234 ANTITOXINS 



PRODUCTION OF TETANUS ANTITOXIN 



The method used in the production of tetanus antitoxin is similar to 

 that employed in producing diphtheria-antitoxin, the horses being 

 inoculated with increasing doses of a strong tetanus toxin. 



Tetanus Toxin. The toxin is secured by inoculating large flasks 

 or tubes of neutral veal broth containing 1 per cent, of sodium chlorid 

 and peptone with abundant tetanus culture, and growing these anae- 

 robically at 37 C. for two weeks. The cultures are then filtered rap- 

 idly through Berkefeld filters, and the toxin preserved in fluid form with 

 the addition of 0.5 per cent, phenol. As previously mentioned, the 

 toxin rapidly deteriorates especially tetanospasmin and for pur- 

 poses of antitoxin standardization it is usually preserved in a dry state 

 after being precipitated with ammonium sulphate. The yellowish, 

 crystalline masses are readily soluble in water or salt solution, and should 

 be used immediately after solution takes place. The strength of the 

 toxin is determined by injecting increasing amounts into white mice or 

 350-gram guinea-pigs. 



Immunizing the Animals. According to Park, the "horses receive 

 5 c.c. as the initial dose of a toxin, of which 1 c.c. kills 250,000 grams of 

 guinea-pig, and along with this twice the amount of antitoxin required 

 to neutralize it. In five days this dose is doubled, and then every five 

 to seven days larger amounts are given. After the third injection the 

 antitoxin is omitted. The dose is increased at first slowly until appre- 

 ciable amounts of antitoxin are found to be present, and then as rapidly 

 as the horses can stand it, until they support 700 to 800 c.c. or more at a 

 time. This amount should not be injected in a single place, or severe 

 local and perhaps fatal tetanus may develop." 



Collecting the Serum. The horses are bled, and the serum is col- 

 lected under strict aseptic precautions, in a manner similar to the col- 

 lection of antidiphtheric serum. The serum should be clear and free 

 from blood, and should be proved sterile by cultural tests. It may be 

 preserved in the liquid state by adding 0.5 per cent, of phenol or 0.4 

 per cent, of tricresol. 



Standardizing the Serum. The official immunity unit of tetanus 

 antitoxin of the United States Government is based largely upon the 

 work of Rosenau and Anderson. These investigators, together with a 

 Committee of the Society of American Bacteriologists, have defined the 

 unit of tetanus antitoxin to be "ten times the least amount of serum neces- 

 sary to save the life of a 350-gram guinea-pig for ninety-six hours against 



