FUNGOUS DISEASES OE THE HONEYBEE 13 



of the cages so that the food could readily be reached by the bees. 

 (PI. 1, A and B.) The number of thicknesses of cheesecloth required 

 depended upon the density of the sirup used. Heavy honeys re- 

 quired but one thickness, but for dilute sugar sirups two or more 

 thicknesses of cloth were necessary to prevent leakage. 



Bees in cages were inoculated by being placed on cultures of the 

 fungi or by mixing the spores with their food. Within the labora- 

 tory, where cultures of fungi were kept, it was unsafe to inoculate 

 bees, by the first of the two methods, with fungi that form dry and 

 dustlike spores ; for by fanning their wings the bees sent a dust of 

 spores into the air. Consequently, inoculations were made outside of 

 the laboratory. When spores of fungi were mixed with the food 

 given to the bees the danger of contamination of cultures within the 

 laboratory was largely avoided. Abundant space outside of the lab- 

 oratory being available, however, the first method of inoculation was 

 generally used, since it probably approached nearest to the natural 

 methods by which bees become infected. Complete inoculations re- 

 sulted from placing the bees for one or more minutes on plate cul- 

 tures that had matured a good crop of dry spores. After inoculation 

 the bees were either removed to sterile dishes or caged, until the 

 experiment was complete, on the culture upon which the inoculation 

 was made. A cone made by cutting off the smaller end of a funnel, 

 so that a hole with a diameter of about 1% inches was left at that 

 end, was used for transferring the bees from the cultures to the 

 cages. The small end was placed over the cage, and the culture dish 

 containing the bees was opened quickly and inverted in the mouth of 

 the funnel, completely closing it. When the bees had entered the 

 cage the cone was removed and the screen cover quickly placed in 

 position and fastened with rubber bands. After a little practice this 

 operation of transferring could be successfully performed without 

 injuring the bees, success depending upon speed and precision of 

 manipulation. 



As a measure of precaution, when several fungi with dustlike 

 spores were being tested at the same time, inoculations on dried 

 cultures were made out of doors and the cages placed in different 

 rooms, or in different parts of the same room. This prevented, to a 

 large extent, infection with pathogenic fungi from other cages. 

 When this precaution was not observed, a high percentage of infec- 

 tion, at times reaching more than 70 per cent, could be traced directly 

 to the crossing over of spores from adjacent cages. When spores of 

 fungi were mixed with the food given to the bees these precautions 

 were unnecessary, 



Sbxection of Bees foe the Experiments 



Old bees from the field force of a colony die rapidly when caged, 

 even under conditions of temperature, moisture, etc., that duplicate 

 those of the hive. Their early death when caged appears to be due 

 in part to continued exertion in an effort to escape. Records of the 

 death rate among infected bees are therefore unsatisfactory when 

 field bees are used. Yoiing bees for caging were obtained by setting 

 aside frames of emerging brood a few days before bees were needed, 

 or by lightly shaking frames of bees to rid them of most of the old 

 bees, as young bees cling more tightly to the combs than do the older 



