THE NATURE OF HEMOLYSINS 95 



The proteins were dissolved in 5 cc. of salt solution and the solution of antigen 

 mixed drop by drop with continual gentle shaking. 



1 cc. of A plus 1 cc. of cell suspension ++ in 20 minutes 



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



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



Control emulsions of like amounts of solutions of broth with the proteins alone 

 showed no hemolytic power. 



All the above mixtures remained perfectly clear, and were inactivated at 65°C. 

 for thirty minutes. Other combinations were tried with amounts of fat antigen 

 varying from 40 mgm. to 120 mgm. per liter, and with quantities of protein 

 varying from 5 mgm. to 20 mgm. per 40 cc. These mixtures also remained per- 

 fectly clear. 



The influence of the colloidal nature of the broth on these 

 artificial hemolysins was so apparent that we were desirous of 

 seeing whether alterations in the broth would affect any par- 

 ticular changes. To this end the ordinary standard broth was 

 passed through a Berkefeld filter before emulsification with the 

 antigens. A control unfiltered broth of pH 7.1 containing 32 

 mgm. per liter of K salt antigen in amounts of 1 cc. produced 

 total hemolysis of 1 cc. of cell suspension in sixty minutes, 

 whereas the filtered broth containing the same amount of the 

 antigen gave no hemolysis whatever. By doubling the amount 

 of antigen added to the filtered broth the hemolysis appeared 

 and was complete in one hour. This experiment, repeated with 

 the Na salt and with the fatty acid antigen, gave similar results, 

 and appeared to indicate that filtration removed from the broth 

 particles of some material instrumental in hemolysin production. 



xVfter it had been found that inactivation of the B. megatherium 

 lysin could be brought about by various adsorbents, to be men- 

 tioned later, we attempted the same procedure with both the 

 natural and artificial streptolysin. The results appear in table 4. 



The results of inactivation by means of the surface of defatted 

 colon bodies were the same as from starch. Also inactivation by 

 the same adsorbents in the ice box over night instead of at 45°C. 

 gave identical results. In short, the artificial antigen was 

 readily inactivated by these methods but the natural lysm was 

 not. We then tested out the inactivation of the artificial lysin 

 when produced in 10 per cent serum broth. As stated pre- 



