Tecnai g2 f20 s twin transmission

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).

Using TEM to observe the reconstruction of fimbriae in vitro, the concentrations of purified mtCfaA-CfaE, mtCfaA-CfaB and CfaC are 0.25 μM, 5 μM and 0.25 μM, respectively, each sample was incubated at 25°C for 24 hours in a buffer containing 10 mM Tris-HCl, pH 7.5 and 20 mM NaCl, negatively stained with 2% (w/v) uranyl acetate for 1 min and imaged using FEI Tecnai G2 F20 S-TWIN microscope. The micrographs were recorded at an accelerating voltage of 200 kV and a magnification of 20,000. To illustrate the effect of different mutations of CfaE on CFA/I fimbriation by TEM, sample cell pastes were resuspended in PBS, pH 7.4 to a density of 107–108 cells mL-1. Formvar/carbon coated 300 mesh copper grids were prepared by adsorbing 2–5 μL bacterial suspensions for 5 minutes. The grids were then negatively stained with 2% uranyl acetate for 1–2 minutes and imaged using a FEI Tecnai G2 F20 S-TWIN transmission electron microscope. Micrographs were recorded at an accelerating voltage of 200 kV.