Sybr green 1 sg

Staining was performed as described previously [25 (link)]. For a working solution, we diluted SYBR Green I (SG) (Invitrogen, USA) 100-fold in anhydrous dimethylsulfoxide (DMSO), and added propidium iodide (PI, 30 mM) to a final PI concentration of 0.6 mM (SGPI). This working solution was stored at -20°C until use. We stained 1 mL of water sample with 10 μL SYBR Green I working solution for enumeration of total bacteria or 10 μL SGPI for enumeration of intact/damaged bacterial cells. Before analysis, samples were incubated in the dark at 37°C for 15 min and bacteria-counting beads (Invitrogen, USA) were added to each sample for calculation. Prior to FCM analysis, water samples were diluted with 0.22 μm filtered bottled water (Evian) to concentrations ranging from 1×10⁵ cells.mL^-1 to 1×10⁶ cells.mL^-1. We performed FCM using a FACSCalibur (BD Biosciences, USA) instrument with CellQuest software equipped with an argon laser emitting light at a fixed wavelength of 488 nm. In the flow cytometer, optical filters were set up so that PI was measured above 630 nm and SYBR Green I at 520 ± 10 nm. The trigger was set for the green fluorescence (520 nm) channel (FL1), and data were acquired on two-parameter dot plots of green fluorescence (520 nm) versus red fluorescence (630 nm); ultrapure water was used as sheath fluid. No compensation was used for any measurement.

For the counting of intact bacterial cells, two fluorescent dyes, SYBR^® Green I (SG) and propidium iodide (PI; Invitrogen, Belgium) were used for staining (Wang et al., 2010 (link)). When necessary, samples were diluted in 0.22 μm filtered bottled mineral water prior to staining. The staining solution was prepared as followed: PI (20 mM in dimethyl sulfoxide, DMSO) was diluted 50 times and SG (10,000 times concentrate in DMSO) was diluted 100 times in sterile DMSO. All samples were stained with 10 μl ml^-1 staining solution and 10 μl ml^-1 EDTA (pH 8, 500 mM) for outer membrane permeabilization. 10 μl ml^-1 CytoCount counting beads (Dako, Belgium) were added as internal standard. Prior to flow cytometry analysis, the stained samples were incubated for 5 min in the dark at 37°C. Flow cytometry was performed using a Cyan^TM ADP LX flow cytometer as described by before (Boon et al., 2006 (link)). Only intact bacterial cells were counted. Dust particles and non-bacterial cells were excluded using the appropriate software.