244 ANTITOXINS 



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 850-gram guinea-pig for ninety-six hours against 

 the official test dose of a standard toxin. This test dose consists of 100 

 minimal lethal doses of a precipitated and dried toxin, tested out 

 against 350-gram pigs, and preserved in the Hygienic Laboratory, 

 from where it is sent to various antitoxin plants for the purpose of secur- 

 ing a uniform method and unit of standardization. 



In standardizing tetanus antitoxin, the L+ dose of toxin is em- 

 ployed. A standard toxin and an antitoxin, arbitrary in their first 

 establishment, are preserved in the Hygienic Laboratory, and are kept 

 constant by making frequent tests one against the other. In determin- 

 ing the L+ dose, increasing amounts of toxin are mixed with a constant 

 amount of antitoxin equal to one-tenth of an immunity unit, and in- 

 jected into 350-gram pigs. The L+ dose must contain just enough 

 toxin to neutralize this amount of antitoxin and kill a pig in four days. 

 This L + dose of toxin is sent out by the Hygienic Laboratory to those 

 interested, commercially or otherwise, in the manufacture of antitoxin 

 for purposes of standardization. 



For determining the strength of an unknown serum a large number 

 of mixtures are made, each containing the L + doses of the toxin and 



