264 Journal oj Agricultural Research vu. 45, No. 5 



become dried without adhering so tenaciously to the cell walls as 

 they do in more humid climates at lower altitudes. These observa- 

 tions iadicate the necessity of further work on the resistance of bees to 

 the disease and variation in virulence of different strains of the 

 organism. 



INOCULATION OF INDIVIDUAL BEE LARVAE WITH DEFINITE 

 NUMBERS OF SPORES OF BACILLUS LARVAE 



In the light of the results of the foregoing experiments, in which 

 colonies were inoculated with presumably a quantity of spores 

 sufficient to produce infection but in which no disease developed, the 

 question arises as to what became of the spores in the sugar sirup, 

 some of which presumably were fed to healthy larvae. In those 

 colonies developing disease that received a minimum number of 

 spores, how many spores did each larva developing the disease receive? 

 In order to obtain information on these points, a preliminary series of 

 experiments was planned in which individual larvae were inoculated 

 with known numbers of spores. 



Touraanoff (29) reports that he was unable to cause infection by 

 giving individual larvae a drop of a rich emulsion of a culture of 

 Bacillus larvae in salt solution. He found that many of the larvae so 

 treated were removed from the cells by the bees, and those remaining 

 failed to develop disease. He further found that larvae given only 

 uninoculated salt solution were also removed in the same way. 

 Therefore, in the present experiments sugar sirup was used instead 

 of salt solution. In a comb from a healthy colony containing numer- 

 ous coiled larvae, a drop of an uninoculated 50 per cent solution of 

 sugar in water was placed in each cell containing a larva, as near the 

 mouth parts of the larva as possible. The rim of each cell so treated 

 was marked with a paint consisting of 1 part of liquid white shellac, 

 1 part of a paint pigment, and 4 parts of ethyl alcohol. The sugar 

 sirup was slightly colored with water-soluble eosin in order to aid 

 m determining the effect. Frequent observations showed that 

 practically all larvae that were fed this colored sugar sirup developed 

 normally and were sealed over, the pigment markings still being 

 present on the edges of the cappings. In most of the cells a residue of 

 colored sirup could be observed for several hours after the larvae 

 had fed. 



A series of 5-frame nuclei was prepared, each containing one or two 

 combs havmg a large number of unsealed larvae. A set of dilutions of 

 spores was made from a stock suspension with a steriUzed 50 per cent 

 sugar su-up m such a way that each 0.01 c c of the dilution would 

 £?^*f 1^ a^ approximate Imown number of spores, as indicated in 

 iable 2. btenhzed 2 c c Luer tuberculin hypodermic syringes Grad- 

 uated m 0.01 c c the needles of which had been blunted, were "used 

 in inoculating the cells containing coiled larvae. Fifty or more 

 coiled larvae at least 4 days old were each given 0.01 c c of a dilution 

 ot spores, each dilution being given to larvae in one comb in a separate 

 colony, and the cells so inoculated were distinctively marked A few 

 larvae that had just been sealed also were inoculated by puncturing 

 the cappmg with the inoculating needle and depositing the 0.01 c c in 

 the ceU Observations were taken at the end of 24 hours and at 

 frequent intervals thereafter until the end of the brood-rearing 



