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

Introduction

Enterobacteriaceae (EB) is a large family of Gram-negative bacteria that includes many important genera such as Escherichia, Salmonella, Shigella, and Klebsiella. While some members are harmless or part of normal gut flora, several are pathogenic or indicative of hygiene lapses in food and water.

ISO 21528-2 — titled Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of Enterobacteriaceae — Part 2: Colony-count method — provides a standardized approach for quantifying EB in food samples. Enumeration is widely used in food safety labs to monitor hygiene, assess processing controls, and comply with regulatory standards.

This guide presents the ISO 21528-2 method in a simplified, step-by-step format, including principle, procedure, result interpretation, recording, web references, and a disclaimer. It is optimized for SEO to help students, food professionals, and laboratory staff access educational information online.

Why Enterobacteriaceae Testing Matters

• Indicator of hygiene: High EB counts suggest poor sanitation, cross-contamination, or post-processing contamination.
• Food safety assessment: Detecting EB helps prevent the presence of potential pathogens.
• Regulatory compliance: Many food safety standards and export requirements set limits for EB levels.
• Public health protection: Early detection reduces the risk of foodborne illness outbreaks.

EB enumeration provides general hygiene insights rather than identifying specific pathogens, making it a useful routine monitoring tool in dairy, meat, produce, and ready-to-eat foods.

Principle of ISO 21528-2

The ISO 21528-2 method is a culture-based plate count technique for quantifying Enterobacteriaceae.

Key steps:

1. Serial dilution of the food sample to achieve countable bacterial concentrations.
2. Plating on selective agar (e.g., Violet Red Bile Glucose Agar – VRBG) that supports EB growth and inhibits non-target bacteria.
3. Incubation at 37 °C for 24–48 hours.
4. Identification of characteristic colonies, typically red to purple with precipitate due to bile reaction.
5. Counting colonies and calculating CFU per gram or milliliter.

The selective medium ensures accurate enumeration of EB while minimizing interference from other microorganisms.

Materials & Equipment

Consumables

• Sterile sample containers (bags or tubes)
• Sterile pipettes and tips
• Sterile diluent (e.g., peptone water or buffered saline)
• VRBG agar plates or equivalent selective medium
• Sterile spreaders

Equipment

• Incubator capable of 37 °C ±1 °C
• Stomacher or vortex mixer
• Colony counter (manual or digital)
• Calibrated pipettes

Personal Protective Equipment (PPE)

• Lab coat, gloves, eye protection
• Adhere to biosafety protocols for handling potential 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 a 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 to prepare further dilutions (10⁻³, 10⁻⁴, etc.), depending on expected EB levels.
3. Ensure thorough mixing at each dilution step.

Serial dilutions ensure that plates will have 20–150 colonies for statistically reliable counts.

3. Inoculation of Agar Plates

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

Proper labeling ensures traceability and avoids errors.

4. Incubation

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

Incubation conditions favor EB growth while suppressing many non-target bacteria.

5. Colony Identification

After incubation:

• Enterobacteriaceae colonies typically appear red to purple with precipitation around the colony due to bile salt reaction.
• Colonies may be round, convex, and 1–3 mm in diameter.
• Only count colonies that exhibit typical morphology for enumeration.

Optional confirmation tests (e.g., oxidase negative, gram stain) can validate presumptive EB colonies.

Calculating Colony Counts

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

Example Calculation

• Colonies on 10⁻³ dilution plate = 80
• Volume plated = 1 mL

CFU/g=80×103=8.0×104 CFU/g

Always select plates with 20–150 colonies for accurate results.

Interpreting Results

CFU/g or CFU/mL	Interpretation	Recommended Action
Low (<10²)	Good hygiene	Routine monitoring
Moderate (10²–10⁴)	Potential hygiene issue	Review sanitation procedures
High (>10⁴)	Hygiene failure or contamination	Immediate corrective action; investigate sources

EB counts provide a hygiene and contamination profile, but do not indicate specific pathogenic organisms.

Practical Applications in Food Safety

• Dairy products: Monitor post-pasteurization hygiene and storage conditions.
• Meat and poultry: Evaluate slaughterhouse, processing, and handling sanitation.
• Ready-to-eat foods: Assess cross-contamination risks from equipment, surfaces, or handlers.
• Produce and beverages: Identify contamination from water, soil, or processing environments.

Routine EB monitoring supports HACCP plans and ISO 22000 compliance, ensuring product safety and consumer protection.

Common Errors & Troubleshooting

Problem	Possible Cause	Recommended Action
No colonies	Over-dilution or non-viable bacteria	Plate lower dilutions or check sample storage
Too many colonies (TMTC)	Under-dilution	Plate higher dilutions
Non-typical colonies	Contaminated media or wrong incubation	Verify media, temperature, and aseptic technique
Inconsistent counts	Uneven sample mixing	Improve homogenization and pipetting accuracy

Recording & Reporting

A complete EB test report should include:

• Sample ID and description
• Date and time of collection
• Dilutions plated
• Colony counts for each plate
• Final CFU/g or CFU/mL result
• Method reference: ISO 21528-2
• Analyst initials or signature

Accurate documentation ensures traceability, reproducibility, and audit compliance.

Tips for Accurate Testing

• Always use fresh, validated VRBG agar plates.
• Maintain correct incubation temperature and time.
• Plate duplicates to improve reliability.
• Record all dilutions and volumes meticulously.
• Monitor trends over time for hygiene and process control insights.

Web References

1. ISO 21528-2:2017 — Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of Enterobacteriaceae — Part 2: Colony-count method
   https://www.iso.org/standard/53328.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) — Enterobacteriaceae and Food Safety
   https://www.cdc.gov/foodsafety

Disclaimer

This blog is intended for educational purposes only. The method described follows ISO 21528-2 principles, but laboratories must use validated standard operating procedures (SOPs), adhere to quality management systems, and comply with local regulations. Results should be interpreted by qualified microbiology personnel. This content does not replace formal laboratory training or accreditation.