Overview
EN ISO 14189:2016 (identical to ISO 14189:2013) - "Water quality - Enumeration of Clostridium perfringens - Method using membrane filtration" is a CEN-adopted standard that specifies a membrane-filtration culture method for enumerating vegetative cells and spores of Clostridium perfringens in water. The method is intended principally for water intended for human consumption, but can be applied to other water types provided samples do not contain particulate or colloidal matter that interferes with filtration.
Key topics and technical requirements
- Principle: Filter a measured sample or dilution through a 0.45 μm membrane to retain clostridial spores and cells, incubate on selective/differential TSC (tryptose-sulfite-cycloserine) agar anaerobically, then count and confirm characteristic colonies.
- Incubation conditions: Anaerobic incubation at (44 ± 1) °C for (21 ± 3) h to improve selectivity for C. perfringens.
- Spore selection: Optional heat pre-treatment at (60 ± 2) °C to inactivate vegetative bacteria when enumeration of spores alone is required.
- Apparatus: Membrane filtration unit, sterile funnels, sterile membrane filters (nominal pore size 0.45 µm), anaerobic jars or gas systems (≈90% H2 / 10% CO2), incubators (36 ± 2 °C and 44 ± 1 °C), autoclave, water bath (optional).
- Culture media & reagents: TSC agar (selective/differential), blood or Columbia agar for confirmations, acid phosphatase reagent for confirmatory testing.
- Results: Expressed as colony count per sample volume; presumptive and confirmed definitions provided.
- Quality & safety: Requires suitably trained personnel and compliance with laboratory safety and quality assurance (media preparation, equipment sterilization, method validation).
Applications and users
This standard is used for:
- Routine monitoring of drinking water quality and treatment performance
- Environmental surveillance of faecal contamination (spores can persist longer than other indicators)
- Assessing treatment stages where spores may resist routine disinfection (e.g., chlorination)
Typical users:
- Public health and municipal water laboratories
- Environmental monitoring and research laboratories
- Water utilities and treatment plant operators
- Regulatory agencies and compliance auditors
Related standards
Normative references cited in EN ISO 14189:2016 include:
- ISO 8199 (enumeration of micro-organisms by culture)
- ISO 19458 (sampling for microbiological analysis)
- ISO/TS 11133‑1 (culture media preparation quality assurance)
- ISO 3696 (water for laboratory use)
- ISO/IEC Guide 2 (standardization vocabulary)
For labs implementing EN ISO 14189:2016, integrate this method into broader water quality monitoring programs to reliably detect and enumerate Clostridium perfringens as an indicator of faecal pollution and treatment efficacy.