[Vol. 2 



786 ANNALS OP THE MISSOURI BOTANICAL GARDEN 



terial for such action were employed. The tissue was added 

 directly to the substrate, or it was extracted with water by 

 the method to be described later and a water-diffusion used of 

 the alcohol precipitate. If the fresh tissue were to be used 

 directly, it was ground in a meat chopper two or three 

 times, then pounded in a large mortar with an equal amount 

 of fine, clean, quartz sand. This treatment gave a very homo- 

 geneous pulp, one in which a large number of the cells were 

 broken down. If desired for future use, the plants were either 

 dried at room temperature or dehydrated by the following 

 modified Buchner "dauerhefe" process: 



3 volumes 95 per cent alcohol for 15 minutes. 

 3 volumes acetone for 15 minutes. 



3 volumes 95 per cent alcohol for 10 minutes. 

 3 volumes acetone for 5 minutes. 



2 volumes absolute alcohol 



or ether for 5 minutes. 



After each treatment, the dehydrating liquid was pressed 

 out through two thicknesses of cheese cloth by making a 

 tourniquet. Upon the removal of the absolute alcohol or 

 ether, the tissue was spread out on adsorbent paper, either 

 filter paper or paper toweling, until all the dehydrating agent 

 had evaporated. A uniformly dry, brittle, easily crushed 

 material usually resulted that was roughly broken up and 

 stored in tightly stoppered bottles for future use. Those 

 plants that were dried at room temperature were simply 

 wrapped in paper or placed in paper bags until needed. 



The crushing of the dry material was accomplished in the 



same manner as was the fresh. Usually it was ground twice 

 or more in an ordinary meal mill, then pounded in a mortar 



with an equal weight of quartz sand until a very fine powder 

 was obtained. The sand was dispensed with if the tissue were 

 easily crushed. 



Methods of isolating the enzymes. — As indicated above, 

 there were two general methods of using the material for 

 enzyme action: first, adding the crushed tissue directly to 

 the substrate, either as fresh pulp or as " dauerhef e ' ' powder ; 



