Axenic (pure) cultures of marine unicellular cyanobacteria of the Prochlorococcus genus grow efficiently only if the inoculation concentration is large; colonies form on semisolid medium at low efficiencies. In this work, we describe a novel method for growing Prochlorococcus colonies on semisolid agar that improves the level of recovery to approximately 100%. Prochlorococcus grows robustly at low cell concentrations, in liquid or on solid medium, when cocultured with marine heterotrophic bacteria. Once the Prochlorococcus cell concentration surpasses a critical threshold, the "helper" heterotrophs can be eliminated with antibiotics to produce axenic cultures. Our preliminary evidence suggests that one mechanism by which the heterotrophs help Prochlorococcus is the reduction of oxidative stress.Members of the genus Prochlorococcus are the most abundant marine photosynthetic organisms and, as such, are major contributors to photosynthesis in the ocean (20). Over 30 strains of Prochlorococcus have been brought into culture, isolated from many locations within the band from 40°N to 40°S, including the North Atlantic, the North and South Pacific Oceans, the Mediterranean Sea, and the Arabian Sea (20). Despite this success, very few pure cultures of Prochlorococcus (e.g., those of strains PCC 9511 and MIT 9313 [18,22]) have been obtained. The vast majority of cultures contain heterotrophic microbes as contaminants; these heterotrophs were cocultured from the marine environment during the isolation procedure, which has relied thus far exclusively on liquid cultivation. While plating for contiguous lawns of Prochlorococcus has proven to be productive (15), attempts at colony formation (by pour plating or surface streak plating) have thus far met with significantly less success. Recovery efficiencies of the pour plating technique of 0.1 to 10% have been reported previously for some strains (15,24), but this technique has yet to produce pure cultures (15). The inability to readily obtain clonal, pure cultures of Prochlorococcus has severely limited progress in the genetic and physiological analysis of this ecologically important lineage.The "helper" phenotype of heterotrophic bacteria. Standard dilution streaking of contaminated Prochlorococcus cultures onto semisolid medium failed to produce axenic colonies. Colonies formed only within a visible mass of the contaminant heterotrophic bacteria; such masses appeared typically at the sites of the earliest, heaviest dilution streaks (data not shown). One interpretation of these results was that Prochlorococcus was able to grow only in the presence of the contaminating bacteria, perhaps because the bacteria provide a growth factor and/or remove an inhibitory factor. Coculturing with heterotrophic bacteria is required for the growth of some bacterial isolates (9) and is known to improve the growth of dinoflagellates (2, 7), suggesting that a similar interaction may help Prochlorococcus. To test this hypothesis, a heterotrophic contaminant (designated EZ55) of a culture of the ...

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