Supplementary Figure 1: Killing of E. coli colonies on nitrocellulose membranes by ciprofloxacin.

a, Killing of wild-type E. coli colonies by ciprofloxacin. Cells were seeded onto nitrocellulose membranes and grown for 24 h, then transferred to LB supplemented with ciprofloxacin for 24 h, and plated (mean ± range; n = 2 or n = 4 biologically independent experiments). b, Same as in a, except colony area was calculated (mean ± s.d.; n = 3 or n = 4 biologically independent experiments). Note that at concentrations as high as 5,000-fold MIC, only ~1 log of cell death occurred with insignificant changes in colony size. c, 24-point MIC curves of bactericidal antibiotics against wild-type E. coli. Overnight cultures were inoculated 1/10,000 into fresh LB media in final volumes of 100 µl in 96-well microtiter plates, and antibiotic was added to the concentrations indicated. Cultures were incubated until untreated control cultures reached stationary phase, and optical density at 600 nm was measured (mean ± range; n = 2 biologically independent experiments). d, 24-point MIC curves of bacteriostatic antibiotics against wild-type E. coli. Overnight cultures were inoculated 1/10,000 into fresh LB media in final volumes of 100 µl in 96-well microtiter plates, and antibiotic was added to the concentrations indicated. Cultures were incubated until untreated control cultures reached stationary phase, and optical density at 600 nm was measured (mean ± range; n = 2 biologically independent experiments). e, Killing of mature wild-type E. coli colonies by diverse antibiotics (top panel; mean ± range; n = 2 biologically independent experiments). Amp, ampicillin; cef, cefsulodin; mec, mecillinam; azt, aztreonam; cip, ciprofloxacin; gen, gentamicin; rif, rifampicin; cm, chloramphenicol; spec, spectinomycin. Numbers show fold-MIC concentrations used. Amp, cef, mec, azt, cip, and gen are regarded as bactericidal against E. coli; rif, cm, and spec are bacteriostatic. The bottom panel shows metabolic staining of wild-type E. coli colonies after treatment with the corresponding antibiotics on the top (mean ± range; n = 2 biologically independent experiments). f, Kinetics of TTC staining of E. coli. Wild-type cells were seeded onto nitrocellulose membranes and grown for 24 h on solid LB under aerobic conditions. Membranes were then transferred to LB under aerobic conditions, LB under anaerobic conditions, or LB supplemented with KCN at the indicated concentrations for 24 h. Membranes were then transferred to LB supplemented with 50 µg/ml TTC (aerobically for aerobic and KCN conditions; anaerobically for anaerobic conditions) and incubated for the noted durations prior to imaging (mean ± range; n = 2 biologically independent experiments). One hour of staining was selected as the experimental endpoint for the Keio screen to acquire a sufficiently high signal while avoiding overstaining. Fluorescence was normalized on the basis of maximum fluorescence (time = 0). g, Bacterial cell killing and metabolic staining of diverse species—E. coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Bacillus simplex, and Mycobacterium smegmatis—subjected to heat treatment at 80 °C for 30 min. Blue bars show CFU quantification of colonies that were not heat treated (mean ± range; n = 2 biologically independent experiments). For CFU quantification of heat-treated samples, asterisks represent cell densities below the detection limit of 10² CFU/ml.

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