FUNGOUS DISEASES OF THE HONEYBEE 9 



tical with these were also prepared to inoculate large quantities of 

 nutrient solution to be used in the preparation of brood-comb 

 cultures. 



PREPARATION OF THE CULTURES 



All of the fungi tested and found to be pathogenic for bees were 

 cultured on one or more of the solid or liquid nutrient media used. 

 Cultures of these fungi were prepared by inoculating the nutrient 

 medium in the culture dish with a single spore or with spores or 

 mycelium from pure cultures. Normal room temperatures were gen- 

 erally used although cold rooms and electric ovens were employed 

 at times. Combs were sterilized in a 20 per cent aqueous Solution of 

 formaldehyde. Two liters of nutrient solution containing spores 

 of the desired organism taken from a pure culture were poured into 

 each comb. These combs were kept in a sterile moist chamber until 

 spores had matured over the greater part of their surface. Prac- 

 tically pure cultures were obtained by this method. The few con- 

 taminating spores which settled on the combs while the spore suspen- 

 sion was being poured were soon overgrown and rarely matured. 



It was found better to keep the moist chambers out of doors while 

 the fungi on the combs were developing, or at least in rooms where 

 beekeeping equipment, particularly extracting and brood combs, 

 were not stored, since under proper atmospheric conditions the combs 

 are overgrown with fungi, principally Penicillia and Aspergilli. 

 An abundance of spores of such pathogenic organisms within the 

 room will add to the danger of spreading the molds to other combs. 



One strain of Aspergillus ocfiraceus was found to produce but 

 slight growth on liquid media. Cultures of this organism on combs 

 produced few or no spores and were usually soon overrun by species 

 of Penicillium. Inoculation of colonies with this organism were 

 generally unsuccessful. This difficulty was overcome by culturing 

 the organism on heavy sheets of reinforced blotting paper. The 

 blotting paper was first sterilized and then saturated with nutrient 

 agar before inoculation by spraying with a suspension of spores. 

 After a crop of spores had matured the sheets of blotting paper were 

 suspended in the hive containing the colony to be inoculated. 



THE MEDIA EMPLOYED 



Several nutrient media, both synthetic and natural, were used dur- 

 ing this work for the artificial culture of the organisms. Solid media 

 which were used extensively for isolation, study, storage, and ship- 

 ping purposes, were prepared by the addition of agar, and less fre- 

 quently gelatin, to the nutrient solutions. The agar and gelatin were 

 omitted in preparing cultures for inoculation purposes when it was 

 desired to have a large quantity of spores that could be readily freed 

 from the medium. Liquid nutrient media were also used when the 

 object was to extract toxic products of pathogenic organisms, and for 

 the culture and study of yeasts. Natural media, such as potato, car- 

 rot, and milk were rarely used. Occasionally liquid media to which 

 <i small quantity of tartaric acid was added were used when it was 

 desired to eliminate bacteria from the yeast cultures. The media 

 used in the course of these investigations are described on the pages 

 that follow. 



59850°— 30 2 



