2.4.2. Minimum Inhibitory Concentration (MIC)

Biocide MICs were determined using broth microdilution. Overnight cultures grown on tryptic soy agar (TSA; Merck KGaA) were adjusted to about 10⁶ CFU/mL in 2-fold concentrated tryptic soy broth (TSB; Merck KGaA). In 96-well microtiter plates (Greiner Bio-One, Frickenhausen, Germany), 50 µL of the bacterial suspension was added to 50 µL of the double-concentrated biocide solution. Plates were incubated at 37 °C for 20 ± 2 h. Optical density at 595 nm (OD595) was measured after 5 s of shaking using the Mithras2 multimode reader (Berthold Technologies, Bad Wildbad, Germany; Software MikroWin 2010 v5.18, German UI). Bacterial growth was compared to a negative control (microtiter well containing biocide solution and tryptic soy broth, Thermo Fisher Scientific) and a ΔOD_{595 nm} of 0.08 was applied as the cut-off value. The MIC was defined as the lowest concentration of a biocide at which no growth was observed. Three independent experiments were performed on different days and the median was considered as the final MIC.