September, 1952] S. B. V. RAO, S. NARAYANAN, D. R. RAMNANI AND J. DAS 219
2. Antigen of Coburn and Stafseth [1931].
Culture media. Beef and chicken infusion agar pH. 7.2 incubated for 72 hours
at 37.5° C. Suspending fluid—Phenol 0.5 per cent in N.S.S. Dye
used. Saturated alcoholic solution of gentian violet 1 c.c. and brilliant
green 5 c.c. of 0.1 per cent solution for every 100 c.c. of antigen.
3. Experimental antigen No. 3.
Glucose glycerine agar for dissociation plates for colony selection, before
charging the Special solid media.
2. Beef infusion agar enriched with glycerine and sodium thiosulphate for roux
flask. pH of media 7.0 and incubated for 72 hours at 37.5°C.
Suspending Fluid. 12 per cent sodium chloride solution containing 0.125 formal-
dehyde (40 per cent) and 0.125 phenol per 100 c.c. of the solution. The pH of final
antigen at the time of test is 8.0 by addition of one per cent sodium-hydroxide
(a final pH of 6.5 has also given good results).
Dye used. Crystal violet 0.03 per cent solution, 3 c.c. per 100 c.c. of the antigen.
Except for the variations as regards media, buffer solutions, dye and pH with respect
to different antigens the methods of washing, collection, preservation and rendering
the product safe for use on poultry farms, were same for the three antigens under
study.
A single strain of S. gallinarum No. 399 was used to prepare the antigens. This
strain was highly specific and had been isolated recently from a virulent outbreak,
and it was being used to eliminate reactor. The organism was found to have a
good antigenic property as revealed by cross agglutination and absorption tests
with the standard S. pullorum stock strain, obtained from Hull, Que, through the
courtesy of the Dominion Animal Pathologist, Canada. The organism was plated
out several times and individual smooth colonies picked out after 48 hours incubation
of the plates at 37.5° C, under low power of the microscope.. Each colony was
seeded into a peptone broth, and those showing even suspension of growth following
24 hours incubation at 37.5° C. without sedimentation were selected for antigen
preparation. 1½ c.c. of the culture was flowed over the surface of the solid media
in each roux flask uniformly. The flasks were paraffin sealed and incubated for
72 hours at 37.5° C., and those that showed a dense uniform growth were washed
with 10 c.c. of their respective Suspending fluids, by gently rocking for five minutes.
The suspension was filtered through sterile absorbent cotton wool into coloured
sterile stoppered bottles and preserved in darkness at 30°F. for a week or more until
the organism was killed as proved by safety test. Dyes were added and density
adjusted 100 times Brown's opacity tube No. 1 with their respective Suspending
fluids shortly before use.
A pen of 28 carrier birds raised for four months in isolation was tested simul-
taneously with the above listed three antigens weather and temperature remaining
constant-during the period.
The details of the test are given in the table.
The low titre birds Nos. 1168, 237, 68, 7082, 1553 and 458 which were negativ
to the first two antigens but positive to the third experimental Nos. 3 were bled fo
10A