228 ANTITOXINS 



sodium chlorid, and 0.1 per cent, dextrose are added. The reaction is 

 again noted, and adjusted to +0.5 per cent. The broth is then filtered 

 through filter-paper into flasks and test-tubes and sterilized in the auto- 

 clave at a temperature of 120 C. for twenty minutes. 



After incubating for from seven to ten days the culture is removed, 

 and its purity having been tested by microscopic and cultural methods, 

 it is rendered sterile by the addition of 10 per cent, of a 5 per cent, solu- 

 tion of carbolic acid. After forty-eight hours the dead bacilli have 

 settled on the bottom of the jar, and the clear fluid above is siphoned 

 off, filtered, and stored in full bottles in a cold place until needed (Fig. 

 69). 





FIG. 68. A FLASK OF DIPHTHERIA CULTURE. 



The bacilli grow on the surface and form a scum. As the culture grows older, 

 the bacilli die and sink to the bottom of the flask. A flask of this shape affords a 

 large surface of culture-medium in contact with oxygen and facilitates toxin pro- 

 duction. 



Testing the Toxin. The strength of the toxin is then tested by 

 injecting a series of guinea-pigs with carefully measured amounts. 

 When injected hypodermically, less than 0.005 c.c. should kill a 250- 

 gram guinea-pig, and a toxin requiring more than 0.01 c.c. to kill a pig 

 of this weight is too weak for present purposes. This preliminary titra- 

 tion of the toxin will suffice for determining the dosage for horses, but 

 in standardizing antitoxin, the technic must necessarily be more ac- 

 curate. 



Immunizing the Animals. The horses used should be young, vig- 

 orous, of fair size, and absolutely healthy. They should be severally 



