Auriga scanning electron microscope

Freshly taken biofilms were fixed in original spring water including 0.1% glutardialdehyde (w/v). Scanning electron microscopy was carried out as described elsewhere (Probst et al., 2014 (link)). Samples were examined using a Zeiss Auriga scanning electron microscope operated at 1–2 kV. For TEM, the sample preparation and procedure is described in Probst et al. (2014 (link)). Samples were examined using a CM12 transmission electron microscope (FEI Co., Eindhoven, The Netherlands) operated at 120 kV. All images were digitally recorded using a slow-scan charge-coupled device camera that was connected to a computer with TVIPS software (TVIPS GmbH, Gauting, Germany).

The cultures used for enzymatic analyses (as described above) after incubation were examined using an AURIGA scanning electron microscope (Carl Zeiss Microscopy, GmbH). The cultures with MgO NPs, SiO₂ NPs, and C6Im were chosen for microscopic analysis. Additionally, the cultures without studied compounds were also investigated as control samples. Each sample was fixed with glutaraldehyde solution by adding 50 μL of glutaraldehyde (50%) to 1 mL of the sample. The fixation was carried out for 10 min. The samples were then centrifuged for 2 min at 8000 × g. Then the residues were gradually suspended in 1 mL of 50%, 70%, and 96% ethyl alcohol solutions. Before this procedure, higher concentrations of alcohol were used; samples were centrifuged for 2 min at 8000 × g, and 5 μL of bacterial suspension in 96% ethanol, from each sample, was applied to a microscope slide, covered with a 20 nm layer of carbon, and left to dry. The samples were then coated with a 20 nm layer of gold. Each layer of carbon was coated by a vacuum coater (Quorum 150T ES; Quorum Technologies, Lewes, UK). Furthermore, carbon tape bridges were made to avoid excessive charge accumulation. The secondary electron-mode photos were taken using a 60 μm aperture and a 20 keV acceleration voltage. The beam intensity was 1.5 nA, and the working distance was chosen at approximately 8 mm.

For SEM, drops of fixed samples (0.1% glutardialdehyde; w/v) were placed onto glass slides, covered with a coverslip, and rapidly frozen with liquid nitrogen. The coverslip was removed with a razor blade and the glass slide was immediately fixed with 2.5% (w/v) glutardialdehyde in fixative buffer, washed, postfixed with 1.0% osmium tetroxide, washed with buffer, followed by deionized water, dehydrated in a graded series of acetone solutions, and critical-point dried after transfer to liquid CO₂. Specimens were mounted on stubs, coated with 3 nm of platinum using a magnetron sputter coater, and examined with a Zeiss Auriga scanning electron microscope operated at 1 kV. For TEM, fresh, unfixed biofilm pieces were deposited on a carbon-coated copper grid and negatively stained with 2% (w/v) uranyl acetate, pH 4.5 or 2.0% (w/v) phosphotungstic acid (PTA), pH 7.0. Samples were examined using a CM12 transmission electron microscope (Philips) operated at 120 keV.

Samples for SEM were dropped onto a glass slide covered with a coverslip, shock frozen with liquid nitrogen, and fixed with 2.5% glutaraldehyde in fixative buffer containing 25 mM cacodylate, 75 mM NaCl, and 2 mM MgCl₂ (pH 7.0). The samples were washed with fixative buffer, postfixed with 1% OsO₄ in fixative buffer, and washed three times with the fixative buffer, followed by ddH₂O. The samples were dehydrated with increasing concentrations of acetone and then critical-point dried with liquid CO₂. The samples were mounted on carbon stubs with conductive silver and sputtered with platinum (3 to 5 nm). High-resolution micrographs were taken with an Auriga scanning electron microscope (Zeiss).

Evaluation of the surfaces of the laser textured samples was performed using a Leica MZ6 stereoscopic microscope (Heerbrugg, Switzerland) with MultiScan 8.0 image analysis software (CSS, Poland). Photos of each sample were taken at 100× and 320× magnification. The images are presented in Figure 4. In addition, the surfaces of the modified samples were assessed on the basis of images taken using an AURIGA scanning electron microscope (Zeiss, Göttingen, Germany). The secondary electron signal was used for imaging. The electron beam had 10 keV of energy. The samples had been previously placed in a vacuum deposition chamber to obtain a conductive surface.
The contact angles for all the samples were determined before and after the surface modification process. Droplets with a volume of 2 μL were placed on the surfaces of each sample. Deionized water was used as the measuring liquid. Photos of the drops were made 5 s after they were placed, determining the contact angle by tangents to the external droplet profile on both sides. In each case, five measurements were made and an average value was calculated. Due to the significant effect of microgeometry on the surface roughness of the samples, no free surface energy was determined.