Quantification of Aquatic Microbiome

Subsamples (2 ml) were fixed with glutaraldehyde (0.5% final concentration), incubated at 4 °C for 15 min in the dark, flash frozen in liquid nitrogen and then stored at − 80 °C [37 (link)]. With scatter diagrams of side scatter vs. red fluorescence and orange fluorescence vs. red fluorescence, the abundances of picoeukaryotes, Synechococcus and Prochlorococcus were directly determined by flow cytometry (Epics Altra II, Beckman Coulter, USA) [38 (link), 39 (link)]. The samples for heterotrophic bacterial counting were stained with 1.0 × 10⁻⁴ SYBR Green I (v/v, final concentration, Molecular Probes), incubated for 15 min in the dark and analysed by flow cytometry with scatter diagrams of side scatter vs. green fluorescence and red fluorescence vs. green fluorescence [38 (link), 39 (link)]. To obtain viral abundance, the samples were diluted with Tris–EDTA buffer (pH 8.0; Sigma), stained with 5.0 × 10⁻⁵ SYBR Green I (v/v, final concentration, Molecular Probes) and then incubated at 80 °C for 10 min in a thermostat water bath (DKB-501A, Shanghai Jinghong, China); abundance was then determined by flow cytometry [40 , 41 (link)]. Data analyses were performed with FCS Express V3 software (De Novo Software, http://www.denovosoftware.com/).