THERAPEUTIC IMMUNIZATION IN MAN 465 



intracutaneous reaction with this amount indicates an absence of 

 antitoxin from the blood, or at least an insufficient protection. The 

 Schick reaction is at present carried out at the New York Depart- 

 ment of Health, under the direction of Park, with 1/5 Oth M L D 

 intracutaneously injected. The dilutions are so made that this 

 quantity is contained in a total volume of 0.1 c. c. 



TETANUS ANTITOXIN AND ITS STANDARDIZATION 



The methods employed in the production and standardization of 

 tetanus toxin are in every way analogous to those used in the case 

 of diphtheria antitoxin. A strong toxin is obtained by growing the 

 organisms under anaerobic conditions on suitable media. Accord- 

 ing to Vaillard and Vincent 37 it is essential that the media upon 

 which the tetanus bacilli are grown should be freshly made and 

 sterilized. Apparently this precaution, which has been similarly 

 recommended by Wladimiroff, ISTovy, and others, is made necessary 

 by the gradual absorption of oxygen which takes place if the media 

 are allowed to stand for a long time without heating. It is further 

 necessary in preparing tetanus toxin that the culture medium should 

 not be acid, and a weakly alkaline initial titre is advised. For the 

 same reason, also, most workers have advised against the use of 

 glucose or other carbohydrates in the media, since the acid formed 

 by the fermentation of these substances inhibits growth and toxin 

 production. Recently Hall 38 has advised the use of a simple meat 

 extract broth to which have been added 1 per cent, of dextrose and 

 0.5 per cent, of finely powdered magnesium carbonate. The last- 

 named substance, by neutralizing any acid that is formed from the 

 glucose, prevents the harmful acidity. Anaerobic conditions are ob- 

 tained by growing the organisms under a layer of oil in tightly stop- 

 pered flasks. 



Although mice were formerly used in the standardization of 

 tetanus toxin and antitoxin, the more recent usage has been to sub- 

 stitute guinea pigs as in diphtheria standardization. According to 

 the recent directions of Rosenau and Anderson 39 the purposes of the 

 standardization are carried out as follows: 



The unit of antitoxin is arbitrarily designated as 10 times the 

 smallest amount of serum necessary to preserve the life of a guinea 

 pig weighing 350 grams for 96 hours, when given together with an 

 official test dose of toxin. The test dose of toxin contains 100 min- 

 imal lethal doses. And the minimal lethal dose is measured against 

 a 350-gram guinea pig. 



37 Yaillard and Vincent. Ann. Past., 1891. 



38 Hall. "Univ. of Cal., Publ. in Path.," Vol. 2, No. 11, 1913. 



39 Rosenau and Anderson. U. S. P. H. Service Hyg. Lab. Bull. 43, 1908. 



