858 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 Coln succeeded in immunizing a goat ina 
month and a half so that the animal finally withstood a dose of 0.06 
gramme, but this animal ceased to give milk, became anemic, 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 (Rohgifte) 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 
