Parameciidae 115 



put in sterile petri dishes where they are cut into pieces % inch long. 

 Each piece is placed in a tube of sterile water. Such tubes are held at 

 room temperature for some time before use, and any showing con- 

 tamination are discarded. 



Special Medium for Paramecia. Two species of Paramecium {P. cauda- 

 tum and P. multimicronucleatum) probably furnish the best examples of 

 free-living Protozoa which, when free of other micro-organisms, require a 

 very special complex nutritive medium (Glaser and Coria, 1933, 1934a). 

 Various workers have successfully freed Paramecia of contaminating 

 bacteria, usually by means of the washing of isolated individuals, but the 

 bacteria-free organisms could not be cultured. Such was at first the 

 experience of Glaser and Coria ( 1930) who used the migration technique 

 to obtain the pure Protozoa. They established a "pure-mixed" culture 

 in basic medium of Paramecium caudatum in association with the yeast, 

 Saccharomyces cervisiae. Since the yeast is non-motile and heavier than 

 water one can readily take advantage of the negative geotropic migration 

 of the Paramecia to secure pure Protozoa. In practise, the "pure-mixed" 

 culture was centrifuged and washed three times in sterile water. The 

 material so obtained was introduced at the base of a pipette in the 

 manner previously described and the Protozoa permitted to migrate 

 through the column of sterile water. Every day for 5 days material from 

 the top of the pipette was removed and plated on dextrose agar. Only in 

 cultures made on the first day were any yeast colonies obtained, showing 

 that after the first day the yeast cells brought to the surface by the Para- 

 mecia had been digested. Upward migration of the Protozoa was com- 

 plete by the fourth day at a temperature of 20 o -2 2° C. Pure Paramecia 

 obtained in this way were finally cultured in a medium consisting of liver 

 extract, killed yeast, and fresh rabbit kidney. Repeated tests (aerobic 

 and anaerobic cultures in a variety of media at room and incubator 

 temperatures) showed that the cultures so obtained were free of other 

 micro-organisms. 



The liver extract consists of a 0.5% solution of Eli Lilly Company's 

 liver extract No. 343 in water. This is filtered through paper and 

 sterilized by filtration through a Berkefeld N filter. This extract has a 

 pH of 6.2 to 6.4 and is placed in 10 cc. amounts in sterile test tubes. 

 Liver extract may also be made in the following way: One hundred grams 

 of finely ground rabbit, beef, or swine liver are infused over night in the 

 refrigerator in 200 cc. of water. The suspension is filtered through cotton, 

 heated over a water bath for about one hour, and then strained through 

 fine gauze, all the fluid being squeezed out of the coagulum. The liquid 

 is further cleared by centrifuging and is then diluted with water to 400 cc. 

 It is warmed to 6o° C. and passed successively through sterile Berke- 

 feld V and N candles and then is tubed in 10 cc. amounts. Heat steriliza- 



