🦠 Staphylococcus aureus in cosmetics — detection after enrichment in a broth with neutralisers and isolation on Baird-Parker agar

Microbiology PN-EN ISO 22718

Overview

The microbiological testing of cosmetics is carried out in accordance with a microbiological risk analysis, which depends on the possible change of the product, on the pathogenicity of the micro-organisms, on the site of application (hair, skin, eyes, mucous membranes) and on the users (adults, children up to 3 years of age). The detection of the skin pathogens — Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans — is important, because they may cause infections of the skin or of the eyes. ISO 22718:2015 (the second edition, a minor revision of ISO 22718:2006) gives general guidelines for the detection and identification of S. aureus in cosmetic products; low-risk products (low water activity, hydroalcoholic products, extreme pH — ISO 29621) may not require testing. The method is based on the detection of S. aureus in a non-selective liquid medium (an enrichment broth) and then on isolation on a selective agar; for certain products (e.g. those not miscible with water) it may be inappropriate — ISO 18415 is then used — and alternative methods (e.g. automated ones) require a demonstration of equivalence. S. aureus are Gram-positive cocci in clusters, with smooth, usually yellow-pigmented colonies, catalase- and coagulase-positive; it is an opportunistic pathogen which may live on the skin of healthy persons and which is undesirable in a cosmetic. In Poland the standard is in force as PN-EN ISO 22718:2016-01 and is one of the basic standards for testing the microbiological purity of cosmetics (alongside PN-EN ISO 21149, 21150, 22717, 18416, 18415).

Method principle

The first stage is enrichment in a non-selective broth, in order to increase the number of micro-organisms without the risk of inhibition by selective components; the second is isolation on a selective medium and identification tests. Any possible inhibition of growth by the sample must be neutralised, and the effectiveness of the neutralisation of the antimicrobial properties of the product checked and demonstrated (clause 11 of the standard, the neutralisers in annex B). The enrichment broth Eugon LT 100 contains neutralising components — lecithin and polysorbate 80 — and the dispersing agent octoxynol 9 (the composition per 1 l: casein digest 15 g, soya digest 5 g, L-cystine 0.7 g, NaCl 4 g, sodium sulfite 0.2 g, glucose 5.5 g, egg lecithin 1 g, polysorbate 80 5 g, octoxynol 9 1 g; pH 7.0 ± 0.2; sterilisation at 121 °C for 15 min); other broths are permitted (annex A) if their suitability has been demonstrated. The selective Baird-Parker agar: the basic medium (casein digest 10 g, yeast extract 1 g, meat extract 5 g, sodium pyruvate 10 g, L-glycine 12 g, lithium chloride 5 g, agar 12–22 g, water to 950 ml; pH 7.2 ± 0.2) with the addition of a potassium tellurite solution (1 g/100 ml, sterilised by filtration at 0.22 µm, stored for up to a month at (3 ± 2) °C) and of an egg yolk emulsion (about 20 %). For the suitability test tryptone sodium chloride solution (tryptone 1 g, NaCl 8.5 g/l; pH 7.0 ± 0.2) and TSA/SCDA agar (pH 7.3 ± 0.2) are used; the general guidelines — ISO 21148, the test strains — EN 12353. Water: distilled or purified in accordance with ISO 21148.

Applications

• Control of the microbiological purity of finished products and of raw materials in the cosmetics industry (testing before the preservation test)
• Cosmetics for the skin, the hair, the eyes and the mucous membranes and products for children up to 3 years of age — a skin pathogen of considerable importance
• Assessment of the hygiene of the manufacturing process (product information file, GMP)
• Complaint testing and batch release; validation of the neutralisation of the preservatives

Key parameters

Standard

Standard number

PN-EN ISO 22718:2016-01 (ISO 22718:2015)

Title (PL)

Kosmetyki — Mikrobiologia — Wykrywanie obecności Staphylococcus aureus

Title (EN)

Cosmetics — Microbiology — Detection of Staphylococcus aureus

Step-by-step procedure

1. Risk analysis and sample

   Establish on the basis of a risk analysis (ISO 29621) whether the product requires testing; take at least 1 g or 1 ml of product as the sample for the initial suspension.

2. Initial suspension and enrichment

   Disperse the sample in a specified volume of the enrichment broth with neutralisers (Eugon LT 100 or another of demonstrated suitability); prepare dilutions if necessary; incubate the broth (clause 9.3 of the standard).

3. Isolation

   From the enrichment culture inoculate Baird-Parker agar (or another medium from annex A) and incubate it; subculturing onto a non-selective medium with subsequent identification by kits is also permitted.

4. Identification

   Confirm the colonies of characteristic appearance on the selective medium by the catalase and the coagulase test (S. aureus: both positive); where there is doubt use identification kits.

5. Suitability test (neutralisation)

   In parallel demonstrate that the product does not inhibit growth: prepare an inoculum of S. aureus in tryptone sodium chloride solution, add it to the sample with the broth and compare the recovery with a control on TSA (clause 11.3); without a confirmation of the neutralisation a negative result is not valid.

6. Result and test report

   State the presence or the absence of S. aureus in the amount of product examined, the broth and the medium used, the manner of neutralisation and the result of the suitability test (clauses 10 and 12).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• S. aureus is an opportunistic pathogen (infections of the skin and of the eyes) — work under the conditions of a microbiological laboratory, decontamination of the cultures
• Potassium tellurite is toxic — weigh it out and prepare the solution wearing gloves, avoid raising dust
• Egg yolk emulsion — sterility and hygiene of the preparation (disinfection of the shells)

Frequently asked questions