I. MEDIA 625 



flask of alkali solution to the stopper of the carboy of medium. When 

 half-strength medium is used or when the reaction is to be kept acid, 5 

 per cent NaOH is preferred; otherwise 10 per cent NaOH is more 

 convenient. 



c. Growing the pneumococci. There is placed in the carboy of me- 

 dium a stopper which carries: (1) A mechanical stirrer of the usual 

 sealed type; cresol solution is used as the sealing liquid. (2) An inlet 

 tube to which the rubber tube from the flask of alkali solution can be 

 attached. (3) An inlet tube through which can be added the glucose so- 

 lution, defibrinated blood, and culture. (4) A sampling tube through 

 which samples may be removed from the carboy without undue risk of 

 contamination. The stirrer, stopper, and tubing are sterilized separately. 



To the medium are added the glucose solution and about 50 cc. of 

 defibrinated blood. The carboy is then placed on a stand so arranged 

 that the stirrer can be driven by a motor and that the flask of alkali 

 solution can be connected with the carboy. Alkali solution is then added 

 to the medium in an amount sufficient to adjust the reaction to a pH of 

 7.0 or of 8.0 to 8.5 depending upon whether acetyl SSS or the ordinary 

 SSS is to be made. The medium should be thoroughly stirred when the 

 alkali is added. 



Very late in the day about 5.0 cc. of an 8-hour culture of Pneumococ- 

 cus is added to each carboy, the mixture stirred a few minutes, and the 

 organisms allowed to grow overnight. By morning the pH will have 

 fallen to about 4.5. The reaction is then readjusted to pH 7.0 or 8.0 to 

 8.5; the medium requires readjustment the same evening and both 

 morning and evening of the second full day of growth, after which time 

 adjustment of the reaction once a day will suffice. In the early stages of 

 growth, a pH of 8.8 will not kill the culture. Appreciable acid produc- 

 tion ceases after about a week. 



The cultures are kept for about two weeks before starting prepara- 

 tion of capsular polysaccharide. The methods of Heidelberger, Avery, 

 and their colleagues are followed in purifying the substance. 



Blood Broth 



Plain broth or hormone medium to which defibrinated rabbit, horse, 

 or human blood is added to 0.05' to 5 per cent concentration when the 

 medium is cool. Rabbit blood is preferable. 



Inulin Serum Water (Rockefeller Monograph No. 7 36 ) 



For the determination of inulin fermentation by Pneumococcus, the 



