Mycobacterium tuberculosis Susceptibility Testing by Flow Cytometry

Ronald F. Schell1, Dean T. Nardelli1, David J. DeCoster1, Scott M. Kirk2, Steven M. Callister3

1 University of Wisconsin, Madison, Wisconsin, 2 Amersham Biosciences, Sunnyvale, California, 3 Gundersen Lutheran Medical Center, LaCrosse, Wisconsin
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.7
DOI:  10.1002/0471142956.cy1107s27
Online Posting Date:  February, 2004
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Abstract

Here is another unit with clinical relevance. Tuberculosis remains a major health problem throughout the world, with approximately one‐quarter of the population being infected. Rapid and accurate susceptibility testing for the tubercle bacillus is essential for control of the disease. Such testing can be accomplished by flow cytometry within twenty‐four hours, instead of the days to weeks required by traditional methods. The use of flow cytometry both improves the quality of susceptibility testing and advances public health measures for the prevention and control of this ancient scourge.

Keywords: flow cytometry; Mycobacterium tuberculosis; susceptibility testing; tuberculosis; public health

     
 
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Table of Contents

  • Basic Protocol 1: Assessing Susceptibility of M. Tuberculosis by Flow Cytometry
  • Support Protocol 1: Preparation of Sterile 7H9 Broth
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Assessing Susceptibility of M. Tuberculosis by Flow Cytometry

  Materials
  • Anti‐tuberculosis agents (ethambutol, isoniazid, and rifampin; see recipe)
  • M. tuberculosis isolates grown on Lowenstein‐Jensen agar, 7H10 agar, or other suitable solid medium
  • McFarland 1.0 standards
  • 7H9 broth (see protocol 2; may also be purchased commercially)
  • 500 ng/ml fluorescein diacetate (FDA; Molecular Probes) in phosphate‐buffered saline, pH 7.4 ( appendix 2A) or 50 µg/ml 5‐chloromethylfluorescein diacetate in dimethyl sulfoxide (DMSO; Sigma)
  • 16 × 125–mm screw‐cap tubes
  • 3‐ or 4‐mm glass beads (Fisher)
  • 0.2‐µm filters
  • 50.0‐ml polypropylene screw‐cap tubes
  • 2.0‐ml screw‐cap microtubes (Sarstedt)
CAUTION: The following procedures must be carried out in a biosafety cabinet contained in a P‐3 biosafety facility. In addition, cap, gown, gloves, and safety glasses must be worn.

Support Protocol 1: Preparation of Sterile 7H9 Broth

  Materials
  • Tween 80 (Sigma)
  • 2.5% ammonium sulfate
  • 2.5% glutamic acid
  • 10% sodium citrate dihydrate
  • 0.1% pyridoxine hydrochloride
  • 0.2% biotin
  • Disodium phosphate, anhydrous
  • Monopotassium phosphate, anhydrous
  • 10% ferric ammonium citrate
  • 1% magnesium sulfate heptahydrate
  • 0.1% calcium chloride dihydrate
  • 0.1% zinc sulfate heptahydrate
  • 0.1% copper sulfate pentahydrate
  • 50% glucose (see recipe)
  • 1000 µg/ml catalase (see recipe)
  • 5% BSA fraction V (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Bownds, S.E., Kurzynski, T.A., Norden, M.A., Dufek, J.L., and Schell, R.F. 1996. Rapid susceptibility testing for non‐tuberculosis mycobacteria using flow cytometry. J. Clin. Microbiol. 34:1386‐1390.
   Centers for Disease Control and Prevention (CDC). 1998. Tuberculosis morbidity—United States, 1997. Morbid. Mortal. Weekly Rep. 47:253‐257.
   CDC. 1996. Tuberculosis morbidity—United States, 1995. Morbid. Mortal. Weekly Rep. 45:365‐370.
   Kirk, S.M., Schell, R.F., Moore, A.V., Callister, S.M., and Mazurek, G.H. 1998. Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid and rifampin in 24 hours. J. Clin. Microbiol. 36:1568‐1573.
   Moore, A.V., Kirk, S.M., Callister, S.M., Mazurek, G.H., and Schell, R.F. 1999. Safe determination of susceptibility of Mycobacterium tuberculosis to antimicrobial agents by flow cytometry. J. Clin. Microbial. 37:479‐483.
   National Committee for Clinical Laboratory Standards (NCCLS). 1995. Antimycobacterial Susceptibility Testing for Mycobacterium tuberculosis. Proposed Standard M24‐T. NCCLS, Villanova, Pa.
   Norden, M.A., Kurzynski, T.A., Bownds, S.E., Callister, S.M., and Schell, R.F. 1995. Rapid susceptibility testing of Mycobacterium tuberculosis (H37Ra) by flow cytometry. J. Clin. Microbiol. 33:1231‐1237.
   Siddiqi, S.H., Hawkins, J.E., and Laszio, A. 1985. Interlaboratory drug susceptibility testing of Mycobacterium tuberculosis by a radiometric procedure and two conventional methods. J. Clin. Microbiol. 22:919‐923.
   Vena, R.M., Munson, E.L., DeCoster, D.J., Feh, D.B., Callister, S.M., and Schell, R.F. 2000. Flow cytometric testing of Mycobacterium avium to amikacin, ciproflaxin, clarithromycin and rifabutin in 24 hours. Clin. Microbiol. Infect. 6:365‐375.
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