Vacuum evaporator

Shape and surface morphology of APIs and MPs were observed by Scanning Electron Microscopy (SEM). Samples were fixed on the sample holder with double-sided adhesive tape, sputter coated with Au/Pd under argon atmosphere performed using a vacuum evaporator (Edwards, Crawley UK) and examined by means of a scanning electron microscope SEM (Philips XL 30) operating at 20.0 kV accelerating voltage. The MPs size distribution was evaluated by sieve analysis, using a vibrating shaker (Octagon Digital, Endecotts, London, UK) and standard sieves (Scientific Instruments, Milan, Italy) of 75, 150, 250, 355, and 500 μm.

For the purpose of evaluating the SEM investigation, the bacteria were incubated overnight (18 h) in TSB at 37 °C. An overnight bacterial inoculum was prepared after 1:100 dilution in TSB. The diluted inoculum was then resuspended and placed in a 12-well plate, with 2 mL of the inoculum added to each well in the presence of sterile ceramic tablets. Duplicate samples were prepared for each variant. The plate was incubated at 37 °C in a static condition for 24 h. After this interval, the laser-treated tablets were washed and fixed with 4% glutaraldehyde in 0.1M Na cacodylate buffer at 4 °C for 2 h. The tablets were then washed again in cacodylate buffer and post-fixed for 1 h using a solution of 1% OsO₄ at 4 °C. After washing twice with cacodylate buffer, a dehydration procedure was performed in a graded ethanol series (30, 50, 70, 80, 90 and 100%) through 15 min time intervals. Finally, the dehydrated tablets were installed on scanning electron microscopy holders and sputter-coated with a layer of gold using a vacuum evaporator (Edwards, Stansted, UK). The observations of the bacterial morphology were made on Lyra I XMU Scanning electron microscope (Tescan, Czech Republic) with an accelerating voltage of 20 kV.

Morphological alterations of bacterial cell surface and biofilm formation in the presence of plant extracts were investigated using SEM. C. violaceum biofilms were incubated on sterile plastic pieces and treated as described above. After the treatment, samples were washed with PBS and fixed with 4% glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.2) and incubated for 2 h at 4 °C. After that, they were washed again in cacodylate buffer and post-fixed for 1 h using a solution of 1% OsO₄ again at 4 °C. A dehydration procedure was performed in a graded ethanol series through 15 min time intervals. Finally, the samples (with duplicates per variant) were mounted on scanning electron microscopy holders and sputtered with gold using a vacuum evaporator (Edwards, Irvine, CA, USA). Observations were made on a Lyra/Tescan scanning electron microscope (TESCAN GROUP a.s., Brno, Czech Republic) with an accelerating voltage of 20 kV.