F. C. MINETT AND M. R. DHANDA 313
The Mukteswar soils were passed first through a coarse sieve, then ground
lightly and put through a 1 mm. sieve ; the sieved soil was then air-dried and
stored in jars at laboratory temperature. At time of use, 25-50 gm. were
transferred to 125 c.c. bottles of 5 cm. diameter, water was added and the
wool-plugged bottles autoclaved for 1-2 hours at 20-25 lb. pressure. Later
in attempting to surmount the difficulty of sterilizing this amount of soil
at one or two heatings in the small bottles, the soil was placed in thin layers
in open beakers and flooded with water before autoclaving. After cooling,
the wet soil was placed in the bottles and re-autoclaved. When cold, sterile
water was added to give the required concentration and indiarubber corks
were fitted to the bottles. (One of the major difficulties in this work has been
to sterilize the soil effectively ; our efforts were not always successful, even
when 25 gm. amounts spread in thin (about 1 cm.) layers and well flooded
with water were autoclaved). Tests were also made with natural (i.e. un-
sterilized) soil. At the conclusion of the experiment, the water content of each
sample was taken as a check.
Water
A few experiments were made to see if B. anthracis and Cl. chauvœi would
grow in tap water (500 c.c. in round flasks), sterilized or natural, over a thin
layer of soil.
Seed
(a) The anthrax strain used had been isolated in Burma from an elephant
and its virulence for sheep and guinea-pigs was established. It had apparently
lost much of its virulence for hill cattle. In preparing the suspension to be
added to the soil, the sporulated growth from an agar slant after 4 or 5 days
at 37°C. was evenly suspended with the aid of glass beads in distilled water
to such a concentration that a platinum loop disappeared from view at about
15 mm. below the surface. The viable count in such a suspension varied
considerably, from 12 to 90 millions per c.c. but was usually between 20 and
40 millions. From this a suitable dilution was prepared, such that 1.0 c.c.
of it added to and then thoroughly mixed with the soil was estimated to give
100-300 organisms, occasionally more, per gm. The actual count was, of
course, estimated at the time of seeding. When samples were taken after
incubation the soil was again thoroughly mixed. With very wet soils, this
mixing was easy but it tended to be rather more difficult with pasty soils.
On a few occasions, heart blood from guinea-pigs freshly dead of anthrax
was used for sowing the bottles.
(b) The Cl. chauvœi strain used was virulent for sheep and guinea-pigs.
Its characters were checked for us by Mr V. R. Rajagopalan, as follows :
subterminal spores swelling the rod, Claudius + , no blackening of chopped¬
meat or brain-liver medium, gelatin liquefied, no liquefaction of solid serum,
no change in litmus milk, glucose, maltose, lactose and sucrose fermented
and salicin and mannitol not fermented. The suspension to be used for seed-
ing the soil was prepared from 3 or 4-day Robertson meat medium cultures,
which at that time contained many spores. The organisms were washed
once in distilled water and suspended in that fluid ; the approximate total