PROTECTIVE INOCULATIONS. 315 



40 C., and then heated to 102 C., it still retained its antitoxic 

 potency. 



Ehrlich, Kossel, and Wasserniann (1894) have made experiments 

 upon goats, which they found very susceptible to the action of the 

 diphtheria poison. Sterilized cultures were first injected in gradually 

 increasing amounts, and later virulent cultures. In this way they ob- 

 tained a serum which has a value sixty times that of Behriug's "nor- 

 mal serum." In a subsequent communication (1894) Wassermann 

 gives an account of his experiments with the milk of immunized 

 goats, which contains the antitoxin in considerable quantity, and 

 from which it was obtained in a concentrated form by the following 

 method : The milk is obtained in sterilized vessels and twenty cubic 

 centimetres of normal hydrochloric acid are added to each litre; a, t 

 sufficient quantity of rennet is then added to coagulate the casein, 

 and this is separated from the liquid, which is then shaken up with 

 chloroform for some time. The liquid is now allowed to stand in 

 order that the butter, which has been dissolved by the chloroform, 

 may sink to the bottom. The clear liquid is then decanted and the 

 antitoxin precipitated from it by means of ammonium sulphate (thirty 

 to thirty-three per cent). The precipitate is rapidly dried upon 

 porous porcelain plates, in vacuo, and then dissolved in water in the 

 proportion of ten parts for one hundred of milk first employed a 

 concentration to one-tenth. Of this solution 0.125 cubic centimetre 

 was found to neutralize 0.9 cubic centimetre of a toxin which killed 

 guinea-pigs weighing five hundred grammes in the dose of 0.1 cubic 

 centimetre. This toxin was an old bouillon culture of the diphtheria 

 bacillus to which 0.5 per cent of carbolic acid had been added to 

 preserve it. In a communication of the same date Ehrlich and Was- 

 sermann report that they have for some time had a cow immunized 

 to such a degree that one cubic centimetre of its milk protects guinea- 

 pigs from the fatal effects of 0.9 cubic centimetre of the above-men- 

 tioned toxin. The antitoxic value of the milk of an immunized cow 

 or goat, as compared with that of its blood, is estimated by Ehrlich 

 and Wassermann as from 1 : 15 to 1 : 30 usually about 1 : 20. 



Aronson, in testing his antitoxin, uses a bouillon culture of the 

 diphtheria bacillus two and one-half months old, which he preserves 

 by the addition of 0.3 per cent of trikresol. He finds that the im- 

 munity which results from injections of the antitoxin is established 

 at once; that it is not accompanied by any reaction or symptom of 

 sickness ; and that it is of comparatively short duration. 



As a result of extended experiments made at the Pasteur Institute 

 in Paris, Koux has perfected the following method for the production 



