,
30
aqueous humor, urine, vegetable infusions, broth, potatoes, gelatine, and
peptonised jelly. It thrives well in neutral urine, but meat broths are
preferable; it does well in gelatine or jellies as well as on potatoes. Air
and free oxygen are necessary to its existence ; it is "Ærobic ;" it cannot
be cultivated in vacuo, nor in the presence of carbonic acid, nitrogen, or
hydrogen. It is not cultivated below 16° C. At 16° C. the bacillus does not
form spores, at least for a very long time. It is cultivated rapidly at from
42° to 45° C., but at the latter temperature spore-formation is prevented
or arrested. It does not grow in chicken broth at a temperature of 45° C.
The temperature most favourable to the rapid development of the bacillus
and the formation of spores is 30° to 35° C. The filaments are killed by
a temperature of 60° C. or by carbonic acid or alcohol. The spores do
not suffer any alteration from contact with the air, either in their vitality
or their virulence; they resist the action of alcohol, carbonic acid, and
vacuum, as well as, for a short time, a temperature of 95° centigrade;
they are, however, killed with certainty at 100° C. When the condi-
tions are favourable, the bacteridium multiplies at first by scission or
segmentation as in the animal body.
The temperature of the blood of birds (41° to 42° C.) hinders multi-
plication, and in order to show that the temperature hindered develop-
ment in the fowl, M. Pasteur cooled the bird by keeping the body in
water, and so, on inoculation, produced the disease. It must be remem-
bered, however, that in some exceptional cases birds may take charbon
without being thus cooled down; for instance, the sparrow, which has
a high temperature, is very susceptible to the virus. By the inverse
process Gibier has been able to produce charbon in frogs and in fishes, by
causing them to live in water at a temperature of 35° C. The blood of
these animals, after inoculation, contained a large number of bacilli.
The culture media should be sown with pure charbonous material
containing nothing but the bacilli, such as the blood, products of the
spleen, ganglia, etc., taken with antiseptic precautions from fresh cada-
vers; and if we are only able to obtain an impure seed, it is necessary
to sow it upon a solid medium, so that we may be able afterwards to
separate the colonies of the different microbes.
Culture of the Bacillus in liquid media.—Broths made from beef,
veal, chicken, etc., and of course sterilized and rendered slightly alkaline,
are very suitable for the culture of the bacillus. We sow them with a
drop of blood, or with the pulp of a charbonous organ, and place them
in a stove or incubator at a constant temperature of 35° to 37° C. Under
these conditions, the microbe of charbon rapidly forms in a few hours
(about 24), numerous filaments, long, undulating, felting together, and
forming snowy or cottony flocculi, easily seen in the culture liquid in
31
which they are held in suspension, and sometimes adhering to the side of
the tube; these flocculi enlarge and become more numerous ; the culture
liquid remains clear and limpid in the parts not occcupied by the flocculi
of filaments. The floccuent appearance, snowy or resembling cotton, is
characteristic. By degrees the culture changes its appearance; after
some days, the liquid becomes slightly brown or yellowish coloured, the
flocculi disintegrate, and a white or slightly yellow powder forms at the
bottom of the culture which spreads through the liquid and renders it
turbid when shaken.
Culture of the bacillus on solid media.—Plate cultivations on
nutrient gelatine may be made. The bacteria form colonies visible to the
naked eye after one or two days, according to the temperature at which
they are maintained. They appear to the naked eye as little white spots
or specks which, when examined with a low power, say 60 diameters,
with small diaphragm, exihibit two distinct forms. One form, on care-
fully focusing, has the appearance of a little compact ball of twisted
thread ; in the other, liquefaction of the gelatine has occurred, and the
bundles are spreading out like locks of hair in the neighbouring gelatine.
These appearances are perfectly characteristic.
Test-tube cultivation in nutrient gelatine.—Typically charac-
teristic appearances are obtained by inoculating a 5 to 8 per cent. nutrient
gelatine. Towards the second or third day the whole of the track of the
puncture assumes a downy aspect, and a whitish coloured line develops,
from which numerous fine filaments spread out in the gelatine. Occa-
sionally a little isolated spot develops, from which rays extend in all
directions, like the silky filaments of thistle down. These filaments are
more easily observed with a magnifying glass. In a more solid nutrient
gelatine the growth appears only as a thick white thread. As lique-
faction of the gelatine progresses, these characters rapidly disappear,
and the growth subsides as a white flocculent mass. In exhausted
culture media, and sometimes in blood, filaments are seen in a state of
degeneration.
Test-tube cultivations in nutrient Agar-Agar.—When a culti-
vation is made on a sloping surface of nutrient Agar-Agar, a viscous
snow-white or creamy layer is formed.
Cultivation on potato.—A very characteristic growth results from
the inoculation of sterilized potatoes. The damp chamber containing
the spores is placed in the incubator, and in about 36 to 48 hours a
creamy, very faintly yellowish layer forms over the inoculated surface,
with usually a very translucent edge. On removing the cover of the
damp chamber a strong penetrating odour of sour milk is encountered.