Quantifying Prokaryotic Abundance in Sediments

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} .