ZI 6 Phylum Protozoa 



tion of the liver extract, either in the autoclave or in the Arnold sterilizer 

 does not give a satisfactory medium. 



Dead yeast is prepared from baker's yeast grown for 5 days on dextrose 

 agar in a Blake bottle. The growth is washed off in sterile water, 

 centrifuged, and again washed and centrifuged. The washed yeasts are 

 suspended in 15 cc. of sterile water and this suspension is distributed in 

 5 cc. amounts to tubes which are then sealed and placed for 30 minutes 

 in a water bath at a temperature of 75 ° to 8o° C. The heated suspen- 

 sions are tested for sterility on dextrose agar slants. Killed cultures of 

 Staphylococcus pyogenes aureus or albus may be used instead of the 

 killed yeast. 



In the final preparation of the medium, pieces of kidney weighing 

 0.2 to 0.5 gms. are aseptically removed from a freshly killed rabbit and 

 placed in tubes of liver extract. To each tube is added 0.1 cc. of heat- 

 killed yeast suspension and also some Paramecia from the surface of a 

 migration pipette. An inoculum of about 870 Paramecia was usually 

 used. In such tubes a luxuriant growth of Paramecium caudatum is pres- 

 ent by the tenth day and successful subcultures are regularly obtained 

 between the seventh and fifteenth day of incubation at room temperatures. 

 Table I shows that the liver extract, killed yeast, and fresh kidney are 

 all essential to the growth of Paramecium caudatum in the absence of 

 other micro-organisms. Very similar results have been obtained with the 

 large Paramecium multimicronucleatum. By the ordinary migration 

 technique this ciliate was easily freed of all but one contaminant, a small 

 motile bacillus. Further migrations were now performed in sterile water 

 containing a few drops of living yeast cell suspension. The bacillus 

 multiplied very slowly in the water, and the Paramecia fed on the yeast 

 cells. These eventually settled out while the Protozoa swam to the top 

 of the long water column. Such daily migrations were repeated over a 

 period of 6 days and finally a migration in sterile water without yeast 

 cells yielded Protozoa free of other micro-organisms. Such Paramecium 

 multimicronucleatum grew in the same medium which was used for 

 P. caudatum. 



Necessity for a Change of Medium. While both species of Paramecia 

 in their special culture medium, and the flagellate Parapolytoma satura on 

 blood agar, seem to grow indefinitely, the other purified Protozoa, when 

 grown continuously in "basic medium" showed a gradually lessening 

 developmental rate and finally died out unless transferred to some other 

 culture medium. Thus after several months in "basic medium," with 

 transfer intervals of 1 to 2 weeks, when the Protozoa had begun to grow 

 less luxuriantly, they were subcultured to potato or carrot water or to 

 basic medium containing sterile rabbit kidney or yeast extract. Such 

 transfers again gave excellent growth, but this again weakened after 



