H4 Phylum Protozoa 



taminated culture of the Spirillum was placed on the surface of sterile 

 tap water in the large arm of a V-tube. Five-tenths of a cc. amounts, 

 withdrawn one hour later from the surface of the small arm, gave pure 

 cultures of the Spirillum upon inoculation into suitable media (see also 

 Part III). 



II. CULTURE MEDIA FOR CERTAIN BACTERIA-FREE PROTOZOA 



"Basic Medium." Among the free-living Protozoa freed of bacteria 

 by any of the above methods some, such as Trichoda pura, were able to 

 grow well in simple media such as peptone water. Even such Protozoa, 

 however, grow better in the so-called "basic medium" of Glaser and 

 Coria (1930). This has been recently modified ( Glaser and Coria, 1934a) 

 to give more nearly uniform results and is now prepared in the following 

 way: Stock Solution A consists of 50 cc. of horse serum in 1000 cc. of 

 well water (or distilled water). This is autoclaved for 30 minutes at 

 15 lbs. and has a pH of 7.0 without adjustment. Stock solution B consists 

 of 50 gms. of timothy hay in 1000 cc. of water. The mixture is infused 

 over night in the refrigerator, filtered through cotton, and the reaction 

 adjusted to pH 7.2-7.4. The stock solutions A and B are stored 

 separately in a refrigerator and when needed are combined with well 

 water in these proportions: 



Solution A 500 cc. 

 Solution B 250 cc. 

 Well water 250 cc. 



The medium is adjusted to pH 7.2-7.4, tubed in 8 or 10 cc. amounts and 

 autoclaved. This final medium was used only for Protozoa free of other 

 micro-organisms. When heavy initial cultures of unpurified Protozoa 

 are desired 2 cc. of the final medium is added to 20 cc. of the original 

 Protozoa-containing water. To make a solid medium, 1.5 to 2% agar is 

 added. For a semi-solid medium similar to Noguchi's Leptospira 

 medium, 100 cc. of the melted solid medium is diluted with 900 cc. of 

 warm water. 



Raw Potato. Some Protozoa, which grew well in basic medium when 

 contaminated with bacteria, did not grow at all in this medium when 

 free of other micro-organisms. Thus a flagellate from the intestine of 

 Lucilia caesar would not grow in basic medium except in the presence of 

 living bacteria. The bacteria-free flagellate would not develop on auto- 

 claved potato, but delicate growths of it were obtained in tubes of raw 

 potato under tap water. Such tubes are prepared as follows: Raw po- 

 tatoes are scrubbed with hot water and partly dried by heat; a part of 

 the surface is washed thoroughly with 70% alcohol and flamed until 

 charred. Cylinders are then cut out with a sterile No. 5 cork borer and 



