ISO 9308-2:2012

Overview

ISO 9308-2:2012 - "Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most probable number method" specifies a standardized Most Probable Number (MPN) liquid-culture method to enumerate E. coli and coliform bacteria in water. The method uses a defined substrate medium (Colilert‑18) and an enumeration device (Quanti‑Tray or Quanti‑Tray/2000) to detect coliforms by color (β‑D‑galactosidase reaction) and E. coli by fluorescence (β‑D‑glucuronidase reaction). Results are reported as MPN per 100 ml using MPN tables or software.

Key technical topics and requirements

• Principle: Growth of target organisms in liquid medium, color and fluorescence differentiation, calculation of MPN/100 ml.
• Medium & reagents: Colilert‑18 snap packs (ready‑to‑use powder) for 100 ml samples; antifoam B optional; sterile, oxidant‑free diluent for marine sample dilutions.
• Apparatus: Quanti‑Tray / Quanti‑Tray 2000, Quanti‑Tray sealer, incubator at (36 ± 2) °C, UV lamp (365 nm), sterile vessels.
• Sample handling: Transport and store samples at (5 ± 3) °C; analyse on day of collection or within 18 h (up to 24 h in exceptional cases), follow ISO 19458 for sampling.
• Procedure: Dissolve Colilert‑18 in 100 ml sample (or dilution), pour into Quanti‑Tray, seal and incubate 18–22 h. Yellow wells = coliforms; yellow + fluorescence = E. coli.
• Limitations:
  • Not for enumeration of coliform bacteria in marine water.
  • For E. coli in marine water, a 1→10 dilution in sterile water is typically required unless validated otherwise.
  • The method detects E. coli via β‑D‑glucuronidase expression and therefore may not detect many enterohaemorrhagic E. coli and a small number of strains lacking the enzyme.
• Results & reporting: Compute MPN using MPN tables or software; include method reference, sample ID, results (MPN/100 ml), and any deviations in the test report.
• Quality assurance: Use positive/negative controls and blanks; maintain a defined QC system.

Practical applications and users

ISO 9308-2:2012 is widely used for:

• Routine water quality monitoring (drinking, recreational, and surface waters)
• Wastewater and effluent testing
• Environmental surveys and source water assessments
• Treatment plant performance verification and distribution system checks

Primary users:

• Accredited water testing laboratories
• Public health and environmental protection agencies
• Water utilities and wastewater operators
• Environmental consultants and researchers

Related standards

• ISO 9308-1: membrane filtration method for waters with low bacterial background flora
• ISO 9308-3: miniaturized MPN method for surface and waste water
• ISO 8199: general guide to enumeration by culture
• ISO 19458: sampling for microbiological analysis

Note: Colilert and Quanti‑Tray are commercial trademarks and may involve patent considerations; laboratories should be aware of intellectual property and validation requirements when implementing the method.