Table 2.

Composition of Middlebrook media used to culture strains of M. avium subsp. paratuberculosis from sheep in this study

IngredientAmt/liter in:
Modified BACTEC 12BaModified 7H10 agarbModified 7H11 agarc
For base medium
 Casein digest667 mge800 mgd800 mg
 Ammonium sulfate333 mg400 mg400 mg
 Monopotassium phosphate667 mg1.2 g1.2 g
 Disodium phosphate1.7 g1.2 g1.2 g
 Sodium citrate67 mg320 mg320 mg
 Magnesium sulfate33 mg20 mg40 mg
 Calcium chloride0.33 mg0.4 mg
 Zinc sulfate0.67 mg0.8 mg
 Copper sulfate0.67 mg0.8 mg
l-Glutamic acid333 mg400 mg400 mg
 Ferric ammonium citrate27 mg32 mg32 mg
 Pyridoxine0.67 mg0.8 mg0.8 mg
 Biotin0.33 mg0.4 mg0.4 mg
 Malachite green0.2 mg0.8 mg
 Bacto Agar12 g12 g
For enrichment
 Oleic acid40 mg
 Albumin fraction V, bovine3.3 ge4.0 g4.0 g
 Dextrose1.6 g1.6 g
 Catalase32,000 Ue2.4 mg3.2 mg
 Sodium chloride680 mg
 C14 palmitic acid667 μCie
As additive
 Egg yolk167 ml200 ml200 ml
 Mycobactin J0.83 mg1.0 mg1.0 mg
 PANTA PLUSf33.3 ml40 ml40 ml
  • a Additives (and 700 μl of water) were added to the enriched BACTEC media at the following rates per 4-ml vial, resulting in a final volume of 6 ml/vial: 1 ml of egg yolk, 5 μg (100 μl of mycobactin J, and 200 μl of PANTA PLUS (4a, 13a).

  • b ADC was used for enrichment.

  • c OADC was used for enrichment.

  • d As added Casitone.

  • e Added by manufacturer to standard Middlebrook 7H9 broth to form BACTEC 12B medium.

  • f Consists of polymyxin B (1,000 U/ml), amphotericin B (100 μg/ml), nalidixic acid (400 μg/ml), trimethoprim (100 μg/ml), azlocillin (100 μg/ml), and polyoxyethylene stearate (4 mg/ml).