Lysogeny broth lb

B. subtilis YC161 strain with $P_{\mathit{spank}}$ -gfp^{27 (link)} were grown in Lysogeny broth (LB; 1% tryptone, 0.5% yeast extract, 0.5% NaCl; Neogen, Lansing, MI, USA) and incubated at 37  ${}^{\circ }C$ at 150 rpm for 5 h. For macrocolony formation, 2.5 μL of starter culture suspension (O.D. 600 nm = 1) was inoculated onto biofilm-promoting LBGM agar medium. LBGM medium was prepared using LB growth medium solidified by the addition of 1.5% (w/v) agar and further supplemented with 1% (v/v) glycerol and 0.1 mM MnSO₄^{28 (link)}. An amount of 2.5 μL chlorhexidine digluconate (CHX) solution at 20% in water (Sigma-Aldrich, St. Louis, MO, United States) was spotted one-sided at various distances (1.0 cm, 1.5 cm, 2 cm) from the bacterial inoculation point, at the time of inoculation. In addition, control B. subtilis biofilm plates were prepared which did not contain CHX. All plates were incubated at 30  ${}^{\circ }C$ for a period of 3 days and images captured daily. Number of repeats were as follows—12, 11, 9, 12 plates for distances 1, 1.5, 2 cm and control, respectively.

The strains used in this study were obtained by experimental evolution as previously described [30 (link)] (Tables S1 and S2, available with the online version of this article). Briefly, P. aeruginosa PAO1 biofilms were repeatedly exposed to the QSI C-30 (100 µg ml⁻¹), tobramycin (20 µg ml⁻¹), or a combination of C-30 and tobramycin, in SCFM2, during 16 cycles. P. aeruginosa was maintained on tryptone soy agar (TSA, Neogen) and all overnight cultures were prepared in Lysogeny Broth (LB, Neogen). For each condition three independent replicate populations (lineages) were used. Whole populations of the evolved lineages were used for subsequent analyses.