Stress condition, inhibitors and antibiotics (correspondence, Naotake Ogasawara)

     B. sutilis 168 and mutant strains were pregrown overnight in liquid LB medium at 37 to stationary phase (ca. 2-8 x 109 cells/ml). Aliquots of cultures were serially diluted (1:100, 1:1,000, 1:10,000 and 1:100,00) and cell suspensions were subsequently transferred to 96-well microtiter plate serving as a master plate. Two microliters of cell suspensions were spotted on plates containing inhibitors or antibiotics. Plates were then incubated at 37 for 36 hrs, and growth and β-galactosidase activity of the mutants was scored by comparison with that in the control condition (LB) or with growth of the control strain (168).

      The following compounds and X-gal (100μg/ml) were added to LB media after autoclaving. LB (LB broth LENNOX) and NB (nutrient broth) were obtained from Difco. Growth conditions are as follows (final concentrations in LB media): H2O2, 0.5mM and 1.3 mM; Paraquat, 10μM and 70μM; EtOH, 2% and 8%; NaCl, 0.5M and 1M; Xylose, 0.5M and 0.8M; SDS, 0.001% and 0.007%; SDC (sodium deoxycholate), 0.001% and 0.007%; CCCP (carbonylcyanide-m-chlorophenylhydrazone), 2μM and 7μM; MC (Mitomycin C), 10 ng/ml and 15 ng/ml; PEN G (Penicillin G), 1 ng/ml and 7 ng/ml; STR (Streptomycin), 2μg/ml and 10μg/ml; TRIM (Trimethoprim), 0.1μg/ml and 1ug/ml.