Measuring Minimum Inhibitory Concentrations in Mycobacteria

  • Frederick A. Sirgel
  • Ian J.F. Wiid
  • Paul D. van HeldenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 465)


An agar dilution method for measuring minimum inhibitory concentrations (MICs) of Mycobacterium tuberculosis, based on the method of proportion, is described. Mycobacterium strains are grown on Middlebrook 7H10 (or 7H11) agar medium with twofold serially diluted drug concentrations in order to determine specific inhibitory values. The proportion of bacilli resistant to a given drug is determined by comparing the number of colony-forming units (CFU) on a drug-free control with those growing in the presence of drug within a specific concentration range. The MIC is defined as the lowest drug concentration that inhibits growth of more than 99% of a bacterial population of M. tuberculosis on solid Middlebrook medium within 21 days of incubation at 37°C. The proportion method, the absolute concentration method, and the resistant ratio method have traditionally been used as standard procedures for antimycobacterial drug-susceptibility testing (DST), and reference data are mainly based on these methods. DST concepts and alternative procedures that have been adopted for DST are also briefly discussed.


agar proportion antimicrobial agents MICs Mycobacterium tuberculosis susceptibility testing 


  1. 1.
    Hawkins JE. Drug susceptibility testing. In Kubica GP, Wayne LG, eds. The Mycobacteria. A Sourcebook. Part A. Marcel Dekker Inc, New York and Basel, 1984:177–193.Google Scholar
  2. 2.
    Heifets LB. Introduction: Drug susceptibility tests and the clinical outcome of chemotherapy. In: Heifets LB, ed. Drug Susceptibility in the Chemotherapy of Mycobacterial Infections. CRC Press, Inc., Boca Raton, FL, 1991:1–11.Google Scholar
  3. 3.
    National Committee for Clinical Laboratory Standards. Susceptibility testing of Mycobacteria, Norcardiae, and Other Aerobic Actinomycetes; Approved Standard. NCCLS Document M24-A. NCCLS, Wayne, PA, 2003;23(18):1–71.Google Scholar
  4. 4.
    Chan ED, Iseman MD. Current medical treatment for tuberculosis. BMJ 2002;325:1282–1286.Google Scholar
  5. 5.
    Heginbothom ML. The relationship between the in vitro drug susceptibility of opportunist mycobacteria and their in vivo response to treatment. Int J Tuberc Lung Dis 2001;5(6):538–545.Google Scholar
  6. 6.
    Heifets L, Desmond E. Clinical mycobacteriology (tuberculosis) laboratory: services and methods in tuberculosis and the tubercle bacillus. In: Cole ST, Eisenach KD, McMurray DN, Jacobs (Jr) eds. Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC, 2005:49–69.Google Scholar
  7. 7.
    Mitchison DA. Drug resistance in tuberculosis. Eur Respir J 2005;25:376–379.PubMedCrossRefGoogle Scholar
  8. 8.
    Dickinson JM, Mitchison DA. In vitro activity of new rifamycins against rifampin-resistant M. tuberculosis and MAIS-complex mycobacteria. Tubercle 1987;68: 177–182.PubMedCrossRefGoogle Scholar
  9. 9.
    MacGowan AP, Wise R. Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. J Antimicrob Chemother 2001;48(Suppl S1):17–28.PubMedCrossRefGoogle Scholar
  10. 10.
    Mueller M, de la Pena A, Derendorf H. Issues in pharmacokinetics and pharmacodynamics of anti-infective agents: Kill curves versus MIC. Antimicrob Agents Chemother 2004;48(2):369–377.PubMedCrossRefGoogle Scholar
  11. 11.
    Siddiqi SH. BACTEC 460 TB system. Product and procedure manual, revision D. Becton Dickinson Microbiology Systems, Sparks, MD, 1995.Google Scholar
  12. 12.
    Heifets LB. Drug susceptibility tests in the management of chemotherapy of tuberculosis. In: Heifets LB, ed. Drug Susceptibility in the Chemotherapy of Mycobacterial Infections. CRC Press, Inc., Boca Raton, FL, 1991:89–121.Google Scholar
  13. 13.
    Bemer P, Bodmer T, Munzinger J, Perrin M, Vincent V, Drugeon H. Multicenter evaluation of the MB/BACT System for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2004;42(3):1030–1034.PubMedCrossRefGoogle Scholar
  14. 14.
    Chew WK, Lasaitis RM, Schio FA, Gilbert GL. Clinical evaluation of the Mycobacteria Growth Indicator Tube (MGIT) compared with radiometric (Bactec) and solid media for isolation of Mycobacterium species. J Med Microbiol 1998;47:821–827.PubMedCrossRefGoogle Scholar
  15. 15.
    Bergmann JS, Woods GL. Evaluation of the ESP Culture System II for testing susceptibilities of Mycobacterium tuberculosis isolates to four primary antituberculous drugs. J Clin Microbiol 1998;36(10):2940–2943.PubMedGoogle Scholar
  16. 16.
    Woods GL, Fish G, Plaunt M, Murphy T. Clinical evaluation of Difco ESP culture system II for growth and detection of mycobacteria. J Clin Microbiol 1997;35(1):121–124.PubMedGoogle Scholar
  17. 17.
    Somoskövi A, Magyar P. Comparison of the Mycobacteria Growth Indicator Tube with MB Redox, Löwenstein-Jensen, and Middlebrook 7H11 media for recovery of mycobacteria in clinical specimens. J Clin Microbiol 1999;37(5):1366–1369.PubMedGoogle Scholar
  18. 18.
    Brunello F, Fontana R. Reliability of the MB/BACT system for testing susceptibility of Mycobacterium tuberculosis complex strains to anti-tuberculosis drugs. J Clin Microbiol 2000;38:872–873.PubMedGoogle Scholar
  19. 19.
    Heifets, LB. Drug susceptibility testing. In: Heifets LB (guest ed.), Clauser G ed. Clinics in Laboratory Medicine, vol 16 (3). W.B. Saunders, Philadelphia, 1996:641–656.Google Scholar
  20. 20.
    Dickinson JM, Allen BW, Mitchison DA. Slide culture sensitivity tests. Tubercle 1989;70:115–121.PubMedCrossRefGoogle Scholar
  21. 21.
    Welch DF, Guruswamy AP, Sides SJ, Shaw CH, Gilchrist MJ. Timely culture for mycobacteria which utilizes a microcolony method. J Clin Microbiol 1993;31(8):2178–2184.PubMedGoogle Scholar
  22. 22.
    Mejia GI, Castrillon L, Trujillo H, Robledo JA. Microcolony detection in 7H11 thin layer culture is an alternative for rapid diagnosis of Mycobacterium tuberculosis infection. Int J Tuberc Lung Dis 1999;3(2):138–142.PubMedGoogle Scholar
  23. 23.
    Park WG, Bishai R, Chaisson E, Dorman SE. Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis. J Clin Microbiol 2002;40(12):4750–4752.PubMedCrossRefGoogle Scholar
  24. 24.
    Wanger A, Mills K. Testing of Mycobacterium tuberculosis susceptibility to ethambutol, isoniazid, rifampicin, and streptomycin by using Etest. J Clin Microbiol 1996;34(7):1672–1676.PubMedGoogle Scholar
  25. 25.
    Nilsson LE, Hoffner SE, Ansehehn S. Rapid susceptibility testing of M. tuberculosis by bioluminescence assay of mycobacterial ATP. Antimicrob Agents Chemother 1988;32:1208–1212.PubMedCrossRefGoogle Scholar
  26. 26.
    Jacobs WR Jr, Barletta RG, Udani R, Chan J, Kalkut G, Sosne G, Kieser T, Sarkis GJ, Hatfull GF, Bloom BR. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 1993;260:819–822.PubMedCrossRefGoogle Scholar
  27. 27.
    Albert H, Trollip A, Seaman T, Mole RJ. Simple, phage based (FastPlaque) technology to determine rifampicin resistance of Mycobacterium tuberculosis directly from sputum. Int J Tuberc Lung Dis 2004;8(9):1114–1119.PubMedGoogle Scholar
  28. 28.
    Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson RM, Gilman RH. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate alamar blue assay. J Clin Microbiol 1998;36(2):362–366.PubMedGoogle Scholar
  29. 29.
    Leite CQF, Beretta ALRZ, Anno IS, Telles MAS. Development of a microdilution method to evaluate Mycobacterium tuberculosis drug susceptibility. J Antimicrob Chemother 2003;52:796–800.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Frederick A. Sirgel
    • 1
  • Ian J.F. Wiid
    • 1
  • Paul D. van Helden
    • 2
    Email author
  1. 1.Division of Molecular Biology and Human GeneticsMRC Centre for Molecular and Cellular Biology, DST/NRF Centre of Excellence for Biomedical TB Research, Stellenbosch UniversityTygerbergSouth Africa
  2. 2.Division of Molecular Biology and Human GeneticsStellenbosch UniversityTygerbergSouth Africa

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