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The processed specimen is now ready for culture and a variety of special media can be utilized. Optimally, at least two solid media types should be used, including one serum- and one egg-based. Typical solid media can include:
  • Middlebrook 7H10 (agar base with 2% glycerol, serum-based medium)
  • Middlebrook or Mitchison 7H11 (selective agar-base with antibiotics)
  • Lowenstein-Jensen (L-J), Gruft (selective, egg-based medium with antibiotics)
  • Mycobactosel agar (L-J medium with antibiotics, BBL Microbiology Systems, Cockeysville, MD)
Broth media can provide a more rapid recovery (approximately 10 days versus 17 days or longer with conventional solid media). Typical broth agar can include 7H9 with TWEEN® 80 added as a surfactant to disperse clumps of AFB for bottles and tubes. This broth media is used in:
  • Manual methods
  • Semi-automated radiometric methods: BACTEC 460 (Becton-Dickinson, Sparks, MD)
  • Automated systems:
    • BACTEC 9000 MB (Becton-Dickinson)
    • BACTEC MGIT 960 (Becton-Dickinson)
    • ESP® Culture System II (Trek Diagnostic Systems, Cleveland, OH)
    • VersaTREK® (Trek Diagnostic Systems, Cleveland, OH)
    • MB/BacT ALERT® 3D system (bioMe′rieux, France)
The BACTEC™ MGIT™ 960 and the BACTEC™ 460 systems range in sensitivity from 81.5% to 85.8% and specificity to detect mycobacteria is 99.6% and 99.9%. Sensitivity is increased to 87.7% and 89.7% when solid media culture is combined with automated testing.
Detection of mycobacteria by automated systems, monitored continuously, is state-of- the-art for culturing and rapid recovery of mycobacteria (10-14 days compared to 18-24 days on egg-based media). Laboratories often combine culture on 7H11 agar plates with automation to see micro-colonies, particularly those of non-tuberculosis mycobacteria, in as few as 11 days. However the method is expensive and shelf-life of the media is only 1 month (refrigerated). Also, contamination is a problem in automated systems (less so in the semi-automated BACTEC 460), as in most agar-based methods. However, the addition of antibiotic supplemented media (Mitchison 7H11, L-J Gruft, and Mycobactosel L-J media) are useful in preventing bacterial overgrowth, but must be used in combination with non-selective media.