🍄 Yeasts and molds in food

Microbiology PN-EN ISO 21527

Overview

Yeasts and molds are ubiquitous organisms responsible for food spoilage — they cause changes in taste, odor, consistency and appearance of food products. Some molds (Aspergillus, Penicillium, Fusarium) produce mycotoxins — secondary metabolites with carcinogenic, hepatotoxic, nephrotoxic and immunosuppressive effects (aflatoxins, ochratoxin A, deoxynivalenol, zearalenone).

The PN-EN ISO 21527 standard consists of two parts, adapted to water activity (aw) of tested product. Part 1 covers products with aw > 0.95 (milk, meat, eggs, vegetables, fruits, fresh pastas) and uses DRBC (Dichloran Rose Bengal Chloramphenicol Agar) medium. Part 2 covers products with aw ≤ 0.95 (dried fruits, cakes, jams, dried meat, salted fish, cereals, flours, nuts, spices) and uses DG18 (Dichloran 18% Glycerol Agar) medium.

In both cases, incubation is at 25 ± 1°C for 5 days (with possible extension to 7 days). Dichloran and rose bengal restrict spreading of fast-growing molds and reduce colony size, enabling counting. Chloramphenicol inhibits bacterial growth. Glycerol in DG18 lowers aw of medium, favoring growth of xerophilic and osmophilic fungi.

The method is widely used in food quality control, environmental monitoring of production facilities and shelf life assessment. It does not include mold spore enumeration or fungal species identification — mycological methods are needed for these purposes.

Method principle

Sample is surface plated on DRBC (aw > 0.95) or DG18 (aw ≤ 0.95) medium. Dichloran (2,6-dichloro-4-nitroaniline) restricts spreading of fast-growing molds (e.g., Rhizopus, Mucor). Rose bengal additionally reduces colony size and facilitates counting. Chloramphenicol inhibits bacterial growth. In DG18, glycerol (18%) lowers medium water activity to approx. 0.955, promoting growth of osmophilic yeasts and xerophilic molds. After 5 days incubation at 25°C, we count separately yeast colonies (smooth, moist, convex) and mold colonies (fluffy, filamentous, colored).

Applications

• Quality control of fruits, vegetables and fruit juices
• Testing cereal products — flour, groats, flakes, bread
• Monitoring dried products — nuts, spices, dried fruits, herbal teas
• Dairy product quality control — yogurts, cheeses, ice cream
• Testing jams, preserves, syrups and honeys
• Environmental monitoring of production facilities (swabs, sedimentation)
• Microbiological shelf life assessment of food products
• Cosmetics and dietary supplements control

Key parameters

Standard

Standard number

PN-EN ISO 21527-1:2009 / PN-EN ISO 21527-2:2009

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby drożdży i pleśni — Część 1: Metoda liczenia kolonii w produktach o aktywności wodnej powyżej 0,95 / Część 2: Metoda liczenia kolonii w produktach o aktywności wodnej ≤ 0,95

Title (EN)

Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of yeasts and moulds — Part 1: Colony count technique in products with water activity greater than 0.95 / Part 2: Colony count technique in products with water activity ≤ 0.95

Step-by-step procedure

1. Product water activity determination

   Measure sample aw using water activity meter. If aw > 0.95 → use DRBC. If aw ≤ 0.95 → use DG18.

   ⏱ Time: 10 min

2. Sample preparation

   Weigh 10 g sample into stomacher bag, add 90 mL buffered peptone water, homogenize for 60–120 s. Prepare decimal dilutions.

   ⏱ Time: 10 min

3. Medium preparation

   Dissolve appropriate amount of medium (DRBC or DG18) in distilled water. Sterilize in autoclave (121°C/15 min). Cool to 47°C. Add chloramphenicol. Pour into plates (18–20 mL). Dry surface.

   ⏱ Time: 45 min • 🌡 Temperature: 47°C

4. Surface plating

   Apply 0.1 mL dilution to plate surface. Spread with Drigalski spatula. Perform duplicates. Leave until inoculum is absorbed.

   ⏱ Time: 15 min

5. Incubation

   Incubate plates in normal position (bottom down) at 25 ± 1°C. DO NOT invert plates — molds would spread on lid.

   ⏱ Time: 5 days • 🌡 Temperature: 25°C

6. Reading after 2–3 days (control)

   Check growth after 2–3 days. Mark fast-growing molds that may hinder counting after 5 days.

   ⏱ Time: 10 min

7. Final reading after 5 days

   Count separately yeast colonies (smooth, moist, creamy) and mold colonies (fluffy, filamentous, colored). Count plates with 10–150 colonies. If growth is weak after 5 days, extend incubation to 7 days.

   ⏱ Time: 20 min

8. Calculations

   Calculate number of yeasts and molds separately (or together) as weighted average from duplicates. Express result in CFU/g.

9. Quality control

   Positive control: Saccharomyces cerevisiae (yeast) and Aspergillus brasiliensis ATCC 16404 (mold). Medium sterility control.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Some molds produce mycotoxins and allergens — work in laminar flow cabinet
• Mold spores easily become airborne — avoid rapid movements when opening plates
• Chloramphenicol — potentially harmful, avoid skin contact and inhalation
• Dichloran — irritating, use gloves and lab coat
• Autoclave used plates with molds before disposal (121°C/20 min)

Frequently asked questions