164 Journal of Agricultural Research voi. xxvm, 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, Mie 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 (6), as follows: "The ability of Bacillus subtilis to break 

 down protein in the presence of fermentable sugar, and in the absence of an added 

 buflfer, 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 metaboUsm 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. 



