Reasoner’s 2A Agar (R2A): Composition, Principle, Preparation, and Uses in Microbiology

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Reasoner’s 2A Agar (R2A): Composition, Principle, Preparation, and Uses in Microbiology

Reasoner’s 2A Agar (R2A) is a low nutrient medium designed specifically for the enumeration and cultivation of heterotrophic bacteria, especially those found in treated potable water, bottled water, and purified water systems. It supports the growth of slow-growing or stressed bacteria that may not develop on nutrient-rich media like Plate Count Agar (PCA).

🔬 Introduction

Reasoner’s 2A Agar (R2A) was developed by Reasoner and Geldreich to promote the recovery of heterotrophic microorganisms from water with low organic content. The low nutrient concentration encourages the growth of bacteria that adapt to oligotrophic (nutrient-poor) environments, ensuring more accurate microbial enumeration in water systems.

This medium is recommended by major pharmacopeias such as USP, EP, and JP for testing Purified Water (PW), Water for Injection (WFI), and potable water.

🧫 Principle of Reasoner’s 2A Agar (R2A)

R2A Agar provides a balanced and diluted nutrient environment, allowing recovery of heterotrophic bacteria that are unable to grow rapidly under nutrient-rich conditions. It uses a combination of peptones, yeast extract, and dextrose to supply essential growth factors and carbon sources, while sodium pyruvate, magnesium sulfate, and dipotassium phosphate enhance bacterial metabolism and buffering.

⚗️ Composition of Reasoner’s 2A Agar (R2A Medium)

Ingredients Concentration (g/L)
Yeast Extract0.5 g
Proteose Peptone0.5 g
Casamino Acids0.5 g
Glucose (Dextrose)0.5 g
Sodium Pyruvate0.3 g
Dipotassium Phosphate0.3 g
Magnesium Sulfate0.05 g
Agar15.0 g
Final pH (at 25°C)7.2 ± 0.2

⚙️ Preparation Procedure of R2A Agar

  1. Weigh 17.65 g of R2A Agar powder (commercial dehydrated medium) and suspend it in 1 liter of distilled water.
  2. Heat gently while stirring until the medium is completely dissolved.
  3. Adjust the pH to 7.2 ± 0.2 if necessary.
  4. Dispense into suitable containers or flasks and sterilize by autoclaving at 121°C for 15 minutes.
  5. Allow the medium to cool to about 45–50°C and pour into sterile Petri dishes under aseptic conditions.
  6. Once solidified, store the plates at 2–8°C in a refrigerator. Avoid dehydration or condensation.

🧫 Appearance and Quality Control

  • Dehydrated medium: Light beige, free-flowing powder.
  • Prepared medium: Clear to slightly opalescent, light amber-colored agar.

Quality Control Test Organisms:

Test Organism Expected Growth
Escherichia coli ATCC 25922Good growth
Pseudomonas aeruginosa ATCC 27853Good growth
Micrococcus luteus ATCC 10240Good growth
Flavobacterium sp.Good growth
Environmental isolatesGood recovery of stressed cells

💡 Applications of Reasoner’s 2A Agar (R2A)

  • Enumeration of heterotrophic bacteria in treated or bottled water.
  • Microbial monitoring of pharmaceutical purified water (PW) and WFI systems.
  • Isolation of slow-growing, oligotrophic bacteria from environmental samples.
  • Used for membrane filtration tests of water and cleanroom samples.
  • Applicable for bioburden testing in low-nutrient environments.

🧠 Advantages of Using R2A Agar

  • Promotes growth of stressed or chlorine-tolerant bacteria.
  • Prevents overgrowth of fast-growing species, improving colony differentiation.
  • Recommended by Standard Methods for the Examination of Water and Wastewater.
  • Supports recovery of a wide range of heterotrophic microorganisms.

📋 Storage and Shelf Life

  • Store dehydrated medium at 10–30°C in a tightly closed container.
  • Prepared plates should be stored at 2–8°C and used within 1–2 weeks.
  • Avoid freezing and exposure to direct sunlight or moisture.

📖 References

  1. Reasoner, D.J., and Geldreich, E.E. (1985). A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology, 49(1): 1–7.
  2. United States Pharmacopeia (USP) – Microbial Enumeration Tests.
  3. Standard Methods for the Examination of Water and Wastewater, APHA.
  4. European Pharmacopoeia (EP) – Water for Pharmaceutical Use.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
Mobile: 09505626106

📱 Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.

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