🦠 β-glucuronidase-positive E. coli in food
Enumeration of β-glucuronidase-positive Escherichia coli in food by pour plate method on TBX agar at 44°C. E. coli colonies form characteristic blue-green coloration.
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
| Parameter | Value |
|---|---|
| Incubation temperature | 44 ± 1°C |
| Incubation time | 18–24 h |
| Medium | TBX Agar (Tryptone Bile X-glucuronide) |
| Limit of quantification | 10–3 × 10⁵ CFU/g (at dilutions 10⁻¹ to 10⁻⁴) |
| Colony type | Blue-green colonies (β-glucuronidase +) |
| Plating method | Pour plate, 1 mL per plate |
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.
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.
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.
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).
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.
6. Incubation
Invert plates and incubate at 44 ± 1°C for 18–24 h.
7. Reading results
Count blue-green colonies typical for β-glucuronidase-positive E. coli. Count plates containing 15–300 colonies total (typical and atypical).
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
| Equipment | Example | Indicative price |
|---|---|---|
| Laboratory incubator 44°C | Binder BD 56/115, Memmert INB 200, POL-EKO CLN 53 | 5,000–18,000 PLN |
| Water bath 44–47°C | Memmert WNB 14, Julabo TW 12, POL-EKO LW-4 | 3,000–10,000 PLN |
| Class II laminar flow cabinet | Thermo Scientific MSC-Advantage, ESCO Airstream | 25,000–60,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65, SMS Prestige Medical | 15,000–45,000 PLN |
| Stomacher / homogenizer | Seward Stomacher 400 Circulator, BagMixer 400 | 12,000–25,000 PLN |
| Colony counter | Interscience Scan 300, BZG Colony Star, Stuart SC6+ | 3,000–15,000 PLN |
| Automatic pipettes 1 mL | Eppendorf Research Plus, Gilson Pipetman, HTL Discovery Comfort | 400–1,200 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| TBX Agar (Tryptone Bile X-glucuronide Agar) | 114162-64-0 | Chromogenic medium containing tryptone, bile salts and X-Gluc; ready-made sachets or plates, 500 g / 20 plates package |
| 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) | 114162-64-0 | Chromogenic substrate for β-glucuronidase, TBX component, 1–5 g package |
| Tryptone (casein peptone) | 91079-40-2 | Nitrogen and amino acid source in TBX medium |
| Bile salts (Bile Salts No. 3) | — | Selective agent inhibiting Gram-positive bacteria, concentration 1.5 g/L |
| Buffered peptone water (BPW) | — | For preparation of decimal dilutions of sample, 500 g package |
| Bacteriological agar | 9002-18-0 | Gelling agent, 500 g package |
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