FUNGOUS DISEASES OP THE HONEYBEE 7 



was not uncommon to find brood attacked during the spring in 

 colonies which, owing to weather conditions or other factors, had 

 a high mortality when normally the population should have been in- 

 creasing. The quantity of diseased brood was found to be less 

 during dry weather. At all times of the year strong colonies were 

 found to be relatively free from pathogenic fungi. 



EXAMINATION OF DISEASED BEES AND ISOLATION OF PATHO- 

 GENIC FUNGI 



The methods employed in the isolation of pathogenic fungi from 

 bees must depend to a considerable degree upon the species of in- 

 fecting organisms and the extent and state of their development. 

 Samples of dead bees or bees in various stages of development of 

 the disease were therefore examined microscopically to determine 

 the stage of growth of the fungus. Isolation of filamentous patho- 

 gens was least dilficult when spores practically free from contami- 

 nating organisms were present upon the body of the bees. The tip 

 of an inoculating needle drawn to a sharp point was moistened by 

 dipping it into sterile agar. The tip of a spore-bearing branch 

 with mature spores was lightly touched and a few spores trans- 

 ferred to an agar plate. Pure cultures of the organism desired were 

 often obtained by this method without additional effort. When 

 more than one fungus was fruiting upon the body of the bee or 

 even when scattered spores of other fungi were present, this method 

 usually resulted in mixed cultures. Pure cultures of the Aspergilli, 

 which are mostly rapid growers, were often obtained from mixed 

 cultures by cutting off the tip of a young mycelium and transferring 

 it with a small quantity of agar to a fresh plate. When contami- 

 nation with other fungi was heavy, or when the contaminating 

 organisms grew more rapidly than the fungus to be isolated, the 

 dilution.-spray method devised by Kauffman {16, p. 364) was em- 

 ployed with good results. A suspension of conidia was first made in 

 a tube of sterile tap water. This was diluted until droplets blown 

 from a glass tube drawn to an extremely fine bore at the tip con- 

 tained an average of about one spore. One or two plates of nutrient 

 agar were sprayed with this suspension, the spray being so regulated 

 that droplets on the agar remained separated by a distance of about 

 3 millimeters. When germinating spores first appeared single spores 

 with germ tubes were cut and transferred with small pieces of agar 

 to separate poured plates. Two or more such cultures were prepared 

 in each instance. When contamination occurred transfers were made 

 of the few hyphae from the single spore mycelium as soon as the 

 contaminating organism became visible. 



When spores were present only within the exoskeleton of the bees, 

 and especially when within the alimentary canal, isolation of pure 

 cultures was difficult on account of the presence of great numbers of 

 bacteria. When these conditions were met with, the acidity of ,the 

 nutrient agar upon which the suspension of spores was sprayed was 

 made slightly greater with tartaric acid, and the cultures were kept 

 at a temperature of about 10° C. until the spores had germinated. 

 Though the growth of the fungus was retarded when kept at this 

 temperature, the growth of bacteria was practically inhibited. 

 Single germinating spores were then cut out filong with any adhering 



