A comparative method for testing the enzymes of living hemolytic streptococci. I. Lipase.

Enzymes capable of digesting proteins, carbohydrates and fats have been demonstrated for the hemolytic streptococcus by Stevens and West who extracted these ferments from dead organisms by a process of grinding. This method is not available for quantitative determinations because of the difficulty of complete extraction by the grinding process. Nor can comparative determinations be made of different strains of the streptococci in culture media because of the variability of different strains in their rapidity of growth and because of the modifying factor of the media itself. It is possibible, however, by the use of “indifferent” suspending fluids such as Locke's solution with 0.1 per cent gelatin or a solution containing 1.0 per cent sodium chloride and 0.05 per cent calcium chloride with 0.1 per cent gelatin and by quantitative determinations of bacterial concentration by means of the Gates turbidimeter, to compare the ferment action of a single organism under varying conditions or of different organisms under identical conditions.

The usual method of determining lipolytic activity by other workers has been to titrate with sodium hydroxide the acid produced in the enzyme substrate mixtures. Dietz has shown, however, that enzyme preparations of different activity, as measured by their velocity constants, reach the same equilibrium point if allowed to continue to completion. Within certain limits, when there is an excess of substrate present, the velocity of the reaction is in direct linear proportion to the quantity of active enzyme present. These facts together with the difficulty of titrating minute quantities of acid produced by small quantities of bacteria suggested the possibility of measuring the velocity of the reaction between certain definite hydrogen ion concentrations.

Standard color tubes were prepared by taking up heat killed organisms, in the same concentration as in the test suspensions, with Clark's buffer solutions at pH levels of 8.0, 7.6 and 7.2 with phenol red as indicator.