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Nanomaterials 2020, 10, 376 5 of 22 2.4. Microbial Strains and Culture Three well-described bacterial species and one clinically relevant yeast were evaluated: Gram-negative bacteria E. coli ATCC 25922 and P. aeruginosa ATCC 27853, the Gram-positive bacterium S. aureus ATCC 25923, and C. albicans SC5314. Briefly, microorganisms were harvested on plate count agar (PCA) for 24 h at 37 ◦C from frozen stock, and inoculum was prepared from single colonies grown to stationary phase in BHI broth at 37 ◦C overnight in an orbital incubator under 100 rpm. Cultures were centrifuged (3000 × g, 10 min) and washed twice in PBS. A cell suspension adjusted to a cell density equivalent to 0.5 McFarland (representing approximately 1–5 × 108 cells/mL) was prepared using sterile saline for bacteria, and C. albicans was adjusted at 1 × 106 cells/mL upon counting cells in a hemocytometer. Viable counts were enumerated after overnight incubation at 37 ◦C in PCA. 2.5. Antimicrobial Activity Assays for GO and GO–AgNP Nanohybrids. Determination of the Minimum Inhibitory Concentrations (MICs) The minimum inhibitory concentration (MIC) is defined as the lowest concentration, recorded in mg/L or μg/mL, of an agent that inhibits the growth of a microorganism. In the present study, the MIC of four microorganisms was determined: C. albicans SC5314, S. aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosa ATCC 27853. The EUCAST broth microdilution method (EUCAST EDef 7.3.1) was used to establish the MICs of GO and GO–AgNPs since it is a reference method for antimicrobial susceptibility testing, and one of its main purposes is to establish the activity of new antimicrobial agents. Stock solutions of graphene derivatives at 200-fold the concentration to be analyzed were prepared in order to maintain the same concentration of culture medium in each well of the microdilution plate. Next, these solutions were diluted 100-fold in the corresponding culture medium, which was previously prepared at 2-fold concentration. The culture medium was RPMI with 2% of glucose (RPMI 2% G) and Mueller–Hinton broth (recommended by EUCAST) for C. albicans and bacteria, respectively. Subsequently, 100 μL of each dilution was added to each well; the final test concentration for each compound ranged from 0.25 μg/mL to 128 μg/mL. Then, the microbial suspension was diluted 1:10 for C. albicans and 1:100 for bacteria in sterile distilled water, and 100 μL was pipetted into each well, obtaining the final inoculum concentration of 5 ×104 CFU/mL and 5 × 105 CFU/mL, respectively. Finally, the microdilution plates were read using a microdilution plate reader (iMark Microplate Reader, BioRad, Hercules, CA, USA) at a wavelength of 450 nm at 0 h and 24 h at 37 ◦C. The MIC corresponds to the concentration of the compound that resulted in an absorbance reduction of 50% or greater with respect to the absorbance found in the wells of the growth control. 2.6. Microbial Growth Kinetics Assay in the Presence of GO–AgNP Nanohybrids The four previously mentioned microorganisms were analyzed at the final inoculum density of 5 × 104 CFU/mL and 5 × 105 CFU/mL for yeast and bacteria, respectively. The highest GO concentration in the MIC determination assay (128 μg/mL) was tested along with three concentrations of GO–AgNPs, selected on the basis of the MIC value of each microorganism: 64 μg/mL, 32 μg/mL, and 16 μg/mL for C. albicans and 128 μg/mL, 64 μg/mL, and 32 μg/mL for S. aureus, E. coli, and P. aeruginosa. Briefly, a microplate was loaded with culture medium, the GO–AgNP hybrid, and the microorganism as mentioned above. The microorganism in the culture medium was considered the growth control (GC), and the different concentrations of the compounds without microorganisms but with the culture medium, the blank. The microplates were placed in a microplate reader (BioScreen C, Labsystem, Helsinki, Finland) configured to read the absorbance at 430–580 nm every hour for 72 hours at 37 ◦C. Five wells for each compound concentration were used, and the assay was performed in duplicate.PDF Image | Graphene Oxide–Silver Nanoparticle Nanohybrids
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