🦠 Osmophilic yeasts and xerophilic moulds in products with aw ≤ 0.95 — spread plate on DG18 agar, 25 °C, 5–7 days

Microbiology PN-ISO 21527-2

Overview

Status of the standard: PN-ISO 21527-2:2009 (which implements ISO 21527-2:2008) was withdrawn by PKN on 25 September 2020 without a successor being indicated; ISO 21527-2:2008 itself remains current.

ISO 21527-2:2008 specifies a horizontal method for the enumeration of viable osmophilic yeasts and xerophilic moulds in products intended for human consumption and for the feeding of animals with a water activity less than or equal to 0.95 (e.g. dried fruit, cakes, jams, dried meat, flour and cereals, spices, nuts, dry feed); it does not allow the spores of moulds to be enumerated, and the identification of the fungi and the testing of mycotoxins lie outside its scope. The method is not suitable for halophilic xerophilic fungi (Polypaecilum pisce, Basipetospora halophila) occurring, for example, in dried fish. Osmophilic yeasts and xerophilic moulds are defined as fungi capable of growing at aw ≤ 0.95; the remaining definitions are taken over from ISO 21527-1. The laboratory sample must not be frozen. In Poland the standard was adopted as PN-ISO 21527-2:2009; PKN withdrew it on 25 September 2020, but ISO 21527-2:2008 is still in force and laboratories determine the number of yeasts and moulds in dry products "in accordance with PN-ISO 21527-2:2009". Part 1 covers products with aw > 0.95 and the DRBC medium.

Method principle

Plates are prepared that have been inoculated by the spread-plate technique with a specified amount of the sample, of the initial suspension or of the dilutions on a selective medium and are incubated aerobically at (25 ± 1) °C for 5–7 days (where necessary for 1–2 days in diffuse daylight); the colonies are counted, the doubtful ones are checked under a binocular magnifier or a microscope, and the number per g or ml is calculated from the plates with a countable number of colonies, counting the yeasts and the moulds separately where this is necessary. For the dilutions a diluent with a sufficient amount of dissolved substance is recommended — a 20–35 % solution of glycerol or of D-glucose — in order to minimise the osmotic shock of the cells; besides that, 0.1 % peptone water (pH 7.0 ± 0.2), optionally with 0.05 % Tween 80. Because of the rapid sedimentation of the spores the pipette is held horizontally and the suspensions are shaken; for the homogenisation a peristaltic homogeniser is preferred. DG18 agar (per 1000 ml): casein digest 5 g, D-glucose 10 g, KH₂PO₄ 1 g, MgSO₄·H₂O 0.5 g, dichloran 0.002 g, anhydrous glycerol 220 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 1 % chloramphenicol in ethanol is added, it is sterilised at 121 °C for 15 min, cooled to 44–47 °C and distributed in 15 ml portions; protect from light. Options: chlortetracycline 50 mg/l + chloramphenicol 50 mg/l (gentamicin is not recommended), trace elements (ZnSO₄·7H₂O 1 g, CuSO₄·5H₂O 0.5 g/100 ml; 1 ml/l). Performance testing: productivity over 5 days at 25 °C with Saccharomyces cerevisiae ATCC 9763, Wallemia sebi ATCC 42694, Aspergillus restrictus ATCC 42693 and Eurotium rubrum ATCC 42690 against SDA (PR ≥ 0.5); selectivity — total inhibition of E. coli ATCC 25922 and Bacillus subtilis ATCC 6633.

Applications

• Dry products and products with a reduced water activity: dried fruit, nuts, cereals and flour, spices, cakes, jams, dried meat and ripened sausages, milk powder
• Dry feed and feed materials
• Assessment of the risk of xerophilic moulds (Eurotium, Aspergillus restrictus, Wallemia) in stored raw materials
• Quality control of mycological media with a reduced aw

Key parameters

Standard

Standard number

PN-ISO 21527-2:2009

Title (PL)

Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby drożdży i pleśni — 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 2: Colony count technique in products with water activity less than or equal to 0,95

Step-by-step procedure

1. Medium

   Suspend the components of DG18 agar without the chloramphenicol, bring them to the boil, adjust the pH (5.6 ± 0.2 after sterilisation), add 10 ml of 1 % chloramphenicol in ethanol, sterilise at 121 °C for 15 min, cool to 44–47 °C, distribute in 15 ml portions, dry the surface and store in the dark.

2. Sample

   Do not freeze the laboratory sample; prepare the initial suspension and the dilutions in accordance with ISO 6887 / ISO 7218 / ISO 8261, preferably in a diluent with 20–35 % glycerol or glucose (or in 0.1 % peptone water), using a peristaltic homogeniser.

3. Spread plating

   Shake the suspensions and hold the pipette horizontally; place a specified amount on the surface of the DG18 plates and spread it with a spreader.

4. Incubation

   Incubate aerobically at (25 ± 1) °C for 5–7 days; where necessary for 1–2 days in diffuse daylight.

5. Counting

   Count the colonies on the plates with a countable number, check the doubtful ones under a binocular magnifier or a microscope; where necessary count the yeasts and the moulds separately.

6. Result

   Calculate the number of yeasts and moulds per g or ml and state the optional additives that have been used and any deviations from the method.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The spores of xerophilic moulds spread easily — work carefully and in a cabinet; protection of the respiratory tract with dry, dusting samples
• Chloramphenicol, chlortetracycline, dichloran — avoid raising dust and contact with the skin
• The medium is sensitive to light — store it in the dark

Frequently asked questions