Resazurin assay for assessment of antimicrobial properties of electrospun nanofiber filtration membranes

Abstract

Abstract We developed a simple and fast microplate assay for evaluation of the antimicrobial activity of electrospun nanofiber filtration membranes or similar porous materials for water treatment technologies. Resazurin (alamarBlue®) was used as an indicator of the amount of viable experimental microorganisms Gram-negative Escherichia coli, Gram-positive Enterococcus faecalis, and natural wastewater treatment plant effluent bacteria. A bacterial inoculum of concentration 1–3 × 105 CFU mL−1 was pipetted onto the surface of assessed both functionalized and respective control membranes and incubated in 12-well plates for 4 h at 37 °C. Kinetics of resazurin metabolization, i.e. its reduction to fluorescent resorufin, was evaluated fluorimetrically (λex520/λem590 nm). A number of viable bacteria on the membranes expressed as CFU mL−1 was calculated from the kinetic curves by using calibration curves that were constructed for both experimental bacterial species. Antimicrobial activities of the membranes were evaluated by either resazurin assay or modified ISO 20743 plate count assay. Results of both assays showed the significant antimicrobial activity of membranes functionalized with silver nanoparticles for both bacterial species and wastewater treatment plant effluent bacteria as well (log CFU reduction compared to control membrane > 4), while membranes containing specific quaternary ammonium salts were inefficient (log CFU reduction < 1). The suitability of resazurin microplate assay for testing nanofiber filtration membranes and analogous matrices has proven to be a faster and less demanding alternative to the traditionally used approach providing comparable results.

Keywords

• Resazurin
• Antimicrobial activity
• Electrospun nanofiber filtration membranes
• Silver nanoparticles
• Quaternary ammonium salts