F. C. MINETT AND M. R.
DHANDA
315
18 c.c. saline. 1.0 c.c. amounts were then seeded into tubes of brain-liver
medium. After 24 and 48 hours at 37°C. those tubes showing gas were noted.
After incubation, a microscopical examination was made from all the culture
tubes and those sown from sterilized soil dilutions were also planted out on
agar to detect any aerobic contamination. It was found that in the con-
ditions of experiment gas production could usually be taken as an index
of growth of chauvœi but results were only recorded as positive in this sense
when gas production was associated with the presence of sporing organisms
like chauvœi. A technique on similar lines was used with water.
GROWTH OF B. ANTHRACIS IN SOIL
A. Sterilized soils
It was necessary, in the first place, to find out whether the organism
would multiply in sterilized soils, in spite of their artificial nature. If evidence
of growth were to be obtained, it would then become necessary to ascertain
suitable temperature and moisture conditions and whether growth could be
improved by the addition of manurial substances. The problem of growth
in natural (unsterilized) soils could then be studied.
Table II shows the results of experiments with a sample (B) of Mukteswar
soil, in which the water content was estimated to vary from 0 to 40 per cent.
The actual water contents, taken at the conclusion of the experiments, varied
from 4 to 35 per cent, the tendency being for very dry soils to absorb some
moisture during sterilization. After being seeded with organisms in numbers
estimated in experiments 1 to 5 to vary from 50 to 315 per gm. of wet soil,
the well-closed bottles were kept at 25°C. (usually) and counts made at inter-
vals up to 35 days. It will be seen, not only that the anthrax bacillus is
capable of multiplying in sterile soil at 25°C., but also that, as would be expect-
ed, the degree of multiplication bears a relationship to the water content.
At water content up to 4 per cent, there was no evidence of growth ; at 5-6
per cent it was slight ; at 10-11 per cent it became very definite but was still
not optimal ; it was only when the amount of water reached 20 per cent or
more that multiplication became at all extensive.
These results have some meaning only in respect of the particular soil
in use, the important thing in general being that the soil should be thoroughly
wet or muddy. In bottled soil in this state, the distribution of water is not
uniform, because the soil tends to become packed at the bottom leaving
the upper layers water-logged. Soils not only vary enormously in their
composition but their water-holding capacity also varies in accordance with
the clay content, coarse or fine texture, etc. The amount of free water will
likewise vary and it is largely upon this that bacterial proliferation will depend.
In this connection, experiments were carried out with sample (B) of Mukteswar
soil, sieved so as to give ' fine soil ' with particles below 0.5 mm. diameter
and ' coarse soil ' with particles from 1-2 mm. diameter. When water to,
say, 25 or 30 per cent of dry soil weight was added to each, the coarse soil
became quite ' slushy ', while the fine soil formed a stiff paste. The results
given in Table II show that the anthrax bacillus grows well in the coarse soil
but not in the fine, owing to the abundance of free water in the former.