Mithras2 multimode reader

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.

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.

(i) MICs. The MICs of the biocides under study were determined by broth microdilution assays. An overnight culture of each isolate grown on tryptic soy agar (TSA; Merck, Darmstadt, Germany) was adjusted to about 10⁶ CFU/ml 2-fold concentrated tryptic soy broth (TSB; Merck). In a 96-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 50 μl of the bacterial solution was added to 50 μl of the double-concentrated biocide. The plate was 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 TSB), and a ΔOD₅₉₅ of 0.1 was considered the cutoff value. The MIC was defined as the lowest concentration of a biocide at which no growth was observed. Biological replicates derived from two independent experiments were conducted on different dates. A MIC variation of one dilution step between the two experiments was accepted. The lower value was defined as the MIC. In case of higher variation, the test was repeated once more, and the median was considered the final MIC.