Regan-Lowe Charcoal Agar Bas

Intended Use

Regan-Lowe Charcoal Agar is a selective medium used for isolation of Bordetella pertussis from clinical specimens. Regan-Lowe Charcoal Agar without Cephalexin is used for the cultivation of B. pertussis from clinical specimens and for subcultures of the bacterium.

Summary and Explanation

Regan-Lowe Charcoal Agar plates are used in clinical laboratories for the isolation of Bordetella pertussis, the etiologic agent of whooping cough, from nasopharyngeal swabs and other sources of pharyngeal exudate. This medium was developed by Regan and Lowe as a transport medium for whooping cough specimens, but proved useful as an enrichment medium for the selective isolation of B. pertussis andB. parapertussis. It consists of charcoal agar as a basal medium supplemented with cephalexin to inhibit bacteria indigenous to the nasopharynx and defibrinated horse blood to support the growth of Bordetella species.1-3
Use of the medium without cephalexin in parallel with Regan-Lowe Charcoal Agar is recommended, since a few strains (<10%) of B. pertussis will not grow on selective plates; also the nonselective medium is used for subcultures to obtain a larger amount of growth for additional testing, such as agglutination or immunofluorescence testing.3,4
The medium in 10 mL prepared tubes (deeps) with screw-caps offers a longer shelf-life than the pre-poured plated medium. To prepare the medium from the agar base, 10% horse blood is added and cephalexin can be added to achieve selectivity.

Principles of the Procedure

Beef extract and enzymatic digest of gelatin provide the amino acids and other complex nitrogenous substances necessary to support bacterial growth. Sodium chloride maintains the osmotic equilibrium. Defibrinated horse blood supplies nutrients required for the cultivation of Bordetella species. Nicotinic acid is a vitamin that promotes growth. Charcoal and starch
neutralize substances toxic to Bordetella species, such as fatty acids and peroxides. Cephalexin is a cephalosporin antibiotic that inhibits most normal flora of the nasopharynx.

User Quality Control

Identity Specifications
Regan-Lowe Charcoal Agar Base
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       5.1% solution, soluble in purified water upon
                                   boiling. Solution is charcoal black, homogeneous,
                                   opaque.
Prepared Appearance:    Charcoal black, homogeneous, opaque.
Reaction of 5.1%
Solution at 25°C:           pH 7.4 ± 0.2
Cultural Response
Regan-Lowe Charcoal Agar Base
Prepare the medium per label directions. Inoculate with fresh broth cultures diluted 1:10 and incubate at 35 ± 2°C for 7 days.

Formula

Regan-Lowe Charcoal Agar Base
Approximate Formula* Per Liter
Beef Extract................................................................ 10.0 g
Pancreatic Digest of Casein........................................... 10.0 g
Soluble Starch............................................................. 10.0 g
Sodium Chloride............................................................ 5.0 g
Charcoal....................................................................... 4.0 g
Niacin.......................................................................... 0.01 g
Agar............................................................................ 12.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 51 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes. DO NOT OVERHEAT.
4. For preparation of blood plates, add 10% sterile, defibrinated horse blood to sterile agar which has been previously melted and cooled to 45-50°C.
5. For selective isolation of B. pertussis and B. parapertussis, add 40 μg of cephalexin per mL.
6. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens. Incubate the plates in an inverted position (agar side up) in a moist chamber at 35°C for 7 days. Colonies of B. pertussis may not be visible without the aid of a microscope for 2-4 days. Plates may be discarded as negative after 7 days of incubation.

Expected Results

Examine the plates daily with and without a dissecting microscope (oblique illumination) to detect the presence of B. pertussis. B. pertussis produces small, domed, glistening, white to gray colonies. To prevent overgrowth by spreading colonies or molds, use a sterile scalpel or needle to remove the portions of the agar that contain these contaminants.
*Store at 2-8°C.

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