4 L. A. JULIANELLE 



aseptically and centrifuged at high speed for 5 minutes. One c c of 

 the clear supernatant fluid was added to 1 c c of a washed 2.5% horse- 

 blood suspension and incubated at 37 C. for 2 hours, at the end of 

 which time the tubes were read for hemolysis. The concentration of 

 blood attempted to approximate as closely as possible the conditions of 

 the blood plate. 



It was found that no estimable hemolysins were produced in broth 

 cultures within 24 hours. In fact, as will be borne out later, no 

 hemolysins were shown to be present until the sixth day. It may be that 

 the discrepancy in time between plate and broth cultures is explainable 

 on the grounds that in the former case the hemolysins are so concen- 

 trated around each colony as to assert themselves at a conspicuously 

 earlier period ; whereas in the latter case the hemolysins go into solution 

 and become too dilute to have any effect on a suspension of blood cells. 



The next experiment was planned to obtain the curve for the produc- 

 tion of hemolysins. The technic employed was the same as in the 

 preceding experiment, except for one detail. The cultures were seeded 

 into tubes containing 10 c c of the serum broth, and at the end of each 

 day one tube was removed from the incubator and used for the tests. 

 Care was taken to keep the volume of the tubes constant by adding 

 sterile salt solution to repair any loss by evaporation. 



Table 1 shows that hemolysins begin to appear on the sixth day, 

 reach a maximum at the ninth and tenth days, and disappear between 

 the thirteenth and sixteenth days. 



With the period of hemolysin production established, the logical 

 sequence was to determine if possible the source or the cause of the 

 production. It was assumed entirely theoretically that hemolysis is 

 caused by one of the following or perhaps combination of factors : 



1 . Reaction : An increase or decrease in hydrogen-ion concentration 

 sufficient to cause hemolysis. 



2. Tonicity : An increase or decrease in the tonicity of the medium 

 sufficient to cause crenation or laking of the blood corpuscles. 



3. Hemotoxin : A hemolytic substance elaborated and secreted by 

 the bacterial cell, causing hemolysis. 



4. Proteolysis: The production by the bacterial cell of some sub- 

 stance for the utilization of the blood protein. Under this head would 

 be included autolytic products also. 



In order to establish experimentally which hypothesis was correct 

 the following procedure was adopted : Coincidental with testing for the 



