^96 J. T. CONNELL AND L. E. HOLLY 



vioiisly it is difficult to produce artificial lysin in the presence of 

 serum, but it is merely a question, of adding larger amounts of 

 antigen to serum-broth than are required to render standard 



TABLE 4 



A. Twelve hour centrifugated Streptococcus culture 



B. Artificial lysin with 60 mgm. K salts Strep, antigen 



C. Artificial lysin with 60 mgm. Na salts Strep, antigen 



D. Artificial lysin with 60 mgm. acids Strep, antigen 



1 cc. of A plus 1 cc. cell suspension ++ in 10 minutes 



1 cc. of B plus 1 cc. cell suspension ++ in 12 minutes 



1 cc. of C plus 1 cc. cell suspension ++ in 14 minutes 



1 cc. of D plus 1 cc. cell suspension ++ in 15 minutes 



To 5 cc. of A, B, C, and D there was added a definite quantity of starch sus- 

 pension and the tubes were placed in the water bath at 45°C. for 1 hour, together 

 with control tubes without the starch suspension. After centrifugation of the 

 starch the lysins were tested as follows: 



Al, Bl, CI, Dl, represent the lysins treated with starch 



A2, B2, C2, D2, represent the lysins untreated 



1 cc. of Al plus 1 cc. cell suspension ++40 minutes 



1 cc. of Bl plus 1 cc. cell suspension — 



1 cc. of CI plus 1 cc. cell suspension — 



1 cc. of Dl plus 1 cc. cell suspension — 



1 cc. of A2 plus 1 cc, cell suspension ++18 minutes 



1 cc. of B2 plus 1 cc. cell suspension ++ 20 minutes 



1 cc. of C2 plus 1 cc. cell suspension ++ 20 minutes 



1 cc. of D2 plus 1 cc. cell suspension ++ 20 minutes 



broth hemolytic. Much larger quantities of antigen can be 

 added to serum-broth, if added in small amounts at a time, 

 without clouding, than to standard broth. As a result of this 

 experiment it was shown that with a Streptococcus serum-broth 

 culture fourteen hours old, centrifugated, 1 cc. of which hemo- 

 lyzed 1 cc. of cell suspension in twenty minutes and with an 

 artificial K salts hemolysin containing 300 mg. antigen per liter, 

 1 cc. of which hemolyzed 1 cc. of cell suspension in five minutes 

 attempted inactivation with the adsorbing substances in the ice 

 box and at 45°C. produced no such effect, i.e., neither natural 

 or artificial hemolysin was inactivated. This seemed to justify 

 the conclusion that the reason we were unable to inactivate the 



