164 
Joumdl of Agricultural Research 
Vol. XXVIII, No. 2 
utilizable by bacteria sufficient growth is necessary by which to produce the 
enzym capable of splitting the complex protein molecule into its simpler amino- 
acid forms. In the healthy larva at the age when Bacillus larvae starts growth in 
the intestine, the remaining sugar in the intestinal contents and other material 
of nitrogenous nature in the food is sufficient, as stated earlier, to produce the 
energy for the initial growth of the organism. By the time the invasion of the 
tissues by the organisms occurs sufficient proteolytic enzym has been produced to 
attack the body proteins. Furthermore, the process of histolysis itself, as stated 
in relation to gelatin liquefaction by ropy material, has probably broken up 
sufficient of the body proteins to serve as food for bacterial metabolism. 
Since Bacillus larvae belongs to the spore-forming group of organisms, its 
processes of metabolism may be similar to those of Bacillus subtilis, as described 
by Berman and Rettger (0), as follows: “The ability of Bacillus subtilis to break 
down protein in the presence of fermentable sugar, and in the absence of an added 
buffer, may be explained as follows. This organism attacks glucose slowly, 
and for this reason it is able to produce its proteolytic enzym before the hydrogen- 
ion concentration reaches a point unfavorable to further growth. When the 
enzym is thus formed the products of the nitrogen metabolism neutralize the 
acid, at least in a measure, and the metabolism therefore continues uninter¬ 
ruptedly.” 
The fact that the production of indol and even of ammonia can be demon¬ 
strated, although they may be produced slowly and in small amounts, indicates 
that even though considerable acid may be produced by the fermentation of 
the carbohydrate in the food and the hydrolysis of the glycogen, Bacillus larvae 
has putrefactive functions which bring about the formation of sufficient alkaline 
protein-digestion products from the larval tissues to neutralize this acid produc¬ 
tion, thereby maintaining the hydrogen-ion concentration at approximately 
Ph=6.8. 
SUMMARY AND CONCLUSIONS 
1. It has been shown by the work of others that the glycogen and fat content 
of the bee larva increases in a definite manner for a time and then decreases. In 
the present work it is shown that the per cent and amount of reducing sugar 
likewise increase after the third day of larval feeding, but decrease rapidly im¬ 
mediately after feeding has ceased, until by the eighth day no reducing sugar 
remains in the larva. 
2. The presence of reducing sugar can be determined only after the progressive 
feeding of the larva begins. 
3. The best method for extracting reducing sugars from the bee larva was 
found to be by the use of 50 per cent alcohol and the removal by filtration of 
the insoluble materials. By this means the interference of glycogen with the 
action of the reducing solutions is prevented. 
4. The constitution of the protein molecule of the normal tissue content of 
the healthy bee larva is complex, containing, however, among other amino-acids, 
tryptophan. 
5. The best medium for the growth of Bacillus larvae so far devised is a yeast- 
extract agar medium to which sterile egg-yolk suspension has been added. The 
optimum reaction for cultural growth is Ph=6.8. 
6. Reducing sugars in the culture medium of more than 3 or 4 per cent usually 
inhibit germination of spores and growth of Bacillus larvae. A few spores may 
germinate at higher concentrations, but the resulting vegetative forms fail to 
increase in number and show granular disintegration due to autolysis. Less than 
2 per cent of reducing sugars seems to stimulate growth of the vegetative forms. 
