37^ Determination of Tryptophan [April 



color developed which reached its maximum intensity in about 

 half an hour. 



Details of the experiments. About 500 c.c. of skimmed milk 

 were diluted in a precipitation jar with five volumes of water. The 

 casein was precipitated by the addition of 12.5 c.c. of 10 per cent. 

 acetic acid Solution. The casein was repeatedly washed with water 

 by decantation and then dissolved in Standard sodium hydroxid Solu- 

 tion (enough to dissolve the casein without leaving a large excess of 

 alkali). The liquid required about 150 c.c. of n/2 sodium hydroxid 

 Solution to produce a permanent alkalinity, using azolitmin paper as 

 indicator. After dilution to a definite volume and filtration, two 

 nitrogen determinations were made by the Kjeldahl method. It was 

 found that each 100 c.c. of the Solution contained 0.8755 ö"^- o^ 

 casein. Of the remaining Solution 500 c.c, were neutralized with 

 phenolthalein as the indicator and treated with 0.4 gm. of sodium 

 carbonate for each 100 c.c. volume of the neutral liquid. Then 

 25 c.c. of a saturated pancreatin (commercial) Solution and xylene, 

 as a preservative, were added. Incubation was continued at 2)7° C. 

 for 24 hours, when an equal portion of the pancreatin Solution was 

 added; a third portion was added at the end of 48 hours. The 

 incubation was then continued for forty-four days. Steam was 

 passed through the alkaline Solution to remove the xylene. The 

 digestion was apparently complete ; common tests for tryptophan 

 indicated its presence. The mixture was neutralized and reinforced 

 with neutral salts, such as Hopkins and Cole used in their work — 5 

 gm. Rochelle salt, 0.2 gm. ammonium phosphate and o.i gm. 

 magnesium sulfate, per liter. No gelatin was added. The total 

 volume was now made up to one liter and divided into four equal 

 portions. Each portion contained cleavage products corresponding 

 to 1.0944 gm. of casein. 



Portion A was sterilized in an autoclave and inoculated with a 

 24 hour slant agar growth of intestinal bacteria.^ Portion B was 



• The method of inoculation was as follows : The bacteria were grown first 

 in ordinary broth inoculated from feces. After 24 hours, agar slants were made 

 in the usual way. When these were 24 hours old, the organisms were detached 

 by means of sterile water and a sterile wire, and the liquid containing them was 

 poured directly into the flask to be inoculated. In order to establish a definite 

 degree of alkalinity, the digestion mixtures, after steam distillation, were 



