🦠 Enterobacteriaceae in food and feed — detection with enrichment in BPW and isolation on VRBG agar

Microbiology PN-EN ISO 21528-1

Overview

ISO 21528-1:2017 specifies a method with enrichment for the detection of Enterobacteriaceae. It is applicable to products intended for human consumption and for the feeding of animals, and to environmental samples from the area of primary production and of the production and the distribution of food. The method is appropriate where the micro-organisms sought are likely to require resuscitation by enrichment and where their expected number is below 100 per millilitre or gram of sample. A limitation is the susceptibility of the method to great variability (clause 11 of the standard). Enumeration can be carried out by the most probable number (MPN) method after incubation in a liquid medium (annex A). The standard has been validated for test portions of 10 g or 10 ml.

A temperature of 37 °C for the enrichment and the isolation is used when Enterobacteriaceae are examined as an indicator of hygiene; 30 °C is chosen for technological purposes, when psychrotrophic Enterobacteriaceae also have to be covered. For the enumeration of Enterobacteriaceae by the colony-count method part 2 of the standard (ISO 21528-2) is used — and it is that part which Regulation (EC) No 2073/2005 names as the reference method in the process hygiene criteria (among others, carcasses after evisceration). In Poland part 1 is in force as PN-EN ISO 21528-1:2017-08; the earlier PN-ISO 21528-1:2005 covered detection and enumeration by the MPN method with pre-enrichment. In the teaching of food microbiology (UWM — University of Warmia and Mazury) the same scheme — buffered peptone water at 37 °C/18 h, isolation on VRBG at 37 °C/24 h, dark red colonies, confirmation by oxidase (no change of colour within 10 s) and by the fermentation of glucose — is described as the standard route for detecting the bacilli of this family.

Method principle

Buffered peptone water (BPW) is inoculated with the test portion and incubated at 37 °C (or 30 °C) for 18 h; typically 10 g of sample is mixed with 90 ml of BPW, giving a tenfold dilution. After the enrichment the culture is streaked with a loop onto the surface of violet red bile glucose agar (VRBG) and incubated at 37 °C; the plates are examined after 24 h for typical colonies of presumptive Enterobacteriaceae — pink to red or purple, with or without a halo of precipitated bile. Selected colonies (at least one typical or suspect colony, and where the result is negative up to four further ones; one of each morphology) are subcultured onto a non-selective medium (e.g. nutrient agar, 37 °C, 24 h ± 2 h) and confirmed by two tests: the oxidase reaction (filter paper impregnated with the oxidase reagent or ready-made discs; the test is negative when no dark blue-violet colour appears within 10 s; with a platinum-iridium loop, never a nickel-chromium one) and the glucose fermentation test — oxidase-negative colonies are stabbed into OF medium with glucose, covered with at least 1 cm of sterile mineral oil and incubated at 37 °C for 24 h ± 2 h; a yellow colour throughout the contents of the tube indicates a positive reaction. Some Enterobacteriaceae decolourise the colonies or the medium, which is why, in the absence of characteristic colonies, whitish colonies are selected for the confirmations. The result is reported as Enterobacteriaceae detected or not detected in a test portion of x g or x ml of product or on the surface swabbed.

Applications

• Food and feed in which the expected number of Enterobacteriaceae is below 100/g or 100/ml or in which the cells require resuscitation (heat-treated, dried and frozen products)
• Environmental samples from primary production and from establishments of the production and the distribution of food (swabs from surfaces and equipment)
• Indicator of process hygiene — enrichment and isolation at 37 °C
• Technological purposes covering psychrotrophic Enterobacteriaceae — the variant at 30 °C
• Semi-quantitatively: MPN determination after incubation in a liquid medium (annex A of the standard)

Key parameters

Standard

Standard number

PN-EN ISO 21528-1:2017-08 (ISO 21528-1:2017)

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania i oznaczania liczby Enterobacteriaceae — Część 1: Wykrywanie Enterobacteriaceae

Title (EN)

Microbiology of the food chain — Horizontal method for the detection and enumeration of Enterobacteriaceae — Part 1: Detection of Enterobacteriaceae

Step-by-step procedure

1. Test portion and initial suspension

   Weigh out the test portion (the standard is validated for 10 g or 10 ml) and mix it with nine times its volume of BPW (e.g. 10 g + 90 ml), giving a tenfold dilution; smaller test portions are permitted under the standard.

2. Enrichment

   Incubate the initial suspension at 37 °C (or at 30 °C for technological purposes) for 18 h ± 2 h.

3. Isolation on VRBG

   With a loop streak the surface of a plate of VRBG agar from the enrichment culture and incubate at 37 °C for 24 h ± 2 h.

4. Selection of the colonies

   Mark the suspect colonies (pink to red or purple, with or without a halo of precipitated bile). Select at least one typical colony; if the confirmation proves negative — up to four further ones. Where there are several morphologies — one of each; in the absence of characteristic colonies — whitish colonies.

5. Subculture

   Streak each selected colony onto a non-selective medium (e.g. nutrient agar) and incubate at 37 °C for 24 h ± 2 h; take a well-isolated colony for the tests.

6. Oxidase test

   With a platinum-iridium loop, a wire or a glass rod smear part of the colony on filter paper impregnated with the oxidase reagent (or on a commercial disc). The reaction is negative when the paper does not turn dark blue-violet within 10 s.

7. Glucose fermentation test

   Stab the oxidase-negative colonies with a wire into tubes of OF medium with glucose, cover them with a layer of at least 1 cm of sterile mineral oil and incubate at 37 °C for 24 h ± 2 h. A yellow colour throughout the contents of the tube = a positive reaction.

8. Result

   Consider colonies that are oxidase-negative and ferment glucose to be Enterobacteriaceae. Report: detected / not detected in a test portion of x g or x ml of product or on the surface swabbed.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Enterobacteriaceae include intestinal pathogens (Salmonella, pathogenic E. coli) — work under the conditions of a microbiological laboratory, disinfection and autoclaving of the spent cultures
• Mineral oil and the oxidase reagent — avoid contact with the skin and the eyes
• Agar media at 47–50 °C — risk of burns when pouring

Frequently asked questions