Performance of Reasoner’s 2 Agar (R2A) medium in enumeration of culturable microorganisms in water intended for human consumption

Abstract Enumeration of the heterotrophic plate counts of culturable microorganisms (HPC) is used for the assessment of water quality before, during and after the drinking water treatment processes and monitoring of integrity of the drinking water distribution systems. Any substantial change in HPC is a warning of potential contamination, treatment failure, or intrusion. This study produced data for international standardization of Reasoner’s 2 Agar (R2A) method. Interlaboratory studies were applied to find out which inoculation technique, spread plate or pour plate, is more applicable, and what is the acceptable transport and storage time between the sampling and initiation of the analysis. Based on the produced interlaboratory data, spread plate inoculation followed by incubation for 7 days at 22 °C was selected as the standard method, and it is recommended to analyse samples as soon as possible, but the samples may be kept at (5 ± 3) °C for up to 24 hours after the sampling prior to examination. Performance characteristics of the method were determined in a single laboratory according to ISO 13843:2017 by using process water from waterworks, bottled water, chlorinated and non-chlorinated tap water, and well water intended for human consumption. For quality assurance of the R2A medium, the use of control strains of Bacillus subtilis subsp. spizizenii , Pseudomonas fluorescens and an additional strain of Sphingomonas paucimobilis was verified during the work. The R2A method is especially suitable for determination of micro-organisms forming colonies after a prolonged incubation time, enabling HPC enumeration from biostable waters with low nutrient levels and low temperature. Such waters usually produce zero counts when nutrient-rich formulations of culture media with short incubation times are employed. Further, the nutrient limited conditions of R2A minimize the colony size and overgrowth in all kinds of water providing improvement to the HPC determinations.