Flow cytometry output (ECOBABe phase 1 experiment - microbial viability)
Microbiota preparations were incubated for 5 minutes in the dark with 5 µM SYTO BC Green Fluorescent Nucleic Acid Stain (Thermo Fisher Scientific, Massachusetts, USA, #S34855) and 10 µg/ml propidium iodide (PI, Thermo Fisher Scientific, Massachusetts, USA, #P3566). Controls included an unstained fraction, separate incubations of SYTO BC and PI (for compensation), and a dead cell fraction (thermal shock at 70°C for 30 minutes, followed by PI staining). Samples were analysed on a LSR II flow cytometer (BD, New Jersey, USA). Microbial cells were filtered by size and gated into three fluorescently-distinct populations: viable cells (SYTO BC positive), dead cells (PI positive), and damaged cells (SYTO BC positive, PI positive) (Figure 1). The proportion of each subpopulation was calculated with reference to the total analysed cell count.