Nuclepore black filters

Total prokaryotic abundance was determined by epifluorescence microscopy^{47 (link)}. Sediment samples were treated three times for 1 min by ultrasounds (Branson Sonifier 2200, 60 W) after addition of 0.2 µm pre-filtered tetrasodium pyrophosphate solution at a final concentration of 5 mM, then properly diluted before filtration onto 0.2 µm pore-size Nuclepore black filters (Whatman). Each filter was then stained with 20 µl of SYBR Green I (Sigma Chemicals, previously diluted 1:20 with 0.2 µm pre-filtered Milli-Q water), washed twice with 3 ml sterilized Milli-Q water and mounted onto microscope slide. Filters were analyzed using epifluorescence microscopy (Zeiss Axioskop 2MOT, magnification 1,000×). At least 20 microscope fields and 400 cells were respectively observed and counted for each filter^{48 (link)}. Prokaryotic abundance was expressed as cells per g of dry sediment, after desiccation at 60 °C for 24 h⁴⁵ . Prokaryotic biomass was determined based on cell size, converted into bio-volume, assuming 310 fg C $\mathrm{\mu }$ m³ as a conversion factor, following standard inter-calibration with Scanning Electron Microscope (SEM)^{45 ,48 (link),49} .

Total prokaryotic abundance was determined by epifluorescence microscopy as described in Danovaro et al. (2001) (link). Briefly, the sediment samples were treated by ultrasounds (Branson Sonifier 2200, 60W) three times for 1 min after addition of 0.2 μm pre-filtered tetrasodium pyrophosphate solution (final concentration, 5 mM), then properly diluted before filtration onto 0.2 μm pore-size Nuclepore black filters (Whatman). Filters were then stained with SYBR Green I (Sigma Chemicals) by adding, on each filter, 20 μl of the stock solution (previously diluted 1:20 with 0.2 μm pre-filtered Milli-Q water), washed twice with 3 ml sterilized Milli-Q water and mounted onto microscope slides. Filters were analyzed using epifluorescence microscopy (Zeiss Axioskop 2MOT, magnification 1,000×). For each filter, at least 20 microscope fields were observed and at least 400 cells counted.