Cooke Rose Bengal Agar Base

Intended Use

Cooke Rose Bengal Agar is used for selective cultivation and isolation of fungi.

Composition

Final pH (at 25°C): 6.0±0.2

Formula adjusted, standardized to suit performance parameters

Directions

Suspend 36.54 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. To increase the selectivity of the medium, add 35µg chlortetracycline per ml of the medium. Mix well and pour into sterile Petri plates.

Principle and Interpretation

Cooke Rose Bengal Agar is a selective medium formulated as per Cooke (1, 2). A variety of inhibitory agents have been used to inhibit bacteria in an attempt to isolate fungi from mixed flora. The Kingdom Fungi includes some of the most important organisms, both in terms of their ecological and economic roles. By breaking down dead organic material into simpler forms, they continue the cycle of elements through ecosystems. In addition, most vascular plants could not grow without a symbiotic association with fungi, or mycorrhizae, that inhabit their roots and supply essential nutrients. Other fungi provide numerous drugs (such as penicillin and other antibiotics), foods like mushrooms, truffles and morels, and the bubbles in bread, champagne, and beer (3). Waksman (4) described an acid medium consisting of peptone, dextrose, inorganic salts and agar for the isolation of fungi from soil. Cooke (1) used the Waksman medium without adjustment for isolation of fungi from sewage. It was discovered that papaic digest of soyabean meal 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.

Smith and Dawson (5) used rose bengal for the inhibition of bacteria in media which has almost neutral reaction concerned with retardation of the development of fungi. Martin (6) used 1: 30,000 Rose bengal and 30µg Streptomycin per ml and found that a wide variety of bacteria are inhibited at reactions between pH 5.5 to 6.5 without inhibiting fungi.

The medium should not be exposed to light as photo-degradation of rose bengal yields compounds that are toxic to fungi (7, 8).

Microscopic examination coupled with biochemical testing using pure cultures is recommended for complete identification. 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 partially inhibited.

Papaic digest of soyabean meal provides nitrogen, carbon and vitamins. 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 moulds. Monopotassium phosphate provides buffering capability. Magnesium sulfate is a source of divalent cations.

Quality Control

Appearance: Light yellow to light pink homogeneous free flowing powder

Gelling: Firm, comparable with 2.0% Agar gel

Colour and Clarity of prepared medium: Pink-red coloured, slightly opalescent forms in Petri plates

Reaction: pH 6.0±0.2 (3.65% w/v aqueous solution at 25°C)

pH: 5.80-6.20

Cultural Response

M499: Cultural characteristics observed after an incubation at 25-30°C for 1-4 days.

Key: *- Formerly known as Aspergillus niger

Storage and Shelf Life

Store below 30°C in tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label.

References

1. Cooke, 1954, Antibiot. Chemother., 4:657.
2. Eaton A. D., Clesceri L. S., Rice E. W., and Greenberg A. E., (Eds.), 2005, Standard Methods for the Examination of Water and Wastewater, 21 st Ed., APHA, Washington, D.C.
3. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D. C.
4. Waksman, 1922, J. Bacteriol., 7:339.
5. Smith and Dawson, 1944, Soil Sci., 58:467.
6. Martin, 1950, Ibid., 69:215.
7. Banks, Board and Paton, 1985, Lett. Appl. Microbiol., 1:7.
8. Downes F. P. and Ito K., (Eds.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th Ed., APHA, Washington, D.C.