R2A Agar principle and cultural response (ideal for water treatment)

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Reasoner’s 2A agar is used for the heterotrophic plate count of treated potable water using longer incubation periods.The heterotrophic plate count (HPC), formerly known as the standard plate count is a procedure for estimating the number of live heterotrophic bacteria in water and measuring changes during water treatment, in distribution systems or in swimming pools. r2a agar plate is a type of culture media or a culture media in microbiology which is recommended by APHA for estimating the heterotrophic plate count by the pour plate, spread plate or membrane filter plate procedure.Injured or stressed  microorganisms during water treatment or water testing methods in microbiology are unable to grow on high nutrient media, since the faster growing organisms outgrow the former .Therefore the use of a low nutrient medium like R-2A media incubated for longer incubation periods allows these stressed organisms to grow well.Many bacteria from natural waters which contain limited nutrients at ambient temperature, grow best on r2a plates with less nutrient levels. They grow better at the temperatures below the routine laboratory incubation temperatures of 35 to 37°C.

Principle of R2a Agar

  • Casein acid hydrolysate, proteose peptone and yeast extract provide nitrogen, vitamins, amino acids, carbon and minerals.
  • Dextrose serves as an energy source.
  • Soluble starch aids in the recovery of injured organisms by absorbing toxic metabolic byproducts while sodium pyruvate increases the recovery of stressed cells.
  • Magnesium sulphate is a source of divalent cations and sulphate.
  • Dipotassium phosphate is used to balance the pH of the medium.
  • The number of colonies on a plate are reported as CFU (Colony Forming Units) per volume of sample.
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Composition of R2a agar

The following are ingredient used in the composition of R2a Agar

IngredientsGrams / Litre
Casein acid hydrolysate0.500
Yeast extract 0.500
Proteose peptone0.500
Starch, soluble0.500
Dipotassium phosphate0.300
Magnesium sulphate 0.024
Sodium pyruvate0.300

How to Prepare R2a agar

  • R2A Agar is a ready to use solid media in glass bottle.
  • The medium is pre-sterilized; hence it does not need media sterilization.
  • Medium in the bottle can be melted either by using a pre-heated water bath or any other method.
  • Slightly loosen the cap before melting.
  • When complete melting of medium is observed dispense the medium as desired and allowed to solidify

Appearance of Medium after preparation

It appears light Amber in colour while in sterile glass bottle it is  slightly opalescent

Uninoculated R2a Agar
Uninoculated R2a Agar
Uninoculated R2a Agar

Uninoculated R2a Agar

Plate reading and Colonies morphology

The following cultural characteristics are obtained after melting the medium and pouring into sterile petri plates. The plates are inoculated with following test organisms and incubation at 35 -37°C for 48-72 hours.

OrganismGrowthInoculum (CFU)
Candida albicansgood-luxuriant50 -100
Enterococcus faecalisgood-luxuriant50 -100
Salmonella Enteritidisgood-luxuriant50 -100
Salmonella Typhigood-luxuriant50 -100
Escherichia coli good-luxuriant50 -100
Pseudomonas spp on R2a
Pseudomonas spp on R2a


  1. Clesceri L. S., Greenberg A. E. and Eaton A. D., (Ed.), 1998, Standard Methods for the Examination of Water and Wastewater, 20th Ed., American Public Health Association, Washington, D.C.
  2. Downes F. P. and Ito K., (Eds.), Compendium of Methods for the Microbiological Examination of Foods, 4th Ed., American Public Health Association, Washington, D.C.
  3. Reasoner D. J. and Geldreich E. E., 1985, Appl. Environ. Microbiol., 49:1.
  4. Collins V. J. and Willoughby J. G., 1962, Arch. Microbiol., 43:294.
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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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