ANTITOXINS 43 



culture and growing it in broth which is about 8 cc. normal 

 soda solution per liter above the neutral point to litmus. 

 The culture fluid should be in comparatively thin layers 

 and in large neck Erlenmeyer flasks, so as to allow a 

 free access of air. The temperature should be about 35 

 to 36 C. The culture, after a week's growth, is removed 

 from the incubator and, having been tested for purity by 

 microscopic and culture tests, is rendered sterile by the 

 addition of 10 per cent of a 5 per cent solution of carbolic 

 acid. After 48 hours the dead bacilli have settled on the 

 bottom of the jar and the clear fluid is filtered through 

 ordinary sterile filter paper and stored in full bottles in a 

 cold place until needed. Its strength is then tested by 

 giving a series of guinea pigs carefully measured amounts. 

 Less than 0.005 cc. when injected hypodermically should 

 kill a 250 gram-guinea pig. 



Immunizing ike Animals. The horses used should 

 be young, vigorous, of fair size, and absolutely healthy. 

 Vicious habits, such as kicking, etc., make no difference 

 except, of course, to those who handle the animals. 



The horses are severally inoculated with 10,000 units 

 of antitoxin so as to allow giving a much larger dose of 

 toxin than would otherwise be safe and thus save several 

 weeks' time. The following figures give the actual inoc- 

 ulation in a horse which produced an unusually high 

 grade of serum. Injections of toxin were given at first 

 every two days and later every three days : 



First injection, 12 cc. toxin (fatal dose -j-J- Q- cc.), the 

 antitoxin having been given the previous day. Second 

 and later injections of toxin without antitoxin were as 

 follows: 15 cc., 20 cc., 30 cc., etc., up to 675 cc. on the 

 6oth day. 



The entire amount should not be injected in one place, 

 but divided into six or eight portions. 



