312 PROTECTIVE INOCULATIONS. 
After the removal of peptone and globulin from the filtered cultures, 
these were evaporated and a precipitate obtained of one of the 
albuminous substances by means of sixty to seventy per cent al- 
cohol. The other substance remained in solution, and was sub- 
sequently obtained by precipitation with absolute alcohol. The 
substance first obtained by this method is toxic, and the other pre- 
cipitate is not. The authors named succeeded in killing rabbits with 
the toxalbumin obtained in this way, but were not able to produce 
immunity in these animals by the injection of non-fatal doses. Fréan- 
kel (1891) had previously reported his failure to immunize guinea-pigs 
by the injection of the dry precipitate, obtained in his experiments 
from diphtheria cultures; but when filtered cultures, or cultures 
sterilized by heat (55° C. for one hour), were injected into these ani- 
mals, they showed an increased resistance to the pathogenic action of 
virulent cultures. Still better results were obtained when ten cubic 
centimetres of a bouillon culture, heated to 100° C., were injected 
subcutaneously, but still this method was not entirely reliable. But 
true immunity was established by injecting into the peritoneal cavity 
ten to twenty ctbic centimetres of a bouillon culture heated to from 65° 
to 70° C. for one hour. The immunity was not fully established until 
about fourteen days after the protective inoculation. Frankel arrives 
at the conclusion that the cultures must contain an immunizing sub- 
stance as well as a toxic proteid, as the diphtheria toxalbumin is de- 
stroyed by the temperature (65° to 70° C.) used in the preparation of 
his cultures for producing immunity. 
Behring, in the same year (1891), commenced his experiments 
upon diphtheria immunity. Guinea-pigs were made immune by the 
use of sterilized cultures, and by inoculations with virulent cultures, 
four weeks old, to which iodine terchloride had been added in the 
proportion of 1:500—the mixture was allowed to stand for sixteen 
hours. Animals were also immunized by injecting beneath the skin 
a virulent culture of the bacillus, and then treating them with sub- 
cutaneous injections of iodine terchloride (two cubic centimetres), 
which was thrown under the skin for three days in succession in the 
vicinity of the point of inoculation. The guinea-pigs treated in this 
way remained sick for some time, but finally recovered and were 
subsequently immune. Still better results were obtained when rab- 
bits were subjected to the same treatment. The animals were im- 
mune against the toxic action of sterilized cultures, as well as against 
infection by virulent diphtheria bacilli. 
In subsequent experiments (1892) Behring and Wernicke used 
cultures which had been attenuated by contact with iodine terchloride 
