A flagellate amoeba, isolated from Ohio River water and identified as (Schardinger), has been maintained in permanent cultures on a buffered sucrose nitrate agar with its original bacterial associate, identified as . On Czapek agar plates, the growth curve of the amoeba at 25-27° was found to consist of a lag phase of about 4 hr., a log growth phase of about 60 hr., and a growth-encystment phase of 2-3 weeks. At 37° the lag phase was shortened by half and the log growth phase by about a third. The generation time was 4 1/2 hr. at 25-27° and 2 1/2 hr. at 37°. Continuous cultivation at 37°, however, resulted in occurrence of abnormal forms. Very poor growth of amoeba on nutrient and tryptose agars was attributed to their high organic nitrogen content, poor growth in liquid media to the difficulty of amoebae to engulf bacteria. Attempts to grow the amoeba on heat-killed bacteria were failures. Redox potential and pH determinations revealed that the mixed fluid cultures were aerobic and the amoebae perished at pH values more acid than 5·6. Replacement of by other bacteria by Oehler's technique was unsuccessful because of swarming but was successful with a sp., which produced a yellow pigment whose antibiotic effect prevented swarming. Permanent cultures of the amoeba with the new bacterial associate were maintained after the amoeba overcame the antibiotic effect of the pigment. Replacement of the sp. in the mixed culture by new species of bacteria was easily accomplished, and permanent cultures were maintained with , 8 species of and 2 of . The amoebae encysted earlier on all these species than on .

Cytological observations include: () appearance of the chromatin material as short rods; () no evidence of thread-like structures radiating from the karyosome or chromatin patches in resting nucleus, nor the centrioles in mitosis; () karyosome origin of both the ‘interzonal body’ and the polar caps.


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