Persister Cell Survival Assay

Persister cell survival was determined by counting the number of colonies that grew on solid media after washing and serially diluting cells exposed to antibiotics, as previously described (Kwan et al. 2013 (link)). Briefly, overnight cultures (16 h) were diluted 1:1000 with fresh LB medium and grown to the desired turbidity (0.8 at 600 nm for the exponential phase and 3–4 at 600 nm for the mid-stationary phase) at 250 rpm. Since pretreatments of bacteriostatic rifampicin (Kwan et al. 2013 (link)) and bactericidal ampicillin and ciprofloxacin (Hu et al. 2015 (link); Kwan et al. 2015 (link)) increased persister cell formation, the three antibiotics have been used in this study. In order to obtain antibiotic-induced persister cultures in buffered LB, cultures were exposed to rifampicin (100 μg mL⁻¹), ampicillin (100 μg mL⁻¹), or ciprofloxacin (0.5 μg mL⁻¹) and incubated for 30 min at 37 °C. Cells were harvested by centrifugation at 4000 rpm for 14 min and washed with fresh LB (turbidity was controlled as 0.5). Cells (0.5 mL) were then transferred to micro-tubes and treated with or without indoles and incubated for 3 h at 37 °C at 250 rpm. DMSO was used as a control. Cell viabilities were determined by serially diluting cells with PBS buffer, plating 100 μL drops on LB agar, and counting colonies.