Cloning, Titration, and Differentiation of Acanthamoeba sp. by Plating

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Abstract
Microscopically visible colonies of Acanthamoeba sp. strains V, R, and S were formed when amoebae and certain yeasts or bacteria were plated in a chemically defined agar medium and incubated at 37 C. The yeasts Candida albicans and Cryptococcus sp. and the bacterium Aerobacter aerogenes were the most satisfactory in supporting growth and colony production by the amoebae. Efficiency of plating for V, R, and S was 0.67 to 0.94 for trophozoites, and 0.24 to 0.84 for fresh cysts. It was shown that a large fraction, often 0.90 to 0.99, of the colonies produced on C. albicans must have been initiated by individual amoebae, and thus were clones. The number of colonies produced by trophozoite and V cyst populations was proportional to the number of potential colony-forming units inoculated thus calculated colony-forming titers of such populations were independent of the amoeba concentration inoculated. A similar relationship was obtained for fresh R and S cysts but only when they were washed before inoculation. This washing was effective because it removed an excystment inhibitor(s) that was present in the medium in which the cysts had been produced. Strain V produced such an inhibitor but was not inhibited by it. By harvesting organisms from atypical colonies, mutants of strains V, R, and S were obtained.