Overview
ISO 14189:2013 specifies a standardized, culture-based membrane filtration method for the enumeration of vegetative cells and spores of Clostridium perfringens in water - primarily for water intended for human consumption but applicable to other water types that do not interfere with filtration. The standard defines the principle, apparatus, media, sample handling, incubation conditions and confirmatory testing required to produce reliable colony counts expressed per unit volume.
Key Topics and Requirements
- Scope: Enumeration of C. perfringens (vegetative cells and spores) by membrane filtration; applicable where particulate/colloidal matter does not prevent filtration.
- Principle: Filter a known volume through a 0.45 µm membrane, incubate on tryptose‑sulfite‑cycloserine (TSC) agar anaerobically and count characteristic colonies; heat pre‑treatment isolates spores when required.
- Sampling & transport: Follow ISO 19458. Cool samples (ideally 5 ± 3 °C). Recommended maximum storage including transport: 12 h for vegetative bacteria and 24 h for spores; absolute maxima: 18 h (vegetative) and 72 h (spores).
- Heat pre‑treatment: To enumerate spores only, heat sample to 60 ± 2 °C for 15 ± 1 min (monitor temperature using a reference bottle).
- Filtration & plating: Filter appropriate volumes (100 ml usual for drinking water). Aim for 10–80 colonies on 47–50 mm membranes. Optionally apply a molten TSC overlay to enhance colony blackening.
- Incubation: Anaerobic atmosphere (~90% H2 / 10% CO2) at 44 ± 1 °C for 21 ± 3 h. Count presumptive colonies (black/grey to yellow‑brown) and perform confirmations.
- Confirmation: Subculture selected colonies onto blood or nutrient agar and perform the acid phosphatase test (purplish colour within ~3–4 min = positive).
- Quality factors: Sterile funnels/filters, validated membrane quality, timely incubation and counting (plates should be read within 30 minutes after incubation as blackening fades).
Applications
- Routine monitoring of drinking water quality and water resources to detect faecal contamination.
- Assessment and verification of water treatment performance and disinfection efficacy.
- Environmental surveillance where spores indicate remote or intermittent faecal pollution (spores persist longer than vegetative indicators).
- Support for regulatory compliance, public health investigations and research into microbial water quality.
Who Uses This Standard
- Water testing laboratories and accredited microbiological labs
- Water utilities and treatment plant operators
- Public health agencies and environmental regulators
- Consultants and researchers involved in water quality monitoring
Related Standards
- ISO 8199 - General guidance on enumeration of micro‑organisms by culture
- ISO 19458 - Water sampling for microbiological analysis
- ISO/TS 11133-1 - Preparation and quality assurance of culture media
Keywords: ISO 14189:2013, Clostridium perfringens, water quality, membrane filtration, TSC agar, enumeration, spores, drinking water monitoring, acid phosphatase, anaerobic incubation.
