ROSE AGAR - Hardy Diagnostics - A66

ROSE AGAR Cat. no. A66 Rose Agar, 15x100mm Plate, 17ml 10 plates/bag Cat. no. J85 Rose / MacConkey Agar, 15x100mm Biplate, 10ml/10ml 10 plates/bag Cat. no. J88 Rose Agar / EMB, 15x100mm Biplate, 10ml/10ml 10 plates/bag INTENDED USE Hardy Diagnostics Rose Agar is recommended for use as a selective growth medium for the cultivation and isolation of gram-positive cocci from clinical and non-clinical specimens which contain mixed flora.

SUMMARY Rose Agar consists of half Columbia CNA Agar and Half Phenylethyl Alcohol Agar Base. The combination of the two basal mediums provides a more selective growth medium for gram-positive cocci than the individual mediums solely provide. Atypical hemolytic reactions may occur with Streptococcus spp., therefore, determination of hemolysis is not recommended. Columbia Blood Agar was first described in 1966 by Ellner, Stoessel, Drakeford, and Vasi who incorporated animal derived peptone, enzymatic digests of casein, and defibrinated sheep blood into one medium.(3) It was found to be an improved form of blood agar, promoting both luxuriant and rapid growth, improved pigment production, typical colony morphology, and sharply defined hemolytic reactions. Ellner, et al. also described the use of nalidixic acid and colistin in Columbia Blood Agar.(3) Columbia CNA Agar was designed to suppress the growth of most gram-negative bacteria, including Klebsiella, Proteus, and Pseudomonas species from mixed flora specimens, thus isolating for gram-positive staphylococci and streptococci.(3) Phenylethyl Alcohol Agar was developed by Brewer and Lilley in 1949 for the selective isolation of gram-positive organisms, particularly gram-positive cocci.(8,9) Phenylethyl alcohol permits the growth of gram-positive organisms while inhibiting most gram-negative organisms, especially swarming Proteus spp. Nitrogen, carbon, sulfur and trace nutrients are made available by the presence of peptones. Osmotic equilibrium is maintained by the addition of sodium chloride. The addition of 5% sheep blood to the basal medium provides many growth factors, however, atypical hemolytic reactions may occur. Therefore, determination of hemolytic reactions should not be made on PEA with 5% sheep blood. The combination of the two formulas provides a rich, stable media with improved performance over the individual formulations. The antimicrobics of Columbia CNA combined with phenylethanol provide inhibition of swarming Proteus and Pseudomonas spp. FORMULA Ingredients per liter of deionized water:* Pancreatic and Enzymatic Digests of Casein 25.0gm Casein Yeast Peptone 10.0gm Sodium Chloride 10.0gm Papaic Digest of Soybean Meal 5.0gm Tryptic Digest of Beef Heart 3.0gm Phenylethanol 2.5gm Corn Starch 1.0gm Colistin Sulfate 8.25mg Nalidixic Acid 4.0mg Sheep Blood 50.0ml Agar 30.0gm Final pH 7.4 +/- 0.3 at 25 degrees C. * Adjusted and/or supplemented as required to meet performance criteria. STORAGE AND SHELF LIFE Storage: Upon receipt store at 2-8 degrees C. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing. The expiration date applies to the product in its intact packaging when stored as directed. This product has the following shelf life from the date of manufacture: 90 Days: A66 Rose Agar J85 Rose / MacConkey Agar, Biplate J88 Rose Agar / EMB, Biplate Refer to the keyword ""Storage"", in the Hardy Diagnostics software program HUGO™, for more information on storing culture media.

PRECAUTIONS This product is for in vitro diagnostic use only and is to be used only by adequately trained and qualified laboratory personnel. Observe approved biohazard precautions and aseptic techniques. All laboratory specimens should be considered infectious and handled according to ""standard precautions"". The ""Guideline for Isolation Precautions"" is available from the Centers of Disease Control and Prevention at www.cdc.gov/ncidod/dhqp/gl_isolation.html. For additional information regarding specific precautions for the prevention of the transmission of all infectious agents from laboratory instruments and materials, and for recommendations for the management of exposure to infectious disease, refer to CLSI document M29. Sterilize all biohazard waste before disposal. Refer to the keyword ""Precautions"", in the Hardy Diagnostics software program HUGO™, for more information regarding general precautions when using culture media. Refer to the keyword ""MSDS"", in the Hardy Diagnostics software program HUGO™, for more information on handling potentially hazardous material.

PROCEDURE Specimen Collection: Consult listed references for information on specimen collection.(1,2,4,6) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Method of Use: Allow the plates to warm to room temperature, and the agar surface to dry before inoculating. Inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface. Streak for isolation with a sterile loop. Incubate plates at 35-37 degrees C. for 24-48 hours in an aerobic atmosphere supplemented with 5-10% CO2. Examine for typical colonial morphology and characteristics.

INTERPRETATION OF RESULTS Consult listed references for the identification of colony morphology and further biochemical tests required for identification.(1,2,4,6) LIMITATIONS It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification. Atypical hemolytic reactions may occur with Streptococcus spp., therefore, determination of hemolysis is not recommended. It is recommended that the organism be subcultured to a Blood Agar Plate (Cat. no. A10) to confirm hemolysis. Some gram-positive organisms may be inhibited by phenylethyl alcohol; additional incubation time may be warranted. Refer to the keyword ""Limitations"", in the Hardy Diagnostics software program HUGO™, for more information regarding general limitations on culture media.

MATERIALS REQUIRED BUT NOT PROVIDED Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided. QUALITY CONTROL The following organisms are routinely used for testing at Hardy Diagnostics: Test Organisms Inoculation Method* Incubation Results Time Temperature Atmosphere Streptococcus pyogenes ATCC® 19615 A 24hr 35°C CO2** Growth; beta-hemolysis may appear atypical Streptococcus pneumoniae ATCC® 6305 A 24hr 35°C CO2** Growth; alpha-hemolysis may appear atypical Staphylococcus aureus ATCC® 25923 A 24hr 35°C CO2** Growth Proteus mirabilis ATCC® 12453 B 24hr 35°C CO2** Partial to complete inhibition Pseudomonas aeruginosa ATCC® 27853 B 24hr 35°C Aerobic Partial to complete inhibition

USER QUALITY CONTROL Check for signs of contamination and deterioration. Users of commercially prepared media may be required to perform quality control testing with at least one known organism to demonstrate growth or a positive reaction; and at least one organism to demonstrate inhibition or a negative reaction (where applicable). Refer to the following keywords, in the Hardy Diagnostics software program HUGO™, for more information on QC: ""Introduction to QC"", ""QC of Finished Product"", and ""The CLSI (NCCLS) Standard and Recommendations for User QC of Media"". Also see listed references for more information.(1,2,4,6,7) * Refer to the keyword ""Inoculation Procedures"", in the Hardy Diagnostics software program HUGO™, for a description of inoculation procedures. ** Atmosphere of incubation is enriched with 5-10% CO2.

PHYSICAL APPEARANCE: Rose Agar should appear opaque, and cherry red in color. Streptococcus pyogenes (ATCC® 19615) colonies growing on Rose Agar (Cat. no. A66). Showing beta-hemolysis. Incubated in CO2 for 24 hours at 35 deg. C. Streptococcus pneumoniae (ATCC® 6305) colonies growing on Rose Agar (Cat. no. A66). Showing alpha-hemolysis. Incubated in CO2 for 24 hours at 35 deg. C. Staphylococcus aureus (ATCC® 25923) colonies growing on Rose Agar (Cat. no. A66). Incubated in CO2 for 24 hours at 35 deg. C. Proteus mirabilis (ATCC® 12453) growth inhibited on Rose Agar (Cat. no. A66). Incubated in CO2 for 24 hours at 35 deg. C. REFERENCES 1. August, M.J., et al. 1990. Cumitech 3A; Quality Control and Quality Assurance Practices in Clinical Microbiology, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C. 2. Forbes, B.A., et al. 1998. Bailey and Scott's Diagnostic Microbiology, 10th ed. C.V. Mosby Company, St. Louis, MO. 3. Ellner, et al. 1966. Am. J. Clin. Path.; 45:502. 4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I & II. American Society for Microbiology, Washington, D.C. 5. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD. 6. Murray, P.R., et al. 1995. Manual of Clinical Microbiology, 6th ed. American Society for Microbiology, Washington, D.C. 7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22-A2, Vol. 16, No. 16. 1996. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA. 8. Brewer, J.H., and B.D. Lilley. Presented before a meeting of the Maryland Association of Medical and Public Health Laboratories, Dec. 2, 1949. 9. Lilley and Brewer. 1953. J. Am. Pharm. Assoc.; 42:6."