🔴 Detection of Listeria monocytogenes
Two-stage selective enrichment and chromogenic plating method for Listeria monocytogenes detection in food. A critical food safety test.
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
| Parameter | Value |
|---|---|
| Result | Detected / Not detected in 25 g |
| Sample size | 25 g (standard) |
| Time to result | 5–7 working days |
| Enrichment I temperature | 30 ± 1°C (Half Fraser) |
| Enrichment II temperature | 37 ± 1°C (Fraser) |
| Limit of detection | 1 cell / 25 g |
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.
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.).
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).
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.
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.
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).
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.
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).
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
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator 30±1°C | Memmert IN110, Binder BD 115 | 5 000–15 000 PLN |
| Incubator 37±1°C | Memmert IN110, POL-EKO ST 2 | 5 000–15 000 PLN |
| Stomacher (homogenizer) | Seward Stomacher 400 Circulator | 12 000–25 000 PLN |
| Autoclave | Tuttnauer 3870ELV, Systec VX-75 | 30 000–80 000 PLN |
| Class II biosafety cabinet | ESCO Airstream, Thermo MSC-Advantage | 25 000–60 000 PLN |
| Microscope with Henry illumination | Olympus CX43, for colony morphology observation | 8 000–20 000 PLN |
| Automatic pipettes | Eppendorf Research Plus 0.1 mL + 1 mL | 800–1 500 PLN/pc. |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Half Fraser broth | — | Fraser base + half concentration of acriflavine (12.5 mg/L) and nalidixic acid (10 mg/L). Merck 1.10398, Oxoid CM0895. |
| Fraser broth (full) | — | Full inhibitor concentration: acriflavine 25 mg/L, nalidixic acid 20 mg/L, lithium chloride 3 g/L, aesculin + ammonium iron(III) citrate. Merck 1.10399. |
| ALOA agar (chromogenic) | — | Chromogenic medium: L. monocytogenes = blue-green colonies with an opaque halo (PI-PLC+). Other Listeria = blue-green WITHOUT halo. Merck 1.00427, bioMérieux. |
| Oxford agar | — | With supplement (cycloheximide, colistin, acriflavine, cefotetan, phosphates). Colonies: black with black halo (aesculin). Oxoid CM0856. |
| TSYEA agar | — | Tryptic Soy Yeast Extract Agar — for pure culture isolation. TSA + 0.6% yeast extract. |
| 5% sheep blood agar | — | For the haemolysis test — L. monocytogenes produces a narrow zone of β-haemolysis. |
| Staphylococcus aureus strain ATCC 25923 | — | For the CAMP test — enhanced haemolysis in the S. aureus zone. |
| Rhodococcus equi strain ATCC 6939 | — | For the CAMP test — no enhancement (differentiation from L. ivanovii). |
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