Reash

The RP437-TC-FliC strain was grown from frozen stocks (made from single colonies) in LB medium (1% w/v Bacto-Tryptone, 0.5% w/v yeast ex-tract and 1% w/v NaCl) overnight at 30 °C. The saturated culture was diluted by 1:100 in M9 medium (M9 minimal supplemented with 0.4% glucose, MEM vitamins (×100) and amino acids (×50)) and grown at 30 °C to an appropriate concentration (OD₆₀₀ = 0.3–0.4). Labeling of the flagella was performed by gently mixing FlAsH or ReAsH (Invitrogen, 2.5 µM) with the diluted E. coli cells and incubated for 10 min at 30 °C. For flagellar growth observations in the flagellin over-expression assay, M9 medium was supplemented with 0.5% glycerol, MEM vitamins and amino acids and ampicillin (50 µg/ml).

Transfection of cells, labeling with resorufin arsenical hairpin binding reagent (ReAsH; Invitrogen) and EM 902 transmission electron microscopy has been described in an earlier study (Tian et al., 2011 (link)).