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picked up and sown on the blood agar slants. The culture was then examined for
capsulated (phase I) cells and if conforming to the desired characters, was inoculated
in nutrient broth tubes, which were incubated at 37ºC for 24 hours.
The roux flasks containing the yeast extract medium were seeded with the
24-hour old broth culture and incubated at 37ºC for a period of 24 hours. The growth
was washed with formol-saline containing 0.25 per cent formalin. The washings
were diluted with the same fluid to an opacity of 6-7 of Brown's scale and the product
was kept overnight in the incubator. In the later brews, however, keeping in the
incubator was eliminated and the vaccine was left at room temperature.
Adjuvants were added to the bacterial suspension on the lines of Bain to give
the following final product:
Bacterial suspension
Liquid paraffin
Anhydrous lanolin
15 parts
10 parts
1 part
The mixture was placed in a waring blender which was run for about five minutes.
The final product was a thick white emulsion which has been found to remain stable
for quite a long time. Contrary to the reports of experience of other workers in
respect of the administration of such a thick vaccine, little difficulty has been en-
countered in drawing in a syringe as well as in injecting this product with wide-bore
(16 gauge) needle. However, in winter months the vaccine had to be sucked in the
syringe directly with the nozzle after pouring it in a petri dish.
(f) Effect of cultural conditions on the growth of Past. septica
The following observations made at the Institute are reproduced as these are of
interest in the context of this work.
P 52 strain of Past. septica was grown on a liquid medium and it was found that
(a) aeration increased the growth about four-fold, but when a stream of pure oxygen
or hydrogen was passed, instead of air, nearly complete inhibition of growth occurred,
(b) growth was not affected appreciably by (i) the complete removal of carbon dioxide
from the environment, (ii) substitution of glucose with other common carbohydrates,
or with some of the organic acids concerned with Kreb's cycle, (iii) addition of small
quantities of certain hormones (thyroxine, stilboestrol, adrenaline, or insulin) and
(iv) supplementation of plain broth with the common amino acids added singly.
In the routine manufacture of the adjuvant vaccine, aeration of the cultures
was not carried out as the process was laborious and time-consuming, and could
hardly be suitable for employing on a large scale without special apparatus. The
yield of the vaccine on the yeast-extract medium even without aeration was quite
satisfactory and at an average 50-60 doses were obtained from a roux flask.
Safety tests: The vaccine was tested for its safety on a very large number of
cattle, buffaloes and calves under experimental as well as field conditions and was
found to be quite safe. However, a small percentage of animals showed a rise
of a degree or two in temperature and slight swelling at the site of inoculation, but
these symptoms generally disappeared within 3 or 4 days.
Potency tests: Series of tests were carried out in cattle with several brews to
determine the immunogenic properties of the vaccine. The results are presented in
Table II.