ISO 16649-1:2018

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.