🍄 Yeasts and molds in food
Enumeration of yeasts and molds in food by surface plating on DRBC agar (products with aw > 0.95) or DG18 agar (products with aw ≤ 0.95) with incubation at 25°C for 5–7 days.
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
| Parameter | Value |
|---|---|
| Incubation temperature | 25 ± 1°C |
| Incubation time | 5 days (possible extension to 7 days) |
| Medium (aw > 0.95) | DRBC Agar (Dichloran Rose Bengal Chloramphenicol) |
| Medium (aw ≤ 0.95) | DG18 Agar (Dichloran 18% Glycerol Agar) |
| Plating method | Surface (spread plate), 0.1 mL per plate |
| Counting range | 10–150 colonies per plate (typical and atypical) |
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.
2. Sample preparation
Weigh 10 g sample into stomacher bag, add 90 mL buffered peptone water, homogenize for 60–120 s. Prepare decimal dilutions.
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.
4. Surface plating
Apply 0.1 mL dilution to plate surface. Spread with Drigalski spatula. Perform duplicates. Leave until inoculum is absorbed.
5. Incubation
Incubate plates in normal position (bottom down) at 25 ± 1°C. DO NOT invert plates — molds would spread on lid.
6. Reading after 2–3 days (control)
Check growth after 2–3 days. Mark fast-growing molds that may hinder counting after 5 days.
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.
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
| Equipment | Example | Indicative price |
|---|---|---|
| Laboratory incubator 25°C | Binder BD 56, Memmert IPP 110, POL-EKO ST 2 | 5,000–18,000 PLN |
| Class II laminar flow cabinet | Thermo Scientific MSC-Advantage, ESCO Airstream AC2 | 25,000–60,000 PLN |
| Stomacher / homogenizer | Seward Stomacher 400 Circulator | 12,000–25,000 PLN |
| Water bath 44–47°C | Memmert WNB 14, Julabo TW 12 | 3,000–10,000 PLN |
| Water activity meter (aw) | Novasina LabMaster-aw, Rotronic HygroLab C1 | 8,000–25,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65 | 15,000–45,000 PLN |
| Colony counter | Interscience Scan 300, Stuart SC6+ | 3,000–15,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| DRBC Agar (Dichloran Rose Bengal Chloramphenicol) | 99-30-9 | Medium for products with aw > 0.95; peptone, glucose, dichloran, rose bengal, chloramphenicol, agar; 500 g package |
| DG18 Agar (Dichloran 18% Glycerol Agar) | 56-81-5 | Medium for products with aw ≤ 0.95; peptone, glucose, dichloran, glycerol 220 g/L, chloramphenicol, agar; 500 g package |
| Dichloran (2,6-dichloro-4-nitroaniline) | 99-30-9 | Restricts spreading of fast-growing molds, concentration 2 mg/L |
| Rose bengal (Rose Bengal) | 632-69-9 | Dye limiting mold colony size, concentration 25 mg/L |
| Chloramphenicol | 56-75-7 | Antibiotic inhibiting bacterial growth; 1% solution in ethanol, final concentration 100 mg/L |
| Glycerol | 56-81-5 | DG18 component lowering medium water activity to ~0.955; 220 g/L |
| Buffered peptone water (BPW) | — | For sample dilutions, 500 g package |
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)