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



active. Bulloch and Hunter showed that while a culture of 

 Ps. pyocyanea is highly alkaline yet when the pH is brought back 

 to near the neutral point it is still hemolytic though less so. 

 This statement has been denied by Jordan who maintains that 

 the hemolysis in this case is due to alkali. 



In view of the light that has been thrown by Warden's work 

 upon the composition of organisms, particularly in respect to 

 their fatty complexes, it seemed logical to us that these fat 

 antigens should be investigated as to the possibility of their 

 playing a part in hemolysin production. This idea seemed 

 particularly attractive because those organisms such as Strepto- 

 coccus and B. inegatherium which produce hemolysin early in 

 their growth, and which also yield the most powerful hemolysins, 

 are Gram positive, and the Gram positiveness of an organism is 

 known to depend upon the presence of unsaturated fats. It is 

 also well known that the unsaturated fatty acids and their salts 

 are much better hemolytic agents than the non-volatile, satu- 

 rated acids. We are aware, also, of the fact that if the fatty 

 acid complexes should play a part in hemolysis the action would 

 not be that following their simple suspension in salt solution 

 because of the factors of a colloid nature introduced by the 

 broth menstrumii. 



With these ideas in mind we decided to see if it were possible 

 to produce an artificial hemolysin, using the fat complexes which 

 were characteristic of the organism whose lysin we were trying 

 to imitate. In order to do this ideally we realized that we must 

 copy as closely as possible the condition existing in the medium 

 at the tune the hemolysin is at its height. The hemolysin first 

 studied was that of the Streptococcus. The medium used 

 throughout this work, called the standard medium, consisted of 

 a veal infusion broth containing 2 per cent bactopepton and 

 0.5 per cent NaCl. The pH was varied from 7.1 to 7.9. In 

 growing the Streptococcus organisms 10 per cent rabbit serum 

 was added before inoculation. The cells used in the hemolytic 

 experiments were fresh rabbit cells washed four times with 0.85 

 per cent salt solution, and made up in a 2 per cent salt solution 

 suspension. 



