R2A AGAR

Cat. no. G03 R2A Agar, 15x60mm Plate, 11ml 10 plates/bag
Cat. no. G54 R2A Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. P32 R2A Agar, 15x60mm Contact Plate, 15ml 10 plates/bag
Cat. no. Q77 R2A Agar, 20x125mm Tube, 18ml 20 tubes/box
Cat. no. U354 R2A Agar, 16oz. Glass Bottle, 400ml 12 bottles/box

INTENDED USE

Hardy Diagnostics R2A Agar is recommended for enumeration of heterotrophic bacteria in water, especially potable water.

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

SUMMARY

Reasoner and Geldreich of the U.S. Environmental Protection Agency developed R2A Agar for the recovery and isolation of aerobic and facultative anaerobic heterotrophic bacteria from treated potable water.(1,2)

R2A Agar, as compared to other media recommended for the heterotrophic plate count (HPC), contains reduced levels of peptone, yeast extract, and dextrose. The decreased nutrient level, along with the addition of sodium pyruvate, enhances the recovery of many stressed and chlorine-tolerant bacteria present in treated waters.(2) Also, the heterotrophic bacteria recovery method using R2A Agar requires incubation temperatures below routine laboratory requirements, which further enhances the recovery of many stressed bacteria.(4,5)

FORMULA

Ingredients per liter of deionized water:*

Casein Acid Hydrolysate 0.5gm
Yeast Extract 0.5gm
Dextrose 0.5gm
Soluble Starch 0.5gm
Dipotassium Phosphate 0.3gm
Sodium Pyruvate 0.3gm
Casein Peptone 0.25gm
Peptic Digest of Animal Tissue 0.25gm
Magnesium Sulfate 0.05gm
Agar 15.0gm

Final pH 7.2 +/- 0.2 at 25°C.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt; store plates at 2-8°C., and store tubes and bottled media at 2-30°C. Products should not be used if there are any signs of contamination, deterioration (shrinking, cracking, or discoloration), or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Before Use of Plates:
It is possible that variation in temperature and pressure during shipping and storage may cause condensation on the innermost bag surrounding the product. If condensation of the packaging or plates is observed, remove the plates from the innermost packaging in a sterile environment and allow them to dry for 10-15 minutes before use.

Specimen Collection: Consult listed references for information on the collection of water samples.(3)

Method of Use: Allow medium to warm to room temperature prior to inoculation. Refer to listed references for standard methods employed in pour plate, spread plate and membrane filter procedures.(3)

For melting bottled media: Autoclave at 121°C. for 1-3 minutes or until melted. Alternatively, a covered, boiling waterbath (100°C.) can be used. There should be enough water in the waterbath to reach the media line. A covered waterbath will help to reach and maintain the temperature. Heat in waterbath until melted. Cool media to 45-50°C. and dispense as desired.

Membrane Filter Method:

For more detailed instructions on sampling and membrane filtration procedures, consult Standard Methods for the Examination of Water and Wastewater.(3)

1. Collect and prepare water samples in accordance with recommended guidelines.

2. After the sample has been filtered, aseptically remove the membrane filter from the filter base and roll it onto R2A Agar. Avoid the formation of bubbles between the membrane and the agar surface.

3. Invert the inoculated plate and incubate.

4. The recommended incubation for R2A Agar is 35°C. for at least 72 hours and up to 7 days. The medium can also be incubated at 20 or 28°C. for not less than 5 days, preferably up to 7 days.

5. After the incubation period, using an illuminated lens with 2-5x magnification, count and record the number of colonies.

Spread Plate Method:

1. Prepare decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

2. Aseptically inoculate agar surface with 0.1ml of well mixed diluted sample.

3. Using a sterile spreader device, distribute the inoculum evenly over the entire agar surface.

4. The recommended incubation for R2A Agar is 35°C. for at least 72 hours and up to 7 days. The medium can also be incubated at 20 or 28°C. for not less than 5 days, preferably up to 7 days.

Pour Plate Method:

1. After autoclaving, cool media to 45-50°C. Maintain in a 45-50° waterbath until ready to pour.

2. Prepare decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

3. Place a 1ml inoculum into a sterile petri plate.

4. Aseptically pour approximately 18ml of the cooled media (45-50°C.) over the inoculum. Carefully swirl the plate to mix the inoculum evenly.

5. Allow plate(s) from step 4 to solidify.

6. The recommended incubation for R2A Agar is 35°C. for at least 72 hours and up to 7 days. The medium can also be incubated at 20 or 28°C. for not less than 5 days, preferably up to 7 days.

INTERPRETATION OF RESULTS

The number of colonies present on the agar medium are reported as colony-forming units per volume of sample. Also reported are: the test method used, incubation temperature and time, and R2A Agar (the test medium used).

The heterotrophic plate count is computed by dividing the total number of colonies or average number, in the case of duplicate plates, by the sample volume.

Consult listed references for standard methods in computing and reporting counts.(3)

LIMITATIONS

The pour plate method is not highly recommended because the recovery of injured bacteria may be lessened by the heat of the media at 45°C. For small sample volumes, the spread plate technique is most effective. When larger amounts of water need to be tested, the membrane filter method is suggested.

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
Escherichia coli
ATCC® 8739
J 1-3 days 35°C Aerobic Growth
Enterococcus faecalis
ATCC® 29212
J 1-3 days 35°C Aerobic Growth
Staphylococcus aureus
ATCC® 6538
J 1-3 days 35°C Aerobic Growth
Aspergillus brasiliensis
formerly A. niger
ATCC® 16404
J 1-5 days 20-25°C Aerobic Growth
Pseudomonas aeruginosa
ATCC® 27853
J 1-5 days 20-25°C Aerobic Growth
Bacillus subtilis
ATCC® 6633
J 1-3 days 35°C Aerobic Growth

* Consult appropriate regulatory agency for user QC requirements.

User Quality Control

PHYSICAL APPEARANCE

R2A Agar should appear clear, slightly opalescent, and light white in color.

E. coli growing on R2A Agar

Escherichia coli (ATCC® 8739) filtered through a black membrane (Cat. no. A045R047A) and growing on R2A Agar (Cat. no. G03). Incubated aerobically for 24 hours at 35oC.

R2A Agar

Uninoculated plate of R2A Agar (Cat. no. G03).

REFERENCES

1. Reasoner, D.J., and Geldreich, E.E. 1979. Paper No. N7, Annual Meeting of The American Society for Microbiology.

2. Reasoner, D.J., and Geldreich, E.E. 1985. Applied and Environmental Microbiology; 49:1-7.

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

4. Stark and McCoy. 1938. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. 2; 98:201.

5. Collins and Willoughby. 1962. Arch. Mikrobiol.; 43:294.

ATCC is a registered trademark of the American Type Culture Collection.

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