HYDROLYSIS OF HIGHER FATS IN EGG-SECRETION. 73 



V. 



With this idea in mind, I prepared a special set of tubes, using 

 two volumes of egg-secretion, one volume of either neutral olive 

 or whale oil, and one volume of a neutral litmus solution. The 

 tubes were " chloroformed " as before, then stoppered and shaken. 



The secretion used in these tests was made with every precaution 

 possible, and I kept a microscopic check on each tube. In a few 

 instances I could find no organisms whatever either at the begin- 

 ning or at the end of the period of digestion; in other cases the 

 number was too slight to account for the results, since control 

 tubes, deliberately infected to the point of cloudiness, required 

 forty-eight hours to produce a barely noticeable reduction of the 

 litmus, whereas in the oil-secretion digests the reduction always 

 began instantaneously. 



Tubes without chloroform became turbid within forty-eight 

 hours and developed the unmistakable rancid odor. In the tubes 

 with bacterial growth inhibited no rancid odor could be noticed on 

 account of the masking effect of the chloroform. This necessi- 

 tated some other indicator for the presence of oleic acid. 



The test finally chosen was based on a statement of Hammar- 

 sten's ('12), and depends on the fact that oleic acid in the presence 

 of cane sugar and sulfuric acid develops a purple color. The usual 

 method was to place a drop of the digest on a slide, to mix with 

 this a drop of saturated solution of cane sugar, and to add from 

 2 to 5 drops of concentrated sulfuric acid. 



Care must be taken not to add the sulfuric too quickly, or to use 

 too much, for if the sugar breaks down suddenly, the carbon set 

 free obscures the reaction in the oleic acid. With these precau- 

 tions, however, the test is delicate and very reliable. The change 

 in the oleic-acid globules can easily be followed under the micro- 

 scope. It begins as a slight discoloration, succeeded by pink- 

 rose and finally a deep purple. At 20 C. the reaction is slow 

 with freshly formed oleic acid, but proceeds more rapidly after the 

 oleic has been exposed for some time to the air. 



By this method it was possible to demonstrate the presence of 

 oleic acid in the digests in which chloroform had masked the rancid 

 odor. Moreover, the concentration of oleic acid, as shown by the 

 number, size, distribution, and depth of color of the purple glob- 



