Introduction

Escherichia coli (E. coli) is a species of bacteria commonly found in the intestines of humans and warm‑blooded animals. While most strains are harmless, some can cause serious foodborne illness. E. coli is also a key indicator organism for fecal contamination and hygiene in food and water.

The ISO 7251 method — titled Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of presumptive Escherichia coli — provides a standardized approach for quantifying E. coli in food products and environmental samples. Accurate enumeration helps food businesses verify hygiene practices, comply with regulatory criteria, and protect consumer health.

This guide simplifies the ISO 7251 method step by step, including principle, test procedure, results interpretation, recording, web references, and disclaimer, all explained in clear, practical language.

Why E. coli Testing Matters

E. coli testing is a crucial part of food safety monitoring:

• Indicator of fecal contamination: High E. coli counts often signal fecal contamination, which may also imply the potential presence of harmful pathogens.
• Hygiene assessment: Helps evaluate the effectiveness of cleaning, handling, and processing controls.
• Regulatory compliance: Many food standards and export requirements include E. coli limits.
• Public health protection: Detecting elevated E. coli prompts corrective actions to prevent foodborne disease.

Unlike total coliform tests (which measure a broad group), E. coli enumeration is more specific and sensitive to fecal contamination and hygiene breakdowns.

Principle of ISO 7251 Method

The ISO 7251 method is a culture‑based technique for estimating presumptive E. coli present in a food or environmental sample.

It involves:

1. Serial dilution of the food sample.
2. Inoculation on a selective and differential medium that favors E. coli growth.
3. Incubation at defined temperatures to allow colony development.
4. Counting colonies with characteristic E. coli morphology.
5. Reporting results as colony‑forming units (CFU) per gram or milliliter.

The medium commonly used is TBX (Tryptone Bile X‑glucuronide agar) or similar, which allows E. coli colonies to appear with distinctive color (e.g., blue‑green) due to glucuronidase activity.

Materials & Equipment

Consumables

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

Equipment

• Incubator capable of 44 °C ±0.5 °C
• Stomacher or vortex mixer
• Colony counter (manual or digital)
• Calibrated pipettes

Personal Protective Equipment (PPE)

• Lab coat, gloves, eye protection
• Follow your lab’s biosafety protocols

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 into a sterile container.
3. Add 90 mL sterile diluent to form the 10⁻¹ dilution.
4. Homogenize thoroughly using a stomacher or vortex for uniform bacterial distribution.

This initial step ensures a consistent starting point for accurate enumeration.

2. Serial Dilutions

Serial dilution reduces bacterial concentration to manageable levels:

1. Pipette 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, 10⁻⁵, etc.) based on expected E. coli levels.

Accurate pipetting and mixing are essential to ensure correct dilution factors.

3. Inoculation of Agar Plates

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

Proper labeling ensures traceability and prevents data errors.

4. Incubation

1. Place plates in the incubator at 44 °C ±0.5 °C.
2. Incubate for 24 hours ±2 hours.
3. Avoid overstacking plates to ensure uniform temperature exposure.

The elevated temperature favors E. coli growth and suppresses many non‑target organisms.

5. Colony Enumeration

1. After incubation, select plates with 20–150 countable colonies.
2. Count colonies showing the typical E. coli appearance (e.g., blue‑green due to glucuronidase activity).
3. Record the number of colonies along with the dilution factor and volume plated.

Only count plates in the reliable range to minimize statistical error.

Calculations & Results

Use this formula: CFU/g (or CFU/mL)=Average colony count×Dilution factorVolume plated (mL)\text{CFU/g (or CFU/mL)} = \frac{\text{Average colony count} \times \text{Dilution factor}}{\text{Volume plated (mL)}}CFU/g (or CFU/mL)=Volume plated (mL)Average colony count×Dilution factor​

Example Calculation

• Colonies on the 10⁻³ plate: 60
• Volume plated: 1 mL

Final result = 60 × 10³ = 6.0 × 10⁴ CFU/g

Report your result clearly, using scientific notation for readability.

Interpreting E. coli Results

Understanding counts in context is critical:

E. coli Level	Interpretation	Suggested Action
Low or Undetectable	Good hygiene and handling	Routine monitoring
Moderate	Possible hygiene concerns	Review cleaning/procedures
High	Clear contamination issue	Immediate corrective action, potential investigation

Important: High E. coli counts do not diagnose specific pathogens but strongly suggest fecal contamination and warrant further investigation.

Practical Applications in Food Safety

E. coli enumeration is used in many situations:

• Dairy products: Post‑pasteurization contamination or unhygienic handling.
• Fresh produce: Irrigation water quality or field handling issues.
• Meat and poultry: Slaughterhouse hygiene and cross‑contamination checks.
• Water used in processing: Indicates fecal contamination risk.

Consistent monitoring over time can reveal trends and process weaknesses before they become safety hazards.

Common Errors and Troubleshooting

Problem	Possible Cause	Solution
No colonies	Over‑dilution	Plate lower dilutions
Too many colonies (TMTC)	Under‑dilution	Plate higher dilutions
Non‑typical colonies	Contaminated media or non‑target organisms	Check media and aseptic technique
Variable results	Inconsistent mixing or pipetting	Improve homogenization and pipette calibration

Accurate technique and attention to detail improve reliability and reproducibility.

Recording and Reporting

A complete E. coli test report should include:

• Sample ID and description
• Date and time of sample collection
• Dilutions tested
• Colony counts and dilution factor
• Final CFU result with units
• Method referenced: ISO 7251
• Analyst name/initials

Good records are essential for audits, traceability, and client reports.

What E. coli Results Tell You in Practice

E. coli enumeration does not replace specific pathogen testing (e.g., for E. coli O157:H7), but it:

• Provides early warnings of fecal contamination
• Supports verification of hygiene controls
• Strengthens food safety management systems (e.g., HACCP)

Consistent tracking of results over time provides trends that help prevent safety incidents.

Web References

1. ISO 7251:2013 — Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of presumptive Escherichia coli
   https://www.iso.org/standard/63577.html
2. World Health Organization (WHO) — Food Safety: Microbiological Hazard Guidance
   https://www.who.int/health-topics/food-safety
3. FAO/WHO Codex Alimentarius — Microbiological Criteria for Foods
   https://www.fao.org/fao-who-codexalimentarius
4. Centers for Disease Control and Prevention (CDC) — Indicator Organisms and Methods
   https://www.cdc.gov/foodsafety/isolates