FUNGOUS DISEASES OF THE HONEYBEE 



27 



The fungous fluids were pressed from the mycelium after" it had been 

 ■washed and ground with sand. Both tlie medium and the fungous 

 fluids were each shaken five times with small volumes of chloroform 

 and separated with a separatory funnel after each operation. After 

 washing in an evaporating dish the chloroform was evaporated to 

 dryness, and the residue from each solution was taken up with about 

 5 cubic centimeters of distilled water and given to worker bees with 

 ah equal volume of honey. In each case the entire quantity of food 

 was consumed without evidence of poisoning. Kepetitions of this 

 experiment at different stages in the development of the lungus gave 

 entirely negative results. It would seem therefore that bases capable 

 of poisoning bees when administered with food are not produced by 

 A. -flavus Ao5c. 



Chemical Nature of the Toxio Sitbstancb 



In most cases chemists have been unable to determine the constitu- 

 tion of toxic substances elaborated by microorganisms ; therefore only 



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PrGUEir 2. — Rate of mortality of honeybees at dally intervals after the 

 commencemexit of feeding with honey containing ether extract of the 

 nutrient miedium in which Aspergillus flavus had' been cultured. First 

 trial made immediately after the extract was prepared, second trial 15 

 daya after the extract was prepared. 



a few tests were made to determine the general nature of this sub- 

 stance. Tests for phenolic compounds were made. by floating small 

 quantities of freshly prepared ether extract on very dilute ferric 

 chloride solution. The absence of color reaction in all cases indicated 

 the absence of such compounds in the extract. When the extract 

 from flask cultures in 5 cubic centimeters of water was mixed with 

 5 cubic centimeters of honey, the resulting solution showed a hydro- 

 gen-ion concentration of about pH 4.6 by the colorimetric method of 

 Clark and Lubs (7) and Clark (6), whereas equal volumes of water 

 and the same honey showed a concentration of about pH 5. 



Something of the nature of the toxic substance is shown by the 

 following experiments: Freshly prepared extract was divided into 

 three equal parts. One part was fed directly to bees in cages. The 

 other two parts were made neutral, or slightly basic, by the addition 

 of potassium hydroxide. One of these parts was immediately ad- 

 justed by hydrochloric acid to a hydrogen-ion concentration of 

 about pH 4. 6. Honey was added to each of these two lots and they 

 were fed to bees. The bees given the original unneutralized extract 

 showed definite evidence of poisoning after 24 hours. The death rate 

 was considerably higher than normal, and all of the bees had died 



