THE NATURE OF HEMOLYSINS 97 



natural streptotysin was because of the presence of the senim 

 and not because of any pecuHarity of the lysm itself. 



Several attempts were made to produce antilysm by injections 

 into animals of the natural streptolysin, the artificial lysin and 

 streptococci themselves, but the results were unsatisfactory. 

 This work is being continued and it is hoped we may report on 

 it later. This difficulty has been commonly encountered by 

 other workers (Besredka, 1903). 



The second microorganism selected for observation was B. 

 megatherium. We chose this bacterium because from the work 

 of Todd (1901) it was known to produce strong hemolysin in a 

 comparatively simple medium, and give rise to good antilysin. 

 The hemolysin of this organism is also very stable which is an 

 advantage over streptolysin. The standard medium was used 

 in all the experiments. We found that the lysin appeared as 

 early as the tenth hour and lasted for weeks. The pH of the 

 cultures, determined at two hour intervals was found to change 

 to 7.2 or 7.3 when the original pH of the medium was 7. 8. The 

 addition of 10 per cent of rabbit serum to the standard medium 

 before inoculation gave very much weaker hemolysin than when 

 the standard broth was used alone. 



The antigen used for the production of the artificial hemolysin 

 was the fat complex found by Warden, Connell and Holly to be 

 characteristic for the B. megatherium. The various solutions of 

 the antigen were made in the sam^e manner as those used in the 

 work on the streptococcus, and the emulsification of the antigen 

 with the broth was carried out similarly. Tests showed that 

 artificial hemolysin containing 40 mgm. K salt antigen per liter 

 gave complete hemolysis, in 1 cc. doses, of 1 cc. of red cell suspen- 

 sion in thu-ty minutes, that of Na. salt antigen of equal strength 

 gave complete hemolysis in the same length of time; and that 

 containing the acid antigen produced, in the same dose, com- 

 plete hemolysis in twenty-five minutes. These artificial lysins 

 were inactivated at 65°C. for thirty minutes. 



The following table is an example of the effect of pH upon the 

 hemolytic power, and upon the inactivation of the K salt and 

 acid antigen of the megatherium. 



