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. Friin- 

 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 cubic 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. Friinkel 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. 



Behriug, 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 bv contact with iodine terchloride 



