R2A Medium

LAB 163

Description

R2A medium was developed to determine the bacterial count in potable waters during treatment and distribution, and has been shown to give significantly higher counts than plate count agar (PCA) or similar high-nutrient media. The standard plate count (SPC) method using PCA provides an enumeration of bacteria which grow best at, or near, body temperature and this estimation at best may correlate to the coliforms present in the sample. However, there will be a population of heterotrophic bacteria which cannot grow at all under the conditions of the SPC method or may grow so slowly that the colonies fail to reach a size detectable to the eye in the 48-h incubation period. In order to enumerate this section of the bacterial population in water, a medium of low nutritional content (R2A) and extended incubation times are required. R2A medium is recommended by the Environment Agency, Methods for the Examination of Waters and Associated Materials, and Standard Methods for the Enumeration of Water and Wastewater.

pH: 7.2 + 0.2

Appearance: Clear opalescent gel

Method for reconstitution

Weigh 18 grams of powder and disperse in 1 litre of deionised water. Swirl to mix and sterilise at 121˚C for 15 minutes. (If required, bring to the boil to dissolve the agar, and pour into smaller volumes before sterilising). Cool to 44-46˚C for not more than 3 hours before use.

Inoculation: Pour 15ml into a Petri dish containing 1ml of sample, mix well and allow to set. Pour a further 10ml as an overlay and again allow to set. Alternatively it may be used as a spread plate, inoculating 0.1ml onto the plate and spreading over the entire surface of the medium. It can also be used with membrane filters if required.

Incubation: When plates have set, incubate at 22˚C for 5-7 days or 30˚C for 3 days. Other incubation temperatures between 20˚C and 28˚C may be used.

Interpretation: Count all colonies and report the number of bacteria in the original sample as the heterotrophic plate count.

Minimum QC organisms:   Pseudomonas fluorescens, Aeromonas hydrophila

References

Reasoner, D.J., Geldreich, E.E. (1985) A New Medium for the Enumeration and Subculture of Bacteria from potable water. App & Env. Microbiol. Jan. 1985 p. 1-7.

American Public Health Association (1985) Standard Methods for the Enumeration of Water and Wastewater. 16th Edition. American Public Health Association Inc. Washington DC.

Environment Agency: The Microbiology of Drinking Water (2002). Methods for the Examination of Water and Associated Materials.