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Joumal of Agricultural Research 
Vol. XXVIII, No 2 
of the eight-day group, averaging 0.13762 gm. in weight, showed a total absence 
of reducing sugar. These larvae 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 (55 ), “Carbohydrate liquid media as ordinarily 
prepared aro 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 ti^me.” 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 biochemical activity 
of the organism can explain. 
From the previous cultural experiments (Table I) it may be seen that apparently 
Bacillus larvae can utilize 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 
Fig. 14.—Healthy larva at 
age of maximum size, just 
after sealing and before the 
start of the cocoon-spin¬ 
ning period (White (56)) 
