Msp 96 target polished steel microscout target plate

Colony material of a 24 h ColbA plate was spotted onto the target plate (MSP 96 target polished steel (MicroScout Target) plate; Bruker Daltonik, Germany). After air drying the spots were overlaid with 1 µl of saturated α-cyano-4-hydroxy-cinnamic acid matrix solution (200 mg in 2.5% trifluoroacetic acid/50% acetonitrile) and dried completely. MALDI-TOF MS analysis was performed using MALDI-TOF Microflex LT (Bruker Daltonics, Germany) using a range of 2,000–20,000 m/z (mass to charge ratio) following the calibration with Bacterial Test Standard (Bruker Daltonics, Germany). For each spectrum 240 laser shots were summed up in 40 shot steps, but at least 80 shots per raster spot from different positions within the sample were acquired by the AutoXecute method using the software FlexAnalysis 3.4. The spectra were compared with the MBT Compass Library, Revision F (Bruker Daltonics, Germany). Each identification obtains a score value. The identification at the species level with a score ≥ 2.000 was considered correct^{16 (link)–18 (link)}.

A single bacterial colony was picked from Löwenstein-Jensen (L-J) slant and used for inoculation of 10 mL of Middlebrook 7H9 medium supplemented with glycerol and OADC (10%) (Becton-Dickinson, Franklin Lakes, USA). The bacteria were cultured at 37°C with shaking for 7 days. Each strain/isolate was cultured independently for three independent replicates. The cells were harvested by brief centrifugation (4,500 rpm, 2 min) and killed by suspending in 300 μL of deionized water and 900 μL of absolute ethanol. After centrifugation (12,000 rpm, 2 min) at room temperature (RT), excessive ethanol was discarded and the samples were air-dried completely. The cell pellet was dissolved in 50 μL of 70% formic acid and 50 μL acetonitril. The samples were then subjected to sonication on ice for 1 min (cycle, 1.0; amplitude, 100%) using a sonicator (UP100H, Hielscher Ultrasound Technology, Teltow, Germany). The suspension was centrifuged at 12,000 rpm for 2 min at RT, and 1.0 μL of the clear supernatant was spotted in triplicate onto the MALDI target (MSP 96 target polished steel (MicroScout Target) plate, Bruker Daltonik, Bremen, Germany). Following air drying each sample was overlaid with 1.0 μL of saturated α-cyano-4-hydroxycinnamic acid matrix solution and allowed to dry completely prior to MALDI-TOF measurement.