ISO 16654:2001

Overview

ISO 16654:2001 specifies a horizontal microbiological method for the detection of Escherichia coli O157 in foods and animal feeding stuffs. The standard defines a stepwise laboratory procedure - enrichment, immunomagnetic separation, selective isolation and confirmation - and includes safety warnings because E. coli O157 has a low infective dose and can cause severe illness. Annex A provides a normative diagram of the procedure.

Key topics and technical requirements

• Scope: Horizontal method applicable to products for human consumption and animal feed (subject to product-specific limitations noted in the Introduction).
• Safety: Laboratory work must be performed by skilled personnel using appropriate containment and biosafety practices.
• Principle: Four main stages
  • Enrichment in modified tryptone soya broth with novobiocin (mTSB + N) - incubation at 41.5 °C ± 1 °C, initial 6 h followed by a further 12–18 h.
  • Immunomagnetic separation (IMS) using magnetic particles coated with anti‑E. coli O157 antibodies to concentrate target cells.
  • Selective isolation by plating IMS material onto cefixime‑tellurite sorbitol MacConkey agar (CT‑SMAC) and a complementary second selective agar chosen by the laboratory.
  • Confirmation of presumptive sorbitol‑nonfermenting colonies by indole production and agglutination with E. coli O157 antiserum; further characterization can be referred to reference laboratories.
• Culture media & reagents: Standard provides compositions and preparation notes for mTSB, CT‑SMAC (base + cefixime and tellurite), nutrient agar, tryptone/tryptophan medium, Kovac’s indole reagent, IMS particles and wash buffer.
• Quality assurance: Inclusion of relevant test strains and adherence to general microbiological practices (see ISO 7218). Sampling and test portion preparation reference ISO 6887‑1.

Applications

• Routine screening and verification testing in food microbiology laboratories for beef, dairy and other food matrices where E. coli O157 is a concern.
• Feed‑stuff testing to ensure animal feed safety.
• Outbreak investigation and public‑health surveillance to isolate and confirm E. coli O157 from food samples.
• Supplier monitoring, HACCP validation and regulatory compliance testing.

Who should use this standard

• Accredited food and veterinary microbiology laboratories
• Public health and reference laboratories
• Food manufacturers, quality assurance teams and regulatory agencies involved in food safety testing

Related standards

• ISO 6887‑1 - sample preparation and initial suspensions for microbiological examination
• ISO 7218 - general rules for microbiological examinations

Keywords: ISO 16654, E. coli O157 detection, CT‑SMAC, immunomagnetic separation, mTSB + N, food microbiology standard, food safety testing, animal feeding stuffs.