Medical millex gs filters

Planktonic bacteria were grown in lysogeny broth (LB) (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, Melford) and plate bacteria were cultured in LB agar plates (LB with 15 g/L agar). E. coli cell line BW25113 was obtained from Dharmacon (GE Healthcare). Single bacterial colonies, picked from a streak plate, were incubated with shaking (200 rpm) in fresh LB (150 mL) over 16 hours at 37 °C. Following incubation, the obtained culture was centrifuged (5 min, 3220 x g) at 20 °C. The supernatant was filtered (2x) using Medical Millex-GS filters (0.22 μm) from Millipore Corp., and then was used to re-suspend the bacterial pellet at the OD₅₉₅ of 75. Single-cell kinetic measurements was performed using the resulting highly concentrated suspension of bacteria, together with M9 minimal medium (1 × M9 salts, 2 mM MgSO₄, 0.1 mM CaCl₂, 1 mg/L thiamine hydrochloride) from Sigma Aldrich, and propidium iodide (PI) from Thermo Fisher Scientific. The protocol of the measurements is described below.