Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 283 



minimum dose of spores of B. larvae that will produce American foul- 

 brood infection must be large. 



The germination of spores of Bacillus larvae and vegetative growth 

 on a suitable artificial culture medium resulting from the inoculation 

 of 556 culture tubes with seedings varying from approximately 

 50,000,000,000 to 500 spores per culture also shows that a certain 

 minimum initial number of spores in the inoculum is necessary in 

 order to produce growth. This minimum number of spores produc- 

 ing vegetative growth on a medium consisting of yeast-carrot extract, 

 egg-yolk suspension, and agar was found to be approximately 50,000 

 in 1 c c of suspension inoculated. 



The production of nitrite in this medium by the vegetative growth 

 of Bacillus larvae serves as a fairly delicate and reliable indicator of 

 such growth. 



There was a tendency for the seedings containing the smaller num- 

 bers of spores of Bacillus larvae to require a longer period of incubation 

 than the larger seedings in order to produce vegetative growth. 

 However, there was a considerable variation in the germination time 

 of many of the seedings of spores, in one case a seeding of 9,000,000 

 spores requiring 27 days' incubation to produce growth and another 

 of 70,000 spores requiring only 6 days. This variation, thought to 

 be due to the variable character known as dormancy in bacterial 

 spores, prevented more than a slight correlation. 



In the group of cultures comprising seedings between 5,000,000,000 

 and 9,000,000 spores, only 1.67 per cent required more than 10 days' 

 incubation to produce vegetative growth, 100 per cent having shown 

 growth after 30 days. In the group of cultures comprising seedings 

 between 8,000,000 and 500,000 spores, 71.93 per cent required more 

 than 10 days' incubation, while 53.81 per cent showed no growth at 

 the end of 30 days' incubation. In the group of cultures comprising 

 seedings between 400,000 and 50,000 spores, 97.18 per cent required 

 more than 10 days' incubation, while 91.55 per cent of the group 

 showed no growth at the end of 30 days. Below 50,000 spores no 

 growth was obtained. In other words, below a seeding of 9,000,000 

 spores an increasing number of the smaller spore seedings required a 

 longer period of incubation. About 80 per cent of all the positive 

 cultures were obtained during the first 10 days of incubation, although 

 this was approximately only 30 per cent of all the cultures made; at 

 the end of 30 days' incubation only about 38 per cent of all the cul- 

 tures had shown any growth. 



It was found possible to demonstrate the presence of spores of 

 Bacillus larvae in 15 out of 187, or in 8 per cent, of the samples of 

 commercial honey examined by means of the centrifuge and the 

 microscope. "The preliminary results indicate that, even though 

 spores of B. larvae may be demonstrated in a certain percentage of 

 samples of commercial honey, in most instances they are probably 

 present in such small numbers as to be less than the minimum 

 number, 50,000,000 per liter, found to be capable of producing dis- 

 ease, and therefore are ineffective in the spread of American foul- 

 brood. 



