280 Journal of Agricultural Research voi. «, No, s 



numbers to recover and identify them under the microscope. Even 

 with a comparatively large filtering surface, the process was so slow 

 that the diluted honey would frequently start to ferment before it 

 had all passed through the fUter. A filter of smaller area would 

 become clogged, preventing the passage of a sufficient quantity of 

 honey. 



Several unsuccessful attempts were made to recover spores of Bacil- 

 lus larvae from honey by centrifuging samples diluted with an equal 

 quantity of water. After considerable experimentation with honey 

 of known spore content, it was found that it was necessary to dilute 

 the honey to a much greater extent — 1 part to at least 9 of water — ia 

 order to throw the spores down with the sediment. Apparently the 

 specific gravity of these spores is so low that on centrifuging they 

 remain in suspension in only slightly diluted honey. 



The procedure finally used for demonstrating the presence of spores 

 of Bacillus larvae in honey is as follows: Five c c of warmed honey 

 is thoroughly mixed with 45 c c of distilled water in a 50 c c cone- 

 shaped centrifuge tube made of heat-resistant glass. Duplicate 

 quantities of each sample of honey are made up for examination. 

 The diluted honey is then centrifuged at 2,000 revolutions per minute 

 for one-half hour. Because of the difficulty of obtaining a satisfac- 

 tory stained smear from the sediment thrown down in the presence 

 of the sugars of the honey solution, all but 2 c c of the solution in each 

 centrifuge tube is drawn off by means of a 50 c c pipette. Another 

 45 c c of distilled water is added , the sediment is thoroughly shaken 

 up in the water, and the tabes are centrifuged again for 20 minutes. 

 After all but 2 c c or less of the wash water has been removed, 

 0.01 c c of the sediment is removed by means of a capillary pipette 

 and smeared on a cover glass over a surface of 1 cm^, a small loopful 

 of carbol fuchsin being mixed with the material before it is allowed 

 to dry. After drying by gentle heat, the cover glass is mounted 

 on a slide by means of a drop of distilled water and the smear is 

 examined with an oil-immersion objective. Spores of B. larvae are 

 identified by their size and shape in conjunction with their distinctive 

 habit of breaking loose from the stained mass of the smear and of 

 showing a delicate Brownian movement in the thin film of water 

 between the two pieces of glass. In a few samples only one or two 

 spores were seen in numerous fields examined or the spores did not 

 have the typical appearance of spores of B. larvae. In such cases 

 another test, in which twice as much honey was used, was made 

 from the sample. 



OBSERVATIONS 



One hundred and ninety-one samples of honey were examined by 

 this method. (Table 7.) Of tlfese, 187 were regular commercial 

 samples purchased in the open market and 2 were from the experi- 

 mental apiary at Laramie. The other two were miscellaneous 

 samples, one of which was obtained from a brood comb from a dis- 

 eased colony and the bther from a cappings melter which had been 

 used with combs from an infected apiary. 



