14 TECHNICAL BULLETIN 149, U. S. DEPT. OP AGEICULTUEE 



ones. Young bees are also more easily handled, since they are less 

 excitable when caged. Young drones and queens, which may or 

 may not have recently emerged, are quite as satisfactory for these 

 experiments as are worker bees, and were frequently included. 



EXPERIMENTS WITH COLONIKS 



Colonies were inoculated by placing matured dry cultures of fungi 

 on brood combs or on strips of blotting paper between the brood 

 combs. One or two of these cultures were put in each of the ex- 

 perimental colonies. Complete infection was obtained somewhat 

 sooner with two comb cultures than with one, though in order to 

 secure complete infection of a colony it was necessary to use cultures 

 with an abundance of dry spores on the greater portion of the sur- 

 face. Spores were soon spread to all parts of the hive by the worker 

 bees and after three or four days were found in the food of the brood 

 and in. the alimentary canal of all the adult bees examined. Colonies 

 were less frequently infected by mixing spores with dilute sugar 

 sirup and confining the bees to the mixture for food. 



BLOOD INOCULATION EXPERIMENTS 



The blood of the honeybee is well protected from the entrance of 

 microorganisms by the tough exoskeleton. As far as is known, in- 

 fection by pathogenic organisms occurs only by way of the alimen- 

 tary canal. Practically nothing has been done to determine the 

 resistance, or lack of resistance, of the blood of the honeybee to 

 infection by microorganisms. A series of blood inoculations of adult 

 bees was therefore made with the fungi used in these investigations. 



While they were being inoculated the bees were held between the 

 thumb and the second finger, with the wings turned upward and 

 held with the fore finger. The propodeum was moistened by lightly 

 rubbing it with a wad of cotton or filter paper saturated with a 

 water suspension of the organism under observation until a film was 

 spread over the surface. This portion of the exoskeleton was care- 

 fully punctured with a fine-pointed sterilized needle. When the 

 needle was withdrawn the water containing the spores was spread 

 into the wound. As an alternative for the first method, the needle 

 was dipped into the suspension of spores and the tip inserted into 

 the wound a second time. 



Checks, to determine the effect of the punctures upon the bees, were 

 prepared by a similar treatment, except that spores were omitted 

 from the water with which the propodeum was moistened or in which 

 the needle was dipped. Inoculations were also made at other points 

 on the body, but fatal injuries resulted more often when inoculations 

 were made in places other than the propodeum. Inoculated and check 

 bees were kept in cages until the experiment was completed. 



EXPERIMENTS WITH BROOD 



In addition to the inoculation of colonies in which spores reached 

 the food of all uncapped brood, uncapped larvae were inoculated 

 directly from plate or tube cultures. Dried cultures in Petri dishes 

 or tubes were scraped with a scalpel or inoculating needle to loosen 



