122 HEPBURN— BIOCHEMICAL STUDIES OF 



Several of the colonies were studied separately (a) by stained 

 smears, and (b) by transfers to lactose bile salt broth. All the 

 microorganisms were rod-like, and a few of them contained spores ; 

 none of the transfers developed gas ; therefore it may be concluded 

 that members of the family Bacteriacecc, other than the colon- 

 aerogenes group were present. 



The liquor in an old pitcher, which was becoming brown at the 

 top, gave a count of 104,000 bacteria per c.c. 



Liquefaction of Gelatin. — The liquor from each of two pitchers 

 was sown on nutrient gelatin ; the bacteria grew and completely 

 liquefied the gelatin in forty-eight hours. 



Tests for the Colon-acrogenes Group. — In two experiments, i 

 c.c. of liquor from a single pitcher was sown in lactose bile salt 

 bouillon ; on incubation at 37° C, gas developed within seventy-two 

 hours, showing the presence of organisms of this group. A com- 

 posite sample of liquor, collected from several pitchers, was sown 

 in this medium in several successive dilutions, the greatest dilution 

 being i : 10,000; gas developed in even the greatest dilution within 

 seventy-two hours, therefore at least 10,000 organisms of the colon- 

 aerogenes group were present in each c.c. of the liquor. 



Certain special media were used in the study of the power of 

 the bacteria to digest proteins, and to decompose simple compounds 

 containing carbon and nitrogen. In the preparation of these media, 

 the directions of Crabill and Reed^^ were follow^ed with modifica- 

 tions. A stock solution of inorganic salts, containing magnesium, 

 ferrous, potassium, chloride, sulphate, and phosphate ions, was 

 prepared as directed by these authors, and was used in the media. 



The liquor from each pitcher was studied separately in all the 

 experiments described below. The temperature of incubation was 

 always 37° C. 



Production of Tryptophane and Indol from Protein. — Protein, 

 obtained from aleuronat, gave a purple color with glyoxylic acid 

 and sulphuric acid, and therefore contained a tryptophane group. A 

 medium, containing 0.4 gram protein, 20 c.c. o.i A^ sodium hy- 

 droxide solution, and 80 c.c. of the stock solution of inorganic salts, 

 was prepared and sterilized. One c.c. of pitcher liquor was sown 



11 Crabill and Reed, Biochcm. Bull., 1915, IV., 30-44- 



