ROSE BENGAL AGAR WITH CHLORAMPHENICOL

Cat. no. W87 Rose Bengal Agar with Chloramphenicol,
15x100mm Plate, 24ml
10 plates/bag
Cat. no. P42 Rose Bengal Agar with Chloramphenicol,
15x60mm Contact Plate, 12ml
10 plates/bag
Cat. no. U308 Rose Bengal Agar with Chloramphenicol,
16oz. Glass Bottle, 400ml
12 bottles/box
Cat. no. Q81 Rose Bengal Agar with 1.5X Chloramphenicol,
20x150mm Tube, 20ml Deep
100 tubes/box

INTENDED USE

Hardy Diagnostics Rose Bengal Agar with Chloramphenicol is recommended for the selective isolation and enumeration of fungi from environmental and food sources.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Fungi are recovered from, air, soil, lakes, ponds, rivers, streams, wastewaters, and well waters. (5) Due to their heterotrophic nature, and their ability to adapt to a wide range of environmental conditions, fungi are also frequently encountered as contaminants in various commodities including foods, inadequately cleaned food processing equipment, and food storage facilities. Since yeasts and molds can initiate growth over a wide pH range and temperature ranges, growth can occur on almost any type of food including processed foods and food ingredients. (3,6)

Traditionally, low pH media have been used to enumerate yeasts and molds from water, soil, and food. Such media are now believed to be inferior to antibiotic supplemented media. The use of antibiotics, rather than acid, for suppressing bacteria results in improved recovery of injured (acid-sensitive) fungal cells, better control of bacteria, and less interference during counting from precipitated food particles. (4) Hardy Diagnostics Rose Bengal Agar with Chloramphenicol contains the antibiotic chloramphenicol, which is added as a selective agent to inhibit most bacterial growth. (3)

In addition to chloramphenicol, rose bengal is added to the media, to increase the selectivity and help control overgrowth by rapidly growing molds such as Neurospora and Rhizopus species. Besides providing better isolation of slow growing fungi, rose bengal dye is also taken up my fungal isolates, thereby aiding in their recognition. Smith and Dawson found that rose bengal added to a near-neutral medium (pH of 6.8), allowed for more colonies to develop than did an acidified medium (pH of 4.2). (8) Hardy Diagnostics Rose Bengal Agar with Chloramphenicol also contains soy peptone as a source of carbon and nitrogen, dextrose as an energy source, and magnesium sulfate to provide trace elements. (2,8)

FORMULA

Ingredients per liter of deionized water:*

Dextrose 15.0gm
Papaic Digest of Soybean Meal 5.0gm
Monopotassium Phosphate 1.0gm
Magnesium Sulfate 0.5gm
Chloramphenicol 0.1gm
Rose Bengal 0.05gm
Agar 15.0gm

Final pH 7.2 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

Note: Rose Bengal Agar with 1.5X Chloramphenicol (Cat. no. Q81) contains 0.15g Chloramphenicol.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

For melting agar deeps: Liquefy the medium by autoclaving at 121°C for 1-3 minutes Cool the medium to 45-50°C and pour into sterile petri dishes. Allow the agar to solidify for at least 30 minutes prior to use. Alternatively, a covered, boiling waterbath (100°C) can be used. There should be enough water in the waterbath to reach the top of the media line. Heat in a waterbath until melted through. A covered waterbath will help to reach and maintain the media temperature prior to dispensing.

Note: After autoclaving, do not heat media using a hot plate, heat block or waterbath for longer than 3 hours at 45-50°C. Melt only enough media that can be poured within a 3 hour time period. For optimal performance, sterile solidified medium should be remelted only once prior to use.

Yeasts and molds should be enumerated by a surface spread-plate technique rather than using pour plate methods. This technique provides maximal exposure of the cells to atmospheric oxygen, avoids heat stress from molten agar, gives more uniform growth and makes colony isolation easier. Agar spread plates should be dried overnight before being inoculated. (3,4,6) Pour plating should only be used when yeast or non-stressed mold cells are being detected. (4)

1. Inoculate 0.1ml of sample, or appropriate dilution, in duplicate onto the agar surface.

2. Spread the inoculum over the entire surface using a sterile bent glass rod, or disposable spreaders. Do not invert the plates.

3. Inoculated plates should be incubated undisturbed in an upright position at 22 to 30ºC for 7 days before colonies are counted. Room temperature incubation can be used if an incubator is not available. (3-5)

Refer to the document " Contact Plate Media " on the Hardy Diagnostics Technical Document website for more information regarding the method of use for Rose Bengal Agar with Chloramphenicol Contact Plate (Cat. no. P42).

INTERPRETATION OF RESULTS

Interpretation of viable yeast and mold counts is often difficult as background data on expected and excessive levels for many foods have not been established. Determining the predominant species is also important. (3) Microscopic examination is recommended for presumptive identification. Biochemical testing using pure cultures is necessary for complete identification.

Plates with 15 to 150 fungal colonies are usually counted. If the mycoflora consists primarily of molds, the lower population range is selected; if primarily yeast colonies, the upper limit is counted. Report counts as colony forming units (CFU) per gram or ml of sample. (3-5) If counting must be delayed temporarily, plates can be held at 4°C for no longer than 24 hours. (5)

LIMITATIONS

Although this medium is selective for fungi, microscopic examination is recommended for presumptive identification.

It is important to not expose this medium to light since photodegradation of rose bengal produces compounds that are toxic to fungi. (3)

Chloramphenicol may not be sufficient to inhibit all bacterial flora. (2)

As fungal colonies take up the rose bengal dye, it may be necessary to subculture onto a secondary medium prior to inoculation onto Rose Bengal Agar. (2)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms* Inoculation Method** Incubation Results
Time Temperature Atmosphere
Candida albicans
ATCC ® 10231
A 48-96hr 15-30°C Aerobic Growth; pink smooth raised colonies
Aspergillus brasiliensis ATCC ® 16404 A 3-5 days 15-30°C Aerobic Growth; white and filamentous, black specks on colonies
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic Inhibited
In addition to the above organisms, Cat. no. Q81 is tested with the following organism:
Staphyloccus aureus
ATCC ® 6538
B 24hr 35°C Aerobic Inhibited

User Quality Control

PHYSICAL APPEARANCE

Rose Bengal Agar with Chloramphenicol should appear slightly opalescent, and bright pink in color.

C. albicans growing on Rose Bengal Agar with Chloramphenicol

Candida albicans (ATCC ® 10231) colonies growing on Rose Bengal Agar with Chloramphenicol (Cat. no. W87). Incubated aerobically for 48 hours at 30ºC.

A. niger growing on Rose Bengal Agar with Chloramphenicol

Aspergillus brasiliensis (ATCC ® 16404) colonies growing on Rose Bengal Agar with Chloramphenicol (Cat. no. W87). Incubated aerobically for 72 hours at 30ºC.



Rose Bengal Agar with Chloramphenicol

Uninoculated plate of Rose Bengal Agar with Chloramphenicol (Cat. no. W87).



REFERENCES

1. Atlas, R.M. 1997. Handbook of Microbiological Media, 2nd ed. CRC Press, Inc., Boca Raton, FL.

2. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

3. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

4. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

5. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

6. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.

7. Waksman, S.A. 1922. A method for counting the number of fungi in the soil. J. Bacteriol.; 7:339-341.

8. Smith, N.R., V.T. Dawson. 1944. The bacteriostatic action of Rose Bengal in media used for plate counts of soil fungi. Soil Sci.; 58: 467-471.

9. Cooke, W.B. 1954. The use of antibiotics in media for the isolation of fungi from polluted water. Antibiotics and Chemotherapy; 4:657-662.

10. Papavizas, G.C., C.B. Davey. 1959. Evaluation of various media and antimicrobial agents for isolation of soil fungi. Soil Sci.; 88:112-117.

11. Jarvis, B. 1973. Comparison of an improved Rose Bengal-Chlortetracycline Agar with other media for the selective isolation and enumeration of moulds and yeasts in foods. J. Appl. Bact.; 36:723-727.


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041116gr