The present study aims to evaluate the influence of extracellular matrix amount on the antifungal activity of silver nanoparticles against single and mixed biofilms of Candida albicans and Candida glabrata. Silver nanoparticles (average diameter 5 nm) will be synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. The single and mixed biofilms of Candida species will be formed (during 48 h) statically (which has little matrix) and with shaking (which produces more matrix) on the surfaces of acrylic resin specimens inside 24-wells microtiter plates. Then, biofilms will be exposed to different concentrations of silver nanoparticles (13.5 and 54 µg/mL) for 24 h. Chlorhexidine digluconate in a concentration of 0.12% will be used as positive control. After being treated, the specimens containing the biofilms will be immersed in PBS (phosphate buffered saline) and sonicated for 30 s at 40 W. The obtained suspensions will be vortexed and, then, serial decimal dilutions will be plated on appropriate medium, and after 24 h the number of colony forming units will be enumerated. Biofilm biomass, obtained after treatment with silver nanoparticles will be evaluated by the crystal violet (CV) staining method. For that, biofilms will be fixed with methanol, stained with CV for 5 minutes and then the CV will be removed by adding of acetic acid into each well. Afterwards, the absorbance of obtained solution will be measured at 570 nm. Also, Candida biofilm structure after treatment with silver nanoparticles will be visualized by scanning electron microscopy. Data will be analyzed to employ the most appropriate statistical test. P < .05 will be considered statistically significant.
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