Standardized SARS-CoV-2 Inoculum Matrices

Low and high inoculum levels were developed in three separate inoculum matrices at a target concentration of 3.5 and 7.0 log PFU on surfaces, respectively. Sterile 1× phosphate-buffered saline (PBS), pH 7.4, was prepared by adding 100 μL of diluted Φ6 stock (high, 10-fold dilution; low, 1,000-fold dilution) to 5 mL of PBS. Similarly, 100 μL of diluted Φ6 stock (high, 10-fold dilution; low, 1,000-fold dilution) was added to 5 mL of artificial saliva consisting of 1.54 mM KH₂PO₄ (Sigma-Aldrich), 2.46 mM K₂HPO₄ (Fisher Scientific, Loughborough, UK), 0.04 mg/L MgCl₂·7H₂O (Alfa Aesar, Ward Hill, MA), 0.11 g/L NH₄Cl (VWR), 0.12 g/L (NH₂)₂CO (VWR), 0.13 g/L CaCl₂ (VWR), 0.19 g/L KSCN (Acros Organics, Carlsbad, CA), 0.42 g/L NaHCO₃ (Fisher Scientific), 0.88 g/L NaCl (VWR), 1.04 g/L KCl (VWR), and 3 g/L mucin (Sigma-Aldrich) at pH 7 (30 , 31 (link)). Lastly, the tripartite matrix (5 mL) was prepared as described in international standard ASTM E2197-17 (24 ) by combining 3.4 mL of PBS containing Φ6 stock (low and high) with a 1.6-mL solution consisting of 0.8 mg/mL bovine mucin (Sigma-Aldrich, St. Louis, MO), 2.5 mg/mL bovine serum albumin (VWR), and 3.5 mg/mL tryptone (VWR) to mimic fluids shed by infected individuals (1 (link), 3 (link), 24 , 32 (link)).