698 Journal oj Agricvltural Research voi. 62, no. 9 



basis of its spore content^that is, a detailed study of the distribution 

 of spores of B. larvae m. the honey from infected hives or apiaries, 

 or in commercial honey obtained on the open market, or of the effect 

 of mixing infected honey with disease-free honey in the course of 

 production or blending and preparation for the market — a more 

 detailed iavestigation has been made of the spore content of honey 

 containing approximately known numbers of spores. This has been 

 accomphshed by an improved and more accurate method of deter- 

 mining the number of spores in such honey, and the accuracy of the 

 results and method has been demonstrated by means of a statistical 

 analysis of the data obtained. 



METHOD OF OBTAINING THE DATA 



PREPARATION OF SAMPLES OF HONEY 



A series of samples of honey containing approximately known 

 numbers of spores per cubic centimeter were prepared in the manner 

 described previously,' by adding to 100-cc quantities of spore-free 

 honey the necessary quantities of various dilutions of a stock suspen- 

 sion of spores of Bacillus larvae containing approximately 5,000,000,000 

 spores per cubic centimeter. Five samples of honey were prepared 

 in this way containing approximately 1,000,000, 800,000, 500,000, 

 300,000, and 50,000 spores per cubic centimeter, respectively. These 

 samples, each considered as a unit and not as a dilution of the 

 1,000,000-spore sample, were heated in a water bath to 120°-130° 

 F., and then thoroughly mixed with a mechanical stirrer for 5 minutes. 

 Duplicate 5-cc quantities of each sample were then placed in 50-cc 

 conical centrifuge tubes, and 45 cc of distilled water of approximately 

 the same temperature was added. When the honey and water were 

 completely mixed, the samples were centrifuged at 2,000 revolutions 

 per minute for 45 minutes. All but about 1 cc of the supernatant 

 honey-water solution of each sample was then removed by means of 

 a pipette and suction. Again approximately 45 cc of distilled water 

 was added, and after thorough mixing the suspensions were centri- 

 fuged for 30 minutes longer. The removal of the supernatant solu- 

 tion was repeated until all but approximately 0.1 cc' of the water 

 had been removed from each centrifuge tube, and each sample of 

 sediment was completely suspended in this remaining quantity of 

 water by blowing gently through a capUlary pipette dipped into the 

 water. Duphcate 0.01-cc quantities of each suspension were then 

 transferred with the capillary pipette (calibrated to deliver 0.01 cc) 

 to microscope cover glasses. Circular cover glasses, size 12, no. 1 

 thickness, having an area of 1.13 cm 2, proved satisfactory for this 

 P^Pj®^" u ^^^'^ ^^ *° ^ "^™) loopful of carbolfuchsm stam was 

 added to the drop of suspension on the cover glass and thoroughly 

 mixed with it. This stained liquid was then spread uniformly over 

 a 1-cm area of the cover glass, a narrow ring at the outside edge 

 being left uncovered. The smears were allowed to dry in the air 

 and were then mounted on microscope slides either with water or pref- 

 erably, with Canada balsam, for examination under the microscope, 

 ihese stamed smears were not washed in water, as this might have 

 caused some spores to be lost. 



• Stuetevant, a. p. See footnote 3. 



' A mark was placed on the outside of the conical centrifuge tubes to indicate the 0.1-co volume. 



