Significance of size and nucleic acid content heterogeneity as measured byflow cytometry in natural planktonic bacteria

Total bacterial abundances estimated with different epifluorescence microsc opy methods (4',6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dea d) and with flow cytometry (Syto13) showed good correspondence throughout t wo microcosm experiments with coastal Mediterranean water. In the Syto13-st ained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, "live" bacteria (determined as such with the Molecular Probes Live/Dead Bac Light bacterial viability kit), and nucleoid-containing bacteria (NuCC) com prised similar fractions of the total bacterial community. Similarly, LDNA bacteria and "dead" bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The ra tes of change of each type of bacteria during the microcosm experiments wer e also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the H DNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA ) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HD NA and LDNA bacteria can be obtained, and given the similarity to the numbe rs of "live" cells and NuCC, the %HDNA is suggested as a reference value fo r the percentage of actively growing bacteria in marine planktonic environm ents.