Quantifying Culturable and VBNC Cells

The counts of culturable cell were determined using the standard plate count method according to Su et al. (2015b) (link). Each sample was serially diluted 10-fold with 0.9% (w/v) physiological saline (sodium chloride solution) and incubated on LB agar at 30°C for 48 h. The culturable cell counts of R. biphenylivorans TG9 were calculated as counts per milliliter (CFU/mL). The viable cells number was measured using a flow cytometer. The 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) (Dojindo, Japan) staining was used to determine the viable cells. Bacteria in the VBNC state still have active respiration. CTC can be reduced by electron transport chains to give off red fluorescence whose intensity reflects the number of viable cells (Zhang et al., 2018 (link)). CTC solution was added to 500 μL of R. biphenylivorans TG9 at a final CTC concentration of 1 mM. Then the suspensions were mixed with 50 μL absolute counting beads (Thermo Scientific, United States). Before analysis, the cell suspensions were incubated at 37°C for 1 h in the dark. When the number of cultured cells had decreased to an undetectable level, the cells still viable were denoted as entering the VBNC state. The VBNC cells number was calculated as the viable cells number minus the culturable cells number (Chen et al., 2019 (link)).