Epics altra 2 flow cytometer

For viral and bacterial counts, 2 ml seawater was fixed at a final concentration of 0.5% glutaraldehyde at 4°C for 20 min and then stored at 4°C. Viral and bacterial abundances were determined using an Epics Altra II flow cytometer (Beckman Coulter, Miami, FL, USA) as described by Brussaard (73 (link)). The fixed samples were stained with SYBR green I (Invitrogen, CA, USA) and enumerated at event rates of 50 to 200 particles/s (bacteria) or 100 to 300 particles/s (viruses). For every sample, 10 μl of 1 mm-diameter fluorescent microspheres (Molecular Probes, Inc., OR, USA) was added as reference beads. Each sample was run twice on the flow cytometer, and the average of count values was taken. The data were analyzed by EXPOTM_32 MultiCOMP software (Beckman Coulter, Miami, FL, USA).

Abundances of Synechococcus were determined using 2-mL seawater aliquots on an Epics Altra II flow cytometer (Beckman Coulter, Brea, CA, United States) with a 306C-5 argon laser (Coherent, Santa Clara, CA, United States), according to the method of Jiao et al. (2002) (link). They were identified and distinguished from other autotrophs based on their positions in plots of side scatter versus red fluorescence and orange fluorescence versus red fluorescence. Event rates were set to 50–200 events/second (0.1–1 mL h^-1) in order to enhance the particle capturing sensitivity. Fluorescent microspheres of 1 μm diameter (Polysciences Inc., Warrington, PA, United States) were added to all samples as an internal standard to calibrate flow rate and cell size. All samples were run in triplicate. The data were analyzed with EXPOTM32 MultiCOMP software (Beckman Coulter, United States).

The enumeration of viral-like particles (VLPs) and picoplankton was according to [61 (link),62 (link),63 (link)]. All samples were added with 1-μm diameter yellow-green fluorescent beads (Molecular Probes, Thermo Fisher, Waltham, MA, USA) as an internal standard with the final concentration of ca. 1% to get a better indication effect. For the enumeration of autotrophic picoplankton including pigmented pico-sized eukaryotes (PPEs), Synechococcus, and Prochlorococcus, no staining step was performed [63 (link)]. Samples for the enumeration of VLPs and heterotrophic prokaryotes were stained with SYBR Green I (Molecular Probes, Thermo Fisher, Waltham, MA, USA) [61 (link),62 (link)]. Briefly, for the enumeration of VLPs, after thawed at 37 °C, diluted with 0.02-μm filtered Tris-EDTA buffer (Sigma-Aldrich, Darmstadt, Germany), and stained with SYBR Green I, VLPs were analyzed at a flow rate of 0.1–1 mL h⁻¹ and identified on the basis of the green fluorescence and side scatter signal [61 (link)]. Autotrophic picoplankton, heterotrophic prokaryotes, and VLPs were analyzed on the same Epics Altra II flow cytometer (Beckman Coulter, Brea, CA, USA). FCS Express V3 software (De Novo Software) was used to obtain VLPs and picoplankton abundance.