92 J. T. CONNELL AND L. E. HOLLY 



The pH of the standard medium was found to change to 7.0 

 at eight hours returning to about the original reaction at from 

 eighteen to twenty hours. The pH of the medium containing 

 glucose rose to 5.58 at twelve hours and returned to 6.10 at 

 twenty hours. 



The striking feature in the chart is seen to be the disadvantage 

 of even small amounts of glucose for the production of hemol- 



TABLE 1 



A. Broth containing 40 mgm. per liter of K salts antigen. 



B. Broth containing 40 mgm. per liter of Na salts antigen 



C. Broth containing 40 mgm. per liter of fatty acid antigen 



1 cc. of A plus 1 cc. of 2 per cent rabbit cell suspension ++20 minutes 



1 cc. of B plus 1 cc. of 2 per cent rabbit cell suspension ++20 minutes 



1 cc. of C plus 1 cc. of 2 per cent rabbit cell suspension ++25 minutes 



+ + indicates complete hemolysis. 



— indicates no hemolysis. 



The above mixtures remained perfectly clear. They were completely inacti- 

 vated upon heating at 65°C. for thirty minutes. 



ysin. Not only was the hemolysin weaker but of much shorter 

 duration, though the specimens were centrifugated in the same 

 centrifuge for the same length of time. 



We also determined the strength and duration of hemolysin, 

 starting with a pH of 7.8 in the standard broth, but found no 

 striking difference from that of the 7.2. In growing these cul- 

 tures and in testing the strength of the hemolysin and the time 

 in which it appeared, two points were impressed upon us, first, 

 that noted by other workers, that the hemolysin occurs earlier 

 and is much stronger if the culture from which the transplant is 

 taken is young, preferably not over twelve hours old, second, 

 that using 0.1 cc. of culture for the transplant instead of a loop- 

 ful caused the hemolysin to appear earlier in the incubation. 



In attempting to produce an artificial hemolysin the standard 

 medium was used. We omitted the serum because it was found 

 that it distinctly interfered with hemolysin production, just as 

 it also interfered with the lytic power of natural hemolysin if 

 added after the lysin appears. We believe that the function of 

 the serum in growing Streptococcus is to insure rapid and abun- 

 dant growth, which is apparently essential for the production of 



