Reviewed by: Darwin Benedict,BSc Biochem, MPH, Co-Editor. January 10, 2026

Introduction

Staphylococcus aureus is a Gram-positive bacterium commonly found on human skin, mucous membranes, and in the environment. Certain strains can produce enterotoxins, leading to foodborne illnesses, commonly referred to as staphylococcal food poisoning. Contaminated food, improper handling, or inadequate hygiene practices can lead to S. aureus contamination.

ISO 6888-1 — titled Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coagulase-positive staphylococci — Part 1: Technique using Baird-Parker agar — is the internationally recognized standard for counting S. aureus in food.

This blog provides a simplified, step-by-step guide for educational purposes, helping food safety students, professionals, and laboratory staff understand the method, interpret results, and maintain accurate records while optimizing for search engines.

Why Staphylococcus aureus Testing Matters

• Food safety: S. aureus produces heat-stable toxins that can survive cooking, making detection in raw and processed foods critical.
• Indicator of hygiene: High counts suggest poor handling, contaminated equipment, or inadequate sanitation.
• Regulatory compliance: Many countries enforce limits on coagulase-positive staphylococci in food products, especially dairy, meat, and ready-to-eat foods.
• Public health protection: Early detection prevents outbreaks and ensures consumer safety.

Unlike general microbial counts, testing specifically for S. aureus identifies the risk of enterotoxin formation.

Principle of ISO 6888-1

The ISO 6888-1 method uses Baird-Parker agar, a selective medium that supports the growth of S. aureus while inhibiting most other bacteria.

Key steps:

1. Sample dilution to obtain countable bacterial levels.
2. Plating on Baird-Parker agar enriched with egg yolk and tellurite.
3. Incubation at a defined temperature, typically 37 °C for 24–48 hours.
4. Identification of presumptive colonies based on characteristic black or grey colonies with clear zones (egg yolk reaction).
5. Confirmation tests such as coagulase or latex agglutination (optional for regulatory testing).
6. Enumeration and reporting as colony-forming units per gram or milliliter (CFU/g or CFU/mL).

Materials & Equipment

Consumables

• Sterile sample containers (bags or tubes)
• Sterile pipettes and tips
• Sterile diluent (e.g., peptone water)
• Baird-Parker agar plates (with egg yolk tellurite enrichment)
• Sterile spreaders

Equipment

• Incubator (37 °C ±1 °C)
• Stomacher or vortex mixer
• Colony counter (manual or digital)
• Calibrated pipettes
• Coagulase test kits or latex agglutination reagents (optional for confirmation)

Personal Protective Equipment (PPE)

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

Step-by-Step Test Procedure

1. Sample Preparation

1. Weigh 10 g of solid food or measure 10 mL of liquid sample.
2. Aseptically place the sample in a sterile container.
3. Add 90 mL sterile diluent to achieve the 10⁻¹ dilution.
4. Homogenize thoroughly using a stomacher or vortex to ensure uniform bacterial distribution.

2. Serial Dilutions

1. Transfer 1 mL from the 10⁻¹ dilution into 9 mL sterile diluent → 10⁻² dilution.
2. Repeat as needed to produce further dilutions (10⁻³, 10⁻⁴, etc.), depending on expected S. aureus levels.
3. Ensure thorough mixing at each dilution step.

3. Inoculation of Baird-Parker Agar Plates

1. Label Petri dishes with sample ID and dilution factor.
2. Pipette 0.1–1 mL of each selected dilution onto Baird-Parker agar.
3. Spread evenly using a sterile spreader.
4. Allow plates to stand for a few minutes to absorb.

4. Incubation

1. Place plates in the incubator at 37 °C ±1 °C.
2. Incubate for 24–48 hours.
3. Avoid stacking plates too tightly to ensure even temperature exposure.

5. Colony Identification

After incubation:

• Presumptive S. aureus colonies appear as black or grey, shiny colonies with clear zones (egg yolk reaction).
• Colonies may be round, convex, and 1–3 mm in diameter.

Optional confirmation tests:

• Coagulase test: Confirms the presence of coagulase-positive S. aureus.
• Latex agglutination: Rapid method for confirmation.

Count only colonies with characteristic morphology for enumeration.

Calculating Colony Counts

Use the standard formula: CFU/g (or CFU/mL)= (Average colony count × Dilution factor) / Volume plated (mL)

Example Calculation:

• Colonies on 10⁻³ dilution plate = 55
• Volume plated = 0.1 mL

CFU/g=55×1030.1=5.5×105 CFU/g

Always select plates with 20–150 colonies for statistical accuracy.

Interpreting Results

CFU/g or CFU/mL	Interpretation	Recommended Action
Low (<10²)	Good hygiene	Routine monitoring
Moderate (10²–10⁴)	Possible contamination	Review cleaning and handling
High (>10⁴)	Significant risk	Immediate corrective action; investigate sources

⚠️ Remember: High S. aureus counts may indicate risk for enterotoxin formation, even if the bacteria are later killed during cooking. Prompt action is essential.

Practical Applications in Food Safety

• Dairy products: Monitoring pasteurized milk, cheeses, and ice cream for post-processing contamination.
• Meat and poultry: Ensuring hygienic handling during slaughter, processing, and storage.
• Ready-to-eat foods: Preventing cross-contamination from equipment, handlers, or surfaces.
• Bakery products and sandwiches: Detecting contamination that could lead to toxin formation.

Consistent monitoring allows early corrective action and supports HACCP and ISO food safety systems.

Common Errors and Troubleshooting

Problem	Possible Cause	Recommended Action
No colonies	Over-dilution	Plate lower dilutions
Too many colonies (TMTC)	Under-dilution	Use higher dilutions
Non-characteristic colonies	Contamination or incorrect media	Verify media and aseptic technique
Variable duplicate counts	Inconsistent sample homogenization	Improve mixing and pipetting technique

Recording and Reporting

A complete S. aureus test report should include:

• Sample ID and description
• Date and time of collection
• Dilutions tested
• Colony counts and dilution factor
• Final CFU/g or CFU/mL result
• Method reference: ISO 6888-1
• Analyst initials or signature

Proper documentation ensures traceability, audit compliance, and reliable client reporting.

Tips for Accurate Testing

• Use fresh, high-quality Baird-Parker agar.
• Incubate at the correct temperature and duration.
• Perform duplicate plating for reliability.
• Record all dilutions and volumes accurately.
• Track trends over time to detect hygiene lapses early.

Web References

1. ISO 6888-1:2021 — Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coagulase-positive staphylococci — Part 1: Technique using Baird-Parker agar
   https://www.iso.org/standard/68691.html
2. FAO/WHO Codex Alimentarius — Microbiological Criteria for Foods
   https://www.fao.org/fao-who-codexalimentarius
3. World Health Organization (WHO) — Food Safety: Microbiological Hazards
   https://www.who.int/health-topics/food-safety
4. Centers for Disease Control and Prevention (CDC) — Staphylococcus aureus and Foodborne Illness
   https://www.cdc.gov/foodsafety