I9I3] Jcsse A. Sanders and Clarence E. May 377 



treated with sodium carbonate (0.4 gm. per 100 c.c), sterilized and 

 inoculated with some o£ the same 24 hour growth of bacteria. 

 Portion C was made alkaline with sodium carbonate to 0.8 per cent. ; 

 portion jD to i.o per cent. Both were inoculated as in A and B. 

 After four days of incubation, the four flasks were reinoculated 

 with fresh 24 hour cuhures of the bacteria and again incubated. 

 After eight days' incubation, flask A was removed from the oven. 

 Active indol-producing bacteria were present in the mixture. The 

 original putrefaction mixture, neutral to litmus and giving a faint 

 odor of indol, was made alkaline with sodium carbonate (to 0.4 per 

 cent.). With steam distillation, all the indol passed into the first 

 300 c.c. of distillate. 



Determination of indol. A Standard indol Solution was made 

 by dissolving 0.25 gm. of the pure substance in a liter of water. 

 Twenty-five c.c. of the original distillate from flask A, diluted to 

 about 90 c.c, were treated with 10 drops of a 0.2 per cent. sodium 

 nitrite Solution, and six drops of conc. sulfuric acid Solution, diluted 

 to 100 c.c, and mixed uniformly. The liquid was allowed to stand 

 until the maximum rose-red color of the nitroso-indol developed, 

 when it showed the same intensity of color as that produced by 1.3 

 c.c. of the Standard indol Solution diluted in the same manner to 

 100 c.c The total distillate contained 3.9 mg. of indol. In flask 

 B, incubation was continued for nine days after the second inocula- 

 tion. At the end of that time, indol-producing bacteria were still 

 active. The reaction-mixture being distinctly alkaline, no alkali was 

 added prior to steam distillation. All the indol appeared in the 

 first 650 c.c of distillate, which was diluted to 700 c.c. and thor- 

 oughly mixed. Of this Solution 50 c.c. contained as much indol as 

 1.2 c.c. of the Standard Solution. The total indol content of flask 

 B was 4.2 mg. Flask C was incubated twenty-six days. The 

 mixture smelled strongly of indol and was alkaline in reaction. No 

 additional alkali was added. A distillate of 1000 c.c. was obtained, 

 each 50 c.c. of which contained as much indol as 2.05 c.c. of the 

 Standard indol Solution. The total content of indol in this putre- 

 faction mixture was 10.25 mg. Flask D was incubated twenty-five 



titrated with n/io hydrochloric acid Solution and then neutralized quantitatively. 

 The required weight of sodium carbonate was then added to the neutral Solu- 

 tion to give the desired alkalinity. 



