Ophryoglenidae 1 1 1 



able as media with which to conduct certain types of experimental in- 

 vestigations because their composition is unknown and uncontrollable and 

 doubtless varies greatly as prepared at different times and places. 



For these reasons we have used the reproducible, non-nutritive medium 

 shown in the following table, supplemented by the addition of a pure 

 strain of bacteria for food. 



Balanced Physiological Medium 



(after Osterhout, 1906) 



67c total salt (0.937 M.) 

 0.937 M. Parts 



NaCl 1 ,000 



MgCl 2 .6H 2 78 



MgS0 4 . 7 H 2 38 



KC1 , 22 



CaClo 10 



This solution is diluted five times to the approximate concentration 

 of pond water, or 0.012% total salt (0.0018 M.). One cc. of M/20 

 NaH 2 P0 4 is added to each 30 cc. of this diluted solution for buffering, 

 and the pH adjusted with suitable quantities of M/20 NaOH. 



Colpoda in this medium are fed upon suspensions of a pure culture 

 of the bacterium Pseudomonas fluorescens. This bacterium is eminently 

 suitable as a food for Colpoda. When grown in suspensions of it, they 

 appear large and well fed, their division rate is equal to or greater than 

 that in the most favorable hay infusions, they continue normally active, 

 and may be kept indefinitely on this single diet without degeneration. 



Pseudomonas fluoresceins may be isolated from soils by the use of a 

 liquid enrichment culture in which 2% asparagine serves as the only 

 source of carbon and nitrogen. The bacterium may be isolated and grows 

 rapidly on ordinary yeast — or peptone-glucose-agar plates at 37 C. or 

 below. If inoculated heavily on such plates, the colonies spread out 

 within a few days into a continuous sheet from which masses of bacteria 

 may be removed with a sterile platinum loop in order to be fed to the 

 Protozoa. 



The density of the bacterial suspension provided for the Protozoa is 

 difficult to define except as that density in which optimal growth occurs. 

 This most favorable density of bacterial suspension will of course depend 

 in part upon the number of Protozoa inoculated into a given volume of 

 the medium. An approximate idea of a favorable suspension density may 

 be gained from the following illustration: the mass of bacteria which will 

 fill a platinum wire loop 1 mm. in diameter is sufficient to make 6 

 suspensions of % cc - into each of which 20 Colpoda are to be inoculated. 



No effort is made to avoid air contaminations of the Pseudomonas 

 suspensions. It was assumed that the suspensions would not be suffi- 



