BAM Media M29a: Abeyta-Hunt Bark Agar

January 2001

Bacteriological Analytical Manual

Abeyta-Hunt Bark Agar

Heart infusion agar (Difco) 40 g
Yeast extract 2 g
Distilled water 950 ml

Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2. Cool and add sodium cefoperazone (6.4 ml if using broth preparation or 4 ml of the agar preparation[below]), 4 ml rifampicin, 4 ml amphotericin B, and 50 ml lysed horse blood.

After pouring plates, dry plates overnight on bench.If plates must be used the same day, place them in 42°C incubator for several hours. Do not dry in a hood with lids open. Even very brief surface drying will inhibit campylobacter growth.

  1. Sodium cefoperazone. Prepare as described for broth for final concentration of 32 mg/liter, adding 6.4 ml to agar. OR dissolve 0.8 g in 100 ml water in a 100 ml volumetric flask, filter and add 4 ml to agar.
  2. Rifampicin. Dissolve 0.25 g slowly into 60-80 ml alcohol in a 100 volumetric, swirling repeatedly. When powder is dissolved completely, bring to the line with distilled water. Store up to 1 year at -20°C. Final concentration is 10 mg/liter.
  3. Amphotericin B, solubilized (Sigma Cat. No. A9528). Dissolve 0.05 g in water in a 100 ml volumetric flask and bring to the line. Filter sterilize and store at -20°C for 1 year. Final concentration is 2 mg/liter. Add 4 ml per liter.
  4. FBP.  Dissolve 6.25 g Sodium pyruvate in 10-20 ml distilled water. Pour into a 100 ml volumetric. Add 6.25 g Ferrous sulfate and 6.25 g Sodium metabisulfite. Bring to the line with distilled water and filter sterilize. Use 4 ml/liter agar.

    Note: FBP is light sensitive and absorbs oxygen rapidly. Prepare only the amount needed. 10-25 ml amounts can be filtered with a 0.22 µm syringe filter. Freeze unused portions in 5 ml amounts at -70°C as soon as possible after preparation. It can be stored at -70°C for 3 mos or -20°C for 1 mo.

Original Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Updated: 29-DEC-2000

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