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BAM Media M28a: Campylobacter enrichment broth (Bolton formula)

January 2001

Bacteriological Analytical Manual

M28a
Campylobacter enrichment broth (Bolton formula)

(Oxoid AM-7526 (manufactured by Med-Ox Chemicals Ltd.) or Malthus Diagnostics Lab)

Enrichment Broth Base

Meat Peptone10 g
Lactalbumin Hydrolysate5 g
Yeast Extract5 g
NaCl5 g
Haemin0.01 g
Sodium Pyruvate0.5 g
Alpha-Ketoglutamic Acid1 g
Sodium Metabisulphite0.5 g
Sodium Carbonate0.6 g
Total Dry Incredients27.61 g
Distilled Water1000 ml

Final pH, 7.4 ± 0.2.

 

THOROUGHLY MIX 27.61 g in 1 liter water. Let soak 10 min. Swirl again and autoclave 15 min at 121°C (in screw-capped bottles if possible). Before use, add 2 rehydrated vials of Campylobacter enrichment broth (Bolton formula) supplement (Oxoid NDX131 or Malthus Diagnostics X-131). If supplement is not available add 4 ml each of antibiotic concentrates (formulas below, solutions made separately).

Store powdered media in a tightly fastened container in a cool, dry area to reduce oxygen infusion and peroxide formation, which can inhibit recovery of microaerophiles. Use prepared broth within 1 month of preparation (preferably less than 2 weeks).

 

ItemStorage
conditions
Shelf life
Complete mediumcool, dry2 weeks - 1 month
Sodium cefoperazone4°C5 days
-20°C14 days
-70°C5 months
Trimethoprim lactate4°C1 year
vancomycin4°C2 months
Cycloheximide*4°C1 year
Substitute solubilized amphotericin B (Sigma Cat. No. A9528) for cycloheximide if cycloheximide not available. See instructions below.

 

Campylobacter Enrichment Broth Supplements
(Prepare each solution separately)

  1. Sodium cefoperazone. Weigh 0.5 g into 100 ml distilled water in volumetric flask and dissolve. Only prepare amount required and filter-sterilize, 0.22 5m, using a syringe filter if 25 ml. Store solution 5 days at 4°C, 14 days at -20°C, and 5 months at -70°C. Freeze in sterile plastic tubes or bottles. Powder may be purchased from Sigma or obtained free from Pfizer by writing Roering Division, Pfizer, 235 E. 42nd St., New York, NY 10017. Request 2-4 g for in vitro use. Final concentration is 20 mg/liter.
  2.   Trimethoprim

    1. Trimethoprim lactate (Sigma Cat. No. T0667). Dissolve 0.66 g in 100 ml distilled water, and filter. May be stored 1 year at 4°C. Add 4 ml/liter to yield final concentration of 20 mg/liter Trimethoprim.

      or

    2. Trimethoprim (Sigma Cat. No. T7883)[a lower cost alternative]. To prepare Trimethoprim (TMP)-HCl: add 0.5 g TMP to 30ml 0.05N HCl at 50°C (stir until dissolved using hot plate with magnetic stirrer). Adjust volume to 100ml with distilled water. Add 4 ml/liter to yield final concentration of 20 mg/liter Trimethoprim.
  3. Vancomycin (Sigma). Dissolve 0.5 g in 100 ml distilled water and filter. Store up to 2 months at 4°C. Because of short shelf life, prepare smaller amounts. Add 4 ml/liter for final concentration of 20 mg/liter
  4. Cycloheximide Dissolve 1.25 g in 20-30 ml ethanol in a 100 ml volumetric flask and bring to line with water. Filter-sterilize. Store at 4°C up to 1 year. Add 4 ml for final a concentration of 50 mg/liter. Use amphotericin B as described below if cycloheximide is not available.

    1. 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.

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