Overview
EN ISO 7899-1:1998 specifies a miniaturized Most Probable Number (MPN) method for the detection and enumeration of intestinal enterococci in surface water and wastewater. The method uses inoculation of diluted samples into dehydrated liquid medium in 96-well microtitre plates and detects enterococci by fluorescence from hydrolysis of 4‑methylumbelliferyl-β-D-glucoside (MUD). Results are reported as MPN per 100 ml. The procedure is intended for environmental monitoring (including waters high in suspended solids) but is not suitable for drinking water or waters with guideline counts below 15/100 ml.
Key topics and technical requirements
- Target organisms: major faecal enterococci (e.g., E. faecalis, E. faecium, E. durans, E. hirae) and occasional related species.
- Principle: fluorogenic substrate (MUD) hydrolysis in the presence of selective agents (thallium acetate, nalidixic acid, TTC) indicates positive wells; reading under UV (366 nm).
- Incubation: 44 °C ±0.5 °C for 36–72 hours.
- Plate format & volumes: sterile, non-fluorescent 96‑well flat-bottom microtitre plates; typically 100 µl medium per well (dehydrated) and 200 µl inoculum per well.
- Diluents & media: Special Diluent (synthetic sea salt-based) and MUD/SF culture medium (prepared from defined solutions A–D). Media sterilization by 0.2 μm filtration; dehydration in controlled conditions.
- Dilution strategy: multiple dilutions depending on expected contamination; results read as counts of positive wells per dilution and converted to MPN.
- Quality & performance: Annexes include performance characteristics, software examples for statistical MPN computation, and normative criteria for manufacturing and calibration of microtitre plates.
- Safety: contains toxic reagents (thallium acetate, N,N-dimethylformamide) and requires UV-protection during reading.
Applications and users
- Routine environmental and bathing water monitoring for fecal contamination and public-health risk assessment.
- Wastewater and effluent testing, particularly where suspended solids interfere with membrane filtration.
- Users: environmental laboratories, water-quality testing labs, public health agencies, wastewater treatment operators, environmental consultancies, and researchers performing microbiological assessments.
- Advantages: miniaturized, high-throughput, suitable for turbid or particulate-rich samples where membrane methods are less practical.
Related standards
- EN ISO 7899-2 (membrane filtration method for enterococci)
- ISO 5667 series (water sampling guidance)
- ISO 8199 (enumeration of microorganisms by culture)
- ISO 3951 (sampling procedures) and ISO/IEC Guide 2 (vocabulary)
Keywords: EN ISO 7899-1:1998, intestinal enterococci, water quality, MPN, microtitre plates, wastewater testing, surface water monitoring.
