52 Phylum Protozoa 



Several investigators (Parpart, 1928; Luck and Sheets, 1931; Hether- 

 ington, 1934) have described relatively simple methods for washing 

 Protozoa free from bacteria. The method of Parpart, a method which 

 offers few technical difficulties, has been used successfully in our labora- 

 tory and elsewhere. In this method it is a comparatively easy matter to 

 free the Protozoa from bacteria, although it is often very difficult to estab- 

 lish thriving cultures of the bacteria-free organisms. 



MEDIA FOR CHLOROPHYLL-BEARING FLAGELLATES 



For obvious reasons, the chlorophyll-bearing flagellates are more 

 easily established in bacteria-free cultures than is any of the other groups 

 of free-living Protozoa. This is evidenced by the fact that approximately 

 40 species of green flagellates have been isolated in such cultures, most of 

 the strains being maintained at the present time in the laboratory of 

 Professor E. G. Pringsheim (Pringsheim, 1928a, 1930). The composi- 

 tion of the various media, so far as inorganic constituents are concerned, 

 is based upon past experience in the growth of algae and green flagellates. 

 With the establishment of bacteria-free cultures, peptones or other organic 

 sources of nitrogen have usually been added to the media for the mainte- 

 nance of rich cultures. Several of the media which have been tried in our 

 laboratory and have given good results with different species of Euglenida 

 and Phytomonadida are listed in Table 1. 



table 1. Media for chlorophyll-bearing flagellates. 



Media 



Constituents ABC D E F 



NH4NO3 0.5 gm 1.0 gm. 



KNO3 0.5 gm 0.5 gm. 



Tryptone 2.5 gm 2.0 gm. . . .5.0 gm. 



Glycine 1 -99 6 gm. 



K 2 HP0 4 0.209 gm. 



KH2PO4 0.5 gm. . . .0.5 gm. . . .0.2 gm. . . .0.25 gm. . .0.5 gm. 



MgS0 4 0.1 gm. . . .0.1 gm. . . .0.2 gm 0.25 gm. . .0.25 gm . .0.048 gm. 



NaCl 0.1 gm. . . .0.1 gm 0.1 gm. 



KC1 0.2 gm .... 0.25 gm. 



FeCI 3 Trace Trace Trace Trace 



Sodium acetate 2.5 gm 2.0 gm 1 .48 gm. 



Dextrose 2.0 gm. 



Distilled water . ...i.ol 1.0 1 1.0 1 1.0 1 0.0 1 1.0 1 



Medium A, adjusted to pH 7.0, has given very good results (Loefer, 

 1934) with Chlorogou'nim euchlorum and C. elongatum, and should be 

 equally satisfactory for other species of Chlamydomonadidae. Medium 

 B, at pH 7.0, has supported growth of Chlorogonium and C. elongatum 

 (Loefer, 1934) in darkness for more than a year, the flagellates retaining 



