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Polymers 2019, 11, 2000 5 of 21 2.4. Antimicrobial Testing (AATCC TM100; ISO20743) Antimicrobial activities of composite materials were screened in vitro against the common reference pathogen, Escherichia coli American Type Culture Collection (ATCC) 25922, which is a gram negative bacterial strain, under aseptic conditions throughout [75,76]. In order to generate reliable and reproducible results, the standard ‘lawn culture’ method was used, where an inoculum is spread over an agar plate using the same inoculation dosage and incubation time, such that all samples are measurable and undergo consistent operational conditions. Bacterial growth was measured by the counted colony forming units (CFU) measuring bacterial growth on agar plates [77]. Experiments were conducted in triplicate and repeated on three separate occasions, both before and after washing, on fresh samples each time. The mean value with a standard error reported in the final results. A fresh E. coli culture was grown on a tryptic soy agar plate (Oxoid) and a single bacterial colony inoculated in 10 mL tryptone soya broth (Oxoid) at 37 ◦C for 24 h with shaking at 250 rpm, prior to harvesting. The bacterial suspension was diluted 1000-fold to obtain an inoculum of ~106 CFU/mL. The inoculum was confirmed by plating 10-fold serial dilutions on tryptone soya agar for viable counts. Likewise, each textile substrate swatch sample (~1 × 1 cm) was inoculated with 25 μL of bacterial suspension at a concentration of 105 CFU/mL and incubated at 37 ± 1 ◦C for 6 h. After incubation, 450 μL saline was added to the inoculated swatch samples, which was followed by vortexing to release the bacterial cells from the substrate swatch. The contents were then diluted to 106 CFU/mL, and plated (100 μL) evenly onto tryptic soy agar plates. Total bacterial count per sample was manually recorded after incubation at 37 ± 1 ◦C for 24 h in the dark. Log reduction of viable bacterial counts and percentage-kill for swatches with different composite materials were subsequently calculated and compared against an untreated (control) sample. The Mann-Whitney U test (a nonparametric test often used to rate pharmaceutical drug efficacy) was used to determine the statistical significance of counted antibacterial results (i.e., (i) an untreated ‘control’ blank substrate sample vs. inoculum, (ii) blank substrate vs. rGO, (iii) blank substrate vs. AgNP, and (iv) blank substrate vs. Ag-rGO), using the R software package. 2.5. Physico-Chemical Nanoparticle (NP) Characterisation NP properties depend on their size, shape, and agglomeration as well as a local physical and chemical environment and detailed characterisations that were carried out to probe these changes. UV–Visible absorbance data was acquired on a Varian Cary 300 Conc UV–Vis Spectrophotometer (Palo Alto, CA, USA), over a 200–800 nm range at a step size of 0.5 nm. Raman spectroscopy data was acquired on a BaySpec Nomadic Raman Microscope (San Jose, CA, USA) using a 785 nm laser excitation source, over the 200–3200 cm−1 range at RTP (room temperature and pressure). Attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy was acquired at a Perkin-Elmer Spectrum 100 (Waltham, MA, USA), over the 650–4000 cm−1 wavenumber range in the transmittance mode, with 0.2 cm/sec scan speed and 20 scans. The polymer composite degradation properties were mapped using thermogravimetric analysis (TGA) in triplicate and differential scanning calorimetry (DSC) cycling tests on a Mettler-Toledo Star e TGA Thermogravimetric Analyzer (Columbus, OH, USA), under N2 (20 mL/min) conditions in dynamic mode, at a 10 ◦C/min ramp rate, over the 50–500 ◦C range. Scanning electron microscopy (SEM) was done on a Field Emission Electron Microscope JEOL JEM-2100F (Akishima-shi, Japan) at accelerating voltages up to 20 kV with fabric samples sputter coated with gold at low-vacuum. Cluster analysis was carried out using the linear intercept method, from 15 separate points at a time, on ImageJ (National Institutes of Health, Bethesda, MD, USA) [78]. Microscopic images of fabrics were taken on a Leica M165C (Wetzlar, Germany). Fibre cross-sectional and longitudinal analysis was carried out on a Nikon Opthiphot-Pol (Tokyo, Japan). Fabric thickness was tested on a Ames Thickness Testor (B.C. Ames, Framingham, MA, USA), using a modified version of the ASTM D1777-96 method, taken at 30 s under applied pressures of 4 gf/cm2 (0.392 kPa, 39.2 mN/cm2) and 30.7 gf/cm2 (3.012 kPa, 301.2 mN/cm2), for fabric samples ~6.5 × 4.5 cm, and the difference between them used to calculate the fabric surface layer thickness [79]. In each case, specimens were tested in triplicate and the average values were reported. Wide angle X-ray diffraction (WAXD) was employedPDF Image | Antimicrobial from Silver-Graphene Coated Medical Textiles
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