150 



Journal of Agricultural Research voi. xxvm, No 2 



Fig. 14.— Healthy larva at 

 age of maximum size, just 

 after sealing and before the 

 start of the cocoon-spin- 

 ning period (White (56)) 



Of the eight-day group, averaging 0.13762 gm. in weight, showed a total absence 



of reducing sugar. These larvs represent the two-day quiescent prepupal stage 

 (fig. 3 and 4). They have stretched out motionless in 

 the cell, the intestines are entirely empty and colorless, 

 and the histolysis of the tissues preliminary to metamor- 

 phosis has begun. 



From these observations it is seen that there is an 

 amount of reducing sugar in the entire actively feeding 

 larva which would seriously interfere with the germina- 

 tion and growth of Bacillus larvae, provided the entire bee 

 larva were to serve as the medium for its growth. Since 

 this reducing sugar does not exist equally distributed 

 throughout the bee larva, and since at this stage the 

 organisms are found almost solely in the intestinal tract, 

 it is certain that the reducing sugar concentration of the 

 intestine is sufficient to prevent the germination of 

 Bacillus larvae, so that death from American foulbrood is 



delayed until after the larva has been sealed in the cell and has become quiescent. 



This will be discussed more in detail later. 



SUPPLEMENTARY STUDIES ON THE BIOCHEMICAL REACTIONS 

 OF BACILLUS LARVAE 



Up to the present time few facts have been determined concerning the 

 biochemical reactions of Bacillus larvae, mainly because of a lack of suitable 

 culture media. White states {65), "Carbohydrate liquid media as ordinarily 

 prepared are not suitable for the growth of Bacillus larvae. In some of these 

 after a considerable period a slight growth may appear at the bottom of the 

 tubes. A little brood-filtrate or egg-suspension added to the media improves it. 

 No visible gas is formed, but in some instances slight acidity Is produced. No 

 growth takes place in plain or in brood-filtrate gelatin at temperatures at which 

 it remains congealed." Maassen states {28) , " The bacillus also grows on nutrient 

 gelatin. Upon a nutrient gelatin medium which had been made from the pre- 

 viously mentioned nutrient liquids, and an almost completely neutralized gelatin 

 (a so-called emulsion of gelatin), there resulted growth although very slowly, 

 from which a quite gradual liquefaction of the gelatin resulted. Liquefaction 

 did not occur in the presence of grape sugar (dextrose) . Through the addition of 

 1 per cent grape sugar the growth-producing ability of the gelatin as well as of other 

 nutrient media was noticeably improved. On the most favorable media no 

 special chemical properties were shown, with the exception of the ability to 

 peptonize. The destruction of the albuminous bodies occurred very slowly 

 and with little characteristic appearance. Only in worn-out cultures could 

 any odor resembling foul glue be detected after a tipie.'' There are, however, 

 certain characteristic manifestations in American foulbrood resulting from the 

 growth and metabolism of Bacillus larvae, aside from the gross symptoms and 

 appearances, which only a more complete knowledge of the biocheniical activity 

 of the organism can explain. 



From the previous cultural experiments (Table I) it may be seen that apparently 

 Bacillus larvae can utiUze in its metabolism a certain amount of reducing sugar 

 (dextrose), although this sugar is not necessary to the development of the 

 organism. In the larva which is attacked by American foulbrood there may be 

 two sources of sugar, that present unassimilated in the intestine and that hydro- 

 lyzed from the stored glycogen. Hydrolysis of glycogen may occur in connection 

 with histolysis of the tissues preparatory to metamorphosis through enzym 



