Changes in community composition during dilution cultures of marine bacterioplankton as assessed by flow cytometric and molecular biological techniques

Dilution experiments do not measure in situ growth, but rather growth patte rns of an enrichment. Furthermore, it was demonstrated that the combination of flow cytometric analysis and sorting combined with FISH and DGGE analys is presented a fairly rapid method of analysing the taxonomic composition o f marine bacterioplankton. Dilution cultures are a common technique for mea suring the growth of bacterioplankton communities. In this study, the taxon omic composition of marine bacterioplankton dilution cultures was followed in water samples from Plymouth Sound and the English Channel (UK). Bacteria l abundances as well as protein and DNA content were closely monitored by f low cytometry. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 18S rDNA fragments and fluorescence in situ hybridization (FISH) were applied directly to the water samples and to cel ls sorted from the dilution cultures based on their protein and DNA content . As expected, a rapid activation of bacteria occurred. However, molecular techniques showed that the community developed in the dilution culture with in 1 day was significantly different from that in the original water sample s. Whereas in the original samples, cells detectable by FISH were dominated by members of the Cytophagal Flavobacterium (CF) cluster, in dilution cult ures, gamma-proteobacteria accounted for the majority of cells detected, fo llowed by alpha-proteobacteria. An actively growing and an apparently non-g rowing population with average cellular protein contents of 24 and 4.5fg re spectively, were sorted by flow cytometry. FISH indicated mostly gamma- (64 %) and alpha-proteobacteria (33%) in the first active fraction and 78% memb ers of the CF cluster in the second fraction. Sequencing of DGGE bands conf irmed the FISH assignments of the latter two groups. The data presented cle arly show that even relatively short-term dilution experiments do not measu re in situ growth, but rather growth patterns of an enrichment. Furthermore , it was demonstrated that the combination of flow cytometric analysis and sorting combined with FISH and DGGE analysis presented a fairly rapid metho d of analysing the taxonomic composition of marine bacterioplankton.