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Rose Bengal Agar Base
Catalog Number: 1008917
Rose Bengal Agar Base
Description: Rose Bengal Agar Base and Chloramphenicol are used in the preparation of Rose Bengal Chloramphenical Agar. The complete medium is recommended in the selective isolation and enumeration of yeasts and molds from environmental materials and foodstuffs.
Rose Bengal Agar Base is a selective basal medium which supports good growth of yeasts and molds. The pH of the medium is near neutrality for improved growth and recovery of acid sensitive strains.4,5,11 The presence of rose bangal in the base suppresses the growth of bacteria and restricts the size and height of mold colonies.8,10,11 This restriction in growth of molds aids in the isolation of slow-growing fungi by preventing their overgrowth by more rapidly growing species. In addition, rose bengal is taken up by yeast and mold colonies thereby facilitating their recognition and enumeration. Supplementation of Rose Bengal Agar Base with chloramphenicol provides for improved inhibition of bacteria present in environmental materials and foodstuffs.
Formulation in g/L:
|Peptone, soybean||5 g||Dextrose||10 g|
|Monopotassium Phosphate||1 g||Magnesium Sulfate||0.5 g|
|Rose Bengal||0.05 g||Agar||15 g|
- Use aseptic technique in rehydrating chloramphenicol and adding it to the base.
- Follow proper, laboratory procedures in handling and disposing of infectious materials.
- Avoid the formation of aerosols.
- Store the Rose Bengal Agar Base below 30oC. The powder is very hygroscopic. Keep container tightly closed.
- Do not use the agar base if it is caked, discolored or shows other signs of deterioration.
- Specimens should be collected in sterile containers and transported immediately to the laboratory in accordance with recommended guidlines.12-14
For the Chloramphenicol supplement:
- Rehydrate 0.05 g of chloromphenicol with 2 ml of ethanol.
- Thoroughly mix to dissolve the powder.
- Do not use the rehydrated chloramphenicol if it is contaminated, partially or completely evaporated or shows other signs of deterioration. It is recommended that solutions be made fresh each time to avoid potential problems.
Preparation of the Rose Bengal Chloramphenicol Agar:
- To rehydrate the base, suspend 16 g Rose Bengal Agar Base in 500 ml of distilled or deionized water and heat to boiling to dissolve completely.
- Sterilize in the autoclave for 15 minutes at 15 lbs psi (121oC).
- Cool to 43-46oC.
- Aseptically add 2 ml of rehydrated chloramphenicol to the molten agar. Mix thoroughly.
Inoculation and Incubation:
Prepare the sample for pour plate inoculation in accordance with recommended guidelines.12-14 Aseptically add 0.1 or 1 ml of each dilution to a sterile 90 mm Petri dish. Add 10-20 ml of sterile molten agar, mix by gently rocking and tilting the plate. Allow the medium to solidify. Incubate plates aerobically at 20-25oC. Examine the plates for growth after 5 to 7 days incubation.
Colonies of molds and yeasts should be apparent within 5 days of incubation. Colonies of yeasts appear pink due to uptake of rose bengal. Determine the yeast or mold count by counting the number of colonies appearing on those plates containing 30 to 300 colony forming units and multiplying that number by the dilution factor. Results may be reported as yeast or mold count per gram or milliliter of sample, as applicable. Appropriate references may be consulted for further information.12-14
User Quality Control:
- Examine the agar base for color and texture. The powder should be beige to faint pink, free-flowing and homogenous.
- Determine the pH of the base after preparation and cooling to 25oC. The pH should be 7.2 + 0.2.
- Examine the lyophilized and rehydrated chloramphenicol for evidence of deterioration.
- Check the performance of the base and chloramphenicol by testing in the complete medium. Plates should be inoculated with approximately 100 colony forming units of the cultures listed below and incubate aerobically at 20-25oC. Examine plates for growth after 5 days incubation. Results should be as stated below:
|Organism||Growth (after 5 days incubation)||Colonial Morphology (after 5 days)|
|Candida albicans ATCC 10231||good||colonies appear pink, smooth, pasty and raised.|
|Aspergillus niger ATCC 1015||good||colonies appear white and filamentous becoming salt and pepper and eventually black.|
|Escherichia coli ATCC 25922||marked to complete inhibition|
|Micrococcus luteus ATCC 10240||marked to complete inhibition|
Limitations of the Procedure:
- Rose Bengal Agar Base and chloramphenicol are intended for use in the preparation of rose bengal chloramphenicol agar. 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. Consult appropriate references for further information.12-14
- 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.
- Waksman, S.A., "A method for counting the number of fungi in the soil." J. Bact., v. 7: 339-341 (1922).
- Koburger, J.A., "Yeasts and molds," p. 225-229. In M.L. Speck (ed.), Compendium of methods for the microbiological examination of foods. American Public Health Association, Inc. Washington, D.C. (1976).
- Mossel, D.A.A., M. Visser, and W.H.J. Mengerink, "A comparison of media for the enumeration of molds and yeasts in foods and beverages." Lab Practice, v. 11: 109-112 (1962).
- Martin, J.P., "Use of acid, rose bengal and streptomycin in the plate method for estimating soil fungi." Soil Sci., v. 69: 215-232 (1950).
- Koburger, J.A., "Fung in foods. IV. Effect of plating medium pH on counts.", J. Milk Food Technol., v. 35: 659-660 (1972).
- Tyner, L.E., "Effect of media compositions on the numbers of bacterial and fungal colonies developing in Petri plates." Soil Sci., v. 57: 271-274 (1944).
- Smith, N.R., and V.T. Dawson, "The bacteriostatic action of rose bengal in media used for th eplate counts of soil fungi." Soil Sci., v. 58: 467-471 (1944).
- Cooke, W.B., "The use of antibiotics in media for the isolation of fungi from polluted water." Antibiotics and Chemotherapy, v. 4: 657-662 (1954).
- Papavizas, G.C., and C.B. Davey, "Evaluation of various media and antimicrobial agents for isolation of soil fungi." Soil Sci., v. 88: 112-117 (1959).
- Overcast, W.W., and D.J. Weakley, "An aureomycin-rose bengal agar for enumeration of yeast and mold in cottage cheese." J. Milk Technol., v. 32: 442-445 (1969).
- Jarvis, B., "Comparison of an improved rose bengal-chlortetracycline agar with other media for the selective isolation and enumeration of molds and yeasts in foods." J. Appl. Bact., v. 36: 723-727 (1973).
- Speck, M.L. (ed.), Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, D.C. (1976).
- Marth, E.H. (ed.), Standard methods for the examination of dairy products, 14th Ed. American Public Health Association, Washington, D.C. (1978).
- Greenberg, A.E., J.J. Connors, and D. Jenkins (ed.), Standard methods for the examination of water and wastewater, 15th ed., American Public Health Association - American Water Works Association - Water Pollution Control Federation, Washington, D.C. (1981).