Sept. 1, 1932 Commercial Honey and Spread oj American Foulbrood 269 



c c flasks bv adding 4 c c of a dilution to 36 c c of sterUe water. The 

 series of dilutions containing gradually decreasing numbers of spores 

 per cubic centimeter to be used in inoculating the culture medium 

 were then prepared as indicated in Table 4. Sterile burettes were 

 used in adding the proper proportions of spore suspension or spore- 

 suspension dilutions to the proper quantities of sterile water in sterile 

 test tubes, in order to make up the desired series of dilutions contain- 

 ing approximately known numbers of spores. 



Inoculation op Culture Medium 



Swann has observed that in old cultures of anthrax a considerable 

 percentage of spores are dead and therefore never germinate. Be- 

 cause of the possibility that some of the 

 spores in the stock suspensions of Bacillus 

 larvae might not be viable, an effort was made 

 to determine the approximate proportions of 

 viable and dead spores in the stock suspen- 

 sions. Since the determination of viable 

 spores of B. larvae by means of plate cultures 

 is difficult because of the opaqueness of the 

 special culture medium that is required, an 

 attempt was made to determine the percent- 

 age of viable spores by the differential stain- 

 ing method of Burke (4) as modified by Koser 

 and MUls (IS). The procedure is as follows : 

 A small quantity of the spore suspension is 

 spread in a thin film on a slide and allowed 

 to dry without heating. The slide, after 

 immersion in a solution of carbol fuchsin at 

 room temperature for two minutes, is washed 

 in water and decolorized with absolute ace- 

 tone for a few seconds, washed again, and 

 immersed in Loefiler's alkaline methylene 

 blue for two minutes, washed, dried, and 

 examined. Very few solid-staining forms 

 were observed in any of the suspensions ex- ^'<'™t^tinrotKii sXensS ^'' 

 amined, possibly one or two spores in several 



fields. It was therefore assumed that the number of nonviable spores 

 could be considered as negligible and probably within the limits of 

 the precision of the measurements as indicated by this procedure. 



One cubic centimeter of each dilution was added to duplicate tubes 

 of the slanted solid medium by means of sterile Ice pipettes, each 

 cubic centimeter of inoculum containing an approximately known 

 number of spores of Bacillus larvae. After inoculation the cultures 

 were incubated at 37° C. In order to prevent the liquid in the tubes 

 from drying out on long incubation, from time to time, as the water 

 of condensation evaporated, 2 or 3 c c of sterile broth similar in com- 

 position to that of the base medium, without the egg, was added to 

 each tube by means of the apparatus shown in Figure 1. A total of 

 556 cultures was made during this series of experiments. 



