PROTECTIVE INOCULATIONS. 313 
for from thirty-six to forty-eight hours, and proved that the method 
could be successfully employed in immunizing sheep; and the fact 
was ascertained that blood serum from an immune animal could be 
used with success in arresting diphtheritic infection in susceptible 
animals. To preserve the serum, which they obtained from immun- 
ized sheep, rabbits, and guinea-pigs, they added to it 0.5 per cent of 
pure carbolic acid. For producing immunity they found that a 
smaller amount of serum was required than was necessary for the 
cure of an animal already infected. If the injection was made imme- 
diately after infection, from one and a half to two times the amount 
was required; eight hours after infection the amount was three times 
as great, and twenty-four to thirty-six hours after infection the dose 
required was eight times the immunizing dose. 
The immunizing value of blood serum from different animals was 
estimated by finding the smallest dose which would protect an animal 
from fatal infection by the minimum lethal dose of a culture the toxic 
potency of which had been carefully determined. The value is ex- 
pressed in figures which give the proportion required compared with 
the body weight of the animal. Thus an immunizing value of one 
hundred would mean that one gramme of the serum is sufficient to 
protect an animal weighing one hundred grammes from the fatal 
effect which would be produced in a control animal of the same weight 
by infection with a virulent culture of the diphtheria bacillus in the 
minimum doses required to produce this result. The cultures em- 
ployed are made in bouillon containing one per cent of peptone; they 
are inoculated from agar cultures and are kept in the incubating oven 
for two days. Cultures prepared in this way were found to be quite 
uniform in their pathogenic virulence as tested upon guinea-pigs. But 
when cultures are kept for some time there is an increase in virulence. 
Thus a culture obtained from a fatal case of diphtheria which in 1890 
killed guinea-pigs in three to four days, when injected subcutaneously 
in the dose of 0.1 cubic centimetre (two-days-old bouillon culture), 
at the end of a year was fatal to these animals in the dose of 0.025 
cubic centimetre. This increase in virulence is ascribed to the fact 
that the cultures were renewed at long intervals. 
More recently (1894) Behring has fixed a standard for what he 
calls a normal therapeutic serum. This is a serum which when in- 
jected into guinea-pigs in the proportion of 1:5,000 of body weight 
saves the animal from the fatal effects of ten times the minimum dose 
of a culture in bouillon, two days old, which would kill a control ani- 
mal not treated. 
In a subsequent communication (November, 1894) Behring states 
