🦠 Enterobacteriaceae in food
In short
Enumeration of Enterobacteriaceae in products intended for human consumption and for the feeding of animals and in environmental samples by pour plating on VRBG agar with an overlay, incubation at 37 °C for 24 h and confirmation by oxidase and glucose fermentation tests. The test is performed according to PN-EN ISO 21528-2:2017-08; Medium: VRBG agar (crystal violet, neutral red, bile, glucose), composition according to Annex A (9.3.2). The procedure comprises 10 steps; it is used for: Products intended for human consumption and for the feeding of animals (clause 1), Environmental samples in the area of primary production, food production and food handling (clause 1), Carcases of cattle, sheep, goats, horses and pigs — process hygiene criteria (Regulation (EC) No 2073/2005, items 2.1.1–2.1.2).
At a glance
- Standard: PN-EN ISO 21528-2:2017-08
- Category: Microbiology
- Procedure steps: 10
- Medium: VRBG agar (crystal violet, neutral red, bile, glucose), composition according to Annex A (9.3.2)
- Inoculation: pour plate: 1 ml of liquid sample or initial suspension per plate, one plate per dilution (two when only the initial suspension is used); about 15 ml of medium at 44–47 °C, at the latest 15 min after inoculation (9.3.1–9.3.2)
- Overlay: about 5–10 ml of the same medium — prevents spreading growth and achieves semi-anaerobic conditions (9.3.3)
Overview
STATUS (as of 02.11.2026). PN-EN ISO 21528-2:2017-08 — English version published on 23.08.2017, Polish version on 18.10.2019; adopts EN ISO 21528-2:2017 and ISO 21528-2:2017 [IDT], replaced PN-ISO 21528-2:2005, without a withdrawal note. ISO 21528-2:2017 (second edition, 16.06.2017) replaced the 2004 edition: in the confirmation, glucose agar was replaced by glucose OF medium and precision data were added (Annex B); the corrected version added a 44–47 °C water bath and lowered the medium temperature in 9.3.2 from 47–50 °C to 44–47 °C. The changes are considered minor (ISO 17468).
SCOPE AND DEFINITION (clause 1, 3.1). Enumeration of Enterobacteriaceae in products intended for human consumption and for the feeding of animals and in environmental samples in the area of primary production, food production and food handling — when the number of colonies sought is expected to exceed 100 per 1 ml or 1 g; for lower numbers the most probable number technique of ISO 21528-1 is generally used. Enterobacteriaceae are microorganisms forming characteristic colonies on violet red bile glucose (VRBG) agar, fermenting glucose and showing a negative oxidase reaction.
TEMPERATURE (4.2, note). 37 °C is generally used when the determination serves as a hygiene indicator; 30 °C can be chosen when the test is conducted for technological purposes and is to include psychrotrophic Enterobacteriaceae.
COUNTING AND CONFIRMATION (9.4–9.6). Characteristic colonies are pink to red or purple, with or without precipitation haloes. They are counted on plates with fewer than 150 characteristic colonies, and five of them are chosen at random from each plate for confirmation (all of them if there are fewer); spreading colonies are counted as single colonies, and when they overgrow more than one quarter of the plate, the count is discarded. Certain Enterobacteriaceae decolourize their colonies or the medium — when no characteristic colonies are present, five whitish colonies are confirmed. Oxidase-negative and glucose-positive colonies are confirmed as Enterobacteriaceae.
REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026) specifies EN ISO 21528-2 in process hygiene criteria: carcases of cattle, sheep, goats and horses — daily mean log m = 1.5, M = 2.5 log cfu/cm² (item 2.1.1), pig carcases — 2.0 and 3.0 log cfu/cm² (item 2.1.2), pasteurised milk and other pasteurised liquid dairy products — 10 cfu/ml (n = 5, c = 0, item 2.2.1), milk powder and whey powder — 10 cfu/g (n = 5, c = 0, item 2.2.7), ice cream and frozen dairy desserts — 10 and 100 cfu/g (n = 5, c = 2, item 2.2.8), egg products — 10 and 100 cfu/g or ml (n = 5, c = 2, item 2.3.1). For dried infant formulae the criterion is “absence in 10 g” and the reference method is EN ISO 21528-1, not this part of the standard (items 2.2.9–2.2.10).
WHAT WE DO NOT GIVE HERE. The preparation of media and reagents (Annex A — VRBG agar, glucose OF medium, oxidase reagent) is not in the available text of the standard — we do not give it; the composition of VRBG agar is given according to the data sheet of the medium made to the standard. The standard does not describe the calculation: it refers to ISO 7218.
Method principle
An initial suspension and decimal dilutions are prepared from the test sample (4.1). VRBG agar is inoculated with a specified quantity of the liquid sample or the initial suspension and further dilutions, covered with an overlay of the same medium and incubated at 37 °C (or 30 °C) for 24 h (4.2). Presumptive colonies are subcultured onto a non-selective medium and confirmed by tests for glucose fermentation and oxidase (4.3); the number of Enterobacteriaceae per 1 ml or 1 g is calculated from the number of confirmed characteristic colonies per dish (4.4).
Applications
- Products intended for human consumption and for the feeding of animals (clause 1)
- Environmental samples in the area of primary production, food production and food handling (clause 1)
- Carcases of cattle, sheep, goats, horses and pigs — process hygiene criteria (Regulation (EC) No 2073/2005, items 2.1.1–2.1.2)
- Pasteurised milk and other pasteurised liquid dairy products, milk powder and whey powder, ice cream and frozen dairy desserts, egg products (Regulation (EC) No 2073/2005, items 2.2.1, 2.2.7, 2.2.8, 2.3.1)
- Tests for technological purposes including psychrotrophic Enterobacteriaceae — incubation at 30 °C (4.2, note)
Key parameters
| Parameter | Value |
|---|---|
| Medium | VRBG agar (crystal violet, neutral red, bile, glucose), composition according to Annex A (9.3.2) |
| Inoculation | pour plate: 1 ml of liquid sample or initial suspension per plate, one plate per dilution (two when only the initial suspension is used); about 15 ml of medium at 44–47 °C, at the latest 15 min after inoculation (9.3.1–9.3.2) |
| Overlay | about 5–10 ml of the same medium — prevents spreading growth and achieves semi-anaerobic conditions (9.3.3) |
| Incubation | 37 °C (or 30 °C), (24 ± 2) h, plates inverted (9.3.4) |
| Characteristic colonies | pink to red or purple, with or without precipitation haloes; plates with fewer than 150 colonies are counted (9.4) |
| Confirmation | 5 random colonies per plate: subculture on non-selective agar, oxidase test (negative), fermentation in glucose OF medium (positive) (9.5–9.6) |
| Result | cfu per 1 g, 1 ml, 1 cm² or sampling device; calculation according to ISO 7218 (clause 10) |
| Repeatability | r = 0.37 as the difference between log10-transformed results (11.2) |
Standard
- Standard number
- PN-EN ISO 21528-2:2017-08
- 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
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Sample and dilutions
The initial suspension and a single decimal dilution series are prepared according to ISO 6887 (9.2). Sampling is not part of the method — recommended standards: ISO/TS 17728, ISO 13307, ISO 17604, ISO 18593 (clause 7).
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Inoculation
Transfer to a sterile Petri dish 1 ml of the liquid sample or initial suspension; repeat with further dilutions, each time with a fresh sterile pipette; when only the initial suspension is used — two plates (9.3.1).
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Pouring the medium
Add about 15 ml of VRBG agar cooled in the water bath to 44–47 °C — at the latest 15 min after inoculating the dishes; mix by horizontal movements and allow to solidify on a cool surface (9.3.2).
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Overlay
After complete solidification add about 5–10 ml of the same medium to prevent spreading growth and achieve semi-anaerobic conditions; allow to solidify (9.3.3).
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Incubation
Invert the dishes and incubate at 37 °C for (24 ± 2) h (9.3.4).
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Counting and selection of colonies
On plates with fewer than 150 characteristic colonies (pink to red or purple, with or without precipitation haloes) count them and choose five at random for confirmation (all of them if fewer); count spreading colonies as single ones, discard the count if more than 1/4 of the plate is overgrown; with no characteristic colonies — choose five whitish colonies (9.4).
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Subculture
Streak the selected colonies on pre-dried non-selective agar so as to obtain well-isolated colonies, incubate at 37 °C for (24 ± 2) h and take a well-isolated colony from each plate (9.5).
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Oxidase test
Transfer part of the colony with a platinum/iridium loop or wire or a glass rod onto filter paper moistened with oxidase reagent; negative reaction — the colour does not turn dark blue-purple within 10 s (9.6.1).
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Glucose fermentation
Stab the oxidase-negative colonies into glucose OF medium, overlay with sterile mineral oil at least 1 cm thick and incubate at 37 °C for (24 ± 2) h; a yellow colour throughout the tube — positive reaction (9.6.2).
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Result
Oxidase-negative and glucose-positive colonies are confirmed as Enterobacteriaceae; the number is reported in cfu per 1 g, 1 ml, 1 cm² or sampling device, calculated according to ISO 7218 (9.6.3, clause 10).
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Apparatus for dry sterilization (oven) or wet sterilization (autoclave) | as specified in ISO 7218 (6.1) | — |
| Incubator | (37 ± 1) °C or (30 ± 1) °C (6.2) | — |
| Drying cabinet ventilated by convection or incubator | 25–50 °C (6.3) | — |
| Water baths | one capable of being maintained at 47–50 °C, another at 44–47 °C (6.4) | — |
| Test tubes or flasks | of appropriate capacity (6.5) | — |
| Petri dishes | glass or plastic, diameter about 90 mm and (optionally) about 140 mm (6.6) | — |
| Loops and wires | loops of about 3 mm diameter and wires of platinum/iridium or nickel/chromium, glass rods, or sterile disposable loops and needles; a nickel/chromium loop or wire is not used for the oxidase test (6.7, 9.6.1) | — |
| Pipettes | graduated or automatic, nominal capacities 10 ml, 1 ml and 0.1 ml (6.8) | — |
| pH meter | accurate to ± 0.1 pH unit at 25 °C (6.9) | — |
| Homogenizer | as specified in ISO 7218 (6.10) | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| VRBG agar (violet red bile glucose agar) | — | medium for pour plating and overlay; composition according to the data sheet of the medium made to the standard: per 1 000 ml, enzymatic digest of animal tissues 7.0 g, yeast extract 3.0 g, sodium chloride 5.0 g, bile salts No. 3 1.5 g, glucose 10.0 g, neutral red 0.03 g, crystal violet 0.002 g, agar 9–18 g, pH 7.4 ± 0.2; dissolved by boiling, without autoclaving; for performance testing the data sheet lists E. coli WDCM 00013 and 00012 (pink to red colonies with or without a precipitation zone) and Enterococcus faecalis (inhibited); preparation according to the standard — Annex A.2, beyond the available text |
| Non-selective agar | — | for subculturing the selected colonies, dried before use (9.5, Annex A.3) |
| Oxidase reagent | — | on filter paper or as commercial discs or sticks; negative reaction — the paper does not turn dark blue-purple within 10 s (9.6.1, Annex A.5) |
| Glucose OF medium | — | for the fermentation test, under a layer of sterile mineral oil at least 1 cm thick (9.6.2, Annex A.4, A.6) |
| Diluent | — | according to ISO 6887 (clause 5, 9.2) |
Health and safety (OHS)
- Tests for Enterobacteriaceae are only undertaken in properly equipped laboratories, under the control of a skilled microbiologist, with great care in the disposal of all incubated materials (warning of the standard)
- The standard does not address all safety aspects — establishing safety and health practices is the responsibility of the user (warning of the standard)
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 21528-2:2017-08 — “Microbiology of the food chain — Horizontal method for the detection and enumeration of Enterobacteriaceae — Part 2: Colony-count technique”.
How does this method work?
An initial suspension and decimal dilutions are prepared from the test sample (4.1).
What is the measuring range and accuracy?
Medium: VRBG agar (crystal violet, neutral red, bile, glucose), composition according to Annex A (9.3.2); Inoculation: pour plate: 1 ml of liquid sample or initial suspension per plate, one plate per dilution (two when only the initial suspension is used); about 15 ml of medium at 44–47 °C, at the latest 15 min after inoculation (9.3.1–9.3.2); Overlay: about 5–10 ml of the same medium — prevents spreading growth and achieves semi-anaerobic conditions (9.3.3); Incubation: 37 °C (or 30 °C), (24 ± 2) h, plates inverted (9.3.4).
How long does the test take?
The procedure comprises 10 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Apparatus for dry sterilization (oven) or wet sterilization (autoclave), Incubator, Drying cabinet ventilated by convection or incubator, Water baths, Test tubes or flasks, Petri dishes, Loops and wires, Pipettes, pH meter, Homogenizer.
Where is this test used?
Products intended for human consumption and for the feeding of animals (clause 1); Environmental samples in the area of primary production, food production and food handling (clause 1); Carcases of cattle, sheep, goats, horses and pigs — process hygiene criteria (Regulation (EC) No 2073/2005, items 2.1.1–2.1.2); Pasteurised milk and other pasteurised liquid dairy products, milk powder and whey powder, ice cream and frozen dairy desserts, egg products (Regulation (EC) No 2073/2005, items 2.2.1, 2.2.7, 2.2.8, 2.3.1); Tests for technological purposes including psychrotrophic Enterobacteriaceae — incubation at 30 °C (4.2, note).
What safety precautions apply?
Tests for Enterobacteriaceae are only undertaken in properly equipped laboratories, under the control of a skilled microbiologist, with great care in the disposal of all incubated materials (warning of the standard); The standard does not address all safety aspects — establishing safety and health practices is the responsibility of the user (warning of the standard).
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 21528-2.