PRODUCTION AND TESTING OF ANTITOXINS 275 



refusal of food, and sometimes muscular twitching. A second injec- 

 tion should never be given until all such symptoms have completely 

 subsided. This being the case, after five to eight days double the 

 original dose is given together with a neutralizing amount of anti- 

 toxin or with the addition of terchlorid of iodin. Again after five 

 to eight days, a larger dose is given and thereafter, at similar 

 intervals, the quantity of toxin is rapidly increased. In America 

 the neutralizing antitoxin is omitted after the third or fourth injec- 

 tion; in v. Behring's laboratory the quantity of terchlorid of iodin 

 is gradually diminished. The increase of dosage is often controlled 

 by the determination of the antitoxin contents of the animal's blood 

 serum. The immunization is increased until enormous doses (500 

 c.c.) of a toxin in which the minimal lethal dose for mice is repre- 

 sented by 0.0001 c.c., or less, is borne by the horse without apparent 

 harm. 



The antitoxic serum is then obtained by bleeding from the jugular 

 vein, as in the case of diphtheria antitoxin. It may be preserved 

 in the liquid state by the addition of five-tenths per cent of carbolic 

 acid or four-tenths per cent of tricresol. 



Standardization. The universal prophylactic use of tetanus anti- 

 toxin has, as in the case of diphtheria antitoxin, necessitated its 

 standardization. A variety of methods are in use in different parts 

 of the world. In the following description the American method 

 only will be considered as laid down under the law of July, 1908, 

 and based upon the work of Rosenau and Anderson 16 at the" United 

 States Hygienic Laboratories at Washington. 



In conjunction with a committee of the Society of American 

 Bacteriologists, these authors have defined the unit of tetanus anti- 

 toxin as follows: 



The unit shall be ten times the least amount of serum necessary 

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

 the official test dose of standard toxin. The test dose consists of 

 100 minimal lethal doses of a precipitated toxin preserved under 

 special conditions at the hygienic laboratory of the Public Health 

 and Marine Hospital Service. (The minimal lethal dose is in this 

 case, unlike v. Behring's minimal lethal dose, measured not against 

 20 gram mice, but against 350 gram guinea-pigs.) 



In the actual standardization of tetanus antitoxin, as in that of 



18 Rosenau and Anderson, Pub. Health and Mar. Hosp. Serv. TJ. S., Hyg. Lab. 

 Bull. 43, 1908. 



