FUNGOUS DISEASES OP THE HONEYBEE 25 



EXTRACTION OF THB TOCSIN FBOM THE MEDIUM 



It appeared, as a result of the experimeBts discussed above, that 

 the toxic substance must be extracted in order to avoid error from 

 other factors that affected the death rate. Since nothing was known 

 of the nature or constitution of the toxic substance, it was necessary 

 to determine the best method for extracting it. 



Alcohol extracts 



Extraction of proteins with alcohol was tried first. The precipi- 

 tates obtained from the medium in which fungi had been cultured 

 and from the juices pressed from the mycelium after grinding it with 

 sand were dissolved in water and fed with dilute honey to bees in 

 cages. The bees took this food readily, but the death rate remained 

 normal throughout two trials. It is not proved by these experiments 

 that a poisonous protein is not produced, since such a poison may 

 be digested to nonpoisonous compounds before absorption, as with 

 higher animals {11, p. 367). On the other hand, absorption by the 

 honeybee of some of the simpler sugars appears to be a question of 

 only from one to a few minutes. 



Ether extract 



The medium from'10-day-old flask cultures of Aspergillus ftaws 

 Ao5c and the fungous juices pressed from the mycelium after it had 

 been washed and ground with sand were shaken five times with small 

 quantities of ether. After each shaking the ether was separated 

 from the medium with a separately funnel. The ether was then 

 washed with a large volume of distilled water, separated, and evap- 

 orated to dryness in evaporating dishes. Brown amorphous residues 

 were obtained in larger quantities from the medium than from the 

 fungous juices. Each of the residues was dissolved in 5 cubic centi- 

 meters of distilled water and added to about 6 cubic centimeters of 

 clover honey. This was given as food to caged worker bees. The 

 bees that received the extract from the fungous juices lived normally; 

 but some of the bees receiving the extract from the culture medium 

 were noticeably affected after 4 hours, and after 10 hours all that had 

 been fed with this extract were dead. The experiment with the 

 culture medium extract was repeated with four lots of bees, the same 

 food being used for each succeeding lot after all of the bees in the 

 previous lot had died. A constant decrease in potency was noticed 

 with each successive trial. With the second trial all of the bees were 

 dead at the end of 18 hours ; with the third, after 30 hours ; with the 

 fourth, after 2 days ; and with the fifth, after 7 days. (Table 1 and 

 fig. 1.) 



This experiment was repeated several times" with similar results 

 except that the earlier deaths were not obtained again in succeeding 

 experiments. Extracts from old cultures that had been kept at 

 room temperature and from young cultures prepared at about the 

 time spores were forming appeared to contain only small quantities 

 of the toxic substance. The greatest accumulation of toxin within 

 the medium seems to be present at about the time the cultures are 



