DIPHTHERIA TOXIN AND ANTITOXIN 483 



especially desirable that the sterilization period of the medium 

 should be as short as possible, to reduce destruction of the ac- 

 cessory factors to a minimum and yet be sufficient to ensure 

 thorough heat penetration. Autoclave sterilization at 120°C. 

 (15 pounds steam pressure) for a period not exceeding thirty 

 minutes has given satisfaction. 



c. Cultivation 



The parent culture of Corynebact. diphtheriae on the Loeffler 

 slants is transplanted through several twenty-four hour genera- 

 tions in tubes containing 10 cc. of bouillon to stimulate maximum 

 pellicle formation The tubes are then used to inoculate small 

 "starter" flasks containing 30 cc. of medium which are also in- 

 cubated for twenty-four hours. The large flasks of broth are 

 now inoculated with the twenty-four hour " starters," allowing 

 one for each large flask. A temperature range of 36° to 38°C. 

 has been found most satisfactory for incubation. Numerous 

 potency tests have demonstrated that at least ten days incuba- 

 tion of the large flasks is necessary to ensure maximum elabora- 

 tion of toxin and a twelve-day period is desirable. As has been 

 shown in a previous publication (Davis, 1918), toxin of appreci- 

 able strength is elaborated by toxicogenic cultures within forty- 

 eight hours. The potency gradually increases to a maximum 

 value about the twelfth day, occasionally sooner. Incubation 

 for an additional period of two weeks, or four weeks altogether, 

 shows no deterioration of the final toxin. That this behavior 

 is not confined specifically to the peptone employed is proved 

 by the fact that, with the procedure as given above, Witte's 

 peptone permits of similar results. In this case, toxin of maxi- 

 mum potency is obtained in the large flasks, only after a two- 

 weeks' incubation. 



When cultivated in plain bouillon under the optimal conditions 

 already described, Corynebact. diphtheriae causes an initial increase 

 in the hydrogen ion concentration of the medium. This is soon 

 followed by a steady decrease until, apparently, a limiting alka- 

 line reaction is attained. The following table, taken from the 



