🦠 Aerobic mesophilic bacteria in cosmetics — colony count on SCDA/TSA agar (32.5 °C, 72 h) or detection after enrichment

Microbiology PN-EN ISO 21149

Overview

ISO 21149:2017 gives general guidelines for the enumeration and detection of aerobic mesophilic bacteria present in cosmetics: by counting colonies on an agar medium after aerobic incubation or by checking the absence of growth of bacteria after enrichment. Because of the diversity of products the method may not be appropriate in every detail (e.g. for products that are not miscible with water); other methods (e.g. automated ones) may be used once equivalence has been demonstrated. Where necessary the micro-organisms counted or detected are identified by tests from the standards listed in the bibliography. 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 ones, those of extreme pH and so on.

The method comprises three variants: plate count, membrane filtration and detection after enrichment. In each of them any inhibition of growth by the product must be neutralised and the effectiveness of the neutralisation checked and demonstrated. In Poland it is in force as PN-EN ISO 21149:2017-07 with amendment A1:2023-01. Laboratories examining cosmetics use it to determine "aerobic mesophilic micro-organisms" on TSA agar (bacteria), alongside PN-EN ISO 16212 on SDA agar (fungi).

Method principle

Plate count: preparation of pour plates or spread plates on a specified medium and inoculation with a specified amount of the initial suspension or of a dilution of it; aerobic incubation at (32.5 ± 2.5) °C for 72 h ± 6 h; counting of the colony-forming units (cfu) and calculation of the number of aerobic mesophilic bacteria 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 an agar medium (in accordance with ISO 21148); incubation as above. Detection after enrichment: incubation of a specified amount of the initial suspension in a non-selective liquid medium with neutralisers and/or dispersing agents at (32.5 ± 2.5) °C for at least 20 h, transfer of a specified amount onto a non-selective agar, aerobic incubation for 48–72 h at the same temperature and a result expressed as the presence/absence of aerobic mesophilic bacteria in the sample S of product. The media: the neutralising diluent SCDLP 20 (pancreatic digest of casein 20.0 g, soya lecithin 5.0 g, polysorbate 20 40.0 ml, water 960 ml; the polysorbate is dissolved in a bath at (49 ± 2) °C and, once the components have been added, is heated for about 30 min; sterilisation at 121 °C for 15 min), soybean-casein digest agar (SCDA) or tryptone soya agar (TSA) for the counting, and Eugon LT 100 broth and agar for the detection.

Applications

• Routine control of the microbiological purity of cosmetics — number of aerobic mesophilic bacteria (cfu/g, cfu/ml)
• Water-based products of high microbiological risk (gels, creams, lotions) — batch release
• Products for which only a "presence/absence" result is required — the variant with enrichment
• Products with a low number of bacteria or products difficult to plate — membrane filtration
• Testing of the effectiveness of preservation (challenge test) — counting of the surviving bacteria

Key parameters

Standard

Standard number

PN-EN ISO 21149:2017-07 + A1:2023-01 (ISO 21149:2017 + Amd 1:2022)

Title (PL)

Kosmetyki — Mikrobiologia — Zliczanie i wykrywanie mezofilnych bakterii tlenowych

Title (EN)

Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria

Step-by-step procedure

1. Initial suspension

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

2. Check of the neutralisation

   Check and demonstrate the effectiveness of the neutralisation of the antibacterial properties of the product (required in every variant of the method).

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 soybean-casein digest agar (SCDA/TSA).

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 in accordance with the procedure and transfer the filter onto the surface of the agar (ISO 21148).

5. Incubation and counting

   Incubate aerobically at (32.5 ± 2.5) °C for 72 h ± 6 h; count the cfu and calculate the number of aerobic mesophilic bacteria per ml or g of product.

6. Variant C — detection after enrichment

   Incubate a specified amount of the initial suspension in a non-selective broth with neutralisers (Eugon LT 100) at (32.5 ± 2.5) °C for at least 20 h, transfer a specified amount onto a non-selective agar and incubate aerobically for 48–72 h; assess the growth.

7. Result

   Report cfu/ml or cfu/g of product or the presence/absence of aerobic mesophilic bacteria in the sample S; where necessary identify the isolates.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Cultures of bacteria of unknown affiliation — work under the conditions of a microbiological laboratory, autoclaving of the waste
• Hot media (bath at 49 °C, autoclave at 121 °C) — risk of burns
• Membrane filtration under vacuum — glassware in good condition

Frequently asked questions