Reviewed by: Darwin Benedict,BSc Biochem, MPH, Co-Editor. January 11, 2026

Introduction

Listeria is a genus of Gram-positive bacteria, with Listeria monocytogenes being the most notable pathogen causing listeriosis — a serious foodborne illness. Listeriosis can be life-threatening, particularly for pregnant women, newborns, elderly individuals, and immunocompromised people.

ISO 11290-2 — titled Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of Listeria spp. — Part 2: Enumeration by plating — is the international standard for detecting and enumerating Listeria spp. in food.

Monitoring Listeria is critical for food safety, regulatory compliance, and public health. This blog provides a step-by-step, simplified guide to ISO 11290-2, suitable for food safety students, laboratory professionals, and quality control personnel.

Why Listeria spp. Testing Matters

• Food safety: Listeria monocytogenes can survive refrigeration and some food processing steps, making detection crucial in ready-to-eat (RTE) foods, dairy, meat, and seafood.
• Indicator of hygiene: Presence of Listeria indicates potential contamination in the production environment.
• Regulatory compliance: Many countries mandate limits for Listeria in RTE foods, with zero tolerance in some cases.
• Public health protection: Early detection prevents outbreaks and ensures consumer safety.

Listeria enumeration differs from general microbial tests as it focuses on specific pathogenic species, rather than general hygiene indicators.

Principle of ISO 11290-2

The ISO 11290-2 method is a culture-based enumeration technique with two main stages:

1. Sample enrichment: Food samples are homogenized and pre-enriched in non-selective broth, then transferred to selective enrichment broth to favor Listeria growth.
2. Plating on selective agar: Samples are plated on Listeria selective agar such as Oxford agar or PALCAM agar, which inhibits most non-target bacteria.
3. Incubation: Plates are incubated at 30 °C for 24–48 hours.
4. Colony identification: Listeria colonies are recognized by characteristic morphology, color, and halo formation.
5. Enumeration: Presumptive colonies are counted and expressed as CFU/g or CFU/mL.

Optional confirmation tests (e.g., catalase test, Gram staining, or biochemical tests) are recommended for regulatory compliance.

Materials & Equipment

Consumables

• Sterile sample containers (bags or tubes)
• Sterile pipettes and tips
• Sterile diluent (e.g., buffered peptone water)
• Non-selective enrichment broth (e.g., Half Fraser broth)
• Selective enrichment broth (e.g., Fraser broth)
• Listeria selective agar plates (Oxford or PALCAM)
• Sterile spreaders

Equipment

• Incubator (30 °C ±1 °C)
• Stomacher or vortex mixer
• Colony counter (manual or digital)
• Calibrated pipettes
• Confirmatory test kits (optional)

Personal Protective Equipment (PPE)

• Lab coat, gloves, eye protection
• Follow biosafety protocols for handling pathogenic bacteria

Step-by-Step Test Procedure

1. Sample Preparation

1. Weigh 25 g of solid food or measure 25 mL of liquid sample.
2. Aseptically place the sample into a sterile stomacher bag.
3. Add 225 mL of Half Fraser broth for a 1:10 dilution.
4. Homogenize using a stomacher for 1–2 minutes.

2. Pre-Enrichment

1. Incubate homogenized samples at 30 °C for 24 ± 2 hours.
2. This allows stressed Listeria cells to recover before selective enrichment.

3. Selective Enrichment

1. Transfer 0.1 mL of pre-enriched sample into 10 mL Fraser broth (selective enrichment).
2. Incubate at 37 °C for 24–48 hours.
3. Selective enrichment suppresses background flora and favors Listeria growth.

4. Plating on Selective Agar

1. Label Petri dishes with sample ID and dilution factor.
2. Pipette 0.1 mL of selective enrichment onto Listeria selective agar (Oxford or PALCAM).
3. Spread evenly using a sterile spreader.
4. Allow plates to absorb before incubation.

5. Incubation

1. Incubate plates at 30 °C ±1 °C for 24–48 hours.
2. Avoid stacking plates too tightly to ensure uniform growth.

6. Colony Identification

• Presumptive Listeria colonies on Oxford agar appear grey-green with black halos due to esculin hydrolysis.
• Colonies on PALCAM agar typically show grey-green colonies with black centers surrounded by inhibitory zones.
• Optional confirmatory tests:
  • Catalase test: Listeria is catalase positive.
  • Gram stain: Small Gram-positive rods.
  • Biochemical tests or PCR for species confirmation.

7. Counting Colonies

1. Count colonies on plates with 10–150 colonies for accuracy.
2. Only include characteristic Listeria colonies.

Calculation Formula: CFU/g (or mL)= (Average colony count×Dilution factor)/Volume plated (mL)

Example Calculation:

• Colonies on 10⁻¹ plate = 25
• Volume plated = 0.1 mL

CFU/g=25×1010.1=2.5×103 CFU/g

Interpreting Results

CFU/g or CFU/mL	Interpretation	Recommended Action
Low or undetectable	Good hygiene and low contamination risk	Routine monitoring
Moderate	Potential contamination	Review sanitation and handling
High	High contamination risk	Immediate corrective action; investigate sources

Listeria results are critical for RTE foods, as even low counts can pose a risk due to the pathogen’s ability to grow at refrigeration temperatures.

Practical Applications in Food Safety

• Ready-to-eat (RTE) foods: Meat, dairy, and salads — zero tolerance for L. monocytogenes in many countries.
• Dairy products: Cheese, milk, and ice cream — monitor post-pasteurization contamination.
• Meat and seafood: Assess contamination during processing, handling, and storage.
• Environmental monitoring: Surfaces, equipment, and water sources in food production.

Consistent monitoring supports HACCP, ISO 22000, and regulatory compliance, protecting consumers and brands.

Common Errors & Troubleshooting

Problem	Possible Cause	Recommended Action
No colonies	Over-dilution, non-viable cells	Plate lower dilutions or check sample storage
Too many colonies	Under-dilution	Plate higher dilutions
Non-characteristic colonies	Contamination or incorrect media	Verify agar and aseptic technique
Inconsistent counts	Uneven mixing or plating errors	Improve homogenization and pipetting accuracy

Recording & Reporting

A complete ISO 11290-2 test report should include:

• Sample ID, description, and collection date
• Dilutions tested
• Colony counts and dilution factor
• Final CFU/g or CFU/mL result
• Method referenced: ISO 11290-2
• Analyst initials or signature

Proper records ensure traceability, reproducibility, and compliance.

Tips for Accurate Testing

• Use fresh, validated selective agar and enrichment broths.
• Maintain precise temperature and incubation times.
• Plate duplicates for reliability.
• Track trends over time to identify hygiene lapses early.
• Include confirmatory testing for regulatory compliance.

Web References

1. ISO 11290-2:2017 — Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of Listeria spp.
   https://www.iso.org/standard/60331.html
2. FAO/WHO Codex Alimentarius — Microbiological Criteria for Foods
   https://www.fao.org/fao-who-codexalimentarius
3. World Health Organization (WHO) — Listeria monocytogenes and Food Safety
   https://www.who.int/health-topics/food-safety
4. Centers for Disease Control and Prevention (CDC) — Listeria
   https://www.cdc.gov/listeria

Disclaimer

This blog is provided for educational purposes only. Although it reflects ISO 11290-2 methodology, laboratories must follow validated SOPs, quality management systems, and local regulations. Results should be interpreted by qualified microbiology personnel. This guide does not replace formal laboratory training or accreditation.