Overview
EN ISO 16654:2001 (also published as ISO 16654:2001) specifies a horizontal laboratory method for the detection of Escherichia coli O157 in food and animal feeding stuffs. The standard is intended for products meant for human consumption or animal feed, though the introduction notes that the horizontal method may not suit every product and product‑specific adaptations can be justified. The document was adopted by CEN and references common microbiology practice standards.
Key topics and technical requirements
- Scope and safety: The standard warns that E. coli O157 can cause severe illness and requires skilled personnel, good laboratory practice and appropriate containment.
- Overall principle: Four successive stages are required - enrichment, immunomagnetic concentration, selective isolation, and confirmation.
- Enrichment: Use modified tryptone soya broth with novobiocin (mTSB + N). Typical incubation described is 41.5 °C ± 1 °C for an initial 6 h followed by a further 12–18 h.
- Immunomagnetic separation (IMS): Concentration and separation of target cells using magnetic particles coated with antibodies specific to E. coli O157.
- Selective isolation: Subculture IMS particles onto cefixime-tellurite sorbitol MacConkey agar (CT‑SMAC) and at least one second selective agar chosen by the laboratory.
- Confirmation: Presumptive sorbitol‑negative colonies (and typical colonies from the second agar) are confirmed by indole production and agglutination with E. coli O157 antiserum. Further characterization (e.g., virulence markers) can be performed by reference laboratories.
- Media and reagents: The standard provides compositions and preparation notes for mTSB, novobiocin solution (final novobiocin 20 mg/L), CT‑SMAC base, and potassium tellurite solution.
- Quality assurance and reporting: Test strains and culture methods for QA are specified; the standard defines how results should be expressed and reported.
Applications and users
- Food microbiology testing laboratories - routine monitoring and outbreak investigation.
- Public health and reference laboratories - confirmation and characterization of isolates.
- Food manufacturers and processors - verification of product safety and supplier testing.
- Regulatory agencies - enforcement testing, surveillance and compliance.
- Feed industry labs - testing of animal feeding stuffs as covered by the scope.
Keywords naturally associated: ISO 16654, EN ISO 16654, E. coli O157 detection, CT‑SMAC, immunomagnetic separation, mTSB + novobiocin, food microbiology, food safety testing.
Related standards (if applicable)
- ISO 6887-1 - Preparation of test samples, initial suspension and decimal dilutions.
- ISO 7218 - General rules for microbiological examinations.
- Note: Annex A of ISO 16654 contains a normative diagram of the procedure.