Mycobacteria library version 4

The identification of all colony-types was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a Bruker Biotyper with the in-house extraction method. Each colony type was suspended in 0.5 mL of sterile deionized water in a 2 mL centrifuge tube to make a turbid suspension, and 1 mL of pure ethanol was added to the tube. The suspension was vortexed at high speed for 2 min and then centrifuged for 2 min at 13,000 rpm. The supernatant was then carefully removed with a pipette. The tube was then re-centrifuged for 30 s and any remaining ethanol removed. Ten sterile glass beads were added to the tube containing the pellet and 50 µL of acetonitrile was added. The tube was then vortexed at high speed for 1 min. A 50 µL aliquot of 70% formic acid was then added and the tube was vortexed for a further 5 s. The tube was then centrifuged for 2 min at 13,000 rpm. Duplicate aliquots of 1.5 µL of supernatent were spotted onto a MALDI stainless steel target and both spots were covered with 1.5 µL of HCCA matrix (Bruker, Coventry, UK) and allowed to dry. Species identification was achieved by matching spectra to those available in the general database and mycobacteria were also matched to the Bruker Mycobacteria Library version 4.0.