358 PROTECTIVE INOCULATIONS. 



mals were chosen which were two or three years old and had given 

 birth to young a few weeks before the inoculations were commenced. 

 It having been previously shown by Ehrlich that the precipitated 

 tetanus toxin from cultures could be successfully used to immunize 

 guinea-pigs, the same substance was employed in these experiments. 

 The treatment was commenced with a dose of 0.00001 gramme, which 

 was carefully increased to 0.00007 gramme, the injections being made 

 at intervals of four days. But this proved to be too much, and the 

 animal died of typical tetanus after the last dose. In a subsequent 

 experiment Brieger and Colin succeeded in immunizing a goat in a 

 month and a half so that the animal finally withstood a dose of 0.06 

 gramme, but this animal ceased to give milk, became anaemic, and 

 finally died. 



The authors therefore resorted to a different method which had 

 previously been successfully employed by Ehrlich, Behring, and 

 others. Cultures of the tetanus bacillus in bouillon were heated to 

 65 C. for half an hour, and then used for immunizing two goats. 

 After five weeks' treatment the animals resisted doses of the precipi- 

 tated toxin, which were gradually increased to ten grammes, at which 

 time the treatment had been carried on for nearly six months and the 

 antitoxic value of the milk was found to be 90,000 immunization units. 



The method of determining antitoxic values adopted by Brieger 

 and Cohn is the following : They had found by carefully conducted 

 experiments that their precipitated toxin (Eohgifte) killed a mouse 

 weighing twenty grammes in the dose of 0.0000003 gramme, but 

 failed to kill when injected in the dose of 0.0000002 gramme. The 

 first-mentioned dose was therefore accepted as the minimum fatal dose 

 for an animal weighing eighteen to twenty grammes, and the object 

 in view was to find the minimum amount of milk required to prevent 

 the toxic action of such a dose. 



The antitoxin was obtained from the goat's milk by precipitation 

 with ammonium sulphate, thirty -two per cent; the precipitate was 

 again dissolved and treated with a solution of basic acetate of lead; 

 this salt does not precipitate the antitoxin when the solution is slightly 

 alkaline; the voluminous precipitate produced by the lead acetate is 

 filtered out and repeatedly washed with water; the filtered fluid and 

 wash water are again treated with ammonium sulphate, added to 

 saturation, and the resulting precipitate is dissolved in a small quan- 

 tity of water; a precipitate is again obtained by saturation with am- 

 monium sulphate, and this is dried upon porcelain plates in a vac- 

 uum. The ammonium sulphate remaining could not be removed by 

 dialysis, as experiment showed that a considerable loss of the antitoxin 



