100
Multiplication of B. anthracis, etc.
Ringer fluid was used as diluent, 1.0 c.c. amounts
of each serial suspension being put into each of
five plates, then eight drops of sterile heated ox
serum and 10 c.c. agar at 48ºC. With anthrax
and blackquarter distilled water or saline was
the diluent; with the former, three or five poured
agar plates and with the latter five tubes of
Robertson's meat-liver medium were sown from
each serial dilution. In the early experiments
a single tube of meat-liver medium was used for
each dilution. (The value of this type of medium
for supporting the growth of very small numbers
of Cl. chauvoei has been shown by Haslam [1920].
The medium was always made up in the same
way and usually by the same assistant, Liver
broth containing about 2.0 per cent sheep serum,
incubated for three days in an anaerobic jar, is
also good, growth appearing at the bottom beneath
the clear broth. The medium, however, is not
quite so good and certainly not so convenient as
the meat-liver medium.) When it was desired
to know if or to what extent the organisms had
sporulated, a portion of the first dilution from the
original suspension was heated at 70ºC. for 30
minutes.
Counts for Pasteurella were made after 48 hours
at 37ºC, deep colonies by that time being elliptical
and whitish and surface ones circular smooth and
with a bluish tint. Anthrax colonies were
counted after 24 hours incubation by which time
they are well-developed and of characteristic
appearance. Both with Pasteurella and anthrax,
plates showing some 100-400 colonies were selected
when possible and the counts averaged. Wide
discrepancies in counts on plates made from the
same dilutions were uncommon and when present
the experiment was rejected.
The growth of
Cl. chauvoei is indicated by gas production and a
change in the colour of the meat particles from
brown to reddish. Gas production appears to be
a particularly reliable index. The final tubes
showing growth were carefully noted after 48
and again after 72 hours incubation, Since
exudates and suspensions of fresh bone marrow
from sheep and goats tend to clot, the original
dilution was usually made up in distilled water
or saline containing 1.0 per cent sodium citrate.
Every endeavour was made to follow the same
technique in dealing with tissues removed from
the same carcase owing to the necessity of getting
comparable results, e.g. with muscular tissue
from anthrax carcases the same muscle from the
two hind limbs was used.
When tissues, other than bone marrow
exudates were cultured, the procedure was
same in principle. In the case of muscle, i
ments totalling 3.0 gm. in weight and as free
possible from macroscopically visible blood ves
were taken, finely minced in a mortar,
thoroughly ground with sand, distilled was
added gradually up to 18 c.c. with contin
grinding; after a few moments of settling
fluid was poured off, the residue again gron
further 9 c.c. water added as before and
mixed with the 18 c.c.
While the identification of Pasteurella
anthrax colonies presents no difficulty, this
not so with Cl. chauvoei. It is necessary th
fore that organisms growing within the can
and assumed to be those of blackquarter sh
be properly identified. This was done in m
cases by cultural and immunity tests.
cultural recognition, blood agar slants
seeded in series starting with a loopful of m
liver culture of the suspected organism, w
was known to be free from aerobic contaminat
The slants were incubated for 24 hours in
anaerobic jar and were then examined in
a hand lens. Colonies of Cl. chauvoei are su
and. flattened, have entire edges and smo
surfaces and on the thinner parts of the st
can be seen to have haemolyzed the blood. 1
immunity tests, guinea-pigs were used w
had received intramuscularly at about
nightly intervals increasing numbers of bla
quarter spores. After withstanding four or
such injections of spores, the guinea-pigs wa
able to support a subcutaneous injection of
c.c. meat-liver culture, 0.2 c.c. 5 per cent cala
chloride, mixed with saline up to 1.0 c.c. T
weeks or so after this injection the guinea-
are ready for use. (A safer method of immum
tion is to begin with formalinized meat-li
culture. To a paper-filtered three-day cult
formalin at six parts per thousand is added
allowed to act for 24 hours at 37ºC. 1º0 c.c.
the vaccine subcutaneously is followed 14
later by a similar injection of 3.0 c.c.)
identification of a culture, one immune and
healthy guinea-pig are given subcutaneo
meat-liver culture of the organism in quesl
mixed with calcium chloride in the quant
just mentioned. If the culture is one of
chauvoei, the healthy guinea-pig should die wit
two or three days with the characteristic les
while the immune should show nothing more t