Supra40vp scanning

Specimen preparation and SEM were performed at the Scientific Instrumentation Centre (CIC) of the University of Granada. Biofilm samples from microaerophilic cultures growing on naphthalene were fixed for 2 h at 4°C in 2.5% glutaraldehyde prepared in cacodylate buffer, pH 7.4. The fixed samples were rinsed three times (15 min each) with the same buffer at 4°C and incubated for 1 h with 1% osmium tetroxide at room temperature, rinsed three times (5 min) with water and dehydrated in an increasing ethanol concentration gradient from 50% to 100%. Samples were further desiccated with carbon dioxide in a Leica EM CPD300 critical point dryer according to Anderson (1951). Samples were carbon coated in an EMTECH K975X evaporator and examined in a Zeiss SUPRA40VP scanning electron microscope equipped with a Schottky type emission gun.

Cells cultured for 48 h in 2D culture plates, or for 14 d in 3DPS, were gently washed once in cell medium and fixated for 1 h at room temperature in 2.5% glutaraldehyde (Product ref G7651-10ML, Sigma-Aldrich Sweden AB, Stockholm, Sweden) diluted in cell medium. Scaffolds were washed once in TBS (50 mM Trizma-HCl, Sigma-Aldrich, Stockholm, Sweden; 150 mM NaCl, Merck, Stockholm, Sweden; pH 7.5) with 0.1 mM CaCl₂ (Merck, Stockholm, Sweden), fixated for 1 h at room temperature in 1% osmium tetroxide (Sigma-Aldrich, Stockholm, Sweden) in TBS (50 mM Trizma-HCl (Sigma-Aldrich, Stockholm, Sweden); 150 mM NaCl, (Merck, Stockholm, Sweden); pH 7.5) with 0.1 mM CaCl₂ (Merck, Stockholm, Sweden), rinsed in deionized water, plunge-frozen in liquid propane (EMS-002 Rapid Immersion Freezer, Electron Microscopy Sciences) and freeze-dried overnight (VirTis Sentry 2.0 Benchtop Freeze Dryer, SP Scientific, Warminster, PA, USA). Samples were mounted on aluminum stubs by adhesive carbon tabs (Agar Scientific) and coated with a 10 nm Au/Pd conducting thin film by sputter-coater (PECS Mod 682, Gatan Inc., Pleasanton, CA, USA). Samples were imaged by Zeiss SUPRA 40VP scanning electron microscope operated in secondary electron mode at 3.0–4.1 kV acceleration voltage, 9–17 mm working distance and 100–250,000× magnification range.

Samples were fixed and processed for SEM as previously described [74 (link)]. Briefly, samples were harvested in 1x PBS (P4417-100TAB, Sigma-Aldrich) at room temperature. The PBS was then replaced with a 4% solution of 50% glutaraldehyde (4995.1, Roth) in 1 x PBS and incubated overnight at 4°C. A dehydration series was then performed using freshly prepared solutions of 30%, 50%, 70%, and 90% ethanol (v/v) at room temperature for 30 min each while being gently rocked. Samples were then incubated in 100% ethanol for 60 min at room temperature. Ethanol was then replaced with 100% ethanol and placed at 4°C. A Leica CPD300 or Quorum K850 were used for critical point drying and samples were coated with gold using an Emitech SC 7640 (Polaron) or Quorum Q150T sputter coater. Samples were imaged using a Hitachi TM 4000Plus or a Supra 40VP scanning electron microscope (Zeiss).