28 TECHNICAL BULLETIN 14 9, U. S. DEPT. OF AGRICULTURE 



before the end of the fifth day. Bees fed upon honey with the ether 

 extract, which had been made neutral with potassium . hydroxide, 

 showed a normal death rate for is days, when all of the food was 

 consumed. Bees given the solution that had been readjusted to about 

 pH 4.6 after neutralization died at about the same rate as bees fed 

 on the ether extract that had not been neutralized. (Table 3 and 

 Fig. 3.) 



Takle 3. — Length of Ufe of lees m cages when ether extract of the culture 

 medium of Aspergillus flcumis Ao5c was mMeedi with their food 



i so 



X so 







m 



1 The total number of bees used in this experiment was also 25, The food containing the fungous extract 

 was consumed by the bees after 14 days, when records of the daily death rate wfte discontinued. 



If, as is indicated by the foregoing experiment, the toxic substance 

 is of basic nature, it should be dissolved out with chloroform if the 

 medium is first made basic. Potassium hydroxide was added to the 



_ medium from 12-day-old cultures 

 until it gave a distinct basic reaction. 

 Chloroform was then shaken with 

 the medium, separated, and evapo- 

 rated to dryness, as was done with 

 ether. A flocculent . precipitate, 

 which formed upon the first addi- 

 tion of chloroform, was separated, 

 drained, and dissolved in a small 

 quantity of water. By the addition 

 of honey to this precipitate a food 

 for bees was prepared. A food was 

 prepared also with the residue left 

 upon evaporation of the chloroform. 



-.t 





FiGnEE 3. — Rate of mortality of honey- 

 bees at daily intervals after the com- 

 mencement of feeding with honey con- 

 taining ether extract of the nutrient 



medium in which Aspergillus flavua -.f . * . in,. 



had been cultured: 1, With freshly JNo poiSOning resulted irom either 



Se'sughtf/'^yic^'wirh^potfssiSm' food. After six days each prepa- 

 rS°^o^W'traSi,'? ^rZit'T'^^, ^^ti«n was adjusted to a pH value 



after it had been made slightly basic OI abOUt 4.0 by the addition of SUl- 



phuric acid and fed to recently 

 caged bees. The death rate in both cages remained normal. The 

 toxic substance, if present at first, appeared to have been entirely 

 destroyed in the basic condition. 



That it was nearly all removed from the medium was shown by 

 adjusting the medium to its original pH value and extracting with 

 ether immediately after the extraction with chloroform was com- 

 plete. When this ether extract was fed to bees the death rate was 

 about normal. 



In order to determine whether the toxic substance could be 

 recovered by the adjustment of the pH value immediately after 



