140 
Journal of Agricultural Research 
Vol. XXVIII, No. 2 
These on cooling are inverted and incubated for 48 hours at 37° C., after which 
counts are made. If the plates are flooded with a dilute solution of fuchsin or 
eosin before counting, the colonies are more easily differentiated for counting in 
the semiopaque medium. Vegetative cultures only were used for plating. A 
suspension of one loopful of culture in 3 cc. of sterile broth is made and one 
loopful of that is used to inoculate each plate. Dilution in sterile water was 
also tried, using 1 cc. of the dilution for each plate, but without success, since 
there seems to be a minimum amount of initial inoculum required, below which 
it is difficult to obtain growth. 
Table II .—Average number of colonies per k-mm. loopful of vegetative culture sus¬ 
pension in broth on plates of varying sugar concentration 
Per cent dextrose 
Average 
number 
of colonies 
Per cent dextrose 
Average 
number 
of colonies 
Control (o) _ 
1,500 
1,590 
1,560 
914 
2.0. 
0. 5... 
2.5. 
150 
1.0 
3.0. 
0 
1 . 5 . 
3. 5... 
0 
OBSERVATIONS 
SPORE GERMINATION AND GROWTH IN RELATION TO SUGAR CONCENTRATION 
At different times during the investigation seven different series of culture 
tubes were made, using as material for inoculation either scales or, in most 
-cases, ropy remains heavily laden with spores of Bacillus Icvrvae , but no vegetative 
rods. This material was taken from six different samples of diseased brood 
from different localities. From these series of cultures, varying in sugar con¬ 
centration from 0.5 per cent to 10 per cent dextrose, it was found that active 
growth occurs up to and including 2.5 per cent dextrose, although some growth 
occurs occasionally up to 3 per cent (Table I). The exact limits varied slightly 
with different strains as well as with variation in the amount of inoculum. Even 
up to 10 per cent dextrose concentration, a varying small number of spores 
germinate, as is demonstrated by stained smears, but they give no further evi¬ 
dence of vegetative growth upon the culture medium. 
GROWTH FROM ACTIVE VEGETATIVE CULTURES 
In a similar manner five different series of tubes with varying sugar con¬ 
centrations were made, using 24-hour cultures of three different characteristic 
vegetative cultures of Bacillus larvae , previously isolated and accustomed to 
growth on artificial culture media for different lengths of time. Good growth 
occurs on the average up to 2.5 per cent to 3 per cent dextrose concentration, 
with evidence of varying slight growth up to 4 per cent and in one case up to 
4.5 per cent (Table I). In the latter case much of the variation is due to varia¬ 
tion in the amount of initial inoculum. If a heavy inoculation is made on the 
surface v of the agar tubes, the upper sugar concentration limits for inhibition of 
growth are increased, although in these cases the growth was meager at best. 
Stained smears, however, made after a few days, from the higher sugar con¬ 
centrations particularly, soon showed the peculiar disintegration of the rods 
noted by White (55) as taking place in old cultures and where spore formation 
is inhibited, such as in the presence of sugar. This, according to observations 
of Sturges and Rettger (44) on other organisms, suggests that this disintegration 
of the rods is the result of autolysis. 
