Oct. 2, 1916 
Effect of Blackrot Fungus on the Apple 
21 
duplicate or triplicate ash determinations, and the remainder preserved 
in a desiccator for subsequent analysis in the manner to be presently 
described. 
The soluble portions of the material were now contained in the alcohol 
originally employed for the preservation of the material, in that used for 
the first extraction and for washing the flasks, in the water-alcohol mix¬ 
ture in which the powdered material had been heated after extraction 
with ether, and in the ether extract. This last was now heated on a 
water bath until nearly all the ether had been driven off, when it was 
taken up with hot 70 per cent alcohol. All the solutions were then com¬ 
bined, whereupon some precipitation occurred. The precipitate was 
brought again into solution by warming the flask and adding sufficient 
boiling water to reduce the alcohol concentration to 70 per cent. When 
a perfect solution had been secured, the solution was transferred to a 
volumetric flask, made up to 2,000 c. c. with 70 per cent alcohol, and 
200 c. c. were removed for the determination of the total solids. The 
remainder of the solution was transferred to beakers, placed upon a 
water bath kept at 75 0 C., and evaporated down to a sirupy consistency, 
a little absolute alcohol being added from time to time. The material 
was finally allowed to become alcohol free and was then taken up with 
sufficient distilled water, in most cases about 700 c. c., to form a perfect 
emulsion, and transferred to a 1,000 c. c. volumetric flask. Twenty 
c. c. of chloroform were added, the flask was vigorously shaken for sev¬ 
eral minutes, and the shaking was continued while 10 c. c. of concen¬ 
trated hydrochloric acid were very gradually added. The flask was 
then filled to the mark with distilled water, stoppered, shaken at short 
intervals for 2 hours, and finally submerged to the neck in cold running 
water for 48 hours. At the end of this time the solution was clear, the 
lipoids having been partially carried down by the chloroform layer, 
while a part adhered to the walls and neck of the flask. 
The solution was now filtered through a dry filter, great care being 
taken to prevent the transfer of the chloroform layer or the precipitated 
material upon the flask walls to the filter paper. After the filter had 
been allowed to drain well, the volume of the filtrate was noted. It 
constitutes the water-soluble portion of the alcohol-ether-water extract, 
and is designated as fraction 2. 
The material precipitated by the chloroform, together with that held 
in the chloroform layer, constituted the lipoid precipitate, fraction 1. 
It was next collected by thoroughly washing the filter paper and the 
neck and walls of the precipitation flask with a large volume of boiling 
95 per cent alcohol from a wash bottle, 500 to 600 c. c. being necessary 
with most samples. When a uniform solution had been secured, the 
flask was transferred to the water bath and kept at 70° C. until the 
chloroform had been completely driven off. The flask was then filled 
to the mark with warm 95 per cent alcohol, and aliquot parts of the 
