140 



Journal of Agricultural Research voi. xxviii, No. i 



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 ^-mpi. loopful of vegetative culture sus- 

 pension in broth on plates of varying sugar concentration 



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 larvae, 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 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 pecuHar disintegration of the rods 

 noted by White {65) 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. 



