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(b) From a recent culture on agar of the bacillus typhosus, bacillus pyocyaneus,
bacillus diphtheriai, staphylococcus aureus and vibrio cholera) respectively a turbid
emulsion was made in broth or in separated milk; this was then applied liberally to the
above materials and allowed to dry in the air of the laboratory. In the later experiments
we made our cultures, not on agar, but direct on gelatine, and incubated the culture tubes
for 24 hours at 37° C., whereby the gelatine became, of course, melted, and turbid from
the growth. For the object of carrying out the experiment we applied the turbid (melted)
gelatine culture liberally on the above materials. On cooling, the gelatine set.
In all these proceedings, therefore, the microbes used were emulsified, not in water, but
in menstrua somewhat thicker, e.g., milk and melted gelatine. In choosing these menstrua we
were guided by the fact that under the conditions obtaining in practice the virus to be disinfected
is not suspended in water but in thicker menstrua, e.g., fluid typhoid stool, pus, blood, fluid cholera
stool, diphtheritic throat secretions, and the same applies to the tubercle bacillus, for this as a
rule is voided by the infected persons in the expectoration, which is muco-purulent, As was
mentioned above, in all our experiments with tubercle we used pulmonary sputum, containing the
tubercle bacilli; this was liberally applied on the materials and allowed to dry in the air of the
laboratory.
The materials, paper, linen, cloth, wood, charged with the above microbes, were then exposed
to the various disinfectants in the following manner—
(A.) In the case of the gaseous disinfectants, i.e., formalin, or sulphurous acid gas, the
materials charged with the microbes were carried in large glass cylinders (plugged) from the
laboratory to the room where the disinfection was to be carried out, and here, having been
taken out, were placed and fixed on a table. Next day they were replaced again in the glass
cylinders and carried to the laboratory. Here the materials separately were placed in a tube
of sterile broth—except the cholera-charged materials, which were placed in peptone salt solution
—in this they were slightly washed, and then transferred to a fresh tube of broth (or peptone
salt solution respectively) and both tubes incubated at 37° C. The first washing was made for
the sake of removing any excess of the disinfectant left in the materials.
The resulting growth was examined microscopically, culturally, and by animal
experiment, in order to identify the microbe with which the material had been charged.
It must be obvious that the materials, not having been sterile, and moreover, having
been exposed after the disinfection, for some hours at least, to the air, would harbour a
variety of microbes deposited on them from the air, and therefore the above broth tube, by
incubation, would, and as a matter of fact did, in some instances, produce growths additional
to, or other than, the microbes with which the materials had been intentionally charged. It
was, therefore, a matter of no small importance to diminish the difficulties of identification by
plate cultures or otherwise, of the original microbe, and this was done by our choosing the
more easily identifiable microbes. In the case of the spores of bacillus anthracis, of staphylococcus
aureus, of vibrio of cholera and of bacillus pyocyaneus the identification by one or
another method (microscopically, culturally, animal experiment) is comparatively simple on
account of the marked characteristics of the microbe. But in the case of the typhoid bacillus
and the bacillus diphtherise the identification was considerably more difficult; the difficulties,
as will be stated further below, were in great part due to a possible confusion with similar
microbes (bacillus coli or bacillus xerosis, respectively) widely diffused in Nature and particularly
in town air and dust.
In the case of tubercular sputum, the materials charged with it were, after the gaseous
disinfection, treated in a different way, viz., the surface layers (dried sputum) were scraped off
and distributed in salt solution, and this was injected in considerable amounts subcutaneously
into the groin of a guinea pig and the result watched. Cultural experiments, owing to the
wall-known enormous difficulties connected with the culture of the tubercle bacilli from
sputum were not attempted, injection into the guinea pig being far the easiest and most reliable
method of discovering whether or not the injected materials contained the tubercle bacillus in a
living state. If the injection into the subcutaneous tissue of the guinea pig is, after several
weeks (6—8), not associated with and not followed by tubercular enlargement of the inguinal
lymph glands of the injected side, and if on a post mortem examination no tubercles or tubercular
changes are found in the viscera (liver, spleen, lung), then we may take it for certain
that the injected matter did not contain living tubercle bacilli, and therefore the disinfection
was successful and positive. But if in these and other experiments (carbolic acid, Condy's
fluid, etc.) the inguinal glands and viscera did show the tubercular changes (anatomically, and
after staining for tubercle bacilli), typical of inoculation tuberculosis, then the conclusion was
drawn that the matter injected did contain tubercle bacilli in a living state, and therefore that
the disinfection was unsuccessful and negative.
(B.) Disinfection with the fluid substances: carbolic acid, perchloride of mercury,
Condy's fluid, bleaching powder, was carried out as follows:—The different materials, wood,
cloth, linen and paper having been charged with the recent and active culture (melted gelatine)
of the microbes (bacillus typhosus, bacillus pyocyaneus, bacillus diphtherise, vibrio choleræ,
spores of bacillus anthracis, staphylococcus aureus) were, after setting of the gelatine in the
air of the laboratory, exposed for the required time in a large test tube to the disinfectant
solution; after that the materials were taken out from these solutions and placed in a tube of
broth (or peptone salt in the case of vibrio choleræ), and from here transferred to a new tube of
the same culture medium and then incubated at 37° C., and treated in the same way as was
mentioned in the case of testing gaseous disinfectants.