Ready-to-use media
R2A Agar EP/USP
Solid medium for the enumeration of heterotrophic microorganisms in treated waters according to the Pharmacopoeial Method.
- Cat. No.
- LC-4711
- Category
- Ready-to-use media
- Format
- 30 water plates, 55 mm
- ISO 17025 quality control
- Yes
- Storage
- 2 to 25 °C
- Minimum guaranteed shelf life
- 2 months
- Standards
- European PharmacopoeiaUSP
- Also available
- Dehydrated medium, 1 format (LC-1071)
R2A Agar was proposed in 1979 by Reasoner and Geldreich and a few years later accepted by the APHA as an alternative medium for the enumeration of stressed cells in treated potable water. The culture medium has also been adopted by the European Pharmacopoeia for the control of purified water.
Key features
Applications
Non-selective enumeration of aerobes.
Industry
Environmental monitoring, pharmaceutical/veterinary, water.
Standards
European Pharmacopoeia, USP.
Presentation
30 prepared plates, 55 mm, for filtration purposes, with 9 ± 1 mL.
Description and technique
Description
The use of nutrient-rich media like PCA or TSA allows the growth of most microbes, but does not permit the recuperation of stressed or chlorine-resistant organisms. Using a medium like R2A with low nutrients in combination with a lower temperature and longer incubation time, it is possible to induce the resuscitation of these damaged cells.
In R2A Agar the source of nitrogen is the peptone and yeast extract supplies the vitamins and growth factors. The source of carbon is dextrose, and magnesium sulfate and potassium phosphate maintain the osmotic pressure. The starch is a detoxifier and sodium pyruvate increases the recuperation of stressed cells. The agar acts as a gelling agent.
Technique
The water sample must be processed as quickly as possible. If it is not possible to process within the first 6 hours, the sample must be refrigerated, but not for more than 30 hours.
Collect, dilute and prepare samples and volumes to be filtered as required according to specifications, directives, official standard regulations and/or expected results.
Filter the sample through a 0.45 µm pore membrane and apply it onto the surface of the agar.
When incubating at 35 °C, an incubation period of 3-5 days is recommended. In most circumstances an incubation temperature of 20-25 °C for 5-7 days is more effective. Plates must be protected against dehydration.
After incubation, enumerate all the colonies that have appeared onto the surface of the membrane.
Calculate total microbial count per mL of sample by multiplying the average number of colonies per plate by the inverse dilution factor. Report results as colony forming units (CFU) per mL along with incubation time and temperature.
Presentation
| Presentation | 30 prepared plates, 55 mm, for filtration purposes |
|---|---|
| Filling | 9 ± 1 mL |
| Packaging | 1 box containing 5 plastic bags with 6 plates of 55 mm/bag. |
Composition
| Ingredient | g/L |
|---|---|
| Proteose peptone | 0.500 |
| Casein hydrolysate (tryptone) | 0.500 |
| Yeast extract | 0.500 |
| D(+)-Glucose | 0.500 |
| Starch | 0.500 |
| Sodium pyruvate | 0.300 |
| Dipotassium hydrogen phosphate | 0.300 |
| Magnesium sulfate (anhydrous) | 0.024 |
| Agar | 15.000 |
Quality control
Physical/chemical control
| Color | Pale yellow |
|---|---|
| pH | 7.2 ± 0.2 at 25 °C |
Microbiological control
Growth Promotion Test according to harmonized pharmacopoeial monographs and test methods and ISO 11133:2014.
Membrane filtration / practical range 100 ± 20 CFU; min. 50 CFU (productivity) / 10⁴-10⁶ CFU for selectivity.
Aerobic. Incubation at 30-35 °C for 24-48 h (bacteria) and 20-25 °C for 3-5 days (moulds and yeast).
| Microorganism | Growth |
|---|---|
| Ps. aeruginosaATCC 9027, WDCM 00026 | Good (≥ 70%) |
| Bacillus subtilisATCC 6633, WDCM 00003 | Good (≥ 70%) |
| Escherichia coliATCC 8739, WDCM 00012 | Good (≥ 70%) |
| Aspergillus brasiliensisATCC 16404, WDCM 00053 | Good (≥ 70%) |
| Candida albicansATCC 10231, WDCM 00054 | Good (≥ 70%) |
| Staphylococcus aureusATCC 6538, WDCM 00032 | Good (≥ 70%) |
Sterility control
Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.
Check at 7 days after incubation in same conditions.
Bibliography
- ATLAS, R.M. (1995) Handbook of Media for Environmental Microbiology. CRC Press. Boca Raton. Fla. USA.
- CLESCERI, L.S., A.E. GREENBERG and A.D. EATON (1998) Standard Methods for the Examination of Water and Wastewater. 20th ed. APHA Washington D.C. USA.
- EATON, A.D., A.E. GREENBERG and L.S. CLESCERI (1995). Standard Methods for the Examination of Water and Wastewater. 19th ed. APHA Washington D.C. USA.
- EUROPEAN PHARMACOPOEIA. 6th ed. Suppl 6.3 (2009) General Monographs. Water for injections. (pg. 4339) EDQM. Council of Europe. Strasbourg.
- GREENBERG, A.E., R.R. TRUSSELL and L.S. CLESCERI (1985). Standard Methods for the Examination of Water and Wastewater. 16th ed. APHA-AWWA-WPCF. Washington D.C. USA.
- REASONER, D.J. and E.E. GELDREICH (1979) A new Medium for the enumeration and subculture of bacteria from potable water. Abstracts of Annual Meeting. ASM 79th Meeting. Paper #N7.
- Van SOETSBERGER, A.A. and C.H. LEE (1969) Pour plates or streak plates? Appl. Microbiol. 18:1092-1094.
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