CONTRACTILE VACUOLE IN PARAMECIUM CAUDATUM. 211 



filtrate was then tested for ammonia and urea as described 

 below. The possible presence of uric acid was not investigated 

 at this time. 



The paramecia were washed as follows: Culture fluid con- 

 taining the organisms was poured into a long-necked bottle of 

 approximately one liter capacity until it was filled to within 

 about 5 cm. of the top. Then spring-water was added carefully 

 so as to avoid mixing until the bottle was entirely full. Within 

 five or ten minutes under these conditions the paramecia usually 

 aggregated in very great numbers near the surface of the spring- 

 water. When they had thus collected at the top of the bottle, 

 the surface water containing them was removed with a pipette, 

 and more spring- water added. This process was repeated until 

 most of the animals had been taken from the culture fluid, usually 

 three or four times. The bottle was then emptied, and the water 

 containing the paramecia put into it, after which it was filled 

 with spring-water. The organisms were removed as before. 

 The paramecia were thus washed in fresh spring-water three or 

 four times, after which they were usually found to be free from 

 all heavy debris and large bacterial masses. Smaller organisms 

 which were removed from the bottle with the paramecia were 

 separated from the paramecia by further washing on filter paper. 

 The paper used was about 20 cm. in diameter, and was selected 

 so that the pores were small enough to retain the paramecia, 

 but large enough to allow the smaller organisms to pass through. 

 A liter of w r ater, or more if necessary, was used for this part of 

 the washing process. 



After the paramecia had been thus washed they were put into 

 a clean glass beaker; then spring-water was added until the 

 number per cubic centimeter was reduced to from 500 to 2,500 

 individuals. This was ascertained by counting the numbers in 

 several one cubic centimeter portions and averaging the results 

 obtained. The paramecia were allowed to remain in this water 

 for periods of time ranging from eighteen to thirty-six hours, 

 after which they were removed by filtration. 



A portion of the filtrate was tested for ammonia by Nessleriza- 

 tion and the rest for urea by methods described below. 



In twenty-two of the twenty-five experiments positive tests 



