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786 ANNALS OF 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 ‘‘dauerhefe’’ powder ; 
