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7H9 (and its solid counterpart, 7H10 agar) are selective media used for the culture of free-living mycobacteria such as M. tuberculosis and M. smegmatis.


(makes 1L medium)

  • Deionized water
  • 4.7g Middlebrook 7H9 powder (DifCo catalog # 0713-01-7)
  • 5mL 10% Tween-80: Dilute 40g Tween-80 (liquid) into final volume 200mL with deionized water. Stir on low heat (~65°C) until completely dissolved (may take ~15 minutes). Filter sterilize with a 0.22μm filter. Store at 4°C wrapped in aluminum foil, as Tween is light sensitive.

For M. tuberculosis:

  • 100mL OADC (Dubos/Middlebrook Oleic Acid Albumin Dextrose Complex)
  • 10mL 50% glycerol
  • (If you are using lung homogenates or the like) 10mL 10mg/mL cyclohexamide

For M. smegmatis:

  • 100mL ADS (Albumin-Dextrose-Saline): 8.1g NaCl, 50g Bovine Serum Albumin Fraction V, 20g anhydrous dextrose in 1L deionized water. Filter sterilize in a 0.22μm filter, and store at 4°C.


  1. Dissolve 4.7g Middlebrook 7H9 poweder in 900mL deionized water. Mix with a magnetic stir bar until soluble components have dissolved.
  2. Add the rest of the ingredients appropriate for the organism at hand and continue stirring until well mixed.
  3. Sterilize by filtration through a 0.22μm pore membrane. Store at 4°C.