114 HEPBURN— BIOCHEMICAL STUDIES OF 



columns, were inserted into the newly opened pitcher ; the cotton 

 plug was introduced, and the pitcher and its contents were shaken 

 thoroughly at intervals during one or more days, taking care not to 

 wet the cotton and thereby lose liquor ; the liquor was finally re- 

 moved and used in digestion experiments. 



The volume of liquor secreted by a single pitcher was always 

 so small that liquor could not be obtained from the same pitcher 

 both before and after stimulation. Since two pitchers rarely ma- 

 tured on the same plant at the same time, it was impossible to make 

 a comparative study of the liquor from both non-stimulated and 

 stimulated pitchers of the same plant. While the differences, due 

 to individual plants, could not be entirely eliminated, the problem 

 was attacked by several methods for the study of proteolysis, and 

 a number of experiments were conducted according to each method; 

 the results obtained by all the methods lead to the same general 

 conclusions. 



Either sodium fluoride or trikresol was used as a bactericide in 

 all the experiments reported below. When sodium fluoride was 

 used, sufficient solid fluoride was added to the mixture of pitcher 

 liquor and substrate to render the final concentration of sodium 

 fluoride one per cent. When trikresol was used, a sufficient volume 

 of a two per cent, aqueous solution of trikresol was added to render 

 the final concentration of trikresol 0.2 per cent. — a concentration 

 which was found satisfactory by Graves and Kober^° in certain of 

 their experiments with proteases. Whenever the mixture of pitcher 

 liquor and substrate was diluted to a definite volume^ the trikresol 

 solution was added before the dilution to the final volume was made. 



Unless otherwise stated, the temperature of incubation was 

 37° C. 



In each experiment, a blank or control experiment was carried 

 out with pitcher liquor which had previously been boiled, then 

 cooled to the temperature of the room ; the control experiment was 

 carried out in exactly the same manner, in all other respects, as the 

 determination proper. The control was always compared with the 

 determination proper ; and due allowance was thus made for the 



10 Graves and Kober, //. Am. Chcm. Soc, 1914, XXXVI., 751-758. 



