THE NATURE OF HEMOLYSINS 93 



lysin, and that the colloidal property of the serum which inter- 

 feres with the action of the streptolysin is undoubtedly destroyed 

 by the growth of the organism. We shall show further on that 

 serum added to a medium in which an organism {B. megatherium) 

 produces strong lysin without it, interferes markedly with lysin 

 production. 



The antigen used was the fat complex, in the form of the fatty 

 acids and their salts, found by Warden to be characteristic for 

 the Streptococcus. The sodium and the potassium salts of the 

 com.plex were made up in alcoholic solutions of such strengths 

 that 1 cc. contained respectively 10 mgm. and the solution of the 

 fatty acids such that 1 cc. contained- 20 mgm. and consequently 

 the amounts of alcohol necessary to add to secure the concen- 

 tration of antigen desired was never sufficient to cause a change 

 in the appearance of the broth or to have any hemolyzing effect 

 on the red cells. The antigens were added by means of 1 cc. 

 pipettes graduated in hundredths and thoroughly mixed with 

 the broth, taking care to avoid foam. The amounts used varied 

 between 32 mgm. and 120 mgm. per liter, these quantities 

 apparently having no appreciable effect on the pH. We noted 

 in some of the broth that clouding appeared after about 45 

 mgm. per liter had been added, whereas other broths remained 

 clear with 60 mgm. per liter. One factor in this regard appeared 

 to be the color of the broth — the darker the color the more anti- 

 gen it would take up without clouding. Another important 

 observation was the variation in the amount of antigen per liter 

 required to make the broth hemolytic, in some instances 30 

 mgm. sufficing, in others 50 mgm. These differences were found 

 to be due to slight variations in the manner of emulsifying and 

 in the time the mixtures were allowed to stand. Table 1 is 

 a specimen protocol of the hemolytic power of the artificial 

 emulsions. 



Table 2 gives an example of the effect of pH upon the hemo- 

 lyzing power and the temperature of inactivation of artificial 

 emulsions made with the K salt and fatty acid antigens. 



We realized that the broth containing the natural hemolysins 

 must be quite different from the artificial emulsions we were 



