September, 1951]
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the means of the two groups, viz. ' sun' and ' shade ' was compared for antitoxin
units per c.c.
Selection of bacteria
According to Minett [1937], a strain of Staphylococcus aureus, which is highly
toxigenic and haemolytic for rabbit erthrocytes at a dilution of at least 1 in 1000,
is considered suitable for immunizing animals, because the toxoid prepared from the
toxin of such a strain could be depended upon to stimulate a good antitoxic res-
ponse in the immunized animals. Hence a large number of strains of S. aureus,
available at the Indian Veterinary Research Institute, Mukteswar, were examined
systematically for their respective cultural, biochemical, haemolytic, dermone-
crotic and lethal properties and finally strain No. 11 was selected for the work.
Haemolytic property
Preparation of toxin. Dolman [1933] described the general methods for the
preparation of staphylococcus toxin, but several modifications have been made
by different workers, specially in respect of the medium used. A partial CO 2
atmosphere is generally used in the production of staphylococcus toxin and there-
fore some kind of anaerobic culture apparatus is necessary. The methods reported
by Dolman [1933], Burnet [1929], Jamieson and Powell [1934], Parish and co-
workers [1932] refer to a McIntosh and Fildes anaerobic apparatus or a Novy jar.
A semi-synthetic infusion-free medium was proposed by Eirkbride, Bertheksen
and Clark [1931] for the production of diptheria toxin and this semi-synthetic medium
was used after slight modification by Leonard and Holm [1935] for the production
of staphylococcus toxin. Minett [1936] adopted a method closely similar to that
of Burnet [1931] for the preparation of staphylococcus toxin and this method with
slight modification was adopted by the author with satisfactory results. According
to this method, cultures were grown for 36 hours at 37°C. on plates of 0.8 per cent
beef heart agar, pH 7.4, containing one per cent Witte peptone and 0.25 per cent
NaCl in an atmosphere containing 25 per cent CO 2 . After 36 hours' incubation,
when the lid of the anaerobic jar was opened, a peculiar disagreeable characteristic
odour was evident signifying the formation of a good toxin. One c.c. sterile nor-
mal saline was then added to each plate of 10 c.c. agar, the semisolid medium was
thoroughly chopped and filtered through a Buchner's funnel, the filtrate was centri-
fuged at high speed—15,000 r. p. n. for 30 minutes, and the clear supernatant
fluid constituting the toxin was pipetted off and kept in the refrigerator for use
without addition of any preservative. It has been found by Minett [1939] that
the haemolytic property of staphylococcus toxin kept in the refrigerator without
any preservative does not deteriorate even after two years.
Varieties of toxin. Since the work of Burnet [1929], evidence has been accu-
mulating which shows that there are at least two or possibly more haemolytic factors
antigenically distinct from each other. Walburn [1921] demonstrated a peculiar
type of haemolysis ' in vitro' when goat red blood corpuscles were used, which
took place in ice box after a preliminary period of incubation at 37°C. This
' hot-cold' lysis was studied by Bigger, Boland, and O'Meara [1927] using sheep
and human cells, and they concluded that the substances responsible for the des-
truction of these cells differed from the rabbit erythrocyte haemolysis. Dolam [1932]