8 TECHNICAL BULLETIN 149, U. S. DEFT. OP AGRICULTURE 



bacteria and transferred to sterile plates. These cultures were kept 

 in the cold room until fungus naycelium had grown to a distance of 

 several millimeters beyond the zone of bacterial growth. The tips 

 of hyphae, apparently free of bacteria, were then cut off and trans- 

 ferred to sterile plates. This process usually resulted in pure cul- 

 tures. Whenever bacteria appeared, the process was repeated as 

 often as was necessary. 



A method modified somewhat from the foregoing was used when 

 only mycelium was present within the tissues of the bees. Small 

 pieces of tissue containing mycelium were placed on plates of nutri- 

 ent agar and kept at 10° C. It was not always necessary to place 

 these cultures in the cold room when they could be closely watched 

 and isolations made from the first hyphae that grew into the agar. 



Hyphae grew out rapidly from the infected tissue, and the tips of 

 the first appearing hyphae were cut off and transferred before bac- 

 teria or mycelium from spores could spread beyond the point of 

 inoculation. Pure cultures were made from these after spores had 

 matured. 



The isolation of yeasts from the alimentary canal of the bee re- 

 quired a more delicate technic than the isolation of filamentous fungi 

 because of the presence of other contaminating forms. When yeasts 

 occurred within the tissues or blood of bees they were readily isolated 

 since they were usually found here in pure or nearly pure culture. 

 In the isolation of yeasts a sufficient quantity of acid was added to 

 the medium to retard the growth of bacteria, thus facilitating the 

 isolation of pure yeast cultures. 



To obtain pure yeast cultures the spray method described above 

 was used. A suspension of yeast cells was sprayed on beer-wort 

 agar or on Leonian's agar in Petri dishes. The location of the 

 droplets after evaporation was marked by a perceptible deposit of 

 lime from the tap water. After from 12 to 24 hours these spots were 

 examined under the microscope, and those that contained yeast 

 colonies developed from single yeast cells were marked by scratch- 

 ing the agar about them with a sterile needle. When these colonies 

 had developed to contain several hundred cells second dilutions and 

 sprays were made from them. From these cultures, or from similar 

 succeeding ones when bacterial contamination was heavy, yeast 

 colonies free from bacteria were obtained. Colonies, the origin of 

 which could be traced to a single cell, were chosen for isolation a 

 second time to avoid the possibility of mixed yeast cultures. When 

 the original culture was obtained from the rectum or ventriculus of a 

 bee contamination with bacteria was usually heavy, and several 

 repetitions of the process were often necessary to obtain bacteria- free 

 cultures. 



CULTURE METHODS 



For the culture of the organisms studied the usual mycological 

 equipment was used. 



Erlenmeyer flasks of 500 and 1,000 cubic centimeter capacity, filled 

 to a depth of about 3 centimeters with liquid nutrient media, were 

 used for the culture of pathogenic organisms in experiments to 

 determine the production of metabolic toxins. Flask cultures iden- 



