152 
Journal of Agricultural Research 
Vol. XXVIII, No. 2 
be found that most of the glycogen has disappeared, although the iodin solu¬ 
tion gives a light yellowish brown color. The presence of a trace of reducing 
sugar also occasionally can be demonstrated with Benedict’s solution in dis¬ 
eased material of this type where vegetative organisms are still actively present. 
In material which has decomposed completely, has reached the dark brown 
ropy stage (fig. 9), and contains only spores of Bacillus larvae , glycogen is found 
to be completely absent, nor can any reducing sugar be demonstrated, the 
sugars having been completely destroyed. 
This type of material stained with Sudan III or osmic acid (82, p. 78) shows 
fat globules in practically the same condition and amount as in healthy larvae, 
so that fat is apparently not acted upon by Bacillus larvae even after drying down 
to the scale stage. 
Glycogen of the fat body of the healthy larva is hydrolyzed to dextrose to be 
used in metamorphosis, by the action of enzyms during the histolytic processes 
subsequent to sealing and prior to metamorphosis. This enzym action is demon¬ 
strated by the following experiments: 
EXPERIMENTAL PROCEDURE 
Several series of 50 healthy prepupae each that had reached the period of 
quiescence were macerated in 25 cubic centimeters of 50 per cent alcohol and 
incubated at 37° C. for from 3 to 24 hours. The extract was then filtered and 
diluted with an equal amount of water. A series of test tubes were prepared, 
using for each tube 5 cubic centimeters of this extract and 5 cubic centimeters of 
0.4 per cent glycogen in water, and also another series using 5 cubic centimeters 
each of a 0.1 per cent soluble starch. Both glycogen and starch were used, since 
it has been shown by Bradley and Kellersberger ( 8 ), as well as by experiments 
by the writer using commercial Taka-diastase, that diastase acts similarly on 
both glycogen and starch. These tubes were incubated for various periods and 
then tested with iodin solution for the presence of glycogen and starch (Table 
VI). Hydrolysis of both glycogen and starch seems to be complete after incuba¬ 
tion for about five hours, and positively complete after incubation overnight, 
demonstrating the presence of diastase in the prepupae. 
In another experiment 50 prepupae were macerated in 50 cc. of water and in¬ 
cubated at 37° C. for 24 hours. Then sufficient 95 per cent alcohol was added to 
precipitate any glycogen present, and the solution was filtered and tested with 
both the qualitative and the quantitative Benedict’s solutions. In both cases 
definite traces of reducing sugar could be demonstrated, none having been present 
in the original solution before incubation, again demonstrating enzym activity 
of the larval tissues. This may have been due to action by bacterial contamina¬ 
tion, but if such had been the case the sugar would probably have been fermented 
and could not have been demonstrated. 
In a similar manner extracts with 50 per cent alcohol were made of ropy dis¬ 
eased material, enzym activity being demonstrated in the same manner as above. 
This, however, does not indicate whether the organism causing the disease has 
any diastatic power or whether the reaction was due to enzyms remaining in 
the decomposed tissues. Further extracts were made with 25 per cent and 50 
per cent alcohol of several 48-hour vegetative cultures of Bacillus larvae grown on 
egg-yolk suspension medium. These extracts showed definite enzym activity 
with glycogen after a few hours’ incubation, and 'more positive activity after 
incubation overnight (Table VI), while with starch marked hydrolysis was shown 
after only a few hours’ incubation. 
