ISO 9308-3:1998

Overview

ISO 9308-3:1998 specifies a miniaturized Most Probable Number (MPN) method for the detection and enumeration of Escherichia coli (E. coli) in surface water and waste water. The method uses inoculation of serial dilutions into dehydrated liquid culture medium in 96‑well microtitre plates and detects β‑D‑glucuronidase activity using the fluorogenic substrate MUG. Results are reported as an MPN per 100 ml with 95% confidence intervals. The method is not intended for drinking water (or other waters with guideline < 15 counts/100 ml) and is specific to E. coli (not general coliform enumeration).

Key topics and technical requirements

• Principle: Inoculate diluted samples into wells containing dehydrated MUG/EC medium; incubate at 44 °C ± 0.5 °C for 36–72 hours; positive wells show blue fluorescence under UV (366 nm) from MUG hydrolysis.
• Sample types: Applicable to all types of surface and waste waters, particularly those rich in suspended matter; separate procedures for fresh/brackish and seawater diluents.
• Apparatus & consumables: sterile 96‑well non‑fluorescent microtitre plates (100 µl dehydrated medium per well), 8‑channel multipipette (200 µl per well), UV observation chamber (Wood’s lamp 366 nm), incubator, sterile seals, sterile diluent or synthetic sea salt.
• Culture media: MUG/EC medium formulation and preparation, sterilization by filtration (0.2 µm), dehydration into microtitre plates; quality criteria and calibration procedures are included.
• Procedure: guidance on choice of dilutions and numbers of wells, inoculation (use of Petri dishes and multichannel pipettes), incubation, and reading in the dark under UV.
• Data analysis: determination of the characteristic number (pattern of positive wells), calculation of MPN with confidence intervals, and example software routines for statistical analysis (included as informative annexes).
• Standards conformity: references ISO 5667 series for sampling and ISO 8199 for general microbial enumeration practices.

Applications and users

• Environmental and public‑health monitoring of bathing waters, surface streams, industrial effluents, and wastewater treatment plant influent/effluent.
• Particularly useful for samples with high suspended solids where membrane filtration is impractical.
• Primary users: environmental testing laboratories, municipal and industrial wastewater labs, regulatory agencies, water quality consultants, and researchers conducting routine E. coli monitoring using MPN methods.

Related standards

• ISO 9308-1: Membrane filtration method (E. coli and coliforms)
• ISO 9308-2: Liquid enrichment MPN method
• ISO 5667 (sampling guidance) and ISO 8199 (microbiological enumeration guidance)

Keywords: ISO 9308-3:1998, E. coli detection, Most Probable Number (MPN), microtitre plates, MUG/EC medium, water quality testing, surface water monitoring, wastewater microbiology.