Methods for inactivation and extraction of acid-fast bacteria for characterization and/or identification using mass spectrometry

A method for inactivation and extraction of acid-fast bacteria from a solid or semi-solid culture medium, and subsequently characterizing and/or identifying said acid-fast bacteria, the method comprising the following sequential steps:

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