Bacterial Viability in Fecal Microbiota

Bacterial viability in fecal microbiota samples from fresh and frozen stool was measured by flow cytometry using LIVE/DEAD BacLight™ Bacterial Viability and Counting Kit (L34856, Molecular Probes) according to manufacturer instructions (Molecular Probes, Oregon, United States). In brief, 977 μL of 0.9% NaCl, 1.5 μL of SYTO9, 1.5 μL of propidium iodide (PI), and 10 μL of the diluted sample were added to the flow cytometry analysis tube. Samples were 10-fold diluted in 0.9% NaCl. The tube was incubated for 15 min in the dark at room temperature and 10 μL of the microsphere suspension (beads) was added to the stained sample. The total volume of the sample in the flow cytometry analysis tube was 1000 μL. The samples were analyzed on an LSR Fortessa flow cytometer (Becton Dickinson, New Jersey, United States) with FACS Diva v8 software (Becton Dickinson). The gating strategy was as described in our first work (Bilinski et al., 2020 (link)). Three main cell populations were observed—alive, dead, and unknown (probably alive, probably dead) with a special not-alive-not-dead group of cells (SYTO9^–PI^–). The number of bacteria per mL in each analyzed gate was counted according to the following formula taken from the manufacturer materials: