Amoebae from natural isolates of , unlike the plasmodial phase, are unable to grow in axenic medium. A mutant strain of amoebae, CLd-AXE, can be cultured in the liquid, semi-defined medium used for plasmodial culture but lacks some of the properties required for studies of development and gene expression. From crosses of CLd-AXE with wild-type amoebae, new amoebal strains able to grow in axenic medium have been isolated; some of these can also undergo the reversible amoeba-flagellate transformation and apogamic plasmodium development in axenic conditions. Amoebae maintained in active growth in liquid culture for several months showed little change in their properties. Subcultures made with diluted inocula indicated that the same growth rate was achieved even when single amoebae were inoculated in liquid medium. All strains produced colonies with high efficiency when replated on bacterial lawns. Measurements of DNA content by flow cytometry indicated that the majority of amoebae in liquid cultures were haploid. Homozygous diploid amoebae constructed from one strain grew less well than the haploid cells. Genetic analysis of crosses suggested that amoebae able to grow in liquid axenic medium fell into one major phenotypic class with respect to growth rate, and that mutation at only one or two loci was necessary to allow amoebae to grow in axenic medium. Diploid, heterozygous amoebae constructed by mating a mutant with a wild-type strain were unable to grow in axenic medium, indicating that at least one of the putative alleles was recessive.


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