🦠 Yeasts and moulds in products of aw > 0.95 — spread plate on DRBC agar, 25 °C, 5 days
In short
The sample or the dilutions in 0. 1 % peptone water are spread onto agar with dichloran, rose bengal and chloramphenicol (DRBC), incubated aerobically for 5 days at (25 ± 1) °C (where necessary for 1–2 days in diffuse daylight), and the colonies of yeasts and moulds are counted, being distinguished from bacteria under a binocular magnifier or a microscope. The test is performed according to PN-ISO 21527-1:2009; Scope: products of aw > 0.95. The procedure comprises 6 steps; it is used for: Products of high water activity: meat and cured meats, eggs and egg products, yoghurts, cheeses, cream, juices and fruit, vegetables, salads, fresh pastes, Assessment of the shelf life and of the hygiene of the production of chilled food; complaint investigations (spoilage by yeasts), Moist feed and raw materials.
At a glance
- Standard: PN-ISO 21527-1:2009
- Category: Microbiology
- Procedure steps: 6
- Scope: products of aw > 0.95
- Incubation: (25 ± 1) °C, 5 days, aerobically; optionally 1–2 days in diffuse daylight
- Medium: DRBC: glucose 10 g, dichloran 2 mg, rose bengal 25 mg, chloramphenicol 0.1 g/l; pH 5.6 ± 0.2; 15 ml per plate; protect from light
Overview
Status of the standard: PN-ISO 21527-1:2009 (which implements ISO 21527-1:2008) was withdrawn by the PKN (the Polish Committee for Standardization) on 25 September 2020 without any successor being indicated; ISO 21527-1:2008 itself remains current.
ISO 21527-1:2008 specifies a horizontal method for the enumeration of viable yeasts and moulds in products intended for human consumption and for the feeding of animals with a water activity greater than 0.95 — eggs, meat, milk products (with the exception of milk powder), fruit, vegetables, fresh pastes and so on — by the colony count technique at (25 ± 1) °C. It does not make it possible to enumerate the spores of moulds; the identification of fungi and the examination for mycotoxins lie outside its scope; the method is not suitable for heat-resistant fungi (Byssochlamys fulva, B. nivea) in preserved fruit and vegetables. Yeasts are defined as mesophilic aerobic micro-organisms forming at 25 °C on a mycological medium dull or shiny round colonies with a regular edge and a convex surface, and moulds as mesophilic aerobic filamentous organisms forming flat or fluffy, spreading colonies, often with coloured sporing structures; the distinction is sometimes a matter of convention. The standard warns that the counting of moulds calls for particular care in order to protect the operator and to prevent the contamination of the air with spores. In Poland it was implemented as PN-ISO 21527-1:2009 (it replaced PN-ISO 7954:1999); the PKN withdrew that standard on 25.09.2020, but ISO 21527-1:2008 is still in force and food laboratories commonly determine the number of yeasts and moulds "according to PN-ISO 21527-1:2009". Part 2 (ISO 21527-2) concerns products of aw ≤ 0.95 and the DG18 medium.
Method principle
Plates are prepared by spreading over the surface a defined amount of the liquid sample, of the initial suspension or of the decimal dilutions on a selective medium; the plates are incubated aerobically at (25 ± 1) °C for 5 days, being left where necessary for 1–2 days in diffuse daylight; the colonies/propagules are counted, and doubtful colonies (yeasts versus bacteria) are checked under a binocular magnifier (6.5–50×) or a microscope (250–1000×); the number of yeasts and moulds per g or ml is calculated from the plates with a countable number of colonies, the moulds and the yeasts being counted separately if necessary. Diluent: 0.1 % peptone water is recommended (hydrolysate 1 g/l, pH 7.0 ± 0.2); 0.05 % Tween 80 may be added to the diluents in order to limit the clumping of the spores. DRBC medium (per 1000 ml): hydrolysate of animal and plant tissues 5 g, D-glucose 10 g, KH₂PO₄ 1 g, MgSO₄·H₂O 0.5 g, dichloran (2,6-dichloro-4-nitroaniline) 0.002 g, rose bengal 0.025 g, agar 12–15 g, chloramphenicol 0.1 g; the components without the chloramphenicol are boiled, the pH after sterilisation is 5.6 ± 0.2, 10 ml of a 1 % solution of chloramphenicol in ethanol is added, it is sterilised at 121 °C for 15 min, cooled to 44–47 °C and dispensed in 15 ml portions into the plates; the medium is protected from light (the cytotoxic products of the decomposition of the rose bengal give underestimated results). Options: chlortetracycline 50 mg/l + chloramphenicol 50 mg/l where bacteria overgrow (e.g. raw meat; gentamicin is not recommended), trace elements (ZnSO₄, CuSO₄) for the full morphology of the moulds, Tergitol 1 ml/l against the overgrowth of Mucoraceae. Performance testing of the medium: productivity over 5 days at 25 °C with Saccharomyces cerevisiae ATCC 9763, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404, Mucor racemosus ATCC 42647 against SDA (PR ≥ 0.5); selectivity — complete inhibition of E. coli ATCC 25922 or Bacillus subtilis ATCC 6633.
Applications
- Products of high water activity: meat and cured meats, eggs and egg products, yoghurts, cheeses, cream, juices and fruit, vegetables, salads, fresh pastes
- Assessment of the shelf life and of the hygiene of the production of chilled food; complaint investigations (spoilage by yeasts)
- Moist feed and raw materials
- Quality control of mycological media (performance testing according to ISO/TS 11133)
Key parameters
| Parameter | Value |
|---|---|
| Scope | products of aw > 0.95 |
| Incubation | (25 ± 1) °C, 5 days, aerobically; optionally 1–2 days in diffuse daylight |
| Medium | DRBC: glucose 10 g, dichloran 2 mg, rose bengal 25 mg, chloramphenicol 0.1 g/l; pH 5.6 ± 0.2; 15 ml per plate; protect from light |
| Plating | spread plate; spreader of a diameter of < 2 mm and a length of 80 mm |
| Diluent | 0.1 % peptone water (pH 7.0 ± 0.2), optionally 0.05 % Tween 80 |
| Optional additions | chlortetracycline 50 mg/l (+ chloramphenicol 50 mg/l); trace elements 1 ml/l; Tergitol 1 ml/l |
| Control of the medium | S. cerevisiae ATCC 9763, C. albicans ATCC 10231, A. niger ATCC 16404, M. racemosus ATCC 42647 — PR ≥ 0.5 vs SDA; E. coli/B. subtilis — complete inhibition |
Standard
- Standard number
- PN-ISO 21527-1:2009
- Title (PL)
- Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby drożdży i pleśni — Część 1: Metoda liczenia kolonii w produktach o aktywności wody wyższej niż 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
Step-by-step procedure
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Preparation of the medium
Suspend the components of the DRBC without the chloramphenicol, bring them to the boil, adjust the pH so that after sterilisation it is 5.6 ± 0.2, add 10 ml of 1 % chloramphenicol in ethanol, sterilise at 121 °C for 15 min, cool to 44–47 °C and dispense in 15 ml portions; dry the surface of the plates according to ISO 7218 and store them in the dark.
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Sample and dilutions
Prepare the initial suspension and the decimal dilutions according to ISO 6887 / ISO 8261; the recommended diluent — 0.1 % peptone water (optionally 0.05 % Tween 80).
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Spread plating
Apply a defined amount of the liquid sample, of the initial suspension or of the dilutions onto the surface of the DRBC plates and spread it with a spreader (< 2 mm in diameter, in order to minimise the amount of sample left on the spreader).
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Incubation
Incubate aerobically at (25 ± 1) °C for 5 days; where necessary leave the plates for 1–2 days in diffuse daylight.
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Counting
Count the colonies/propagules on the plates with a countable number; check doubtful colonies (yeasts vs bacteria) under a binocular magnifier or a microscope; where necessary count the moulds and the yeasts separately.
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Result
Calculate the number of yeasts and moulds per g or ml from the plates of the chosen dilutions; give the optional additions used in the medium and any deviations.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator at (25 ± 1) °C | — | |
| Water bath at 44–47 °C | cooling of the medium | — |
| Sterile pipettes of 1 ml (0.1 ml), Petri dishes of 90–100 mm, glass or plastic spreaders (< 2 mm, 80 mm) | — | |
| Binocular magnifier (6.5–50×) and microscope (250–1000×) | distinguishing yeasts from bacteria and moulds from yeasts | — |
| pH meter (± 0.1 pH at 25 °C), bottles, flasks, tubes | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| DRBC agar (dichloran, rose bengal, chloramphenicol) | — | a selective mycological medium limiting the spreading of the moulds and inhibiting the bacteria |
| Chloramphenicol — a 1 % solution in ethanol (10 ml/l) | — | inhibition of the bacteria |
| 0.1 % peptone water | — | diluent |
| Optionally: chlortetracycline HCl 0.1 % (fresh, filtered), a solution of ZnSO₄·7H₂O 1 g + CuSO₄·5H₂O 0.5 g/100 ml, Tergitol, Tween 80 | — | additions according to 5.2.1.2.2–5.2.1.2.4 of the standard |
| Control strains (ATCC 9763, 10231, 16404, 42647, 25922, 6633) | — | performance testing of the medium |
Health and safety (OHS)
- Spores of moulds — work with the greatest care, preferably in a cabinet, in order to protect the operator and not to contaminate the air of the laboratory
- Chloramphenicol and chlortetracycline — antibiotics; avoid raising dust and contact with the skin; dichloran and rose bengal — weigh them out wearing gloves
- The medium is sensitive to light — the products of its decomposition are cytotoxic
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-ISO 21527-1:2009 — “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”.
How does this method work?
Plates are prepared by spreading over the surface a defined amount of the liquid sample, of the initial suspension or of the decimal dilutions on a selective medium; the plates are incubated aerobically at (25 ± 1) °C for 5 days, being left where necessary for 1–2 days in diffuse daylight; the colonies/propagules are counted, and doubtful colonies (yeasts versus bacteria) are checked under a binocular magnifier (6.5–50×) or a microscope (250–1000×); the number of yeasts and moulds per g or ml is calculated from the plates with a countable number of colonies, the moulds and the yeasts being counted separately if necessary.
What is the measuring range and accuracy?
Scope: products of aw > 0.95; Incubation: (25 ± 1) °C, 5 days, aerobically; optionally 1–2 days in diffuse daylight; Medium: DRBC: glucose 10 g, dichloran 2 mg, rose bengal 25 mg, chloramphenicol 0.1 g/l; pH 5.6 ± 0.2; 15 ml per plate; protect from light; Plating: spread plate; spreader of a diameter of < 2 mm and a length of 80 mm.
How long does the test take?
The procedure comprises 6 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Incubator at (25 ± 1) °C, Water bath at 44–47 °C, Sterile pipettes of 1 ml (0.1 ml), Petri dishes of 90–100 mm, glass or plastic spreaders (< 2 mm, 80 mm), Binocular magnifier (6.5–50×) and microscope (250–1000×), pH meter (± 0.1 pH at 25 °C), bottles, flasks, tubes.
Where is this test used?
Products of high water activity: meat and cured meats, eggs and egg products, yoghurts, cheeses, cream, juices and fruit, vegetables, salads, fresh pastes; Assessment of the shelf life and of the hygiene of the production of chilled food; complaint investigations (spoilage by yeasts); Moist feed and raw materials; Quality control of mycological media (performance testing according to ISO/TS 11133).
What safety precautions apply?
Spores of moulds — work with the greatest care, preferably in a cabinet, in order to protect the operator and not to contaminate the air of the laboratory; Chloramphenicol and chlortetracycline — antibiotics; avoid raising dust and contact with the skin; dichloran and rose bengal — weigh them out wearing gloves; The medium is sensitive to light — the products of its decomposition are cytotoxic.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-ISO 21527-1.