Cooke Rose Bengal Agar

Intended Use

Cooke Rose Bengal Agar is used with or without Antimicrobic Vial A in isolating fungi from environmental and food specimens.

Summary and Explanation

Cooke Rose Bengal Agar is a selective medium for the isolation of fungi prepared according to the formula of Cooke.^1,2 Selectivity of the medium is increased by the addition of antibiotics.
A variety of materials and methods have been used to inhibit bacteria in an attempt to isolate fungi from mixed flora. Fungi are extremely successful organisms, as evidenced by their ubiquity in nature.³ Waksman⁴ described an acid medium consisting of peptone, dextrose, inorganic salts and agar for the isolation of fungi from soil. Cooke¹ used the Waksman⁴ medium without adjustment to investigate the isolation of fungi from sewage. It was discovered that soy peptone was particularly suitable for use in this medium and that the combination of chlortetracycline, or oxytetracycline, with rose bengal increased the selectivity of the medium.

Principles of the Procedure

Peptone provides nitrogen, carbon and vitamins in Cooke Rose Bengal Agar. Dextrose is an energy source. Rose bengal and chlortetracycline selectively inhibit bacterial growth and restrict the size and height of colonies of more rapidly growing molds. Monopotassium phosphate provides buffering capability. Magnesium sulfate is a source of divalent cations. Agar is the solidifying agent.

User Quality Control

Identity Specifications
Cooke Rose Bengal Agar
Dehydrated Appearance: Pinkish-tan, free-flowing, homogeneous.
Solution:                        3.6% solution, soluble in purified water upon
                                     boiling. Solution is pinkish red, slightly opalescent.
Prepared Appearance:    Deep pink, slightly opalescent.
Reaction of 3.6%
Solution at 25°C:            pH 6.0 ± 0.2
Cultural Response
Prepare the medium with 35 μg per mL chlortetracycline (Antimicrobic Vial A added aseptically) per label directions. Inoculate and incubate at 25-30°C for up to 72 hours.

Formula

Cooke Rose Bengal Agar
Approximate Formula* Per Liter
Soy Peptone................................................................. 5.0 g
Dextrose.................................................................... 10.0 g
Monopotassium Phosphate............................................. 1.0 g
Magnesium Sulfate........................................................ 0.5 g
Agar.......................................................................... 20.0 g
Rose Bengal............................................................... 35.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

Cooke Rose Bengal Agar
1. Suspend 36 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. Cool to 45°C.
4. Aseptically add 14 mL of rehydrated Antimicrobic Vial A (final concentration of 35 μg chlortetracycline per mL) or appropriate amount of another antibiotic.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Refer to appropriate references for specific procedures on the isolation and cultivation of fungi.

Expected Results

Refer to appropriate references and procedures for results.

Limitations of the Procedure

1. Although this medium is selective primarily for fungi, microscopic examination is recommended for presumptive identification. Biochemical testing using pure cultures is      required for complete identification.
2. Due to the selective properties of this medium and the type of specimen being cultured, some strains of fungi may be encountered that fail to grow or grow poorly on the complete medium; similarly, some strains of bacteria may be encountered that are not inhibited or only partially inhibited.
3. Care should be taken not to expose this medium to light, since photo-degradation of rose bengal yields compounds that are toxic to fungi.^6,7

*Store at 2-8° C.

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