PRODUCTION OF TETANUS ANTITOXIN 243 



nfotf of a unit. These mixtures are made in small test-tubes, the cotton stoppers 

 paraffined, and the tubes incubated for three hours and placed in the refrigerator for 

 twenty-one hours, after which 0.2 c.c. of each is injected into guinea-pigs (prepared 

 by pulling out the hairs); several injections may be made in each pig. 



When the L n dose of the toxin has been determined this amount is mixed in a 

 similar manner with varying amounts of the patient's serum being tested. The 

 amount of serum just neutralizing the toxin contains y^Vir of a unit of antitoxin from 

 which the amount of antitoxin per cubic centimeter of serum may be computed. For 

 example I have found that 0.003 c.c. of serum of a person reacting negatively to the 

 Schick test neutralized this amount of toxin; therefore each cubic centimeter of 

 this person's serum contained 0.33 unit of antitoxin. 



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. Anderson and Leake 1 prepare 100 

 liters of broth with 50 kilograms of minced round steak and add 0.5 

 per cent, sodium chlorid and 1 per cent, peptone; after steaming for an 

 hour the reaction is made neutral to phenolphthalein and the broth 

 filtered through paper into liter Erlenmeyer flasks, followed by steaming 

 without pressure for one and one-half hours. The broth may be stored 

 for a period of two weeks or less. Just before inoculating, a 1 per cent, 

 solution of C. P. glucose (powdered) is added and the medium again 

 heated for one and one-half hours without pressure, cooled to 40 C., 

 and immediately inoculated a few centimeters below the surface with 

 1 c.c. of a twenty-four-hour broth culture of tetanus bacilli which has 

 been subcultured daily in 1 per cent, glucose broth for one to three weeks. 

 No oil or other means is used to secure anaerobiasis; incubation is al- 

 lowed to go on undisturbed for fifteen days at 37 C. The cultures are 

 then filtered rapidly 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 purposes of antitoxin standardization it is usually preserved in a 

 dry state after being precipitated with ammonium sulphate. The yel- 

 lowish, 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. 



1 Jour. Med. Research, 1915, 33, 239. 



