STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 281 
The volume of the alcoholic filtrate containing the fat is 
measured, and a measured portion of it is evaporated on a 
water-bath until all the alcohol i& driven off. The watery 
residue, containing the fats in the form of soaps, is treated in 
a long-necked flask with 40 percent, sulphuric acid, by which 
the fatty acids are liberated from the soaps. A measured 
quantity of petroleum ether is added and shaken up with the 
mixture vigorously for an hour. A measured portion of the 
petroleum is pipetted off and evaporated to dryness in a 
weighed beaker. The beaker containing the solid residue of 
fatty acid is weighed again, and thus the amount of fatty 
acid present in the original filtrate can be calculated. 
Since the liver of a single crab does not furnish a sufficient 
quantity of glycogen for estimation, it is necessary to pool the 
livers of several crabs. For the purpose of the experiments 
two categories of crabs was used for comparison — normal 
male crabs with hard skins, and crabs infected with Saccu- 
lina. For each experiment livers of four or five normal males 
were taken and treated together, but with the infected crabs, 
owing to their smaller size, it was found necessary to take the 
livers of six to ten individuals for each separate determina- 
tion. The percentages given in the following* table are there- 
fore each based, not on determinations made on single indi- 
viduals, but on several taken together, viz. four or five in the 
case of normal males, and six to ten in the case of infected 
individuals. 
In order to get over the effect of food recently taken 
and to test the effect of starvation, the crabs were starved 
tor varying periods beginning at 24 hours and going up to 
532 hours. 
In the table given below the results of glycogen and fat 
estimation are given in percentages calculated as sugar and 
fatt}^ acid respectively. In the first column the number of 
hours during which the crabs were starved is given, and the 
results of the determinations of glycogen and fat are entered in 
the other columns opposite, according to the length of time the 
animals from which the livers were taken were starved. The 
