CULTURAL REQUIREMENTS OF BACTERL^ 313 

 METHODS 



Preparation of cultures 



Three types of pneumococci, I, II and IIA, together with a 

 strain of Streptococcus hemohjlicus, were used. The pneumo- 

 coccus strains were each passed through two mice, and the 

 streptococcus through one mouse and stock cultures were made 

 from the heart blood of the second mouse into small tubes con- 

 taining about 1 cc. of sterile human blood. The latter was ob- 

 tained in the usual way from the median basilic vein and trans- 

 ferred from a svTinge to small sterile tubes, each tube containing 

 two or three glass beads. The tubes were than shaken until 

 defibrination was complete, and incubated twenty-four hours to 

 insure steriUty before being inoculated from the heart blood of 

 the mice. After inoculation the tubes were incubated eight to 

 ten hours until smears showed that the organisms were multi- 

 plying, and they were then stored in the ice box. For trans- 

 plants, ordinary meat infusion peptone broth containing 0.1 

 per cent glucose and brought to pH 7.4 to 7.8, were used. A 

 small loopfuU of the blood culture was transferred to the broth, 

 and after incubating eighteen to twenty-four hours, the culture 

 was used, in the case of the pneumococci for the inoculation of 

 a second similar tube of meat infusion broth. These two cul- 

 tures are called respectively the A and B cultures. Experi- 

 mental media were inoculated from the B cultures in the case 

 of pneumococci, and from the A tubes with streptococci. In 

 this way it is believed that the food requirements of the bac- 

 teria remain reasonably constant. The stock culture remains 

 alive for long periods of time, up to six months or more. After 

 being opened repeatedly, old tubes gradually dry up, and such 

 cultures have been transferred to fresh blood tubes, using the 

 same technic, and always passing the strain through one mouse 

 to guard against a gradual change in cultural requirements in 

 spite of the blood media. It is not unlikely that significant 

 quantities of blood may be carried over into the test media with 

 only a single passage intervening, but it certainly is not enough 

 to produce growth on unsuitable media, although it cannot be 

 overlooked in estimating the value of results obtained. 



