Standardized MIC Determination of Biocides

MIC values were determined for the initial strain panel using a broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [15 ] and ISO 20776-1 [12 ]. Testing was carried out as previously published [16 (link)] except for MH broth (Thermo Fisher Diagnostics GmbH Microbiology) used instead of tryptic soy broth. In compliance with EN 1276, standardized hard water was used to freshly prepare all stock solutions and to subsequently adjust the desired concentrations. The MIC was defined as the lowest biocide concentration completely inhibiting bacterial growth, which was determined after additional visual inspection (data not shown). Optical density was measured with a Mithras² Multimode Reader (Berthold Technologies, Bad Wildbad, Germany). OD₅₉₅ = 0.04 and 0.08 were considered as cut-off values for enterococci and E. coli, respectively. Experiments were repeated on three different days in three technical replicates per day.