310 NINETEENTH REPORT. 



alcohol added to the filtrate. It was washed several times with alcohol, 

 dried in a desiccator, and redissolved in 60 cc. of distilled water. Alcohol 

 was again added to make a concentration of 80% and the resulting 

 precipitate together witli that formed in the first filtrate, filtered off. 

 After wasliing and drying it was again dissolved by repeatedly passing 

 30 cc. of distilled water through the filtrate. To this was added 0.6 cc. 

 of toluol as a preservative. For convenience in tabulating the data whicli 

 folh)ws, this will be called preparation "A." 



Tlic bulk of the alcoholic precipitate consisted of protein and brown 

 |)ignunt matter. It was assumed that any enzyme which might be present 

 in solution would be precipitated by the alcohol. If any white enzj^mic 

 precijjitate was present, there was no macroscopic method of determining 

 this, because of the presence of the brown colored impurities, which the 

 above treatment failed to remove. 



Preparation "B": — The fungus growth from a clover juice culture of 

 the same age as the preceding was filtered out through pajier and then 

 through a sterile Berkfeld filter. To this filtrate 2% toluol was also 

 added. 



Preparation "C" : — The fungus growth which had been removed from 

 the above cultures and that from two others of the same age was washed 

 in a little running tap water and the superficial moisture removed by 

 pressing gently between sheets of filter paper. In order to insure rapid 

 and thorough drying, the fungous growth was then placed in a desiccator 

 containing CaCK and the air exhaust attached. When desiccated it was 

 ground with clean quartz sand until practically no entire spores or 

 mycelial cells remained. This ground mass together with 60 cc. of water 

 plus 2% toluol was put into a bottle and agitated for one hour in the 

 shaking machine. The extraction was prolonged for 18 hours more with 

 an occasional shaking by hand. The sand and fungus particles were 

 removed by filtering through cotton, and the' filtrate used for the experi- 

 ment. 



Preparation "D": — Sterile clover juice medium plus 2% toluol. 



These preparations were tested upon a lot of clover plants all of which 

 had been planted in the greenhouse at the same time, from the same lot 

 of seeds. They were all in normal healthy condition, and well watered 

 during the entire experiment. The Van Tieghem cell arrangement, above 

 described was used; likewise all other technique was the same. Each 

 number again refers to a leaf with each of the three leaflets bearing a 

 cell containing the same preparation. 



The following table explains the procedure and the results after 48 

 hours : 



