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

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

• (a) transferring a test sample from a liquid culture medium containing acid-fast bacteria to a first tube, wherein the first tube comprises a body, a first end to the body having an opening, and a second end to the body having a frustoconical portion ending in a concave tip;
• (b) centrifuging the first tube to pellet the acid-fast bacteria in the concave tip and subsequently decanting a first supernatant;
• (c) resuspending the acid-fast bacteria pellet in alcohol to generate a suspension;
• (d) transferring the suspension to a second tube containing beads for mechanical disruption;
• (e) incubating the suspension in alcohol to inactivate the acid-fast bacteria;
• (f) transferring the suspension to a third tube;
• (g) centrifuging the third tube to pellet the inactivated acid-fast bacteria and subsequently removing a second supernatant;
• (h) resuspending the inactivated acid-fast bacteria pellet to generate a solution comprising the inactivated acid-fast bacteria;
• (i) extracting cellular proteins from the solution comprising inactivated acid-fast bacteria and centrifuging the solution to pellet the cellular debris;
• (j) transferring an aliquot of a third supernatant from step (i) to a mass spectrometry target slide;
• and
• (k) interrogating the mass spectrometry target slide by mass spectrometry to acquire one or more mass spectra of protein profiles of the acid-fast bacteria 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 of protein profiles.

A kit for inactivation and identification of acid-fast bacteria in a test sample, the kit comprising:

• a first tube having a body, a first end to the body having an opening, and a second end to the body having a frustoconical portion ending in a concave tip, wherein the first tube has a volume of at least 5 mL;
• a solution of alcohol;
• a solution of formic acid;
• and
• a solution of acetonitrile.

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

• (a) transferring a test sample from a liquid culture containing acid-fast bacteria to a first tube, wherein the first tube comprises a body, a first end to the body having an opening, and a second end to the body having a frustoconical portion ending in a concave tip;
• (b) centrifuging the first tube to pellet the acid-fast bacteria in the concave tip and subsequently decanting a first supernatant, wherein the frustoconical portion ending in the concave tip is configured to retain the pellet of acid-fast bacteria in the concave tip while decanting at least 90% of the first supernatant;
• (c) resuspending the acid-fast bacteria pellet in alcohol in a second tube;
• (d) agitating the second tube to break up clumps and/or disrupt acid-fast bacteria cells;
• and (e) identifying the acid-fast bacteria via mass spectrometry.