Peptolytic Enzymes of Hemolytic Streptococci. 235 



solutions may be obtained without difficulty. All procedures were 

 sterile. Beef infusion has been used as a base for the media. It 

 has been prepared in the usual way except for the addition of 0.1 

 per cent, dextrose. After the ingredients were dissolved in the 

 infusion, the broth was titrated to Ph 8.5 or 9.0 and boiled until 

 the phosphates were precipitated out. It was then filtered, ad- 

 justed to P H 8.0 and autoclaved. Precipitation during the steri- 

 •lization was avoided by the preliminary boiling in alkaline solu- 

 tion. The glucose was apparently not decomposed to an extent 

 sufficient to interfere with the growth of the bacteria. The acidity 

 developed in the growth of the culture (about P H 6.5) causes the 

 spontaneous agglutination of the streptococci, yet it is not de- 

 trimental to the production or the life of the enzyme. 



Flasks of 6 L. volume were seeded with a broth culture of 

 Strain Py 3, a beta type streptococcus of human origin. After 12 

 hours, the clear supernatant broth was decanted by siphon and 

 the agglutinated streptococci were centrifuged, washed repeatedly 

 in physiological salt solution and resuspended in 15 c.c. of Af/15 

 phosphate of P H 7-0. They were then dried in an agate mortar in 

 vacuum after the addition of 2 grams of powdered glass. Grinding 

 was carried out until there was a minimum amount of Gram 

 positive material in the smear. When they were well macerated, 

 the bacterial mass was taken up in 50 c.c. of distilled water, and 

 allowed to stand under toluol at 10 degrees C. for 12 to 24 hours. 

 The fluid pipetted from beneath the layer of toluol was centri- 

 fuged. It contained the active enzyme and was sterile. The 

 sediment contained no demonstrable peptolytic substance. 



The substrate used in the experiments was a 1 per cent, solu- 

 tion of peptone prepared especially for this work by Fairchild 

 Bros, and Foster; this peptone contained 12.5 per cent, nitrogen 

 of which approximately one fifth could be determined as amino 

 nitrogen according to Van Slyke. The solutions of peptone were 

 sterilized in the Arnold sterilizer. To determine the effects of 

 H-ion concentration, heat and enzyme concentration series of 

 20 c.c. volumetric flasks were prepared with the enzyme, M/15 

 buffer mixtures (phosphate or citrate) and sufficient substrate to 

 bring the final concentration to 1.0 per cent. Duplicate flasks 

 with boiled enzyme served as a control for each determination 



