🦠 Yeasts and moulds in cosmetics — colony count on Sabouraud agar with chloramphenicol (25 °C, 3–5 days)

Microbiology PN-EN ISO 16212

Overview

ISO 16212:2017 gives general guidelines for the enumeration of the yeasts and moulds present in cosmetics by counting the colonies on a selective agar medium after aerobic incubation. The standard recommends a microbiological risk analysis in order to establish to which products it is to be applied; low-risk products (ISO 29621) are those of low water activity, hydroalcoholic products, products of extreme pH and so on. Because of the diversity of the products the method may not fit in every detail (for example certain products that are not miscible with water); other methods (e.g. automated ones) are allowed once their equivalence has been demonstrated. The yeasts that have been counted can be identified by the tests of the standards listed in the bibliography, the moulds by other suitable methods. The standard defines yeasts as unicellular fungi that reproduce mainly vegetatively by budding, and moulds as microfungi that form a mycelium, together with their spores and conidia, capable of growing under the conditions of the test.

In Poland it is in force as PN-EN ISO 16212:2017-08 with amendment A1:2023-01. Laboratories that test cosmetics use it for the enumeration of fungi on SDA in parallel with PN-EN ISO 21149 (bacteria on TSA) within the parameter "aerobic mesophilic micro-organisms".

Method principle

The method consists in counting the colonies on a selective agar medium; any inhibition of the growth of the fungi by the sample has to be neutralised, and the neutralisation of the antifungal properties of the product has to be checked and demonstrated. Plate count: preparation of pour plates or spread plates on the specified medium and inoculation with a specified amount of the initial suspension or of a dilution; aerobic incubation at (25 ± 2.5) °C for 3–5 days; counting of the cfu and calculation of the number of yeasts and moulds per millilitre or gram of product. Membrane filtration: a suitable amount of the prepared sample is transferred into a filtration apparatus moistened with sterile diluent, filtered at once and rinsed, and the filter is transferred onto the surface of the medium (according to ISO 21148) and incubated as above. The alternative condition is (22.5 ± 2.5) °C for 5–7 days on a medium without the antibiotic. Media: the neutralising diluent SCDLP 20 (pancreatic digest of casein 20.0 g, soya lecithin 5.0 g, polysorbate 20 40 ml, water 960 ml; the polysorbate is dissolved in a bath at (49 ± 2) °C and, once the components have been added, the mixture is heated for about 30 min; sterilisation at 121 °C for 15 min; pH 7.3 ± 0.2), fluid A (animal peptone 1.0 g/l, pH 7.1 ± 0.2), tryptone sodium chloride solution for the yeast suspension (tryptone 1.00 g, NaCl 8.50 g, pH 7.0 ± 0.2), Sabouraud dextrose agar with chloramphenicol (SDCA: dextrose 40.0 g, animal peptone 5.0 g, pancreatic digest of casein 5.0 g, chloramphenicol 0.050 g, agar 15.0 g, water 1000 ml; sterilisation at 121 °C for 15 min; pH 5.6 ± 0.2) and Sabouraud dextrose agar (SDA, without chloramphenicol) for culturing the reference strain; for known products that are not contaminated with bacteria the media are used without chloramphenicol. Other diluents, neutralisers and media may be used once their suitability has been demonstrated; dehydrated and ready-to-use media of comparable composition or performance are allowed.

Applications

• Routine control of the microbiological purity of cosmetics — the number of yeasts and moulds (cfu/g, cfu/ml)
• High-risk water-based products (creams, gels, lotions) — batch release
• Products with a low number of fungi — the variant with membrane filtration
• Studies of the effectiveness of preservation — counting of the surviving yeasts and moulds
• Known products that are not contaminated with bacteria — a medium without chloramphenicol, the variant of 22.5 °C / 5–7 days

Key parameters

Standard

Standard number

PN-EN ISO 16212:2017-08 + A1:2023-01 (ISO 16212:2017 + Amd 1:2022)

Title (PL)

Kosmetyki — Mikrobiologia — Oznaczanie liczby drożdży i pleśni

Title (EN)

Cosmetics — Microbiology — Enumeration of yeast and mould

Step-by-step procedure

1. Initial suspension

   Take at least 1 g or 1 ml of the product and prepare the initial suspension in a neutralising diluent (e.g. SCDLP 20); prepare the dilutions.

2. Check of the neutralisation

   Check and demonstrate the effectiveness of the neutralisation of the antifungal properties of the product (clause 12 of the standard, annex D — neutralisers).

3. Variant A — plate count

   Inoculate a specified amount of the initial suspension or of a dilution by the pour-plate or the spread-plate technique onto SDCA agar (for products that are not contaminated with bacteria — without chloramphenicol).

4. Variant B — membrane filtration

   Transfer a suitable amount of the sample into a filtration apparatus moistened with sterile diluent, filter it at once, rinse it according to the procedure and transfer the filter onto the surface of the agar (ISO 21148).

5. Incubation

   Incubate aerobically at (25 ± 2.5) °C for 3–5 days; alternatively at (22.5 ± 2.5) °C for 5–7 days on a medium without the antibiotic.

6. Counting and result

   Count the cfu and calculate the number of yeasts and moulds per ml or g of product; where necessary identify the yeasts (the tests of the standards in the bibliography) or the moulds (other suitable methods).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Moulds produce spores — open the plates carefully and work in a cabinet; people who are allergic should avoid exposure
• Chloramphenicol in the medium — avoid inhaling the powder and contact with the skin when weighing it out
• Hot media (bath, autoclave) — risk of burns

Frequently asked questions