💊 Microbiological purity of cosmetics
Testing microbiological purity of cosmetic products according to ISO 17516. Determination of total microbial count and detection of pathogens (E. coli, P. aeruginosa, S. aureus, C. albicans).
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
| Parameter | Value |
|---|---|
| TAMC (general products) | ≤1 × 10³ CFU/g or mL |
| TAMC (eye area, children <3 years, mucous membranes) | ≤1 × 10² CFU/g or mL |
| Pathogens — absence in 0.1 g/mL | E. coli, P. aeruginosa, S. aureus, C. albicans |
| Incubation temperature (bacteria) | 30–35°C, 48–72 h |
| Incubation temperature (fungi) | 20–25°C, 5–7 days |
| Sample volume | 1 g or 1 mL (with 1:10 dilution) |
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.
2. Homogenization
Homogenize sample in stomacher (1–2 min, 230 rpm) or by vigorous shaking. Obtain homogeneous 1:10 suspension.
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.
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.
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.
6. P. aeruginosa detection
Enrichment in cetrimide broth, 24–48 h, 30–35°C. Plating on cetrimide agar. Confirm (oxidase+, pigment, API/Vitek).
7. S. aureus detection
Enrichment in casein-soy broth, 24–48 h, 30–35°C. Plating on Baird-Parker agar. Confirm (coagulase+, catalase+).
8. C. albicans detection
Enrichment in Sabouraud broth, 48 h, 30–35°C. Plating on SDA agar. Confirm (germ tube test, API AUX/ID32C).
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.
10. Report and compliance assessment
Prepare report with results: TAMC, TYMC, pathogens (present/absent). Issue compliance assessment with ISO 17516. Maintain laboratory documentation.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Class II laminar cabinet | Thermo Fisher MSC-Advantage, Esco Labculture, FASTER BH-EN | 25 000–60 000 PLN |
| Bacteriological incubator 30–35°C | Memmert IN110, Binder BD115, POL-EKO CLN 53 | 8 000–20 000 PLN |
| Mycological incubator 20–25°C | Memmert IPP110, Binder KB115, with Peltier cooling | 10 000–25 000 PLN |
| Laboratory autoclave | Tuttnauer 3870ELV, Systec VX-75, 121°C / 15 min | 20 000–60 000 PLN |
| Analytical balance (0.01 g) | Mettler Toledo ME204, Sartorius Quintix 224 | 5 000–12 000 PLN |
| Stomacher / homogenizer | Seward Stomacher 400, BagMixer 400 | 10 000–25 000 PLN |
| Colony counter | Interscience Scan 300/500, BZG 30 Koloniezähler | 5 000–20 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Casein-soy agar (TSA) | 91079-40-2 | Medium for TAMC determination, ready plates or powder for preparation, pack 500 g |
| Sabouraud agar with chloramphenicol (SDA) | — | Medium for yeasts and molds determination (TYMC), incubation 20–25°C, 5–7 days |
| Diluting buffer with neutralizers | — | Peptone-salt solution + Tween 80 (3%) + lecithin (0.3%) + sodium thiosulfate (0.5%) — preservative neutralization |
| MacConkey agar | — | Selective medium for E. coli and enterobacteria detection |
| Cetrimide agar | — | Selective medium for Pseudomonas aeruginosa detection |
| Baird-Parker agar | — | Selective medium for Staphylococcus aureus detection, with egg yolk emulsion and potassium tellurite |
| Sabouraud Dextrose agar (SDA) | — | Medium for Candida albicans detection, with chloramphenicol |
| Stomacher bags | — | Sterile filter bags for sample homogenization, disposable |
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