ISO 16649-1:2018 PDF

Microbiology of the food chain — Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli — Part 1: Colony-count technique at 44 degrees C using membranes and 5-bromo-4-chloro-3-indolyl beta-D-glucuronide

Title (English):

ISO 16649-1:2018

Document status:: Active
Format:: Electronic (PDF)
Number of pages:: 11
Publication date:: April 26, 2018
Edition:: ISO IS 16649 edition 2 version 1
ICS:: 07.100.30

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Description (English):

ISO 16649-1:2018 specifies a horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli by colony-count technique after resuscitation using membranes and incubation at 44 °C on a solid medium containing a chromogenic ingredient for detection of the enzyme β-glucuronidase[9][10][13][14][17][18][19][20]. It is applicable to - products intended for human consumption, - products intended for feeding animals, - environmental samples in the area of food production and food handling, and - samples from the primary production stage such as animal faeces, dust, and swabs.

Technical details

Technical committee: ISO/TC 34/SC 9 - Microbiology
SKU: ISO 16649-1:2018

Abstract

Overview

ISO 16649-1:2018 specifies a horizontal method for the enumeration of β‑glucuronidase‑positive Escherichia coli using a membrane-based colony‑count technique. The method combines a short resuscitation step on minerals‑modified glutamate agar (MMGA) with transfer to tryptone‑bile X‑glucuronide agar (TBX) containing the chromogenic substrate 5‑bromo‑4‑chloro‑3‑indolyl β‑D‑glucuronide (BCIG). Incubation is performed at 44 °C, and results are reported as colony‑forming units (cfu) per gram, millilitre, square centimetre or per sampling device.

Key topics and technical requirements

Scope and applicability: Applicable to foods and feeds, environmental samples in food production/handling, and primary production samples (animal faeces, dust, swabs).
Resuscitation step: Membrane filtration onto MMGA with a resuscitation incubation (document specifies 37 °C for 4 h) to recover sub‑lethally injured cells before selective culture.
Selective culture and detection: Transfer membranes to TBX containing BCIG and incubate at 44 °C for 20–24 h; β‑glucuronidase‑positive E. coli form typical blue or blue‑green colonies.
Membranes and equipment: Use sterile, non‑inhibitory membranes (cellulose acetate or mixed esters), pore size 0.45–1.2 µm, diameter ~85 mm; standard laboratory incubators, sterile pipettes, forceps and spreaders are specified.
Counting and calculation: Count typical colonies and calculate cfu per unit sample according to standard dilution procedures (see ISO 6887 guidance).
Quality and safety: Guidance on culture media composition, performance testing (ISO 11133), and avoidance of hazardous solvents (DMSO no longer recommended for BCIG dissolution).
Limitations: Some E. coli strains (notably certain O157 serotypes) may be β‑glucuronidase‑negative or grow poorly at 44 °C; consequently, pathogenic strains can be missed by this method. Other Enterobacteriaceae (e.g., Shigella, Salmonella) may show β‑glucuronidase activity and require confirmatory testing.

Applications and users

Routine food microbiology testing in industry quality control and external testing laboratories.
Regulatory and public‑health laboratories monitoring food safety and contamination.
Feed producers, primary producers and environmental monitoring teams in food chains.
Laboratories validating culture media and membrane performance for E. coli enumeration.

Related standards

ISO 16649 series (other parts for alternative enumeration techniques)
ISO 6887 (sample preparation and dilutions)
ISO 7218 (general microbiological requirements)
ISO 11133 (culture media preparation and performance)
Sampling standards referenced (e.g., ISO 707, ISO 18593, ISO 17604)

ISO 16649-1:2018 is a practical, standardized method for E. coli enumeration, optimized for food‑chain applications where recovery of injured cells is important and where chromogenic detection streamlines colony recognition.

Related standards

Standards mentioned in the description

Abstract

Overview

Key topics and technical requirements

Scope and applicability: Applicable to foods and feeds, environmental samples in food production/handling, and primary production samples (animal faeces, dust, swabs).
Resuscitation step: Membrane filtration onto MMGA with a resuscitation incubation (document specifies 37 °C for 4 h) to recover sub‑lethally injured cells before selective culture.
Selective culture and detection: Transfer membranes to TBX containing BCIG and incubate at 44 °C for 20–24 h; β‑glucuronidase‑positive E. coli form typical blue or blue‑green colonies.
Membranes and equipment: Use sterile, non‑inhibitory membranes (cellulose acetate or mixed esters), pore size 0.45–1.2 µm, diameter ~85 mm; standard laboratory incubators, sterile pipettes, forceps and spreaders are specified.
Counting and calculation: Count typical colonies and calculate cfu per unit sample according to standard dilution procedures (see ISO 6887 guidance).
Quality and safety: Guidance on culture media composition, performance testing (ISO 11133), and avoidance of hazardous solvents (DMSO no longer recommended for BCIG dissolution).
Limitations: Some E. coli strains (notably certain O157 serotypes) may be β‑glucuronidase‑negative or grow poorly at 44 °C; consequently, pathogenic strains can be missed by this method. Other Enterobacteriaceae (e.g., Shigella, Salmonella) may show β‑glucuronidase activity and require confirmatory testing.

Applications and users

Routine food microbiology testing in industry quality control and external testing laboratories.
Regulatory and public‑health laboratories monitoring food safety and contamination.
Feed producers, primary producers and environmental monitoring teams in food chains.
Laboratories validating culture media and membrane performance for E. coli enumeration.

Related standards

ISO 16649 series (other parts for alternative enumeration techniques)
ISO 6887 (sample preparation and dilutions)
ISO 7218 (general microbiological requirements)
ISO 11133 (culture media preparation and performance)
Sampling standards referenced (e.g., ISO 707, ISO 18593, ISO 17604)

ISO 16649-1:2018 PDF

Technical details

Abstract

Overview

Key topics and technical requirements

Applications and users

Related standards

Related standards

Abstract

Overview

Key topics and technical requirements

Applications and users

Related standards

Related standards

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