280 
GEOFFREY SMEIH. 
3. Qtjan'J'itative Chemical Methods. 
We have relied at present upon microscopical investigation 
for obtaining information as to the relative amount of fat and 
of glycogen in the livers of normal and infected individuals, 
but it is clearly desirable to check these results by a more 
■accurate quantitative method. For this purpose the following 
procedure has been followed, the method for glycogen estima- 
tion being PflugeFs (7), and for fat a modification of Leathes’ 
( 8 ). A weighed quantity of liver, 12-20 grm. total wet 
weight, is obtained from crabs of a particular category, and 
is boiled on a water-bath for three hours with an equal weight 
•of 60 per cent, caustic potash solution. The resulting mixture 
is washed out into a beaker with distilled water, and cooled, 
and to it is added three times the volume of 96 per cent, 
alcohol, by which the glycogen is precipitated. The pre- 
cipitate is collected in a Gooch filter, and thoroughly washed 
with 70 per cent, alcohol. The alcoholic filtrate, which contains 
the fat, is set apart for further treatment. The glycogen 
precipitate is dissolved in boiling water, and the glycogen 
solution so obtained is acidified with liydrochloric acid, so 
that the strength of the acid in the solution is about 2 per 
cent. The acidified solution is boiled on a water-bath for two 
hours to convert the glycogen into sugar. The strength of 
the sugar solution, which is made up to a known volume, is 
then estimated by Pavy’s method of titrating against copper 
hydrate solution. In this way the quantity of glycogen in 
the liver taken can be determined.^ 
' A serious source of error in the titration of the sugar solution with 
copper hydrate must he gaiarded against. The solution of glycogen dis- 
solved ill hot water always contains some other organic material including 
amides derived from the hreakdown of the proteid. These amides, if 
present in sufficient quantity, will form a stalile greenish compound with 
the copper hydrate, which prevents the disappearance of the colour on 
titration and thus destroys the value of the copper as an indicator. This 
difficulty can he got rid of hy evaporating the sugar solution to lie tested 
nearly to dryness on a water hath and re-dissolving in water, after which 
treatment the action of the amides on the copper hydrate no longer 
occurs while the reducing power of the sugar is not interfered with. 
The difficulty only arises in an acute form in testing weak sugar solutions. 
