🦠 β-glucuronidase-positive E. coli in food

Microbiology PN-ISO 16649-2

Overview

STATUS (as of 02.11.2026). PN-ISO 16649-2:2004 — Polish version, published on 22.03.2004, adopts ISO 16649-2:2001 [IDT]; the PKN catalogue card has no withdrawal note and no “Replaced by” field. ISO 16649-2:2001 (first edition, 03.05.2001) is current, without amendments or corrigenda. The same subject is covered by ISO 16649-1 (method using membranes, recommended for foods that may contain severely stressed cells) and ISO 16649-3 (most probable number technique).

SCOPE (clause 1, 3.1). Enumeration of β-glucuronidase-positive Escherichia coli in products intended for human consumption or for the feeding of animals, by a colony-count technique at 44 °C on a solid medium containing a chromogenic ingredient for detection of the enzyme β-glucuronidase. β-glucuronidase-positive E. coli are bacteria which at 44 °C form typical blue colonies on TBX medium. Warning of the standard: strains of E. coli which do not grow at 44 °C and, in particular, β-glucuronidase-negative strains such as E. coli O157 will not be detected — the detection of Shiga toxin-producing E. coli is described in ISO/TS 13136 (a separate Knowledge Base entry).

TBX MEDIUM (5.2). Per 1 000 ml of water: enzymatic digest of casein 20.0 g, bile salts No. 3 1.5 g, 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (BCIG) 144 µmol (e.g. 0.075 g of the cyclohexylammonium salt), dimethyl sulfoxide (DMSO) 3 ml, agar 9–18 g; pH after sterilization 7.2 ± 0.2 at 25 °C. BCIG is dissolved in DMSO or in the diluent recommended by the manufacturer; the medium is sterilized for 15 min at 121 °C and immediately cooled in a water bath to 44–47 °C. The data sheet of a TBX medium manufactured in accordance with the standard states that the bile salts inhibit Gram-positive bacteria and that the β-glucuronidase of E. coli releases from the substrate a chromophore that colours the colonies; β-glucuronidase is present in 97 % of E. coli strains, and certain Salmonella species are also β-glucuronidase positive. For performance testing the data sheet lists, among others, E. coli ATCC 25922 (WDCM 00013), which gives blue colonies.

COUNTING AND RESULT (9.3, 10.2, 10.3). After incubation, typical blue colonies are counted on each plate containing fewer than 150 typical colonies and fewer than 300 colonies in total (typical and non-typical). A result is valid when at least one plate from two successive dilutions contains at least 15 blue colonies; the number N per 1 ml or 1 g is calculated as the weighted mean N = Σa / [V × (n₁ + 0.1·n₂) × d] and rounded to two significant figures. When both plates of the lowest dilution contain fewer than 15 blue colonies, an estimated number NE = Σc / (V × n × d) is reported, and when they contain no blue colony — the result “less than 1/d”.

REGULATIONS. In Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026) the reference method of the process hygiene criteria for E. coli is “ISO 16649-1 or 2” (n = 5, c = 2): minced meat and mechanically separated meat — m = 50, M = 500 cfu/g (items 2.1.6–2.1.7), meat preparations — 500 and 5 000 cfu/g or cm² (item 2.1.8), cheeses made from milk or whey that has undergone heat treatment — 100 and 1 000 cfu/g (item 2.2.2), butter and cream made from raw milk or milk that has undergone a lower heat treatment than pasteurisation — 10 and 100 cfu/g (item 2.2.6), pre-cut fruit and vegetables and unpasteurised fruit and vegetable juices, ready-to-eat — 100 and 1 000 cfu/g (items 2.5.1–2.5.2). In the criteria for meat E. coli is used as an indicator of faecal contamination, and for cheeses, butter and cream — as an indicator of the level of hygiene. For live bivalve molluscs (item 1.25) and shelled and shucked products of cooked crustaceans and molluscan shellfish (item 2.4.1) the regulation specifies the most probable number technique (ISO 16649-3), and for water intended for human consumption the reference methods are PN-EN ISO 9308-1 and PN-EN ISO 9308-2 (Journal of Laws 2026, item 748, Annex 4 Part A). According to chapter 4 of the BAM manual (FDA), E. coli indicates recent faecal contamination or unsanitary processing.

WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends at 10.3.3; the rest of clause 10 and the test report (clause 11) are not in it. The standard does not describe the preparation of the test sample, initial suspension and dilutions — it refers to ISO 6887-1 and to the standards for the product concerned, and for laboratory practice — to ISO 7218. Performance testing of the medium, control strains and measurement uncertainty are not covered by the available text.

Method principle

Two plates per dilution are poured with TBX medium containing the liquid sample or the initial suspension and further decimal dilutions, incubated for 18–24 h at (44 ± 1) °C and examined for colonies which, from their characteristics, are considered to be β-glucuronidase-positive E. coli (4.1). Bacteria with β-glucuronidase cleave the chromogenic substrate BCIG contained in the medium and form blue colonies (3.1, 5.2); the bile salts inhibit Gram-positive bacteria (data sheet of a TBX medium manufactured in accordance with the standard). The number of cfu per 1 g or 1 ml of sample is calculated according to clause 10 (4.2).

Applications

• Products intended for human consumption and for the feeding of animals (clause 1)
• Minced meat, mechanically separated meat and meat preparations — process hygiene criteria with the method “ISO 16649-1 or 2” (Regulation (EC) No 2073/2005, items 2.1.6–2.1.8)
• Cheeses made from milk or whey that has undergone heat treatment and butter and cream made from raw milk or milk that has undergone a lower heat treatment than pasteurisation (Regulation (EC) No 2073/2005, items 2.2.2 and 2.2.6)
• Pre-cut fruit and vegetables and unpasteurised fruit and vegetable juices, ready-to-eat (Regulation (EC) No 2073/2005, items 2.5.1–2.5.2)
• Testing when validating or verifying procedures based on HACCP principles and good hygiene practice (Regulation (EC) No 2073/2005, Article 4)

Key parameters

Standard

Standard number

PN-ISO 16649-2:2004

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby β-glukuronidazo-dodatnich Escherichia coli — Część 2: Metoda płytkowa w temperaturze 44 °C z zastosowaniem 5-bromo-4-chloro-3-indolilo β-D-glukuronidu

Title (EN)

Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli — Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide

Step-by-step procedure

1. Sample and initial suspension

   The test sample, initial suspension and decimal dilutions are prepared according to ISO 6887-1 and the standard for the product concerned (clause 8, 9.1). Sampling is not part of the method (clause 7).

2. Inoculation

   Transfer to a sterile Petri dish 1 ml of the liquid sample or 1 ml of the initial suspension (10⁻¹), two plates per dilution; repeat with further dilutions if necessary, each time with a new sterile pipette (9.2.1).

3. Pouring the medium

   Pour into each dish about 15 ml of TBX medium cooled in the water bath to 44–47 °C, carefully mix with the inoculum and allow to solidify on a cool horizontal surface. No more than 15 min may elapse between distributing the inoculum and pouring the medium (9.2.2).

   🌡 Temperature: 44–47 °C

4. Incubation

   Invert the dishes bottom uppermost and incubate at 44 °C for 18–24 h; the total incubation time shall not exceed 24 h (9.2.3).

   🌡 Temperature: 44 ± 1 °C

5. Stressed cells

   If the presence of stressed cells is suspected, incubate the plates first for 4 h at 37 °C, then for 18–24 h at 44 °C; the incubation temperature shall not exceed 45 °C (warning in 9.2.3).

   🌡 Temperature: 37 °C → 44 °C

6. Counting colonies

   Count typical blue colonies on each plate containing fewer than 150 typical colonies and fewer than 300 colonies in total; plates with 0 typical colonies are taken into account in the calculation if they are among the retained plates (9.3).

7. Calculation

   N = Σa / [V × (n₁ + 0.1·n₂) × d] — from two successive dilutions, when at least one plate contains at least 15 blue colonies; round the result to two significant figures and report it in cfu/ml or cfu/g (10.2).

8. Low numbers

   When both plates of the lowest dilution contain fewer than 15 blue colonies — estimated number NE = Σc / (V × n × d); when there is no blue colony — “less than 1/d” per 1 ml or 1 g; the case of more than 300 colonies at the first dilution and no blue colonies at the next one — according to 10.3.3.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Dimethyl sulfoxide is harmful by inhalation and contact — the use of a fume cupboard is advised; because of this toxicity, a diluent recommended by the manufacturer may be used (5.2.1, footnote b)
• For laboratory practice the standard refers to ISO 7218 (clauses 5 and 6)

Frequently asked questions