Vapor H2O2 sterilization as a decontamination method for the reuse of N95 respirators in the COVID-19 emergency

Ebru Oral; Keith K Wannomae; Rachel L Connolly; Joseph A Gardecki; Hui Min Leung; Orhun K Muratoglu; Anthony Griffiths; Anna N Honko; Laura E Avena; Lindsay GA McKay; Nick Flynn; Nadia Storm; Sierra N Downs; Ralph Jones; Brandon Emmal

2.3 Virus titration by plaque assay Samples were diluted in DMEM and 10% HI-FBS (Gibco™) with Antibiotic-Antimycotic (Gibco™) and 200µL added to confluent monolayers of NR-596 Vero E6 cells in duplicate. Samples were adsorbed to the cell monolayers for 1 h at 37 °C and 5% CO 2 with gentle rocking approximately every 15 minutes to prevent monolayer drying. The monolayers were then overlaid with a 1:1 mix of 2.5% Avicel® RC-591 microcrystalline cellulose and carboxymethylcellulose sodium (DuPont Nutrition & Biosciences, Wilmington, DE) mixed with 2X Modified Eagle Medium (Temin's modification, Gibco™) supplemented with 2X Antibiotic-Antimycotic (Gibco™), 2X GlutaMAX (Gibco™) and 10% HI-FBS (Gibco™). Cells were incubated at 37 °C and 5% CO 2 for 2 days, fixed with 10% neutral buffered formalin before removing from containment, then stained with 0.2% aqueous Gentian Violet (RICCA Chemicals, Arlington, TX) in 10% neutral buffered formalin for 30 min, rinsed, and plaques were enumerated.