Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 281 



Table 7. — Results of the examination of samples of honey for the presence of spores 



of Bacillus larvae 



" 29 of these samples were doubtful on the first examination, but repeated examinations gave negative 

 Tesults in each case. 



Of the 187 samples of commercial honey obtained from 30 different 

 States or Territories, 15, or 8 per cent, showed the presence of a suf- 

 ficient number of spores resembling spores of Bacillus larvae to be 

 designated as positive. In 29 of the commercial samples, or 15.5 per 

 cent, one or two doubtful spores were seen in each case, but on 

 repeated examinations none of these samples could be considered 

 positive. Two of the four miscellaneous samples from infected 

 sources were also found to contain spores of B. larvae. 



Five of the samples showing the presence of spores of Bacillus 

 larvae were fed to healthy 5-frame colonies during the summer of 1930. 

 These samples consisted of from a pint to a quart of honey. No evi- 

 dence of American foulbrood appeared in any of the five colonies 

 during the entire brood-rearing season. 



In order to determine the approximate number of spores in the 

 samples of honey in which the presence of Bacillus larvae was demon- 

 strated, a series of dilutions of spores was prepared as described for 

 the work with cultures. A stained smear was made of 0.01 c c of 

 each dilution spread over a 1-cm^ surface of cover glass mounted with 

 water and examined with the oil-immersion objective. By this 

 means a definitely recognizable number of spores could be found 

 down to the dilution of 2,000,000 spores per cubic centimeter, with a 

 few single spores seen in occasional fields down to the dilution of 

 500,000 spores per cubic centimeter. (Table 8.) Then 1 c c of each 

 dilution was added to 5 c c of distilled water in 15 c c centrifuge tubes 

 and centrifuged at 2,000 revolutions per minute for 20 minutes. A 

 stained smear made from 0.01 c c of each sediment showed a definitely 

 recognizable number of spores down to the 5,000-spore dilution, with 

 one or two doubtful spores in several fields from the 500-spore dilu- 

 tion. The sample containing the 50,000-spore dilution, which would 

 be comparable to the sugar sirup containing the minimum number of 

 spores per cubic centimeter fed to colonies in the spore-feeding experi- 

 ments that produced infection, showed a great many more spores in 

 each field examined by this method than did the sample of commer- 

 cial honey that showed the greatest number of spores. Therefore, 

 until a better quantitative method is devised, it seems reasonable to 

 believe, from the indications of the preliminary work on this problem, 

 that, even though the presence of a few spores of B. larvae may be 



