314
Multiplication of B. Anthracis and Cl. Chauvœi
count was estimated from breed smears and the actual viable count of the
dilution used for the soil or water was obtained by culture in tubes of brain¬
liver medium [Haslam, 1920].
Estimation of growth
After incubation at temperatures of 20°C., 25°C. 30°C. and occasionally
37°C. for periods of 5 days and upwards the samples were examined for evi-
dence of growth.
(a) Sterile soils which had been seeded with anthrax spores were plated
in agar ; natural soils were examined by inoculation of guinea-pigs or, occa-
sionally mice. By means of a sterile spoon, the soil was thoroughly mixed
with any supernatant water and a sample usually exceeding 4.0 gm. and
often amounting to 6 or 7 gm., was removed from the bottle to a watch
glass. As much soil as was reasonably possible was removed in order to
minimize the sampling error. After further mixing on the watch glass, 4.0
gm. were transferred to 20 c.c. sterile saline (dil. a) and from this tenfold
falling dilutions (dil. b, dil c, etc.—1 c.c. to 9 c.c. saline) were made. Before
removing 1.0 c.c. from dil. a, the soil was very well washed in the saline by
repeated inversion of the tube and every effort made to keep the soil moving
with the pipette and mixed as uniformly as possible, while 1 c.c. for trans-
fer to dil. b was rapidly taken. Plates were sown from dils. b, c. etc., but
not from dil. a, since the rapid sedimentation of most of the soil in this
tube made the count of no absolute or relative value. The same technique
was invariably followed and was carried out by the same operator, so that, it
is believed, the counts on different samples of soil are reasonably comparable.
The counts were made after 24 hours at 37°C. (The limitations of plating
methods, as emphasized in the case of soil by Thornton [1922], are appre-
ciated but we are not concerned with absolute numbers so much as with
relative numbers. On several occasions, the soil samples have been tho-
roughly ground in a sterile mortar before suspending them in saline but no
evidence could be found that the bacterial count was increased thereby).
As a rule, anthrax colonies are easily recognized; surface ones in
particular present the characteristic ground-glass appearance with ragged
edges or featheyr outgrowths (Plate XXI). Deep colonies, though less deve-
loped, can likewise be recognized by their filamentous character. In the
early stages of the work, a number of colonies were picked for identification
by animal inoculation. Much difficulty was caused at times by spreading
surface contamination and many plates had to be discarded for this reason.
With natural soils a similar procedure was followed, but 1.0 c.c. amounts
of appropriate dilutions were inoculated subcutaneously to guinea-pigs or, in
the earlier stages, to white mice. Mice, however, were not found to be
uniformly susceptible and were therefore unsuitable.
(b) In the examination of soil for growth of Cl. chauvœi, the same
technique was used both with sterilized and natural soil. After thorough
mixing, amounts in excess of 4.0 gm. were removed to large sterile tubes
and weighed. Sterile saline was then added at the rate of 20 c.c. to 4.0 gm.,
the soil well mixed and dilutions made with separate pipettes in sterile
saline made as before, except that, for dil. b, 2 c.c. dil. a were mixed with