🔴 Detection of Listeria monocytogenes

Microbiology PN-EN ISO 11290-1

Overview

Listeria monocytogenes is a Gram-positive rod and one of the most dangerous foodborne pathogens. It causes listeriosis — a disease with 20–30% mortality (the highest among foodborne illnesses). Particularly at risk are pregnant women (miscarriages, neonatal infections), the elderly, and immunocompromised individuals.

L. monocytogenes is exceptionally hazardous because it: multiplies at refrigeration temperatures (2–4°C), tolerates high salt concentrations (up to 10% NaCl), and survives across a wide pH range (4.4–9.6). It forms biofilms on food production facility surfaces, making its elimination difficult.

Standard PN-EN ISO 11290-1 is the reference method for L. monocytogenes detection in all types of food and feed. Required by Regulation (EC) 2073/2005 — safety criterion: absent in 25 g for ready-to-eat food intended for infants and for medical purposes, and ≤100 CFU/g in ready-to-eat food during its shelf life.

Method principle

Two-stage selective enrichment method:

1. **Primary enrichment (Half Fraser)** — 25 g sample + 225 mL Half Fraser broth (half concentration of acriflavine and nalidixic acid). Incubation 30°C / 24–26 h. Mild selection allowing regeneration of injured cells. 2. **Secondary enrichment (full Fraser)** — 0.1 mL → 10 mL full Fraser broth (full inhibitor concentration). Incubation 37°C / 24–48 h. Darkening of broth (aesculin hydrolysis → black precipitate) suggests the presence of Listeria. 3. **Plating on selective media** — ALOA (chromogenic) and Oxford/PALCAM. L. monocytogenes colonies on ALOA: blue-green with an opaque halo (phosphatidylinositol-specific phospholipase C, PI-PLC). 4. **Confirmation** — haemolysis on blood agar, CAMP test, motility at 25°C (umbrella pattern), sugar fermentation (rhamnose+, xylose–).

Applications

• Ready-to-eat foods — soft cheese, deli meats, smoked fish
• Infant foods and foods for special medical purposes
• Meat and meat products (poultry, pork)
• Fish products — raw and smoked (salmon, mackerel)
• Dairy products — cheese, ice cream, pasteurized milk
• Ready-to-eat vegetables and fruits (salads, smoothies)
• Environmental monitoring of facilities (surface, drain, and cold room swabs)

Key parameters

Standard

Standard number

PN-EN ISO 11290-1:2017

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania i oznaczania liczby Listeria monocytogenes i Listeria spp. — Część 1: Metoda wykrywania

Title (EN)

Microbiology of the food chain — Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. — Part 1: Detection method

Step-by-step procedure

1. Sample preparation

   Weigh 25 g of sample into a stomacher bag. Add 225 mL of Half Fraser broth. Homogenize for 1–2 min.

   ⏱ Time: 5 min

2. Enrichment I — Half Fraser

   Incubate the bag with sample at 30±1°C for 24–26 hours. After incubation, observe the broth — darkening suggests aesculin hydrolysis (presence of Listeria spp.).

   ⏱ Time: 24–26 h • 🌡 Temperature: 30°C

3. Enrichment II — full Fraser

   Transfer 0.1 mL of Half Fraser enrichment to 10 mL of full Fraser broth. Incubate at 37±1°C / 24 hours (first reading) and up to 48 hours (second reading).

   ⏱ Time: 24–48 h • 🌡 Temperature: 37°C

4. Plating from Half Fraser (24 h)

   From the Half Fraser enrichment, streak with a loop onto ALOA agar and a second medium (Oxford or PALCAM). Incubate at 37°C / 24–48 h.

   🌡 Temperature: 37°C

5. Plating from Fraser (24 h and 48 h)

   After 24 h and 48 h of Fraser broth, streak with a loop onto ALOA agar and a second medium. Incubate at 37°C / 24–48 h.

   🌡 Temperature: 37°C

6. Plate reading

   On ALOA: L. monocytogenes colonies = blue-green with an opaque halo (PI-PLC+). On Oxford: black colonies with a black halo (aesculin+).

7. Pure culture isolation

   Select min. 5 suspect colonies. Subculture onto TSYEA agar. Incubate at 37°C / 18–24 h. Observe colonies: L. monocytogenes — small, greyish, glistening under Henry illumination (blue-grey sheen).

   ⏱ Time: 24 h • 🌡 Temperature: 37°C

8. Haemolysis test

   From pure culture, streak onto 5% sheep blood agar. Incubate at 37°C/24 h. L. monocytogenes: narrow zone of β-haemolysis (clear halo around colonies).

9. CAMP test

   Streak S. aureus and R. equi vertically on blood agar. Streak the test isolate perpendicularly. Incubate at 37°C/18–24 h. L. monocytogenes: enhanced haemolysis near S. aureus, no enhancement near R. equi.

   🌡 Temperature: 37°C

10. Motility test

    Inoculate semi-solid medium (0.4% motility agar). Incubate at 25°C/48 h. L. monocytogenes: "umbrella" growth pattern 2–5 mm below the surface (motile at 25°C, non-motile at 37°C).

    🌡 Temperature: 25°C

11. Sugar fermentation

    Rhamnose: POSITIVE (L. monocytogenes). Xylose: NEGATIVE. Mannitol: NEGATIVE. These 3 tests differentiate L. monocytogenes from other Listeria species.

12. Interpretation and reporting

    Confirmation = β-haemolysis + CAMP+ near S. aureus + umbrella motility + rhamnose+ xylose– → "Listeria monocytogenes DETECTED in 25 g". Absence = "NOT DETECTED".

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Listeria monocytogenes — BSL-2 pathogen! Work exclusively in a laminar flow cabinet
• Especially dangerous for pregnant women — pregnant personnel must be excluded from work with L. monocytogenes
• Nitrile gloves, laboratory coat, and safety glasses mandatory
• All waste must be autoclaved (121°C/20 min) before disposal
• Workstation disinfection after use: 70% ethanol + 0.5% sodium hypochlorite solution
• Sheep blood — potential allergen source, handle with caution

Frequently asked questions