HYDROGEN ION CONCENTRATION AND PROTEOLYSIS. 167 



means of a platinum loop. This process of subinoculation was continued 

 from one flask to another every twenty-four hours, until the fifth flask 

 had been inoculated and incubated. From this flask a platinum loopful 

 of the culture was transferred into a dilution flask, which contained 500 

 cubic centimeters of the same medium. At the same time, three agar 

 plates were made by placing a loopful from the dilution into the first agar 

 tube, and from the first agar tube to the second, and from the second to 

 the third. After these plates had developed for forty-eight hours at 37° C, 

 one entire colony of a medium size was selected and transferred into the 

 sixth flask of the above series by means of a small platinum spatula. This 

 flask had been placed in the incubator for twenty-four hours at 37° C. 

 before using it for inoculation. 



Inoculation of Media. 



Each of a series of flasks (1 to 13), already prepared and containing 

 399 cubic centimeters of Medium IV. of various Ph, was inoculated with 

 1 cubic centimeter of the acchmatized culture. These flasks were then 

 placed in the incubator at 37° C. Waxed paper had been placed over the 

 cotton plug and bound by a rubber band for protection against evapora- 

 tion. Samples of 50 cubic centimeters for determinations were removed 

 from time to time by means of a sterilized pipette. 



RESULTS. 



Testing the Vitality of the Organism. 



Hydrogen ion concentration has a decidedly inliibitory and lethal 

 influence upon microorganisms; accordingly, a crude test was introduced 

 to follow this. Streak cultures on an agar plate were made at each 

 determination of the hydrogen ion concentration. 



— no growth. 



+ positive growth. 



