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To explore the possibility of calculating phylotype-specific growth rates of marine bac- teria, we optimized a protocol that combines bromodeoxyuridine (BrdU) immunocytochemistry with fluorescence in situ hybridization (FISH). The protocol worked well with 8 isolates tested, showing a significant positive correlation between average cellular fluorescence intensity and cell-specific BrdU contents. The BrdU immunocytochemistry-FISH (BIC-FISH) was then applied to neritic sea- water in Hiroshima Bay, western Japan. Rhodobacter/Roseobacter and Bacteroidetes groups were dominant in BrdU-positive cells (22 ± 8 and 26 ± 8%), suggesting a significant contribution to bacte- rial community productivity in the water. Analysis of single-cell immunofluorescence showed higher BrdU incorporation rates in the Gammaproteobacteria, SAR86, and Vibrio groups, suggesting that these less abundant groups were growing more rapidly than other phylotypes. Our study suggests that BIC-FISH has the potential to estimate phylotype-specific variability of bacterial productivity in aquatic systems.

Phylotype-specific growth rates of marine bacteria measured by bromodeoxyuridine immunocytochemistry and fluorescence in situ hybridization