Microorganism suspension preparations, titer determination, and identification

American Type Culture Collection (ATCC) strains (Table 1) were purchased from Microbiologics (St. Cloud, MN, USA) and supplied as quantitative lyophilized pellets. Stock suspensions of all microorganisms with a theoretical concentration of 10 ≤ CFU/mL <100 and subsequent four dilutions were prepared in peptone water (Becton Dickinson [BD], Franklin Lakes, NJ, USA). Before and during the validation phases, viability, purity, and titer determination were assessed by plating 100 μL of each diluted suspension in triplicate on TSA (for aerobic suspensions), Columbia agar with 5% sheep blood (for anaerobic suspensions), and Sabouraud dextrose agar (SDA; for fungal suspensions) plates (all from BD). Plates were incubated at 32.5°C ± 2.5°C for bacteria and 22.5°C ± 2.5°C for fungi. Before incubation, agar blood plates with the anaerobic microorganisms were placed inside resealable GasPak EZ anaerobe pouch systems (BD). The CFU were quantified after 2–3 days for bacteria and 4–5 days for yeast and mold. For each experiment, the theoretical titer of each diluted suspension was confirmed by calculating the arithmetic average of the CFU values obtained. Purity and identity were determined as described elsewhere.33