💊 Microbiological purity of cosmetics

Pharma and Cosmetics PN-EN ISO 17516

Overview

Microbiological purity is one of the key safety parameters of cosmetic products. The PN-EN ISO 17516 standard establishes microbiological limits for cosmetics, ensuring that products do not contain excessive numbers of microorganisms or specified pathogenic microorganisms that could adversely affect product quality or consumer safety.

The standard defines two product categories with different limits. For general use products (body creams, shampoos, shower gels), the acceptable number of aerobic mesophilic microorganisms is ≤1×10³ CFU/g (or mL). For products intended for use around eyes, in children under 3 years, and on mucous membranes — the limit is tightened to ≤1×10² CFU/g (or mL). In both categories, absence of pathogens is required: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in 0.1 g (or 0.1 mL) of product.

Microbiological testing of cosmetics is performed according to accompanying standard series: ISO 21148 (general test instructions), ISO 21149 (determination of mesophilic aerobic bacteria), ISO 22717 (Pseudomonas aeruginosa), ISO 22718 (Staphylococcus aureus), ISO 18416 (Candida albicans), and ISO 16212 (yeasts and molds). These methods describe sample preparation, selection of culture media, incubation conditions, and result interpretation.

The cosmetics manufacturer is responsible for ensuring microbiological purity of the product throughout its shelf life. Regular microbiological testing is an element of the quality assurance system required by Regulation (EC) No 1223/2009 on cosmetic products.

Method principle

The test involves taking a representative sample of cosmetic product, suspending it in diluting solution (peptone-salt buffer with surfactant neutralizing preservative action — e.g., Tween 80, lecithin, sodium thiosulfate), and then plating on appropriate culture media. Total aerobic microbial count (TAMC) is determined by plate method (pour or surface plating) on casein-soy agar (TSA), with incubation 48–72 h at 30–35°C. Yeasts and molds (TYMC) are cultured on Sabouraud agar with chloramphenicol, 5–7 days at 20–25°C. Pathogens are detected by enrichment in selective media (MacConkey, Cetrimide, Baird-Parker, SDA) with biochemical confirmation or API/Vitek test.

Applications

• Quality control of finished cosmetic products (creams, balms, shampoos)
• Testing cosmetic raw materials (plant extracts, floral waters)
• Control of packaging and packaging materials purity
• Production environment monitoring (cosmetic GMP)
• Microbiological stability testing as part of shelf life assessment
• Verification of disinfection and washing process effectiveness of production lines
• Safety assessment of products under Safety Assessment (Regulation 1223/2009)
• Complaint testing — analysis of microbiological contamination causes

Key parameters

Standard

Standard number

PN-EN ISO 17516:2014

Title (PL)

Kosmetyki — Mikrobiologia — Limity mikrobiologiczne

Title (EN)

Cosmetics — Microbiology — Microbiological limits

Step-by-step procedure

1. Sampling and sample preparation

   Aseptically collect 1 g (or 1 mL) of cosmetic product. Transfer to stomacher bag with 9 mL diluting solution with neutralizers.

   ⏱ Time: 5 min

2. Homogenization

   Homogenize sample in stomacher (1–2 min, 230 rpm) or by vigorous shaking. Obtain homogeneous 1:10 suspension.

   ⏱ Time: 2 min

3. TAMC determination (aerobic mesophilic bacteria)

   Inoculate 1 mL of 1:10 suspension on TSA plates (pour or surface method). Perform in duplicate. Incubate 48–72 h at 30–35°C.

   ⏱ Time: 5 min + 72 h incubation • 🌡 Temperature: 30–35°C

4. TYMC determination (yeasts and molds)

   Inoculate 1 mL of 1:10 suspension on SDA + chloramphenicol plates. Incubate 5–7 days at 20–25°C.

   ⏱ Time: 5 min + 7 days incubation • 🌡 Temperature: 20–25°C

5. E. coli detection

   Transfer 1 mL suspension to 9 mL MacConkey broth. Incubate 24–48 h at 30–35°C. Plate on MacConkey agar. Confirm biochemically.

   ⏱ Time: 48–96 h • 🌡 Temperature: 30–35°C

6. P. aeruginosa detection

   Enrichment in cetrimide broth, 24–48 h, 30–35°C. Plating on cetrimide agar. Confirm (oxidase+, pigment, API/Vitek).

   ⏱ Time: 48–96 h • 🌡 Temperature: 30–35°C

7. S. aureus detection

   Enrichment in casein-soy broth, 24–48 h, 30–35°C. Plating on Baird-Parker agar. Confirm (coagulase+, catalase+).

   ⏱ Time: 48–96 h • 🌡 Temperature: 30–35°C

8. C. albicans detection

   Enrichment in Sabouraud broth, 48 h, 30–35°C. Plating on SDA agar. Confirm (germ tube test, API AUX/ID32C).

   ⏱ Time: 48–96 h • 🌡 Temperature: 30–35°C

9. Result reading and calculation

   Count colonies on TAMC and TYMC plates. Calculate CFU/g = colony count × dilution. Assess presence/absence of pathogens. Compare with ISO 17516 limits.

   ⏱ Time: 30 min

10. Report and compliance assessment

    Prepare report with results: TAMC, TYMC, pathogens (present/absent). Issue compliance assessment with ISO 17516. Maintain laboratory documentation.

    ⏱ Time: 30 min

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Work with microorganisms — class II laminar cabinet mandatory, BSL-1/BSL-2 level
• Pathogens (P. aeruginosa, S. aureus, E. coli) — risk of infection, nitrile gloves, hand disinfection
• Autoclaving biological waste before disposal (121°C, 15 min)
• Disinfection of work surfaces with 70% alcohol or sodium hypochlorite solution
• Safety glasses and laboratory coat mandatory

Frequently asked questions