1913] Jesse A, Sanders and Clarence E. May 375 



General method. We studied the production of indol in the 

 tryptic digestion of casein, the tryptophan yield from which is known 

 approximately. We used small amounts (i. 0-1.75 gm.) of casein, 

 digesting them with strong pancreatin Solutions free from trypto- 

 phan, as determined by negative response to the glyoxylic-sulfuric 

 acid test. The digestive periods differed in length. At the end 

 of each, the mixture was neutralized, reinforced with neutral salts, 

 and then made alkaline to one of several degrees of alkalinity. 

 After sterilization in an autoclave, the mixtures were inoculated 

 with mixed fecal bacteria from the stools of an individual on a 

 mixed diet. No bacteria were isolated for purposes of Identifica- 

 tion. The organisms were allowed to develop in the digestion 

 mixtures at 37° C. for periods of different length. The reaction 

 mixtures, after neutralizing and making them alkaline with a known 

 amount of alkali, were distilled with steam until about 700 c.c. of 

 distillate had been obtained. The distillate was diluted to 1,000 c.c. 

 and an aliquot portion was treated with 0.2 per cent. sodium nitrite 

 and conc. sulfuric acid solutions. A control Solution containing 

 0.25 per cent. of indol (Kahlbaum) was treated in the same manner. 

 Each nitroso-indol Solution was then allowed to stand until the 

 maximum color developed.^ We used the Wolf colorimeter for 

 the tinctorial comparisons, and found that even with the small 

 amounts of indol obtained (see figures later) an error of 3 per cent. 

 was very easily detected by difference in the intensity of the resulting 

 colorations. 



We experienced some difficulty, at first, in mixing definite 

 amounts of the indol Solution with the nitrite and sulfuric acid Solu- 

 tions, and water, which would give uniform shade and intensity of 

 color. Later we obtained very constant results by taking an aliquot 

 portion of the indol Solution, adding the nitrite Solution and enough 

 water to fill the cylinder of the apparatus almost to the 100 mark, 

 then adding the conc. sulfuric acid Solution and sufficient water to 

 fill to the mark. On mixing uniformly, a very faint though distinct 



' Moraczewski : Zeit. f. physiol. Chem., 1908, Iv, pp. 42-47; Chem. Ahstr., 

 1908, ii, p. 2578. (Colorimetric determination of indol in feces. The abstract 

 of the original article contains an error that should be corrected: the sodium 

 nitrite Solution has a concentration of 0.2 per cent. instead of 2.0 per cent. 

 See also, Levene and Rouiller : Jour. of Biol. Chem., 1906-7, ii, p. 481. A 

 bromine-tryptophan colorimetric method for the determination of tryptophan.) 



