244 Vniversitij of California PuNicatioiis in Zoology ["^'ol. 16 



the University of California. Small amoinits of soil, about ten cubic 

 centimeters, were placed in previously sterilized, covered glass dishes 

 of creek water or culture medium, the latter giving a greater abundance 

 in a shorter time. In a week amoebas were found in the scum which 

 formed on the surface. Prom these cultures were started, each being 

 made from a single individual amoeba with bacteria of the culture. 



At first a mechanical pipette was used for isolation, but the amoeba 

 stuck to the sides so that an ordinary pipette which has been drawn 

 out very small was used in all subsequent work. "With this the amoeba 

 can be ejected before it has had time to adhere to the glass. The 

 flagellate stage is much more easily handled and so it has been used 

 a great deal for inoculation of cultures. Subcultures were also made 

 from cultures which were pure by the transfer to the new medium of 

 a cover-glass which had been floated on the given culture. In this way 

 numerous amoebas may be obtained more quickly than by the use of 

 a single individual. It has been possible by this method to keep "pure 

 mixed" cultures of amoebas and of the bacteria which form their food 

 in great numbers continuously in the laboratory for the past two years. 



The medium used was boiled, filtered creek water to which one per 

 cent of a mixture in water of ground cabbage and cracker in equal 

 proportions was added. The mixture was then sterilized in the dishes 

 used for cultures for from forty-five minutes to one and a half hours, 

 usually the latter, at eighteen pounds presssure in a steam autoclave. 

 Enoiigh water is always used in the cabbage-cracker mixture to make 

 it thin enough to pour; and it is kept indefinitely in a flask plugged 

 with cotton, being sterilized each time after being opened. Agar plates 

 have also been employed, but the films for study and fixation are much 

 more easilj' made from the liquid medium and the amoebas are more 

 abundant and better distributed here, so that the eulti;re fluid has been 

 used for the most part rather than the agar. 



The method has been the moist-film one. A cover is floated on the 

 bacterial film on the surface of the culture, left for from a few seconds 

 to a minute or two, removed, drained of excess water by standing on 

 edge on a blotter or by merely shaking, then dropped film-side down on 

 the fixing fluid. It is not necessary to use any fixation to make the 

 film adhere to the glass. After it has been on the surface of the fixative 

 for about a half minute or longer, the cover is turned over and allowed 

 to sink. 



The following fixing fluids were ased: Bouin's, Carnoy's. piero- 

 mercuric, carbo-formalin-aeeto-methyl-alcohol, and alcoholic sublimate 



