The peptolytic enzymes of hemolytic streptococci;methods

Abstract

During the study of the virulence of hemolytic streptococci, it has been necessary to understand the action of the cocci on certain protein fractions. On account of the structure of these bacteria, the methods required to obtain the active proteolytic substance from the bacterial cell, and to accomplish the sterilization of the solution containing the enzyme were at first consuming and laborious. With the procedure outlined active 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 P_H 8.5 or 9.0 and boiled until the phosphates were precipitated out. It was then filtered, adjusted to P_H 8.0 and autoclaved. Precipitation during the sterilization was avoided by the preliminary boiling in alkaline solution. 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 detrimental 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 M/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.