🍄 Yeasts and moulds in food

Microbiology PN-ISO 21527

Overview

STATUS (as of 02.11.2026). PN-ISO 21527-1:2009 (Polish version, published on 08.06.2009, adopts ISO 21527-1:2008 [IDT], replaced PN-ISO 7954:1999) and PN-ISO 21527-2:2009 (Polish version, published on 08.06.2009, adopts ISO 21527-2:2008 [IDT], replaced PN-ISO 7698:2004) carry a withdrawal date of 25.09.2020 on their PKN catalogue cards and have no “Replaced by” field. ISO 21527-1:2008 and ISO 21527-2:2008 (first edition, 01.07.2008; they replaced ISO 7698:1990, ISO 7954:1987 and ISO 13681:1995) are current. The description gives the content of the ISO documents.

SCOPE OF PART 1 (clause 1). Enumeration of viable yeasts and moulds in products with a water activity greater than 0.95 — eggs, meat, dairy products (except milk powder), fruits, vegetables, fresh pastes etc. — by a colony-count technique at (25 ± 1) °C. The method does not allow the enumeration of mould spores, does not cover the identification of fungal flora or the examination of mycotoxins and is not suitable for the enumeration of heat-resistant fungi, such as Byssochlamys fulva and Byssochlamys nivea, in canned or bottled fruit and vegetables. According to chapter 18 of the BAM manual (FDA), yeasts and moulds cause deterioration and decomposition of foods, and some moulds produce toxic metabolites — mycotoxins.

SCOPE OF PART 2 (clause 1). Enumeration of viable osmophilic yeasts and xerophilic moulds in products with a water activity less than or equal to 0.95 — dry fruits, cakes, jams, dried meat, salted fish, grains, cereals and cereal products, flours, nuts, spices and condiments etc. — by a colony-count technique at (25 ± 1) °C. Part 2 does not apply to dehydrated products with a water activity less than or equal to 0.60 (dehydrated cereals, oleaginous products, spices, leguminous plants, seeds, powders for instant drinks, dry products for domestic animals) and is not suitable for the enumeration of halophilic xerophilic fungi, e.g. in dried fish.

MEDIA (parts 1 and 2, 5.2). DRBC per 1 000 ml: enzymatic digest of animal and plant tissues 5.0 g, D-glucose 10.0 g, KH₂PO₄ 1.0 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 (10 ml of a 1 % solution in ethanol); pH 5.6 ± 0.2. DG18 — casein enzymatic digest 5.0 g, no rose bengal, with 220 g of anhydrous glycerol, the other ingredients as in DRBC. Sterilization 15 min at 121 °C, cooling to 44–47 °C, 15 ml per plate; the media are not exposed to light, because cytotoxic breakdown products lead to underestimation. Where bacterial overgrowth may be a problem (e.g. raw meats), chloramphenicol 50 mg/l and chlortetracycline 50 mg/l are recommended; gentamicin is not recommended, as it inhibits some yeasts. According to the data sheet of DRBC agar made to the standard, dichloran reduces the colony diameter of spreading fungi, and rose bengal suppresses bacteria and restricts the size of colonies of the more rapidly growing moulds; according to chapter 18 of the BAM manual (FDA), the water activity of DG18 agar is 0.955.

DILUENT AND SAMPLE (5.1, clause 7, 9.1 of part 2). 0.1 % peptone water is recommended (1.0 g of enzymatic digest of animal or vegetal tissues in 1 000 ml, pH 7.0 ± 0.2), and for products of low water activity — a diluent with 20–35 % glycerol or D-glucose, which minimizes osmotic shock. A surface-active agent (e.g. polysorbate 80, 0.05 %) may be added to reduce clumping of spores. Part 2 recommends a peristaltic homogenizer in preference to a blender or shaker, the pipette with the suspension is held horizontally (spores sediment rapidly), and the suspensions are shaken before sampling; the laboratory sample is not frozen.

REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026) contains no criteria for yeasts and moulds.

WHAT WE DO NOT GIVE HERE. The available texts end at clause 6 (part 1) and 9.1 (part 2). We do not give the inoculation volume, the position of the plates during incubation, the counting and selection of plates, the calculation, the confirmation of doubtful colonies or the precision data. For the inoculation, incubation and counting steps we give, for comparison, the provisions of the related method in chapter 18 of the BAM manual (FDA) — they are not part of the standard.

Method principle

Plates with a selective medium are surface-inoculated with a specified quantity of the liquid sample, the initial suspension or their decimal dilutions, depending on the expected number of colonies (4.1). The plates are incubated aerobically at (25 ± 1) °C for 5 d (part 1) or 5–7 d (part 2); if necessary, they are left to stand in diffuse daylight for 1–2 d (4.2). Colonies are counted, doubtful ones being checked if necessary with a binocular magnifier or microscope, and the number of yeasts and moulds per 1 g or 1 ml is calculated from plates at dilution levels producing countable colonies — yeasts and moulds separately, if necessary (4.3–4.4).

Applications

• Products with a water activity greater than 0.95: eggs, meat, dairy products (except milk powder), fruits, vegetables, fresh pastes (part 1, clause 1)
• Products with a water activity less than or equal to 0.95: dry fruits, cakes, jams, dried meat, salted fish, cereals and cereal products, flours, nuts, spices — excluding dehydrated products with a water activity less than or equal to 0.60 (part 2, clause 1)
• Not for: enumeration of mould spores, identification of fungi and examination of mycotoxins, heat-resistant fungi in canned fruit and vegetables and halophilic xerophilic fungi (clause 1 of both parts)

Key parameters

Standard

Standard number

PN-ISO 21527-1:2009, PN-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 wody wyższej niż 0,95 / Część 2: Metoda liczenia kolonii w produktach o aktywności wody niższej lub równej 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. Choice of the part of the standard

   Part 1 (DRBC agar) — products with a water activity greater than 0.95; part 2 (DG18 agar) — products with a water activity less than or equal to 0.95, except dehydrated products with a water activity less than or equal to 0.60 (clause 1).

2. Sample

   The laboratory sample shall not be frozen (part 2, clause 7); the test sample, initial suspension and dilutions are prepared according to ISO 6887, ISO 7218, ISO 8261 and the standard for the product concerned (clause 8, 9.1).

3. Dilutions

   The diluent is usually 0.1 % peptone water; a peristaltic homogenizer is preferred to a blender or shaker; the pipette with the suspension is held horizontally, and the suspension and dilutions are shaken before sampling, because spores sediment rapidly (part 2, 9.1).

4. Preparation of the medium

   Bring the ingredients except chloramphenicol to the boil to dissolve, add 10 ml of a 1 % solution of chloramphenicol in ethanol, sterilize for 15 min at 121 °C, cool in a water bath to 44–47 °C and below 50 °C dispense 15 ml per plate; protect the medium from light (5.2.1.2).

   🌡 Temperature: 121 °C / 44–47 °C

5. Inoculation

   Plates are surface-inoculated with a specified quantity of the liquid sample, the initial suspension or dilutions, depending on the expected number of colonies (4.1); the inoculation volume and number of plates are given in clause 9 of the standard, beyond the available text. In the BAM method (chapter 18), 0.1 ml of each dilution is spread onto DRBC plates.

6. Incubation

   Aerobically at (25 ± 1) °C for 5 d (part 1) or 5–7 d (part 2); if necessary the plates are left to stand in diffuse daylight for 1–2 d (4.2). In the BAM method the plates are not inverted, and if there is no growth after 5 days they are incubated for a further 48 h.

   🌡 Temperature: 25 ± 1 °C

7. Counting

   Count the colonies (propagules); check doubtful colonies — to distinguish yeasts from bacteria — with a binocular magnifier or microscope. Yeasts and moulds are counted separately, if necessary (4.3–4.4). In the BAM method, plates with 10–150 colonies are counted.

8. Result

   The number of yeasts and moulds per 1 g or 1 ml is calculated from plates at dilution levels producing countable colonies (4.4).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The enumeration of moulds is carried out with the greatest care to protect the operator and to prevent contamination of the atmosphere with mould spores (warning in part 1)
• Prepared media are stored in the dark — exposure to light produces cytotoxic breakdown products and leads to underestimation (caution in 5.2.1.2.1)

Frequently asked questions