Biofilm Formation Quantification Assay

Cell morphology was observed using a Tecnai G2 F20 S–TWIN transmission electron microscope (TEM, FEI Company, Hillsboro, OR, USA). The ability of biofilm formation was assayed by the classical crystal violet staining, as described elsewhere, with minor modification [23 (link)]. Briefly, B. altitudinis WR10 cells were first grown in 5 mL of LB broth to exponential phase. Then, 20 μL cultures were added into the 96-well clear polystyrene microplates (Cat. 3370, Corning, NY, USA) filled with 180 μL of LBGM broth, or LBGM supplemented with different concentrations of NaCl (100, 200, 400 mM). The LBGM, a LB based medium supplemented with 1% of glycerol and 200 μM MnSO₄ was used in this study, as it promotes biofilm formation in Bacillus spp [24 (link)]. Plates were sealed with lid and incubated statically at 30 °C for 48 h. Biofilm was stained with 20 μL of a 0.1% (w/v) crystal violet solution. Non-adherent bacteria were removed by washing with phosphate buffered saline (PBS). At last, biofilms stained crystal violet was released by the addition of 200 μL of 100% ethanol and biofilm formation was quantified by measuring absorbance at 562 nm in plate reader (Multiskan FC, Thermo, Germany).