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Molecules 2020, 25, 2023 11 of 14 3.2.2. ζ-Potential and DYNAMIC Light Scattering (DLS) The measurement of ζ-potential and the hydrodynamic diameter of the nanoparticles were determined by Dynamic Light Scattering (DLS) utilizing a Zetasizer (Malvern Instruments, Malvern, UK). Samples were prepared at pH 6, in Milli-Q water. For ζ-potential determination, each sample was measured at 25 ◦C, three times, combining 10 runs per measurement. In the case of DLS, each sample was measured at 25 ◦C, 10 times, combining five runs per measurement. Results were treated using the Malvern Zetasizer software (Malvern, UK). 3.2.3. Small Angle X-ray Scattering (SAXS) An model Ultima IV X-ray powder diffractometer (Rigaku, Tokyo, Japan) using CuKα = 1.5418 Å radiation was used for the small angle X-ray scattering measurements. They were made at low angle in Bragg-Brentano geometry, applying an offset of 0.08o in order to get the SAXS signal, on nanoparticle deposits on silicon substrate, with measurement ranges of q = 0.05 to 1.50 Å−1. 3.2.4. Confocal Raman Microscopy An aliquot of PchNPs was deposited on an aluminum support and dried at room temperature for a further analysis by confocal Raman microscopy. The measurements were made on an Alpha 300 RA WITec Raman microscope (WITec GmbH, Ulm, Germany) using a λ = 532 nm excitation laser wavelength and focused through a 100× objective. 3.2.5. Colloidal Stability Assays The colloidal stability of PchNPs was studied at different and ionic strength (10–500 mM NaCl) and pH (3–10) conditions by the measurement of the absorbance spectrum in the range of 200–800 nm. 3.3. Antimicrobial Activity of PchNPs 3.3.1. Antibacterial Activity against E. coli In order to determine the antibacterial activity of PchNPs, a bacterial inoculum (1 × 106 CFU/mL) of Escherichia coli ATCC 25922 in LB media was supplemented with different concentrations of PchNPs and a blank sample (bacteria without PchNPs) was also included in the assay as negative control, in a 96-well plate. Once the microbial cultures had been grown for a total of 24 h, 30 μL of 0.1 mg/mL resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) in LB media was added to each well and incubated in the dark at 37 ◦C for 1 h under stirring. Enviromental Scanning Electron Microscopy (ESEM) E. coli cells were incubated with PchNPs (0.12 and 0.25 nM), in the same way as in the resazurin assay. Then, three wells of 200 μL each were mixed into an Eppendorf and centrifuged at 1400 rpm (300 G) for 10 min. The supernatant was removed and, for fixation of the cells, the pellet was resuspended into 1.5 mL of 2.5% glutaraldehyde in phosphate buffer 10 mM pH 7.2. The solutions were left for 2 h in the wheel. Then, the cells were washed once with 1.5 mL of sterile PBS and three times with sterile distilled water to remove glutaraldehyde. Finally, the pellets were resuspended in 200 μL of sterile MilliQ water. Data were collected on a Quanta FEG-250 (FEI Company, Hillsboro, OR, USA.) field emission SEM for high-resolution imaging working in ESEM mode using a GSED detector under high relative humidity conditions. Transmission Electron Microscopy (TEM) Samples were prepared as in the ESEM assay, including the PchNPs concentrations. The pellets were resuspended in sterile distilled water, 4 μL of the sample was deposited onto a carbon-coatedPDF Image | Biofilm Eradication Using Biogenic Silver Nanoparticles
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