Yeast and Mold Enumeration Using ISO 6611: Step-by-Step Educational Guide

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

Introduction

Yeasts and molds are fungal microorganisms commonly found in food, beverages, and the environment. While some yeasts are beneficial in fermentation (e.g., baking, brewing), undesirable yeasts and molds can cause spoilage, off-flavors, and foodborne illnesses. Some molds produce mycotoxins, which pose serious health risks.

ISO 6611 — titled Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of yeasts and molds — Colony-count technique at 25 °C — is the international standard for enumerating yeasts and molds in food. Accurate testing allows food safety laboratories to monitor product quality, detect contamination, and ensure regulatory compliance.

This guide presents a simplified, step-by-step approach to ISO 6611, including principle, materials, procedure, results interpretation, recording, web references, and a disclaimer. It is optimized for SEO, targeting professionals, students, and laboratory personnel.

Why Yeast and Mold Testing Matters

• Food spoilage prevention: High counts of yeasts and molds reduce shelf life and quality.
• Food safety: Some molds produce mycotoxins, which are toxic even at low levels.
• Hygiene monitoring: Presence of yeasts and molds may indicate poor sanitation, air contamination, or water quality issues.
• Regulatory compliance: Many countries have specific limits for yeast and mold counts in dairy, bakery, beverages, and packaged foods.

Unlike bacterial indicators, yeast and mold testing focuses on fungal contamination, which requires specialized media and incubation conditions.

Principle of ISO 6611

The ISO 6611 method is a culture-based enumeration technique that uses selective conditions to grow and count yeasts and molds:

1. Sample dilution: Food samples are diluted in a sterile medium to achieve countable fungal populations.
2. Plating on selective agar: Samples are inoculated on differential or non-selective media such as DRBC (Dichloran Rose Bengal Chloramphenicol agar) or PDA (Potato Dextrose Agar) with chloramphenicol.
3. Incubation at 25 °C: Lower temperature favors fungal growth while suppressing bacterial growth.
4. Colony identification: Yeasts appear as smooth, often pigmented colonies, while molds form filamentous, fuzzy colonies.
5. Enumeration: Count colonies and report as CFU/g or CFU/mL.

The method allows laboratories to assess overall fungal load in foods and beverages while differentiating between yeasts and molds.

Materials & Equipment

Consumables

• Sterile sample containers (bags, tubes)
• Sterile pipettes and tips
• Sterile diluent (peptone water or buffered saline)
• Selective agar plates: DRBC, PDA with chloramphenicol, or other validated media
• Sterile spreaders

Equipment

• Incubator set at 25 °C ±1 °C
• Stomacher or vortex mixer
• Colony counter (manual or digital)
• Calibrated pipettes

Personal Protective Equipment (PPE)

• Lab coat, gloves, eye protection
• Follow biosafety protocols when handling molds, which can produce spores and allergens

Step-by-Step Test Procedure

1. Sample Preparation

1. Weigh 10 g of solid food or measure 10 mL of liquid sample.
2. Place the sample into a sterile container or stomacher bag.
3. Add 90 mL of sterile diluent for a 1:10 dilution.
4. Homogenize thoroughly using a stomacher or vortex to ensure uniform fungal 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.).
3. Ensure proper mixing at each dilution step.

Serial dilutions are critical to achieve plates with 10–150 colonies for accurate enumeration.

3. Inoculation of Agar Plates

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

Proper labeling ensures traceability and reduces the risk of data errors.

4. Incubation

1. Place plates in an incubator at 25 °C ±1 °C.
2. Incubate for 5–7 days.
3. Avoid stacking plates tightly to ensure uniform growth.
4. Check plates periodically for fungal growth.

Lower temperature and longer incubation favor slow-growing molds and yeasts, which may not appear at higher temperatures.

5. Colony Identification

• Yeasts: Smooth, creamy or pigmented colonies, typically round, 1–5 mm in diameter.
• Molds: Filamentous colonies with fuzzy or powdery appearance; colors vary (white, green, black, yellow).
• Selective media indicators: DRBC agar suppresses bacterial growth and allows better differentiation between yeasts and molds.

Optional microscopic confirmation can identify molds by spore structures and hyphae.

6. Counting Colonies

1. Select plates with 10–150 countable colonies.
2. Count yeasts and molds separately if differentiation is needed.

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

Example Calculation:

• Colonies on 10⁻² plate = 45
• Volume plated = 0.1 mL

CFU/g=45×1020.1=4.5×104 CFU/g

Interpreting Results

CFU/g or CFU/mL	Interpretation	Recommended Action
Low (<10²)	Acceptable hygiene	Routine monitoring
Moderate (10²–10⁴)	Potential spoilage risk	Review cleaning and handling
High (>10⁴)	High contamination	Immediate corrective action; assess storage and production conditions

High fungal counts indicate potential spoilage, reduced shelf-life, and contamination risks.

Practical Applications in Food Safety

• Bakery products: Monitor molds that spoil bread and pastries.
• Dairy products: Prevent spoilage in cheeses, yogurts, and creams.
• Beverages: Detect yeast in juices, soft drinks, and alcohol.
• Packaged foods: Assess storage conditions and potential cross-contamination.

Routine yeast and mold monitoring supports HACCP plans, regulatory compliance, and consumer safety.

Common Errors & Troubleshooting

Problem	Possible Cause	Recommended Action
No colonies	Over-dilution or non-viable fungi	Plate lower dilutions or check storage
Too many colonies (TMTC)	Under-dilution	Plate higher dilutions
Non-characteristic colonies	Contamination or wrong medium	Verify agar and aseptic technique
Variable counts	Uneven mixing or plating	Improve homogenization and pipetting accuracy

Recording & Reporting

A complete ISO 6611 test report should include:

• Sample ID, description, and collection date
• Dilutions plated
• Colony counts for yeasts and molds separately (if required)
• Final CFU/g or CFU/mL result
• Method reference: ISO 6611
• Analyst initials or signature

Good documentation ensures traceability, reproducibility, and audit compliance.

Tips for Accurate Testing

• Use fresh, validated media for accurate fungal growth.
• Maintain precise temperature and incubation conditions.
• Plate duplicates to ensure reproducibility.
• Track trends over time to detect hygiene and storage issues early.
• Use microscopic confirmation for molds to identify spoilage species if necessary.

Web References

1. ISO 6611:2018 — Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of yeasts and molds — Colony-count technique at 25 °C
   https://www.iso.org/standard/69924.html
2. FAO/WHO Codex Alimentarius — Microbiological Criteria for Foods
   https://www.fao.org/fao-who-codexalimentarius
3. World Health Organization (WHO) — Food Safety: Microbial Contamination
   https://www.who.int/health-topics/food-safety
4. Centers for Disease Control and Prevention (CDC) — Fungal Contamination in Foods
   https://www.cdc.gov/fungal

Disclaimer

This blog is provided for educational purposes only. Although it reflects ISO 6611 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 requirements.