Em ace600

A scanning electron microscope MIRA3 (TESCAN, Brno, Czech Republic) was used to study the morphology and adhesion of the prepared bilayers. Images were taken in a secondary electron emission mode, the scan mode was DEPTH, the beam density was 10 and the high voltage was 10 kV. The working distance was set to 15 mm. The surface of the samples was coated with a 20 nm thin layer of Au/Pd using EM ACE 600 (Leica Microsystems, Wetzlar, Germany). The pore size was characterized from SEM images using ImageJ software and SEM Image Pore Extractor (SEMIPE). A minimum of five and a maximum of ten images with the same resolution were taken from each sample. From each image, 40–90 pores were measured. Data were evaluated using a 2-sample T-test, which assumes unequal variances and unequal sample sizes. The level of significance was set at *p < 0.05 **p < 0.01and ***p < 0.001.

Leptospira strains were cultured as described in the biofilm formation section with minor modifications. Inoculation was performed by adding one mL of diluted culture in a 24-well plate (flat bottom, with low evaporation lid, Corning) each well containing a sterile 12 mm Glass coverslips (12 × 12 mm, Menzel-Glaser, Braunschweig, Germany). The supernatant was removed and biofilm was rinsed once in PBS, then fixed in 4% PFA / 1% glutaraldehyde for 15 min. Coverslips were rinsed again in PBS and stained with 1% osmium tetroxide (OsO₄ in PBS, Acros Organics) for 1 h. After staining, samples were dehydrated through a series of ethanol concentrations (25, 50, 70, 90, and 100% for 10 min each) before being desiccated using hexamethyldisilazane (HMDS, Acros Organics). HMDS drying is a reliable alternative to critical point drying in the preparation of the samples with no differences in term of distortion and shrinkage between the two methods regarding cellular ultrastructure^{54 (link)}. Before visualization, samples were submitted to carbon sputtering to increase their electrical conductance (Em ACE600, Carbon Coater, Leica Microsystems, Mannheim, Germany) and examined under a Jeol JSM IT-300 IntouchScope microscope (SED detector, 10 kV, Jeol LTD, Tokyo, Japan).

Cells were fixed overnight at 4 °C in 2.5% glutaraldehyde in phosphate buffer on 0.2 µg/ml HEL coated on glass and 100 µg/ml HEL coated on PAA gel. They were dehydrated in a graded series of ethanol solutions, then dried by the CO₂ critical-point method, with an EM CPD300 (Leica microsystems). Samples were mounted on an aluminum stub with silver lacquer and sputter-coated with a 5 nm layer of platinum, with an EM ACE600 (Leica Microsystems). Images were acquired with a GeminiSEM 500 (Zeiss).

The surface morphology of both sides and cross-sectional view of the freestanding films, and also the element composition of the films and BGNPs, was assessed using the SEM JSM-6010 LV (JEOL, Tokyo, Japan) coupled with EDS (INCAx-Act, PentaFET Precision, Oxford Instruments, Abingdon, UK). For the observation of the cross-section, the films were fractured using nitrogen. Cross-Sectional SEM views were obtained from the LbL films before and after cross-linking with genipin, and the correspondent film thickness was estimated using the software ImageJ. The surface morphology of the BGNPs was characterized by a high-resolution field emission SEM with focused ion beam (AURIGA Compact FIB-SEM, Carl Zeiss, Oberkochen, Germany). Before each analysis, except for EDS, the samples were sputtered with a gold layer, using a sputter coater EM ACE600 (Leica Microsystems, Wetzlar, Germany). The specific surface area of the BGNPs was measured by determining the N₂-gas adsorption isotherms using a TriStar II 3020 surface area and porosity analyzer (Micromeritics Instrument Corporation, Norcross, GA, USA). The samples were degassed at 130 °C overnight before measurements.

The biofilms were cultured in the analogical manner as described in Section 4.2, with such a difference that the polystyrene coupons were placed in the bottom of the 24-well plate and served as the surface for staphylococcal biofilm growth. Next, the samples were gently cleansed in PBS (Sigma-Aldrich, Darmstad, Germany) buffer; fixed in glutaraldehyde [28 (link)] (POCH, Wroclaw, Poland) and dried in a critical point dryer EM CPD300 (Leica Microsystems, Wetzlar, Germany). Subsequently, the samples were subjected to sputtering with Au/Pd (60:40) using EM ACE600, Leica sputter (Leica Microsystems, Wetzlar, Germany). The sputtered samples were examined using a scanning electron microscope (SEM, Auriga 60, Zeiss, Germany).

The S. aureus ATCC 6538 and R1 biofilm strains were visualized with a Scanning Electron Microscope (SEM, Auriga 60, ZEISS, Germany). Firstly, 1 mL of 2% (w/v) Bacteriological Lab Agar was poured into a 24-well plate and left for solidification. Next, 500 µL of the appropriate medium (TSB or IVWM) was poured into the wells with agar, and the plate was kept refrigerated for 24 h in order to soak the agar surface with the medium. After this time, the medium was removed from above the agar.
Subsequently, the bacterial suspensions were prepared in saline and adjusted to 0.5 MF (McFarland, 1.5 × 10⁸ CFU/mL (Colony-Forming Unit)) using a densitometer and diluted thousand times in TSB or IVWM. The suspensions were added in the amount of 500 µL to the agar wells. The plate was incubated for 24 h at 37 °C under static conditions. The medium was then removed, and 500 µL of 4.5% (v/v) glutaraldehyde was poured. The biofilms were visualised according to the methodology presented in our previous study^{49 (link)}. Drying of the samples was performed with a critical point dryer EM CPD300 (Leica Microsystems, Germany). Next, with the use of EM ACE600, Leica sputter (Leica Microsystems, Germany), the samples were sputtered with Au/Pd (60:40). They were then analyzed with a Scanning Electron Microscope^{49 (link)}.