This study addresses the limitations of Plate Count Agar (PCA), the standard medium for bacterial plate counts in water and wastewater, noting its failure to support the growth of many indigenous bacteria found in potable water. A new medium, designated R2A, was developed for use in heterotrophic plate count (HPC) analyses and subculture of isolates from treated potable water supplies. R2A contains a rich blend of yeast extract, peptones, Casamino Acids, glucose, soluble starch, pyruvate, and salts (pH 7.2). Parallel comparative tests utilizing spread, membrane filter, and pour plate procedures demonstrated that R2A yielded significantly higher bacterial counts than PCA. Furthermore, the magnitude of the count was inversely proportional to the incubation temperature. While maximal counts were achieved after 14 days of incubation at 20°C, the study recommends R2A spread or membrane filter plates incubated at 28°C for 5 to 7 days for practical monitoring of heterotrophic bacterial populations in water treatment and distribution systems.
A New Medium for the Enumeration and Subculture of Bacteria from Potable Water
Standard Medium Limitation: Plate Count Agar (PCA), the previously recommended standard for bacterial plate counts (35°C, 48-h incubation), does not effectively permit the growth of many bacteria present in treated potable water supplies.
R2A Medium Development: A new medium, designated R2A, was developed specifically for heterotrophic plate count (HPC) analysis and subculture of bacteria isolated from potable water.
R2A Formulation: Key ingredients per liter include 0.5 g each of yeast extract, Difco Proteose Peptone no. 3, Casamino Acids, glucose, and soluble starch, along with salts (K2HPO4, MgSO4), 0.3 g of sodium pyruvate, and 15 g of agar. The pH is adjusted to 7.2.
Comparative Results: Parallel studies using spread, membrane filter, and pour plate procedures consistently showed that R2A medium yielded significantly higher bacterial counts compared to PCA.
Temperature Effects: The study found that the magnitude of the bacterial count was inversely proportional to the incubation temperature.
Optimal Conditions (Highest Count): Maximal bacterial counts were obtained after incubation at 20°C for 14 days. Longer incubation (up to 14 days) also increased the detection of pigmented bacteria.
Recommended Monitoring Procedure: For routine monitoring of heterotrophic populations in water treatment and distribution systems, R2A spread or membrane filter plates incubated at 28°C for 5 to 7 days is recommended.