Culture

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The page below is a sample from the LabCE course Tracking Antibiotic-Resistant Tuberculosis. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Culture

The processed specimen, used for culture, is inoculated on media. 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.
  • Automation is expensive and the shelf-life of the media is sometimes only one month (refrigerated).
  • Contamination may be 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.
9. Wikimedia. Caption: "Culture on Lowenstein-Jensen medium revealed typical dry, heaped-up yellow to buff-colored colonies of Mycobacterium tuberculosis." Wikimedia Commons website, public domain image. 2010. Accessed May 11, 2022. File:M-tuberculosis-on-Lowenstein-Jensen.jpg - Wikimedia Commons

MTB on LJ slants. (9)