Overview
EN ISO 16266:2008 (ISO 16266:2006) specifies a standardized membrane filtration method for the detection and enumeration of Pseudomonas aeruginosa in bottled water and other waters with low background flora (for example, pool waters and waters intended for human consumption). The standard describes sampling volumes, culture media, incubation, colony enumeration and confirmation tests to reliably identify P. aeruginosa for water quality and public-health monitoring.
Key Topics and Requirements
- Principle: Filter a measured volume (or dilution) through a 0.45 µm membrane filter, place the filter onto selective medium, incubate and count characteristic colonies.
- Target organism definition: Colonies producing pyocyanin, fluorescing under UV (360 ± 20 nm), oxidase-positive and able to produce ammonia from acetamide are considered P. aeruginosa.
- Selective medium: Pseudomonas agar base (CN-agar) with CN supplement (cetrimide 0.2 g/L and nalidixic acid 0.015 g/L) is specified; detailed preparation, pH and storage conditions are given.
- Confirmation steps: Subculture suspicious colonies onto nutrient agar or King’s B; perform oxidase test, fluorescence checks and acetamide utilization (acetamide broth) to confirm identity.
- Laboratory practice: Media preparation (autoclave at 121 ± 3 °C for 15 min), use of analytical-grade reagents, incubation conditions, and plate storage limits are specified.
- Sampling guidance: Typical regulatory expectations cited - bottled and spring waters should be free of P. aeruginosa in any 250 ml sample; other waters often tested in 100 ml volumes.
- Quality assurance & safety: Tests must be carried out by trained personnel; precautions for handling hazardous reagents (e.g., acetamide) and following safety and QA procedures are emphasized.
Applications and Who Uses It
- Water testing laboratories performing routine microbiological analysis for bottled water, municipal supply or recreational waters.
- Bottled water and spring water producers confirming compliance with regulatory limits (absence in 250 ml).
- Public health and regulatory authorities evaluating water safety and enforcing standards.
- Pool operators and facility managers monitoring recreational waters with low background flora.
- Manufacturers of water treatment and packaging equipment needing validated microbiological methods for product verification.
Related Standards
Keywords: EN ISO 16266:2008, ISO 16266, Pseudomonas aeruginosa detection, membrane filtration method, water quality testing, bottled water microbiology.
