20 
Journal of Agricultural Research 
Vol. VII, No. i 
flasks, and the material was transferred by filtering the alcohol used for 
preservation repeatedly through the thimbles until it had been freed 
from solid particles. As the alcohol used in preserving the samples 
contained such constituents as were readily soluble in cold alcohol, it 
constituted a cold extract and was preserved for addition to the products 
of the subsequent extractions. 
The extraction flasks were prepared by placing about 75 c. c. of redis¬ 
tilled 95 per cent alcohol in each, fitting the condensers, and warming 
gently on an asbestos plate over a gas flame. As soon as all dripping 
from the filled extraction cups had ceased, the material in each was pressed 
down with a glass rod, a weighed circle of ashless filter paper was fitted 
carefully over the top to prevent loss of finer particles, the cups were sus¬ 
pended from the condensers, and the flame so regulated that the filling 
and emptying of the siphon cups occurred about three times per minute. 
The extraction was continued for 12 hours; but at intervals of 3 to 4 hours 
the flame was turned out, the solutions collected from the extraction 
flasks, and equal quantities of 95 per cent alcohol introduced. This pro¬ 
cedure has the twofold advantage that it removes the possibility of altera¬ 
tion in the dissolved materials by prolonged boiling in alcohol, while it 
at the same time prevents the slowing down of the extraction through 
an elevation of the boiling point of the solvent. 
At the close of the alcohol extraction, the flasks were emptied, washed 
out with boiling 95 per cent alcohol, the material in the cups was pressed 
with a rod to remove the alcohol as completely as possible, and the 
extracts and washings combined and preserved. The extraction flasks 
now received 75 c. c. of ether each, and a 12-hour extraction with ether 
was made. While ether removes only very small quantities of material, 
which, except in the case of very heavily cutinized tissues, is quanti¬ 
tatively removed in the first two hours of the extraction, long-continued 
treatment with boiling ether greatly facilitates subsequent comminution 
of the tissues. 
At the end of the ether extraction the material was warmed to drive 
off the ether, removed from the cups to a mortar, and ground into a 
fine powder. Bits of cuticle and fragments of vascular tissue were cut 
into fine bits with scissors and separately ground. The powdered mate¬ 
rial was then transferred to a stoppered flask with 100 c. c. of distilled 
water, placed on a boiling water bath, and heated with frequent shaking 
for 12 hours. Enough 95 per cent alcohol was then added to make the 
concentration of the solution 85 per cent, and the heating on the water 
bath was continued for a second 12-hour period. The solids were then 
collected into the original extraction thimbles by filtering the solution 
repeatedly through them, and a final alcohol extraction of 12 hours* 
duration was made. The residue remaining afjter this extraction con¬ 
stituted the alcohol-ether-water-insoluble fraction 3. It was dried to 
constant weight in the extraction thimbles, an aliquot part taken for 
