THE NATURE OF HEMOLYSINS 99 



centrifugation at 1800 revolutions per minute to remove the 

 adsorbent the fluids were tested for hemolytic power. Those 

 that had been treated with adsorbent were completely inactive 

 while the controls hemolyzed promptly in fifteen and in ten 

 minutes. 



We then tested the natural hemolysin of the B. megatherium 

 to see if it could also be inactivated in the same manner. The 

 culture used was an eighteen hour standard broth growth centri- 

 fu gated at 1800 revolutions per minute and the clear super- 

 natant fluid pipetted off. 0.25 cc. of this hemolysin hemolyzed 

 1 cc. of cell suspension in twenty minutes. The procedure with 

 the previous adsorbents was then repeated with this natural 

 lysin, with the result that the treated portions were found to be 

 completely inactivated while the untreated controls gave com- 

 plete hemolysis in fifteen minutes. In other words we found it 

 possible to inactivate the artificial and the natural lysin by 

 adsorption upon surfaces. Inactivation in this manner can be 

 rapidly effected by heating to 45°C. for one hour, a temperature 

 at which ordinary organisms do not grow, or more slowly by 

 allowing the mixtures to stand in the ice box over night. Appar- 

 ently the pancreatin powder acts, not by breaking up the fats 

 but rather by simple adsorption. Attention has been previously 

 called by others to the fact that pepsin and trypsin destroy the 

 lysin of Ps. pyocyanea. 



It was also found that previous emulsification of the B. mega- 

 therium antigen mixtures with hemoglobin, casein and typhoid 

 protein before their addition to the broth gave results quite in 

 accord with those obtained with the Streptococcus antigen. 



Todd showed, as mentioned previously, that the lysin pro- 

 duced by B. megatherium when injected into annuals gave rise 

 to antilysin. We injected several groups of rabbits, some with 

 natural lysin, others with the artificial hemolysin. These ani- 

 mals were given six injections subcutaneously, three at daily 

 intervals and then, after an interval of four days, three more at 

 daily intervals. On the seventh day after the last injection the 

 animals were bled from the heart and the serums allowed to 

 separate in the ice box over night. The serums were inacti- 



