270 Journal of Agricultural Research voi. 45, No. 5 



Method of Making Observations 



The culture tubes were incubated for 30 days at 37° C. Each tube 

 was examined usually every 24 hours during this period. The pres- 

 ence or absence of vegetative growth was noted at each observation, 

 and ia cases of slight or doubtful growth the vegetative growth was 

 checked both by microscopic examination of a stained smear and by 

 testing for nitrite production in the culture medium by the sulphanUic 

 acid and alpha-naphthylamine acetate test. After a large number of 

 such observations had been made, it was found that vegetative ger- 

 mination of spores of Bacillus larvae, almost too slight to be seen, 

 would give a definite pink color on the addition of the reagents. 



Lochhead {17, p. 14) sta,tes: 



It was found, however, that ordinary nitrate-reducing species, such as B. 

 cereus or Es. coli, which are able to form nitrites readily in nitrate media, were 

 unable to produce nitrites in recognizable amount in the peptone-carrot media, 

 though capable of doing so upon the addition of nitrates. Bacillus larvse under 

 the same condition readily forms nitrites without the addition of nitrate to the 

 medium. 



Despite this statement, a series of miscellaneous organisms was 

 tested in standard nitrate broth, in carrot-extract broth, and on 

 carrot-extract agar. Several organisms that commonly reduce 

 nitrates and a few that do not were used . (Table 3.) Observations 

 were made at short intervals during the first 24 hours. Most of 

 these organisms gave positive nitrite tests within a few hours after 

 inoculation in all the media used, but in the carrot-extract medium 

 the nitrate had apparently disappeared in most cases after 24 hours' 

 incubation, and in all cases after 48 hours. The same organisms 

 on standard nitrate medium still gave positive tests after 48 hours' 

 incubation. A positive nitrite test was obtained in cultures of 

 Bacillus larvae that were incubated for 5 days and in one culture 

 that was incubated for 4 days and then allowed to stand at room 

 temperature for 16 days more before testing. Therefore, it appears 

 probable — at least the results in Table 3 indicate — that in the case 

 of many contaminating organisms having the power to reduce nitrite 

 that might get into the culture tubes inoculated with spores of B. 

 larvae the nitrite, if produced by the contaminating organism, would 

 have disappeared after 48 hours' incubation, leaving contamination 

 to be determined by gross appearence of the culture and microscopic 

 examination. Nevertheless, in order to be sure that contaminating 

 growth of any kind was not giving erroneous results with the nitrite 

 test when this was used alone, any suspicious-looking growth in the 

 culture tubes was examined under the microscope before it was tested 

 with the reagents for nitrite production. Even though a positive 

 mtrite test might be observed in some cases, the contaminations were 

 recorded only as such. 



OBSERVATIONS AND EESULTS 



In no instance was positive growth obtained in cultures moculated 

 with less than 50,000 spores, even after 30 days' incubation, and 

 growth with 50,000 spores was obtained from only two of the eight 

 lots of spores used, namely, Nos. 19 and 23. (Table 4.) In the 

 other six strains the mimmum number of spores that produced positive 

 growth ranged from 5,000,000 to 70,000. 



