An improved isolation method for attached-living Planctomycetes of the genus Rhodopirellula

Rhodopirellula baltica, an attached-living marine bacterium, was so far isolated as aerobic, heterotrophic bacterium forming pink-to-red colonies on ampicillin-containing plates. But many bacteria grow in the presence of ampicillin: marine samples from European Seas contained on average 10,365 colony forming bacteria per ml water sample or sediment. Therefore we developed an improved enrichment protocol to isolate Rhodopirellula strains. To include attached-living bacteria, particles, plankton catches or sediment grains were spread on the plate surface. This was a necessity to reliably obtain new strains. Cycloserine or streptomycin served as additional selective agents. The number of colony forming cells resistant to ampicillin and cycloserine or streptomycin was low, 262 and 107 cfu/ml sample, respectively. Rhodopirellula colonies were identified with a newly developed specific PCR reaction detecting a part of the 16S rRNA gene. The phylogenetic tree of over 60 isolates was established with new primers outside of the 16S rRNA gene and revealed a diversity on the species level in European Seas.