Mtp 384 ground steel plate

The bacterial colonies on Ashdown's agar were picked into 900 µl of water and suspended with 300 µl of ethanol. Bacterial cells were harvested by centrifugation. The cell pellets were then resuspended and vigorously mixed with MALDI matrix solution consisting of 10 mg sinapinic acid in 1 ml of 50% acetonitrile containing 2.5% trifluoroacetic acid. A 2-μL of the mixture was directly spotted onto a MALDI target plate (MTP 384 ground steel plate, Bruker Daltonik, GmbH, Bremen, Germany) and allowed to air dry. Twenty replicates (n = 20) of each bacterial lysate were spotted onto the target plate in the same mass spectrometer run in order to examine data reproducibility.

The microbial samples for MALDI-TOF analysis were prepared using previously described method [36 (link)]. In brief, the colonies which were grown on Ashdown’s agar plate were transferred into 900 μL of water and then deactivated with 300 μL of ethanol. The pellet was collected by centrifugation and mixed with a matrix solution containing 10 mg sinapinic acid in 1 mL of 50% acetonitrile with 2.5% trifluoroacetic acid. Two microliters of bacterial extract, with concentration approximately 0.3–0.5 μg/μL, were spotted on a MALDI steel target plate (MTP 384 ground steel plate, Bruker Daltonik, GmbH, Bremen, Germany) and were dried at room temperature. The Escherichia coli DH5α was used as a positive control and the matrix solution without bacterial cells was used as a negative control. Twenty-four spots (n = 24) from each sample were deposited on a target plate for determination of experimental reproducibility, thus each isolate was repeatedly examined twenty-four times. After drying, the target plate was subjected to analysis in the MALDI-TOF instrument.