V. R. RAJAGOPALAN and V. R. GOPALAKRISHNAN           229

previous workers as well. Magnusson [ 1923 ] has reported his failure to
obtain agglutination of the bacillus with the blood of sick animals and
also from rabbits which had received repeated intravenous injections
of the culture. Bull [ 1924 ] also failed to demonstrate agglutinins in
a horse injected subcutaneously with an emulsion of the culture. Dimock
and Edwards [ 1931 ] however, claim to have produced agglutinating serum
(titre 100), by giving the horse four injections of culture with increasing
dosage at intervals of seven days.

(2) Complement fixation test.—As the agglutination tests were of no
avail in proving the identity of the two organisms, recourse was then had
to the complement fixation test, and cross complement fixation tests.

The sera from the two immunised rabbits, already mentioned, were used
as the source of amboceptors. The Kolmer's technique was followed in
these tests, using two full units of complement in one c.c. volume and 0.5 c.c.
quantities of each of the four other components.

The haemolytic serum was titrated using falling quantities of a 1 in 1,000
diluted serum, fixed quantities of complement, i.e., 0.3 c.c. and½ c.c. quantities
of a two per cent suspension of sheep red blood corpuscles. The mixture
was incubated for one hour at 37°C. and the titre at which haemolysis occurred
was read off. The stock haemolysin was then diluted for the test accordingly,
so that 0.5 c.c. contained two units of haemolysin.

Serum for complement was obtained from a healthy guinea-pig and was
diluted initially 1 in 30. Dilutions of complement from 0.1 to 0.5 c.c. with
a graded increase of 0.05 c.c. were made in normal saline solution to which
two units of haemolysin and two per cent suspension of sheep R. B. C, each
in half c.c. quantities were added and incubated for one hour at 37°C. The
smallest amount of complement giving complete haemolysis was noted and
the next higher, which is the full unit was taken as a guide to prepare a dilution
to contain two full units in one c.c. quantities.

Graded dilutions, in geometrical proportion, were made of the antigen,
for titration, from 2 to 32. Half c.c. quantities of a 1 in 10 dilution of a heated
healthy serum and one c.c. quantities of complement containing two full
units were added. After two hours' incubation at 37°C, haemolysin and
R. B. C. in required quantities were added followed by a further incubation
of one hour at 37°C. The smallest amount of antigen producing some in-
hibition of haemolysis was noted and the stock antigen diluted for the test
in such a way that one-third of the anti-complementary unit was contained
in ½ c.c. volume.

Having titrated the components the complement fixation test was con-
ducted using the sera of the two rabbits which had been immunised against
C. equi and the buffalo diphtheroid respectively. The sera were heated
overnight at 55°C. Dilutions of the serum from 1 in 10 to 1 in 800 were made,
and the titrated quantities of antigen and complement were subsequently