Glutaraldehyde

The SEM analyses of bacteria grown on different culture media (A–C) were performed according to the method described by Xu et al. [45 (link)] with a few modifications as described in our previous work [44 (link)]. In brief, suspensions (2A–C) were adjusted to the concentration equivalent to no. 0.5 of the McFarland scale, centrifuged (3000× g for 10 min), fixed with glutaraldehyde (2.5%, v/v; Chempur, Piekary Slaskie, Poland), and dehydrated in a gradient ranging ethanol (30–100%, v/v; Chempur, Poland). After this, the ethanol was replaced with tert-butanol (Chempur, Poland) at room temperature. The staphylococcal samples were dried for 8 h, and coated with gold for 90 s. Bacterial cells (the average size of bacterial cells) were observed using the scanning electron microscope (Vega 3 LMU, Tescan, Brno, Czech Republic) and analyzed using Tescan Essence^TM software (Tescan, Brno, Czechia).

Immobilization of gentamicin (Sigma-Aldrich, USA) was performed by simple soaking of graft pieces (pristine and polycatecholamine-coated ones) in 1 mg/mL gentamicin solution in 0.1 M Britton-Robinson buffer pH 8.5 for 24 h, typically at 25 °C, 37 °C and 50 °C, depending on the particular experiment. Then the samples were washed twice in distilled water and dried at 37 °C. The amount of immobilized drug was calculated from gentamicin concentrations in the buffer before and after the process and evaluated quantitatively after drug derivatization with phthaldialdehyde (Sigma-Aldrich), as described elsewhere [21 (link)]. For comparison, the reference method of gentamicin immobilization on protein-sealed vascular prostheses using 0.5–2.5% glutaraldehyde (Chempur, Piekary Śląskie, Poland) was performed on FlowNit Bioseal^® collagen-sealed polyester knitted prosthesis, according to a procedure described elsewhere [5 (link)].
Samples of grafts obtained in optimized coating conditions and with immobilized gentamicin were selected for further experiments. They are listed in Table 1.