128
INFLUENCE OF SOLAR RADIATION ON IMMUNITY, I [Vol. XXI, Part III,
postulated the presence of a multiplicity of haemolysins, because he found no paral-
lelism between the titres shown by different toxins against the erythrocytes of
different species of animals. It remained for Glenny and Stevens [1935] to show
that some filtrates contained two antigenically separate substances which they
called Alpha toxin and Beta toxin. Their Alpha toxin haemolysed rabbit and sheep
corpuscles at 37°C., their Beta toxin haemolysed sheep corpuscles only when their
preliminary incubation at 37°C. was followed by cooling, and did not lyse rabbit
corpuscles. The toxin formed by the method, described above, from strain No. 11
was found to be of Alpha variety, as it haemolysed rabbit and sheep corpuscles
at 37°C.
Direct haemolysin titrations. Method adopted by Minett [1936] was followed.
In 10 per cent of broth saline 1 c.c. of a series of dilutions (physiological saline con-
taining 10 per cent of beef infusion broth) was mixed with 0.1 c.c. of 10 per cent
suspension of washed rabbit red cells and the tubes incubated in a water bath
at 37°C. for one hour, with a shake after the first 30 minutes. Readings were taken
just before removing the tubes from the bath and finally after one hour's standing
on the bench. The tubes were shaken after each reading. The end-point was
taken as the dilution showing ' partial' or approximately 50 per cent haemolysis,
as judged by the colour of prepared standard and the minimal haemolytic dose
(M.H.D.) of toxin was determined by adopting the ' Burnet unit of haemolysin'
—(this is defined as the smallest quantity of toxin which will produce 50 per cent
haemolysis of 1 c.c. of 1 per cent cells). Toxin formed from strain No. 11 gave a titre
of 3,200 M.H.D. per c.c.
Preparation of toxoid. The conversion of staphylococcus toxin into toxoid
was effected by the addition of 0.4 per cent formalin and incubating the mixture at
37°C. for 2 days, after which it was found to be completely ' inactivated' i.e. it
no longer haemolysed red cells, failed to cause necrosis on intradermal injection
in guinea pigs and also found to be no longer lethal for rabbits and guinea pigs.
Smith [1936] found out that the mean antitoxin produced in a group of 12 guinea
pigs receiving two spaced injections of toxoid to which potassium alum had been
added was 0.99 unit, while in a group of 12 receiving the same dose of untreated
toxoid under identical conditions, the mean antitoxin was 0.42 unit. Minett
[1939] tried alum-precipitated toxoid in sheep prepared by addition of 2.0 per cent
potash alum, and found that it gave a rather better immunity response than two
doses of unprecipitated toxoid. Hence, in the immunization of rabbits, alum-
precipitated toxoid, prepared in the above way, was used.
SELECTION OF RABBITS
Care was taken to select rabbits free from any disease and more specially diseases
affecting the skin. Other factors taken into consideration were coat colour, size,
age and sex. Of these, coat colour is the most important factor in the experiment
as the degree of photosensitization of the rabbits exposed to the sun directly depends
on the nature of the coat colour. Reemerschmid and Elder [1945] reported wide
variations between breeds of cattle in the amounts of solar radiation absorbed
by the skin. They found that absorption was markedly dependant upon colour,
with white Zulu coat abosrbing only 49 per cent of the rays and the black Angus
coat 89 per cent. A comparison of different shades of red showed percentages of