🦠 β-glucuronidase-positive E. coli in food

Microbiology PN-EN ISO 16649-2

Overview

Escherichia coli is the most important indicator of fecal contamination in food. The presence of E. coli in food products indicates inadequate production hygiene, improper heat treatment, or secondary contamination after pasteurization. The PN-EN ISO 16649-2 standard specifies a horizontal method for enumerating β-glucuronidase-positive E. coli, utilizing the ability of these bacteria to degrade the chromogenic substrate X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide).

The method is based on pour plating on TBX (Tryptone Bile X-glucuronide Agar) medium and incubation at 44°C for 18–24 h. Under these conditions, E. coli forms characteristic blue-green colonies due to β-glucuronidase enzyme activity, which hydrolyzes the chromogenic substrate. Approximately 96–97% of E. coli strains exhibit β-glucuronidase activity, making this method highly specific.

A significant limitation of the method is that E. coli O157:H7 strains — among the most dangerous foodborne pathogens — are predominantly β-glucuronidase-negative and are not detected by this method. Therefore, separate methods are used for STEC/VTEC detection (e.g., PN-EN ISO 13136). The method is also not suitable for all E. coli strains that may grow poorly at 44°C.

This test is required by Polish and EU food safety regulations, including Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. It is widely applied in quality control of dairy products, meat, vegetables, seafood, and drinking water.

Method principle

The method utilizes the ability of E. coli to produce β-glucuronidase (GUD) enzyme, which hydrolyzes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) contained in TBX medium. Hydrolysis releases indoxyl, which undergoes oxidation and dimerization to form an insoluble blue pigment (5,5'-dibromo-4,4'-dichloro-indigo). E. coli colonies acquire characteristic blue-green coloration. Bile salts present in the medium inhibit growth of Gram-positive bacteria, ensuring selectivity. Incubation at 44°C further restricts growth of non-E. coli organisms.

Applications

• Microbiological quality control of food (meat, dairy, vegetables, seafood)
• Compliance with Regulation (EC) No 2073/2005 — microbiological criteria
• Monitoring hygiene of production processes in food plants
• Testing drinking water and water used in food production
• Verification of pasteurization and heat treatment effectiveness
• HACCP and GHP/GMP systems verification
• Official control by Sanitary Inspection and Veterinary Inspection

Key parameters

Standard

Standard number

PN-EN ISO 16649-2:2004

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby β-glukuronidazo-dodatnich Escherichia coli — Część 2: Metoda liczenia kolonii w temperaturze 44°C z zastosowaniem 5-bromo-4-chloro-3-indolylo-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 preparation

   Weigh 25 g (or 10 g) of sample into stomacher bag. Add 225 mL (or 90 mL) buffered peptone water (BPW). Homogenize in stomacher for 60–120 s. This gives 10⁻¹ dilution.

   ⏱ Time: 5 min

2. Preparation of dilutions

   Prepare decimal dilution series (10⁻², 10⁻³, 10⁻⁴) by transferring 1 mL to 9 mL of diluent. Use new pipette for each dilution.

   ⏱ Time: 5 min

3. Pour plating

   Transfer 1 mL of each dilution to two sterile Petri dishes (duplicates). Within 15 min, pour approx. 15 mL of TBX medium cooled to 44–47°C.

   ⏱ Time: 10 min • 🌡 Temperature: 44–47°C

4. Mixing and solidification

   Gently mix inoculum with medium using circular and oscillating movements. Leave plates on horizontal surface until agar solidifies (approx. 10–15 min).

   ⏱ Time: 15 min

5. Resuscitation (optional)

   If you suspect damaged cells (e.g., frozen samples), incubate plates for 4 h at 37°C before transferring to 44°C to allow resuscitation.

   ⏱ Time: 4 h • 🌡 Temperature: 37°C

6. Incubation

   Invert plates and incubate at 44 ± 1°C for 18–24 h.

   ⏱ Time: 18–24 h • 🌡 Temperature: 44°C

7. Reading results

   Count blue-green colonies typical for β-glucuronidase-positive E. coli. Count plates containing 15–300 colonies total (typical and atypical).

   ⏱ Time: 15 min

8. Calculations

   Calculate the number of E. coli as weighted average from two consecutive dilutions according to formula: N = ΣC / (V × 1.1 × d), where ΣC — sum of colonies, V — plating volume, d — dilution. Express result in CFU/g or CFU/mL.

9. Quality control

   Perform negative control (medium without inoculum) and positive control (reference strain E. coli ATCC 25922) in parallel. Check sterility of peptone water.

10. Documentation and reporting

    Record results in laboratory notebook, include sample identification, batch numbers of media, incubation temperature and calculations. Report result as CFU/g with measurement uncertainty.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Work with potentially infectious material — use disposable gloves, laboratory coat and safety glasses
• Perform all procedures with open sample in class II laminar flow cabinet
• Autoclave used media and samples (121°C, 15 min) before disposal
• Disinfect work surfaces with 70% ethanol or chlorine-based preparation
• Follow aseptic principles — risk of enteropathogenic E. coli infection

Frequently asked questions