ISO 16649-2:2001

Overview

ISO 16649-2:2001 specifies a horizontal colony-count method for the enumeration of beta‑glucuronidase‑positive Escherichia coli in food and animal feeding stuffs. The method uses a chromogenic solid medium (Tryptone‑Bile‑Glucuronide, TBX) containing 5‑bromo‑4‑chloro‑3‑indolyl β‑D‑glucuronide (BCIG) and a pour‑plate technique with incubation at 44 °C to produce typical blue colonies that are counted as presumptive E. coli.

Key topics and requirements

• Scope: Enumeration of β‑glucuronidase‑positive E. coli for products intended for human or animal consumption; not suitable for β‑glucuronidase‑negative strains (e.g., E. coli O157) or strains that do not grow at 44 °C.
• Medium and reagent: TBX (tryptone digest, bile salts, agar) with BCIG as chromogen. BCIG is dissolved in DMSO or a manufacturer‑recommended diluent; DMSO handling precautions apply.
• Sample handling: Follow ISO 6887‑1 and ISO 7218 for sample preparation, dilutions and general microbiological practice.
• Plating: Inoculate duplicate plates (1 ml inoculum per plate), pour ~15 ml cooled TBX (44–47 °C) and mix. Use decimal dilutions and two plates per dilution.
• Incubation: Invert plates and incubate 18–24 h at 44 °C ±1 °C. If stressed cells are suspected, an initial 4 h at 37 °C followed by 18–24 h at 44 °C is recommended.
• Counting rules: Count typical blue colonies on plates containing <150 typical CFU and <300 total CFU. Specific procedures are defined for low counts, overloaded plates, and special cases.
• Result calculation: Use a weighted‑mean calculation from two successive dilutions (or arithmetic mean for low counts). Results are rounded to two significant figures and reported per g or per ml.

Practical applications

• Food safety testing: Routine enumeration of presumptive E. coli in raw materials, finished foods, and feed to monitor hygiene and process control.
• Quality control: Verification of sanitation, process validation and batch release testing in food production and feed manufacturing.
• Regulatory and surveillance labs: Standardized method for comparable results across laboratories and for regulatory compliance.

Who should use this standard

• Microbiology laboratories in food and feed industries
• Quality assurance / QC teams and production microbiologists
• Government and third‑party testing laboratories
• Standards and regulatory professionals evaluating microbiological criteria

Related standards

• ISO 16649‑1: colony‑count technique using membranes and BCIG (for stressed cells)
• ISO 16649‑3: Most probable number (MPN) technique
• ISO 6887‑1: sample preparation and dilutions
• ISO 7218: general rules for microbiological examinations

Keywords: ISO 16649-2, beta-glucuronidase-positive Escherichia coli, TBX, BCIG, colony-count technique, food microbiology, E. coli enumeration, microbiological testing, food safety.