Amoebidae 85 



and reproduction of this form. It may be cultured in any concentration 

 from a five-fold dilution to a concentration so high that some of the 

 salts begin to precipitate. The optimum concentration is about 10% 

 distilled water and 90% seawater. The salt ratios may be altered con- 

 siderably and still the amoebae will grow and reproduce. 



The hydrogen-ion concentration. The hydrogen-ion concentration 

 seems to have but little influence on reproduction. It is possible to cul- 

 ture F. mira at any concentration between pH 3.0 and 9.0. Reproduc- 

 tion, however, seems to be more rapid in the ranges pH 8.0-9.0, and pH 

 5.0-7.0, than in the range pH 7.0-8.0. In artificial or natural seawater 

 cultures the hydrogen-ion concentration remains fairly constant, varying 

 within the range pH 8.0 to pH 8.4. 



The culture of F. mira on solid media. Rice (1935) has made use 

 of silica gel and Bacto agar plates in culturing F. mira. The Bacto agar 

 method may be described as follows: Place 100 gm. wheat in 1000 cc. 

 of artificial seawater and autoclave for 20 minutes at a pressure of 15 

 pounds and a temperature of 125 C. Strain through a cheesecloth, 

 and then filter through a coarse filter paper. Now add 15 gms. Bacto 

 agar and enough artificial seawater to bring the volume back to 1000 cc. 

 Autoclave again at the same temperature and pressure for 30 to 60 

 minutes. The resulting agar medium is then tubed, plugged with cotton, 

 sterilized again, and set aside for future use. When ready to culture the 

 amoebae on this medium, melt the agar in a tube, pour into a sterile 

 petri dish, cover, and allow to cool and solidify. Then transfer a small 

 drop of liquid culture medium containing amoebae and associated bac- 

 teria to the center of the plate, or scatter small drops about over the 

 entire surface of the plate. The water will be absorbed by the agar 

 and the amoebae and bacteria will grow and develop on the surface. 

 The bacteria and consequently the amoebae will become very abundant 

 and concentrated. Often the amoebae become so thick that they form 

 a sort of epithelial layer over the surface of the agar. To subculture, 

 amoebae and bacteria are transferred with a platinum loop to freshly 

 poured plates. 



The culture of F. mira on pure strains of marine bacteria. By the 

 proper bacteriological methods isolate bacteria from good cultures of F. 

 mira. Then pour artificial seawater-wheat-extract-agar plates and inocu- 

 late only in the center with amoebae and associated bacteria. Now with 

 a platinum loop make a smear of the previously isolated marine bac- 

 terium in a circle about 1 cm. from the center inoculated with amoebae. 

 Some of the amoebae in traveling the 1 cm. distance from the point of 

 their inoculation over a sterile surface lose contaminating bacteria so 

 that when they enter the smear of the pure strain of bacteria they are 

 sterile. (Some, however, may carry bacteria even this distance.) They 



