452 HEPBURN AND ST. JOHN— BIOCHEMICAL STUDIES 



In a few experiments, plates of plain nutrient gelatin were also sown 

 with the pitcher liquor, in order to detect the presence of liquefying 

 (proteolytic) micro-organisms. 



To test for the Hberation of tryptophane and the formation of indol, 

 a hquid medium was prepared, containing 0.4 gram protein (from aleuro- 

 nat), 20 cc. 0.1 iV sodium hydroxide solution, and 80 cc. of the stock 

 solution of inorganic salts already mentioned. The resulting suspension 

 of protein was placed in tubes (10 cc. to a tube), and was sterilized. 

 The protein gave a purple color with glyoxylic acid and sulphuric acid 

 (reaction of Hopkins and Cole), and therefore contained a tryptophane 

 group in its molecule. 



The formation of basic compounds (e.g. ammonia) from simple or- 

 ganic compounds of nitrogen was also studied, using as substrates: — 

 glycocoU (an amino acid), acetamide (an acid amide), asparagin (which 

 is both an amino acid and an acid amide), and ammonium lactate (an 

 ammonium salt of an organic acid). For this study, recourse was had 

 to solid media, prepared by addition of one of the compounds just named 

 to the stock agar. One percent, of asparagin was used, and the other 

 compounds in molecular concentration equal to that of the asparagin. 

 One-half percent., by volume, of a two percent, solution of rosolic acid 

 in sixty percent, alcohol was added to each medium as an indicator. 

 These media were always sterilized by the discontinuous method. The 

 production of basic compounds by bacteria growing on these media 

 was indicated by a red color of the medium beneath and surrounding the 

 colony. Sterile plates of the rosolic acid media were always poured as 

 controls, to be used in determining the changes in color in the experiments 

 proper. 



In inoculating all of the special media just described, 1 cc. of the 

 undiluted pitcher liquor was sown into each plate or tube. 



Lactose bile-salt broth was used to test for the presence of members 

 of the colon -aerogenes group of bacteria. 



The temperature of incubation was always 37°C., except for the 

 gelatin plates which were kept at 20° C. 



Sources of the Pitcher Liquor Examined 



In the majority of the experiments, the liquor was obtained either 

 from unopened pitchers or from active, open pitchers containing insect 

 remains. A few experiments were conducted on liquor from pitchers 

 partly opened and not yet invaded by insects. The liquor from each pitch- 

 er was studied as a separate experiment. 



