Pneumococcus Grouping on a Thousand Cases. 369 



late, is slight but definite with the pneumococcus on blood plates. 

 Brown 2 in his study upon the hemolyzing properties of the strep- 

 tococcus, ascribes a hemolyzing action to the pneumococcus simi- 

 lar to that shown by his alpha type of streptococcus. As evident, 

 considerable lack of agreement exists among different authorities 

 concerning hemolysin production by the pneumococcus on blood- 

 culture plates. The question remains open for further investiga- 

 tion. 



Fortunately, fishings of colonies from this particular strain had 

 been made and cultured upon blood-agar slants. In order to de- 

 termine if hemolysis is a common property of the pneumococcus, 

 under certain circumstances, tests were performed under varying 

 conditions with this, and other immunological types of the pneu- 

 mococcus isolated at that time. A series of preliminary tests 

 were first carried out as a guide in studying the problem. The 

 results of these tests showed that under certain conditions the 

 pneumococcus colony produced a hemolysis of the cells in the 

 immediately surrounding blood-agar medium. The principal 

 tests undertaken concerned in particular the question of medium 

 reaction and the influence of temperature upon possible hemolysin 

 production. The medium was prepared from a meat-infusion 

 broth (500 gm. to a liter), to which was added 1 per cent, peptone 

 (Fairchild's), and 2 per cent, shredded agar. The reaction of one 

 portion was adjusted to P H 7.0 to 7.1, and the other to P H 8.0 to 

 8.1. It was tubed in 6 c.c. and 12. c.c. amounts and sterilized 

 in the autoclave at 15 pounds' pressure for 30 minutes. To the 

 agar medium at the time of plating the organism, sufficient freshly 

 drawn, defibrinated, human blood was added to produce a 5 per 

 cent, concentration in Petri dishes (9 cm. diameter), and the whole 

 evenly distributed. By using different amounts of the medium, 

 or slanting the dish, the poured inoculated medium gave layers 

 varying from 0.5 mm. to 3.0 mm. in depth. Pneumococci repre- 

 senting all groups were grown on slanted 5 per cent, human blood 

 beef-extract agar, and used for plating purposes after two to four 

 days' incubation. A small amount of growth was removed and 

 evenly suspended in plain broth. Inoculations were made di- 

 rectly from this into the special agar medium, poured, and mixed 



2 Monographs of the Rockefeller Institute for Medical Research, 1919, No. 9 

 P- 23- 



