28 
Two cc. of 2X hydrochloric acid was then added to tube (2), after which 
it was heated to boiling for a few minutes. The tube was then cooled, 
and the contents made alkaline with sodium hydroxide, when the char- 
acteristic red coloration of phenolphthalein was obtained. 
Action of bacteria . — As already pointed out in the above, the fresh 
urine of the guinea pig that had received the phenolphthalein intra- 
peritoneally had the same color as the normal urine of the animal, vary- 
ing from colorless to a decided }mllow. When preserved under toluene 
several specimens of this urine retained their normal color. When 
kept without toluene, however, they gradually took on the purplish- 
red color characteristic of phenolphthalein in alkaline solution. 
That the development of the red color of this urine on standing is 
really due to the action of micro-organisms is evident from the follow- 
ing observation: 
On October 31 two tubes, (1) and (2), were prepared, each containing 
5 cc. of the fresh urine of the pig that had received phenolphthalein. 
Both tubes were plugged with cotton wool and (2) was boiled for sev- 
eral minutes. Both tubes were then allowed to stand at ordinary tem- 
perature. On November 2 the contents of the tube (1) were found to 
have the red color characteristic of phenolphthalein, whereas the con- 
tents of tube (2) were colorless. On November 6 the red color of (1) 
had greatly increased in depth, whereas (2) still remained colorless. 
It is evident from the results of these observations that the bacteria 
normally present in the urine of the guinea pig have the power of 
decomposing the compound of phenolphthalein present in the urine. 
That the difference here observable in the color of the two tubes is not 
due simply to an increase in the alkalinhy of the urine is shown by 
the fact that the phenolphthalein compound present in the urine is not 
hydrolyzed even by strong alkali. 
Action of ferments . — The fact that the phenolphthalein compound 
produced in the animal organism can be hydrolyzed by certain bacteria 
suggested that possibly it could also be hydrolyzed by the unorganized 
ferments. Up to the present only a few observations bearing on this 
point have been made. These are as follows: 
One cc. of the fresh urine containing the phenolphthalein compound 
was mixed with 5 cc. of a clear lipo^tic extract of the liver of a guinea 
pig, prepared according to the methods previously described by Kastle, 
Johnston, and Elvove, a and the mixture kept at 40° C. for four hours. 
It was then made alkaline with sodium hydroxide. No pink or red 
coloration was observable. This experiment was repeated, using the 
fresh aqueous extract of the liver of the guinea pig with like negative 
results. Apparently, therefore, the ferments of the liver can not 
effect the hydrolysis of the compound of phenolphthalein produced in 
«Am. Chem. Jour., XXXI, p. 525. 
