🦠 Listeria monocytogenes and Listeria spp. in food — enumeration on ALOA agar (Ottaviani-Agosti)

Microbiology PN-EN ISO 11290-2

Overview

ISO 11290-2:2017 specifies a horizontal method for the enumeration of L. monocytogenes and of Listeria spp. (including L. monocytogenes) in products intended for human consumption and for the feeding of animals and in environmental samples from the production and the distribution of food; some newly described species of Listeria may not be enumerated or confirmed by this method. The second edition replaced ISO 11290-2:1998 with amendment A1:2004; the main changes: the initial suspension in buffered peptone water or in half-Fraser broth (with or without supplements) and in the diluents of ISO 6887, the removal of the resuscitation stage, the microscopic, catalase and CAMP tests as optional, new performance characteristics and the inclusion of the enumeration of Listeria spp. in the scope. The changes were regarded as significant (ISO 17468) but without any appreciable effect on the characteristics of the method. Commission Regulation (EC) No 2073/2005 indicates EN/ISO 11290-2 as the reference method for the safety criterion of 100 cfu/g for ready-to-eat food (in which the growth of L. monocytogenes is possible or is not possible; a plan of 5 samples, c = 0). Listeria are Gram-positive short rods without spores or a capsule, facultatively anaerobic and psychrotrophic, motile at 20–25 °C, catalase-positive and oxidase-negative; L. monocytogenes grows from −2 °C to 45 °C and at pH 4.4–9, and listeriosis has a mortality of 20–30 % — the examinations must be carried out in laboratories of biological safety level 2, and pregnant persons and persons with impaired cell-mediated immunity should not work with the cultures.

Method principle

The enumeration requires five successive stages (the scheme is in annex A): preparation of the initial suspension in a diluent suitable for the sample; spread plating onto Listeria agar according to Ottaviani and Agosti of a defined amount of the liquid sample or of the initial suspension and, where necessary, of the decimal dilutions; incubation at 37 °C with assessment after 24 h and after a further 24 h; confirmation of the presumptive colonies by morphological and biochemical tests; calculation of the number per g, ml, cm² or sampling device. As the diluent for the initial suspension buffered peptone water or another diluent of ISO 6887 is used; where detection according to ISO 11290-1 is also carried out on the same sample, half-Fraser broth may be used (the selective agents being added preferably after the plating for the enumeration; with supplemented half-Fraser the plates are to be inoculated within 45 min). 0.1 ml is plated onto a small plate (90 mm); for low numbers the limit of detection is lowered tenfold by examining 1 ml spread over a large plate (140 mm) or over three small ones (in duplicate where only the initial suspension is used); the inoculum is spread quickly with a spreader without touching the edges and is left for about 15 min to be absorbed. The plates are incubated inverted at 37 °C for 24 h ± 2 h and for a further 24 h ± 2 h. L. monocytogenes forms blue-green colonies surrounded by an opaque (opalescent) zone (so does L. ivanovii; strains after acid stress may have a weak halo or none; rare strains with a slow PIPLC activity appear after > 4 days); Listeria spp. — blue-green with or without a halo (other organisms may give blue colonies — annex C). Plates with < 150 characteristic colonies (< 360 on 140 mm) are counted, and with mixed cultures or large overlapping halos — < 100 (< 240). For the confirmation 5 colonies representative of the types are taken from each plate, subcultured onto a non-selective medium (blood agar, nutrient agar, TSYEA) and incubated at 37 °C for 18–24 h. The tests obligatory for L. monocytogenes: β-haemolysis +, L-rhamnose +, D-xylose −; the optional ones: the microscopic picture (thin short rods/coccobacilli), catalase +, motility at 25 °C +, the CAMP test +; rare non-haemolytic strains require additional tests (Gram, catalase, motility, CAMP, PCR). Miniaturised biochemical galleries and alternative procedures according to ISO 7218 are allowed once their suitability has been verified.

Applications

• The safety criterion of 100 cfu/g of L. monocytogenes in ready-to-eat food — the reference method of Regulation (EC) No 2073/2005 (categories 1.2 and 1.3)
• Cheeses and milk products, ready-to-eat cured and meat products, smoked fish, salads, chilled products with a long shelf life
• Environmental samples (surfaces, equipment) in establishments producing RTE food — monitoring of Listeria spp. as an indicator
• Feed and raw materials; shelf-life studies (challenge tests) requiring a number of Listeria
• Combined examination for detection (ISO 11290-1) and enumeration from a single suspension in half-Fraser broth

Key parameters

Standard

Standard number

PN-EN ISO 11290-2:2017-07 (ISO 11290-2:2017)

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania i oznaczania liczby Listeria monocytogenes i innych Listeria spp. — Część 2: Metoda oznaczania liczby

Title (EN)

Microbiology of the food chain — Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. — Part 2: Enumeration method

Step-by-step procedure

1. Sample and suspension

   Prepare the sample according to ISO 6887 (or ISO 18593 for surfaces); prepare the initial suspension in buffered peptone water or in another diluent of ISO 6887, or in half-Fraser broth where you are at the same time carrying out detection according to ISO 11290-1 (add the supplement after the plating; with the supplement plate within 45 min).

2. Spread plating

   Apply 0.1 ml of the suspension (or of the liquid sample) and 0.1 ml of the successive dilutions onto 90 mm ALOA plates; for low numbers — 1 ml onto a 140 mm plate or onto three 90 mm plates (dried), in duplicate where you use only the initial suspension. Spread it quickly with a spreader (a new one for each dilution or the same one starting from the highest dilution) and leave it for about 15 min to be absorbed.

3. Incubation

   Incubate the inverted plates at 37 °C for 24 h ± 2 h, assess them and incubate them for a further 24 h ± 2 h.

4. Counting

   After 24 h (before the halos overlap) and after 48 h count the blue-green colonies with an opaque halo (presumptive L. monocytogenes) and the blue-green ones with or without a halo (presumptive Listeria spp.) on the plates with < 150 (90 mm) or < 360 (140 mm) characteristic colonies; with mixed cultures or large halos — < 100 / < 240. Treat three 90 mm plates with 1 ml as one.

5. Confirmation

   From each plate (dilution) take 5 colonies representative of the types (where there are < 5 — all of them), subculture them onto a non-selective medium (L. monocytogenes: blood agar, nutrient agar or TSYEA; Listeria spp.: TSYEA) and incubate them at 37 °C for 18–24 h. Carry out the obligatory tests: β-haemolysis, L-rhamnose, D-xylose (with controls), optionally catalase, motility at 25 °C, CAMP and the microscopic picture; with typical colonies without haemolysis — additional tests (Gram, catalase, motility, CAMP, PCR).

6. Calculation and test report

   From the number of confirmed colonies calculate the number of L. monocytogenes and/or of Listeria spp. per g, ml, cm² or sampling device; compare it with the criterion of 100 cfu/g (Regulation 2073/2005) and describe any deviations.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• L. monocytogenes — work exclusively in laboratories of biological safety level 2, under the supervision of a microbiologist, with careful disposal of the incubated materials
• Pregnant persons and persons with diseases impairing cell-mediated immunity should not work with cultures of Listeria (a risk to the foetus)
• CAMP tests with S. aureus and R. equi and blood agar — handle as infectious material

Frequently asked questions