ISO 16649-2:2001 PDF

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