🦠 Enterobacteriaceae in food
Enumeration of Enterobacteriaceae in food by pour plating on VRBG (Violet Red Bile Glucose) agar with overlay, incubation at 37°C for 24 h.
Overview
Enterobacteriaceae is a large family of Gram-negative bacteria including many genera of sanitary and pathogenic significance — Escherichia, Salmonella, Klebsiella, Enterobacter, Citrobacter, Proteus, Serratia, Cronobacter and others. They are a recognized indicator of general food production hygiene status, complementing E. coli determination (fecal contamination indicator).
The PN-EN ISO 21528-2 standard describes a horizontal method for enumerating Enterobacteriaceae in food and environmental samples. The method uses VRBG (Violet Red Bile Glucose Agar) — selective-differential medium in which crystal violet and bile salts inhibit growth of Gram-positive bacteria and other non-Enterobacteriaceae organisms. Glucose replaces lactose (used in VRBL), allowing the medium to detect all Enterobacteriaceae members, not just coliform bacteria.
Enterobacteriaceae colonies on VRBG are red to dark purple, surrounded by bile salt precipitation zone. Pour plate method with additional overlay agar layer ensures near-anaerobic conditions, inhibiting growth of non-fermenting Gram-negative bacteria and promoting glucose fermentation.
The method is used as hygiene indicator in Regulation (EC) No 2073/2005, including for milk powder, infant foods and ready-to-eat products. It is particularly important in dairy industry, where Enterobacteriaceae indicate potential post-pasteurization contamination.
Method principle
Sample is pour plated on VRBG medium. Bile salts and crystal violet inhibit Gram-positive bacteria. Enterobacteriaceae ferment glucose to acids, which with neutral red stain colonies red-purple. Acid also causes bile salt precipitation around colonies (halo). After pouring, an additional VRBG agar layer (overlay) is applied, limiting oxygen access and inhibiting aerobic non-fermenting bacteria growth. Incubation at 37°C for 24 h.
Applications
- Food production hygiene indicator — Regulation (EC) No 2073/2005
- Milk powder and infant formula quality control
- Post-pasteurization contamination monitoring
- Ready-to-eat (RTE) product testing
- Meat and meat products quality control
- Environmental monitoring of production facilities (surfaces, water)
- Washing and disinfection effectiveness verification of production lines
Key parameters
| Parameter | Value |
|---|---|
| Incubation temperature | 37 ± 1°C |
| Incubation time | 24 ± 2 h |
| Medium | VRBG Agar (Violet Red Bile Glucose Agar) |
| Colony type | Red to dark purple, with bile salt precipitation halo |
| Plating method | Pour plate with overlay |
| Confirmation | Oxidase test (negative) + glucose fermentation test (positive) |
Standard
- Standard number
- PN-EN ISO 21528-2:2017
- Title (PL)
- Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania i oznaczania liczby Enterobacteriaceae — Część 2: Metoda liczenia kolonii
- Title (EN)
- Microbiology of the food chain — Horizontal method for the detection and enumeration of Enterobacteriaceae — Part 2: Colony-count technique
Step-by-step procedure
1. Sample preparation
Weigh 10 g sample into stomacher bag, add 90 mL BPW, homogenize for 60–120 s. Prepare decimal dilutions.
2. Pour plating
Transfer 1 mL of each dilution to two sterile Petri dishes. Within 15 min, pour approx. 10 mL VRBG medium cooled to 44–47°C. Mix gently with circular movements. Leave to solidify.
3. Overlay
After agar solidifies, apply additional layer of approx. 10 mL VRBG medium (44–47°C) to surface. Leave to solidify. Overlay limits oxygen access.
4. Incubation
Invert plates and incubate at 37 ± 1°C for 24 ± 2 h.
5. Colony reading
Count typical colonies — red to dark purple, diameter 0.5 mm or larger, with or without precipitation halo. Count plates with 15–300 colonies total.
6. Confirmation (optional)
Select min. 5 typical colonies. Perform oxidase test (negative result = Enterobacteriaceae). Inoculate glucose broth with Durham tube — incubate 24 h at 37°C (result: glucose fermentation + gas).
7. Calculations
Calculate number of Enterobacteriaceae as weighted average. If confirmation performed, take into account percentage of confirmed colonies. Express result in CFU/g.
8. Quality control
Positive control: E. coli ATCC 25922 (typical colonies, oxidase-negative). Negative control: Pseudomonas aeruginosa ATCC 27853 (no growth or atypical colonies, oxidase-positive). Medium sterility control.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Laboratory incubator 37°C | Binder BD 56, 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 |
| Stomacher / homogenizer | Seward Stomacher 400, Interscience BagMixer | 12,000–25,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65 | 15,000–45,000 PLN |
| Colony counter | Interscience Scan 300, Stuart SC6+ | 3,000–15,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| VRBG Agar (Violet Red Bile Glucose Agar) | 548-62-9 | Selective-differential medium: gelatin peptone, yeast extract, bile salts, glucose, crystal violet, neutral red, NaCl, agar; 500 g package |
| Crystal violet (Crystal Violet) | 548-62-9 | Dye inhibiting Gram-positive bacteria; concentration 0.002 g/L |
| Bile salts (Bile Salts Mixture) | — | Selective agent inhibiting Gram-positive and non-fermenting bacteria; 1.5 g/L |
| Neutral red (Neutral Red) | 553-24-2 | pH indicator — stains glucose-fermenting colonies red; 0.03 g/L |
| D-Glucose | 50-99-7 | Carbon source fermented by Enterobacteriaceae; 10 g/L |
| Glucose broth (Glucose Broth) | — | For glucose fermentation test (confirmation), with Durham tube |
| Oxidase reagent (Kovács) | — | For oxidase test — Enterobacteriaceae oxidase-negative; strips or drops |
| Buffered peptone water (BPW) | — | For sample dilutions, 500 g package |
Health and safety (OHS)
- Enterobacteriaceae family includes pathogens (Salmonella, E. coli) — use PPE
- Work in class II laminar flow cabinet mandatory
- Crystal violet — stains skin, use gloves
- Autoclave used materials before disposal (121°C/20 min)
- Surface disinfection with 70% ethanol or sodium hypochlorite