Introduction

Pseudomonas species are Gram-negative, rod-shaped bacteria commonly found in water, soil, air, and food processing environments. Although many Pseudomonas species are not primary human pathogens, they are major spoilage organisms and important indicators of hygiene and cold-chain integrity, especially in refrigerated foods.

ISO 11059:2009 provides a horizontal method for the detection and enumeration of Pseudomonas spp. in food and animal feeding stuffs. The method is particularly relevant for chilled and minimally processed foods where Pseudomonas can grow at low temperatures and cause spoilage.

This guide presents a simplified, educational overview of ISO 11059:2009, covering the principle, materials, procedure, interpretation of results, reporting, and practical precautions.

Why Pseudomonas Testing Matters

Testing for Pseudomonas spp. is important because:

• Spoilage detection – Pseudomonas is a leading cause of spoilage in meat, fish, milk, and refrigerated foods.
• Cold-chain verification – Their presence in high numbers often indicates temperature abuse during storage or transport.
• Water and environment monitoring – Indicates contamination from water, surfaces, or processing environments.
• Shelf-life assessment – High Pseudomonas counts are associated with off-odors, slime formation, and discoloration.
• Quality control – Helps food processors monitor hygiene and sanitation effectiveness.

Common Pseudomonas Species in Foods

Species	Common Source	Significance
Pseudomonas fluorescens	Milk, water, chilled foods	Spoilage, off-flavors
Pseudomonas aeruginosa	Water, equipment, hands	Opportunistic pathogen, hygiene indicator
Pseudomonas putida	Meat, fish, vegetables	Spoilage during refrigeration

ISO 11059 focuses on enumeration of presumptive Pseudomonas spp., not species-level identification.

Principle of the ISO 11059:2009 Method

The ISO 11059 method is a culture-based enumeration technique:

• Food samples are homogenized and serially diluted.
• Dilutions are plated on selective agar for Pseudomonas (e.g. Pseudomonas Agar Base with CFC supplement).
• Plates are incubated at 25–30 °C.
• Colonies with typical Pseudomonas morphology are counted.
• Results are expressed as CFU/g or CFU/mL.

The selective agents suppress competing flora, allowing Pseudomonas spp. to grow and form characteristic colonies.

Materials & Equipment

Consumables

• Sterile sample bags or containers
• Sterile pipettes and pipette tips
• Sterile diluent (buffered peptone water, saline, or phosphate buffer)
• Pseudomonas selective agar (e.g. Pseudomonas Agar Base + CFC supplement)
• Sterile Petri dishes
• Sterile spreaders

Equipment

• Incubator (25–30 °C)
• Stomacher or vortex mixer
• Colony counter (manual or digital)
• Sterile dilution tubes or bottles

Personal Protective Equipment (PPE)

• Lab coat
• Gloves
• Eye protection

Follow biosafety and aseptic handling procedures at all times.

Step-by-Step Test Procedure

1. Sample Preparation

• Weigh 10 g of solid food or measure 10 mL of liquid sample.
• Transfer into a sterile container.
• Add 90 mL sterile diluent to obtain a 10⁻¹ dilution.
• Homogenize thoroughly using a stomacher or vortex mixer.

Proper homogenization ensures even distribution of bacteria.

2. Serial Dilutions

• Transfer 1 mL of the 10⁻¹ dilution into 9 mL sterile diluent → 10⁻².
• Continue preparing further dilutions (10⁻³, 10⁻⁴, etc.) as needed.
• Mix each dilution thoroughly.

Tip: Select dilutions likely to give 20–300 colonies per plate.

3. Inoculation of Agar Plates

• Label plates with sample ID and dilution.
• Pipette 0.1 mL or 1 mL (depending on lab SOP) onto selective agar.
• Spread evenly using a sterile spreader.
• Allow the inoculum to absorb into the agar surface.

4. Incubation

• Incubate plates at 25–30 °C for 44–48 hours.
• Plates should be inverted to prevent condensation.
• Avoid stacking too many plates together.

5. Colony Examination & Counting

• Select plates with countable colonies.
• Typical Pseudomonas colonies may appear:
  • Flat to slightly raised
  • Pale, greenish, or translucent
  • Sometimes fluorescent under UV light (depending on species and medium)

Count all presumptive Pseudomonas colonies.

Calculation of Pseudomonas Count

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

Example:

• Colonies counted at 10⁻² dilution = 60
• Volume plated = 1 mL

CFU/g = 60 × 10² = 6.0 × 10³ CFU/g

Results Interpretation

Pseudomonas Count	Interpretation	Recommended Action
Low (<10² CFU/g)	Good hygiene and storage	Routine monitoring
Moderate (10²–10⁴ CFU/g)	Acceptable but trending upward	Review storage and sanitation
High (>10⁴ CFU/g)	Poor hygiene or cold-chain failure	Immediate investigation

⚠️ High counts indicate quality and shelf-life issues, not necessarily foodborne illness.

Practical Applications in Foods

• Fresh Meat & Fish: Indicates spoilage and temperature abuse.
• Milk & Dairy Products: Suggests post-pasteurization contamination.
• Ready-to-Eat Foods: Signals cross-contamination from equipment or water.
• Vegetables & Salads: Reflects environmental or wash-water contamination.

Common Errors & Troubleshooting

Issue	Likely Cause	Corrective Action
No growth	Over-selective medium or stressed cells	Plate lower dilution or review sample handling
Too many colonies	Under-dilution	Prepare higher dilutions
Spreading growth	Over-inoculation	Reduce volume plated
Variable counts	Poor homogenization	Improve mixing and pipetting accuracy

Recording & Reporting

A Pseudomonas test report should include:

• Sample identification and description
• Date of analysis
• Dilutions tested
• Colony counts
• Final CFU/g or CFU/mL
• Method reference: ISO 11059:2009
• Analyst name and signature

Accurate reporting supports traceability and quality assurance.

Practical Tips for Reliable Results

• Use freshly prepared selective media.
• Verify incubator temperature regularly.
• Plate samples in duplicates for accuracy.
• Trend results over time to detect hygiene issues early.

References

• ISO 11059:2009 — Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of Pseudomonas spp.
  https://www.iso.org/standard/46325.html
• FAO/WHO Codex Alimentarius — Microbiological Criteria & Hygiene
  https://www.fao.org/fao-who-codexalimentarius/en/
• CDC — Pseudomonas aeruginosa Overview
  https://www.cdc.gov/pseudomonas
• EFSA — Foodborne & Environmental Bacteria
  https://www.efsa.europa.eu/en/topics/topic/foodborne-zoonotic-diseases