Methods for Inactiviation And Extraction of Acid-Fast Bacteria for Characterization And/Or Identification Using Mass Spectrometry

A method for inactivation and extraction acid-fast bacteria in a test sample, the method comprising the following sequential steps:

• (a) acquiring a test sample from a solid or semi-solid culture medium known to contain or that may contain acid-fast bacteria and suspending the test sample in a container containing 70% ethanol and 0.5 mm glass beads;
• (b) bead beating and/or vortexing the container to break up clumps and/or disrupt mycobacteria cells in the container;
• (c) subsequently incubating the suspension for at least about 5 minutes at room temperature to inactivate any acid-fast bacteria contained in the test sample;
• (d) centrifuging the container to pellet the acid-fast bacteria and removing the supernatant;
• (e) resuspending the acid-fast bacteria pellet in at least 3 μL with formic acid;
• (f) adding at least 3 μL acetonitrile to the container;
• (g) transferring an aliquot of the supernatant from step (f) to a mass spectrometry target slide and adding a matrix solution to the supernatant;
• and
• (h) interrogating the test sample on the slide or plate by mass spectrometry to acquire one or more mass spectra of the acid-fast bacteria and characterizing and/or identifying said acid-fast bacteria by comparison of the measured mass spectrum with one or more reference mass spectra.

A method for inactivation and extraction of acid-fast bacteria in a test sample, the method comprising the following sequential steps:

• (a) acquiring a test sample from a liquid culture medium known to contain or that may contain acid-fast bacteria and adding the test sample to a container, centrifuging the container to pellet the acid-fast bacteria in the test sample and subsequently removing the supernatant;
• (b) resuspending the acid-fast bacteria pellet in ethanol;
• (c) adding glass beads to the container;
• (d) bead beating and/or vortexing the container to break up clumps and/or disrupt acid-fast bacteria cells in the container;
• (e) subsequently incubating the suspension for at least about 5 minutes at room temperature to inactivate any acid-fast bacteria contained in the test sample;
• (f) centrifuging the container to pellet the inactivated acid-fast bacteria and subsequently remove the supernatant;
• (g) resuspending the acid-fast bacteria pellet in formic acid;
• and
• (h) subsequently adding acetonitrile to the container;
• (i) transferring an aliquot of the supernatant from step (h) to a mass spectrometry target slide and adding a matrix solution to the supernatant;
• and
• (j) interrogating the test sample on the slide or plate by mass spectrometry to acquire one or more mass spectra of the mycobacteria and characterizing and/or identifying said acid-fast bacteria in the test sample by comparison of the measured one or more mass spectra with one or more reference mass spectra.