🦠 Escherichia coli in cosmetics — detection after enrichment in Eugon LT 100 broth and isolation on MacConkey agar

Microbiology PN-EN ISO 21150

Overview

ISO 21150:2015 gives general guidelines for the detection and identification of Escherichia coli — a "specified" micro-organism in cosmetic products; the list of specified micro-organisms may differ between countries depending on practice and legislation. The method is based on the detection of E. coli in a non-selective liquid medium (an enrichment broth) and then on isolation on a selective agar medium; other methods may be appropriate depending on the level of detection required (e.g. subculturing onto non-selective media and identification with kits). Because of the diversity of cosmetics the method may not suit certain products in every detail (e.g. products that are not miscible with water) — ISO 18415 may then be appropriate; alternative methods (e.g. automated ones) may be used once equivalence has been demonstrated. The standard recommends a microbiological risk analysis in order to establish to which products it is to be applied; low-risk products (ISO 29621) include those of low water activity, hydroalcoholic ones and those of extreme pH.

In Poland it is in force as PN-EN ISO 21150:2016-01 with amendment A1:2023-03 (it replaced PN-EN ISO 21150:2010). In the practice of laboratories examining the microbiological purity of cosmetics the standard belongs to the set of PN-EN ISO standards (21149, 16212, 22718, 22717, 18416) used for quantitative and qualitative testing; to verify the presence of E. coli a plate is inoculated on the selective-differential MacConkey medium, on which these bacilli form dark pink colonies with a dark pink rim.

Method principle

The first stage is enrichment in a non-selective broth, which increases the number of micro-organisms without the risk of inhibition by the selective components of selective-differential media; the second stage (isolation) is carried out on a selective medium, after which identification tests are performed. Any inhibition of the growth of micro-organisms by the sample must be neutralised in order to make the detection of viable cells possible — in every case and whatever the methodology the neutralisation of the antibacterial properties of the product has to be checked and demonstrated (clause 11 of the standard, the suitability test on soybean-casein digest agar). The sample is at least 1 g or 1 ml of product used to prepare the initial suspension. The media listed in the standard: tryptone sodium chloride solution as the diluent for the bacterial suspension, soybean-casein digest agar for the suitability test, Eugon LT 100 broth for the enrichment (other enrichment broths are permitted), MacConkey agar for the isolation of E. coli (it contains, among other things, 30.0 mg of neutral red and 1.0 mg of crystal violet per litre; after dissolution it is boiled for 1 min and sterilised at 121 °C for 15 min) and Levine's eosin methylene blue (EMB) agar for the confirmation (400 mg of eosin Y and 65 mg of methylene blue per litre; to each 100 ml of the melted agar 5 ml of a 20 % lactose solution, 2 ml of 2 % eosin Y and 2 ml of 0.033 % methylene blue are added; sterilisation at 121 °C for 15 min). All the media are sterilised in an autoclave at 121 °C for 15 min.

Applications

• Control of the microbiological purity of cosmetics — detection of E. coli as a specified micro-organism (water-based products: creams, lotions, gels)
• Batch release of cosmetic products and testing of raw materials
• Testing of microbiological stability and of the effectiveness of preservation (together with PN-EN ISO 21149, 16212, 22717, 22718, 18416)
• Contract and in-house laboratories of the cosmetics industry; low-risk products (ISO 29621) after a risk analysis

Key parameters

Standard

Standard number

PN-EN ISO 21150:2016-01 + A1:2023-03 (ISO 21150:2015 + Amd 1:2022)

Title (PL)

Kosmetyki — Mikrobiologia — Wykrywanie obecności Escherichia coli

Title (EN)

Cosmetics — Microbiology — Detection of Escherichia coli

Step-by-step procedure

1. Risk analysis and preparation of the sample

   Establish whether the product is subject to testing (microbiological risk analysis, ISO 29621). Take at least 1 g or 1 ml of product and prepare the initial suspension in the enrichment broth with neutralisers.

2. Suitability test (neutralisation)

   Demonstrate that the antibacterial properties of the product have been neutralised (clause 11 of the standard) — using a suspension of the reference strain in tryptone sodium chloride solution and soybean-casein digest agar.

3. Enrichment

   Incubate the initial suspension in non-selective Eugon LT 100 broth (or another enrichment broth permitted by the standard) under the conditions given in the standard.

4. Isolation

   From the enrichment culture inoculate MacConkey agar and incubate it; look for colonies characteristic of E. coli (dark pink with a dark pink rim).

5. Confirmation and identification

   Subculture the suspect colonies onto Levine's EMB agar and carry out identification tests (e.g. identification kits after subculturing onto a non-selective medium).

6. Result

   Report the presence or absence of E. coli in the sample of product examined (1 g or 1 ml), together with the information that the neutralisation has been demonstrated.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• E. coli may include pathogenic strains — work under the conditions of a microbiological laboratory, autoclaving of the cultures
• The dyes of the media (crystal violet, eosin, methylene blue) — avoid contact with the skin, gloves
• Hot media after boiling and autoclaving — risk of burns

Frequently asked questions