Sc7640

Scanning electron microscopy (SEM) was used to observe bacterial behavior in solution with EOs and molecules samples. For this purpose, 7.5 µL of each sample dilution were placed in a 96-well microplate containing 142.5 µL of a 0.5 McFarland standard of C. violaceum diluted to reach 10⁶ CFU·mL⁻¹. A plastic lid with 96 identical pegs protruding downward (MBEC™ High Throughput (HTP) Assay Innovotech Inc., Edmonton, AB, Canada) was placed on top. These pegs allow bacterial development in a biofilm. The microplates were shaken at 30 °C for 36 hours. The pegs were immersed in a 2.5% glutaraldehyde (Electron Microscopy Science™, Hatfield, PA, USA) fixation solution in sodium cacodylate (Sigma-Aldrich™, St. Louis, MO, USA) buffer 0.1 M (pH 7.2) for 2 to 3 h at 4 °C. They were washed in two successive 10 min baths of sodium cacodylate buffer, dried for 120 h, and then coated with gold/palladium sputter coating (Quorum Technologies SC7640, Lewes, UK). The pegs were then examined under a scanning electron microscope (Hitachi™ S3400N, Tokyo, Japan) with an accelerating voltage of 5 or 10 kV. Each test was performed in triplicate and the images are representative of the bacterial distribution on the whole peg surface.

After each indentation experiment, samples were cut, vacuumed for 24 h and sputter-coated with gold (thickness of 5 nm; 5 × 10⁻² mbar, 10–20 mA, 90 s; sputter coater SC7640, Quorum Technologies Ltd., Lewes, UK). The thickness of the different PACA films was examined by scanning electron microscopy (Phenom G2 pro, Phenom-World B.V, Eindhoven, The Netherlands).

Gold (Au) film was deposited on glass cover slips (24 mm × 24 mm × 0.1 mm, Menzel-Glaser, Braunscheig, Germany). The glass slip was cleaned prior to the deposition process. Typically, the slip underwent ultra-sonication in 50% solution of acetone for 5 min followed by rinsing in concentrated acetone and deionised water. This is to remove dirt and any trace of impurities on the surface of the glass slide before any coating process. The glass slide was then coated with a thin film of gold layer using a sputter coater (model SC7640, Quorum Technologies, Newhaven, East Sussex, UK) at 20 mA for 67 s based on previous experience [23 (link)]. The gold-coated substrate was then stored in EKB-103 storage box. Subsequently, the thin film of the ternary composites was deposited on top of the gold-coated substrate by preparing 15 mg/mL solution in 1-Methyl-2-pyrrolidinone (NMP). The deposition was conducted using POLOS™ spin coater at 6000 rpm for 30 s. The Au-coated glass substrate with the thin film of the ternary composite deposited on it was then kept in an oven at 40 °C for 1 h to allow the solvent to evaporate.

Micrographs of the prepared samples were taken by the scanning electron microscope Nova NanoSEM 450 (FEI, Brno, Czech Republic) with a Schottky field emission electron source operated at an acceleration voltage ranging from 200 V to 30 kV and a low-vacuum SED (LVD) detector. A coating with a thin layer of gold was performed by a sputter coater SC 7640 (Quorum Technologies, Newhaven, East Sussex, UK).

Micrographs of the prepared samples were taken by a Nova NanoSEM 450 (FEI, Brno, Czech Republic) scanning electron microscope with Schottky field emission electron source operated at acceleration voltage ranging from 200 V to 30 kV and low-vacuum SED (LVD) detector. A coating with a thin layer of gold was performed by a sputter coater SC 7640 (Quorum Technologies, Newhaven, East Sussex, UK). Pore size and surface void percentage were determined using the Fiji biological image analysis software [93 (link)]. Average pore sizes were obtained by measuring ten randomly selected pores from SEM images and void percentage was calculated by processing SEM images to obtain 8-bit (black and white) micrographs of the surface of the scaffold and then calculating a percentage of void representing pixels using Equation (4), where V_p is the number of void pixels (black) and S_p (white) is the number of surface pixels: