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 25ºC.

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

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), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

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 35-37ºC 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

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.

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

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

** Atmosphere of incubation is enriched with 5-10% CO2.

USER QUALITY CONTROL

Physical Appearance:

Rose Agar should appear opaque, and cherry red in color.

S. pyogenes growing on Rose Agar

Streptococcus pyogenes (ATCC® 19615) colonies growing on Rose Agar (Cat. no. A66). Showing beta-hemolysis. Incubated in CO2 for 24 hours at 35ºC.

S. pneumoniae growing on Rose Agar

Streptococcus pneumoniae (ATCC® 6305) colonies growing on Rose Agar (Cat. no. A66). Showing alpha-hemolysis. Incubated in CO2 for 24 hours at 35ºC.

S. aureus growing on Rose Agar

Staphylococcus aureus (ATCC® 25923) colonies growing on Rose Agar (Cat. no. A66). Incubated in CO2 for 24 hours at 35ºC.

P. mirabilis inhibited on Rose Agar

Proteus mirabilis (ATCC® 12453) growth inhibited on Rose Agar (Cat. no. A66). Incubated in CO2 for 24 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Ellner, et al. Am. J. Clin. Path.; 45:502.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. MacFaddin, J.F. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

6. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, 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.


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041116gr