ISO 16649-3-2015 (2016) PDF

Full title and description

Microbiology of the food chain — Horizontal method for the enumeration of beta‑glucuronidase‑positive Escherichia coli — Part 3: Detection and most probable number technique using 5‑bromo‑4‑chloro‑3‑indolyl‑β‑D‑glucuronide. This International Standard specifies a liquid‑medium culture (MPN) technique for detection and enumeration of β‑glucuronidase‑positive E. coli in food, feed and related environmental samples.

Abstract

ISO 16649‑3:2015 defines a horizontal method to detect and enumerate β‑glucuronidase‑positive Escherichia coli by a most‑probable‑number (MPN) liquid‑culture technique using the chromogenic substrate 5‑bromo‑4‑chloro‑3‑indolyl‑β‑D‑glucuronide (BCIG). Incubation steps are at (37 ± 1) °C followed by (44 ± 1) °C; the method is aimed at samples from the food chain (products for human consumption, animal feed and environmental samples). The standard notes limitations (method variability and incomplete validation for some matrices such as milk).

General information

• Status: Published (International Standard, edition 1).
• Publication date: 18 May 2015 (ISO edition 1; corrected version published December 2016).
• Publisher: International Organization for Standardization (ISO).
• ICS / categories: 07.100.30 (Food microbiology).
• Edition / version: Edition 1 — 2015 (corrected version December 2016).
• Number of pages: 10 (ISO published document page count).

Key bibliographic and lifecycle details from the ISO bibliographic record and adopted European national publications.

Scope

This part of ISO 16649 specifies a horizontal method for the detection and enumeration of β‑glucuronidase‑positive Escherichia coli by the liquid‑medium culture technique and calculation of the most probable number (MPN) after incubation at (37 ± 1) °C and then at (44 ± 1) °C. It is intended for products intended for human consumption, feedingstuffs and environmental samples in food production/handling areas. The standard emphasises known limitations: susceptibility to variability, incomplete evaluation for all matrices (for example milk and milk products are not fully validated by this method — ISO 7251 is recommended for milk), and that some pathogenic E. coli strains (e.g., some O157) may not be detected because they are β‑glucuronidase negative or do not grow at 44 °C.

Key topics and requirements

• Detection and enumeration of β‑glucuronidase‑positive Escherichia coli using an MPN (most probable number) liquid culture approach.
• Use of the chromogenic substrate 5‑bromo‑4‑chloro‑3‑indolyl‑β‑D‑glucuronide (BCIG) to indicate β‑glucuronidase activity.
• Incubation regime: primary incubation at (37 ± 1) °C followed by (44 ± 1) °C, with interpretation rules for positive/negative tubes and MPN calculation.
• Applicability to food, animal feed and environmental samples in the food chain; stated limitations for some matrices (e.g., milk) and known sources of variability.
• Guidance that results should be interpreted in the context of Clause 11 (method variability and interpretation notes) and that alternative standards (ISO 7251) apply for specific matrices like milk.

Typical use and users

Routine and reference microbiology laboratories in the food and feed sectors, regulatory agencies, quality‑control and assurance teams in food production, contract testing laboratories and research groups conducting method comparison or validation work. The standard is used where an MPN liquid‑culture approach is preferred or required for stressed cells or particular sample types.

Related standards

This part is one element of the ISO 16649 series on enumeration of β‑glucuronidase‑positive E. coli; earlier technical specification ISO/TS 16649‑3:2005 was withdrawn and superseded by ISO 16649‑3:2015. Other related standards include ISO 16649‑1 and ISO 16649‑2 (other enumeration/detection methods for E. coli) and ISO 7251 (enumeration of presumptive E. coli — recommended for milk and milk products). National adoptions exist as EN/ISO derivatives and corrected national publications.

Keywords

Escherichia coli; β‑glucuronidase; BCIG; 5‑bromo‑4‑chloro‑3‑indolyl‑β‑D‑glucuronide; MPN; most probable number; food microbiology; enumeration; horizontal method; ISO 16649.

FAQ

Q: What is this standard?

A: ISO 16649‑3:2015 is an international standard prescribing a horizontal liquid‑medium MPN method for detection and enumeration of β‑glucuronidase‑positive Escherichia coli in food, feed and related environmental samples.

Q: What does it cover?

A: It covers the laboratory procedure (media, substrate, incubation temperatures and times, interpretation of positive/negative tubes), calculation of the most probable number (MPN), applicability and limitations (including method variability and matrices not fully validated such as milk). The method uses BCIG as the chromogenic substrate to detect β‑glucuronidase activity.

Q: Who typically uses it?

A: Food and feed microbiology laboratories, regulatory testing bodies, industry quality control departments, contract testing labs and researchers evaluating E. coli enumeration methods. It is selected when an MPN approach or the BCIG substrate method is required or preferred.

Q: Is it current or superseded?

A: ISO 16649‑3:2015 (edition 1) is the published replacement of ISO/TS 16649‑3:2005; a corrected version of the 2015 text was issued in December 2016. As published by ISO, the 2015 edition is the active international standard for this method. Users should verify national adoption status and any subsequent amendments or confirmations when applying the method.

Q: Is it part of a series?

A: Yes — it is Part 3 of the ISO 16649 series which addresses horizontal methods for enumeration of β‑glucuronidase‑positive Escherichia coli; other parts cover different enumeration/detection techniques. ISO 16649‑3:2015 supersedes the earlier ISO/TS 16649‑3:2005.

Q: What are the key keywords?

A: Escherichia coli, β‑glucuronidase, BCIG, 5‑bromo‑4‑chloro‑3‑indolyl‑β‑D‑glucuronide, MPN, most probable number, food microbiology, enumeration, ISO 16649.