58 BACTERIOLOGY. 



active substance was precipitated by alcohol. Behring, Kitasato, and 

 Schiitz experimented with a view to conferring immunity upon 

 horses. The cultures were mixed with terchloride of iodine, and 

 injected at intervals of eight days, and the antitoxic power tested 

 on mice. By using increasingly virulent cultures, the blood became 

 increasingly antitoxic. 



Vaillard filtered tetanus cultures through porcelain, and heated 

 the filtrate at gradually diminishing temperatures. The first in- 

 jections were made with 10 cc., which had been raised to 60 C. for 

 an hour, then a filtrate was used which had been heated to 55 C., 

 and lastly, a filtrate which had been heated to 50 C. The blood 

 became antitoxic, and by injecting increasing quantities of virulent 

 filtrates the antitoxic power was rapidly intensified, and animals 

 which were injected with antitoxin of full strength possessed 

 immunity many months afterwards. 



Roux and Vaillard introduced another method. Virulent cultures 

 were filtered through porcelain, and the filtrate mixed with Gram's 

 solution of iodine in iodide of potassium. To give immunity to a 

 rabbit, 3 cc. of toxin with 1 cc. of Gram's solution were injected on 

 the first day, and increasing doses of toxin mixed with increasing 

 doses of Gram's solution on the following days. The same method 

 was applied to horses, sheep, and cattle. The antitoxin was found 

 not only in the blood, but in the urine and saliva, and in the 

 milk in cows. With cows and goats it is necessary to proceed 

 with the utmost care ; while horses, on the other hand, bear the 

 injections well, and are therefore more suitable for this purpose. 

 It is also very easy to obtain large quantities of blood from the 

 horse by inserting a trocar and cannula into the jugular vein. 



Frankel was the first to produce immunity against diphtheria 

 by injecting guinea-pigs with toxin which had been heated to 70 C. 

 Behring mixed the toxin with terchloride of iodine, or employed small 

 doses of pure toxin. Horses, sheep, goats, and dogs were rendered 

 immune. 



PREPARATION OF DIPHTHERIA ANTITOXIN. 



For the preparation of diphtheria antitoxin Roux cultivates the 

 diphtheria bacillus in alkaline broth with 2 per cent, of peptone, and 

 by preference, in flasks in which the cultivating liquid can be exposed 

 to a current of moist air at 37 C. After about three weeks the 

 culture is filtered through a Chamberland filter, and if tested on 

 a guinea-pig it will be found that y 1 ^ of a cc. will kill an animal 

 weighing five hundred grammes in forty-eight hours. The diphtheria 



