🦠 Pseudomonas aeruginosa in cosmetics — detection after enrichment in Eugon LT 100 broth and isolation on cetrimide agar
In short
The cosmetic sample (≥ 1 g or 1 ml) is enriched in non-selective Eugon LT 100 broth, P. The test is performed according to PN-EN ISO 22717:2016-01 + A1:2023-03 (ISO 22717:2015 + Amd 1:2022); Sample: at least 1 g or 1 ml of product. The procedure comprises 6 steps; it is used for: Control of the microbiological purity of cosmetics — detection of P. aeruginosa as a specified micro-organism, Water-based products of high risk (creams, gels, lotions, eye products) — batch release, Testing of raw materials, of process water and of products during work on preservation.
At a glance
- Standard: PN-EN ISO 22717:2016-01 + A1:2023-03 (ISO 22717:2015 + Amd 1:2022)
- Category: Microbiology
- Procedure steps: 6
- Sample: at least 1 g or 1 ml of product
- Stages: non-selective enrichment → selective isolation → identification
- Enrichment broth: Eugon LT 100 (or another in accordance with the standard)
Overview
ISO 22717:2015 gives general guidelines for the detection and identification of Pseudomonas aeruginosa — 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 P. aeruginosa in a non-selective liquid medium (an enrichment broth) and then on isolation on a selective agar medium; depending on the level of detection required other methods may be appropriate (e.g. subculturing onto non-selective media and identification with kits). For products that are not miscible with water ISO 18415 may be appropriate; alternative methods (e.g. automated ones) are permitted once equivalence has been demonstrated. The standard recommends a microbiological risk analysis; low-risk products (ISO 29621) include those of low water activity, hydroalcoholic ones and those of extreme pH. The main identifying characteristics of P. aeruginosa according to the standard include growth on selective cetrimide agar.
In Poland it is in force as PN-EN ISO 22717:2016-01 with amendment A1:2023-03 (it replaced PN-EN ISO 22717:2010). In laboratories examining the microbiological purity of cosmetics the selective cetrimide medium (Cetrimide Agar) is used to observe the presence of P. aeruginosa — in accordance with the set of PN-EN ISO standards used for the quantitative and qualitative testing of cosmetics.
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, and the neutralisation of the antibacterial properties of the product must 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 for the initial suspension. The media according to 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 broths are permitted), cetrimide agar for the isolation of P. aeruginosa (it contains, among other things, cetrimide, that is cetyltrimethylammonium bromide, 0.3 g and glycerol 10.0 ml per 1000 ml; after dissolution it is boiled for 1 min and sterilised at 121 °C for 15 min) and Pseudomonas agar P for the detection of pyocyanin (with glycerol 10.0 ml/l; boiling for 1 min, sterilisation at 121 °C for 15 min) for the confirmation.
Applications
- Control of the microbiological purity of cosmetics — detection of P. aeruginosa as a specified micro-organism
- Water-based products of high risk (creams, gels, lotions, eye products) — batch release
- Testing of raw materials, of process water and of products during work on preservation
- Contract and in-house laboratories of the cosmetics industry (the set PN-EN ISO 21149/16212/21150/22717/22718/18416)
Key parameters
| Parameter | Value |
|---|---|
| Sample | at least 1 g or 1 ml of product |
| Stages | non-selective enrichment → selective isolation → identification |
| Enrichment broth | Eugon LT 100 (or another in accordance with the standard) |
| Isolation agar | cetrimide agar: cetrimide 0.3 g/l, glycerol 10 ml/l |
| Confirmation agar | Pseudomonas agar P (pyocyanin), glycerol 10 ml/l |
| Condition of validity | demonstrated neutralisation of the antibacterial properties (clause 11) |
Standard
- Standard number
- PN-EN ISO 22717:2016-01 + A1:2023-03 (ISO 22717:2015 + Amd 1:2022)
- Title (PL)
- Kosmetyki — Mikrobiologia — Wykrywanie obecności Pseudomonas aeruginosa
- Title (EN)
- Cosmetics — Microbiology — Detection of Pseudomonas aeruginosa
Step-by-step procedure
-
Risk analysis and sample
Establish whether the product is subject to testing (ISO 29621). Take at least 1 g or 1 ml of product and prepare the initial suspension in the enrichment broth.
-
Suitability test
Demonstrate the neutralisation of the antibacterial properties of the product (clause 11) using a suspension of the reference strain in tryptone sodium chloride solution and soybean-casein digest agar.
-
Enrichment
Incubate the initial suspension in non-selective Eugon LT 100 broth under the conditions given in the standard.
-
Isolation
From the culture inoculate cetrimide agar and incubate it; assess the growth of colonies characteristic of P. aeruginosa.
-
Confirmation and identification
Subculture the suspect colonies onto Pseudomonas agar P (production of pyocyanin) and carry out identification tests.
-
Result
Report the presence or absence of P. aeruginosa 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
| Equipment | Example | Indicative price |
|---|---|---|
| Autoclave 121 °C | sterilisation of the media for 15 min | — |
| Incubator for the incubation of the cultures | conditions according to the procedure clauses of the standard | — |
| Water bath / hot plate | boiling of the media for 1 min before sterilisation | — |
| Petri dishes, flasks, sterile pipettes, loop | isolation and confirmation inocula | — |
| Identification kits | identification of the isolates (according to the standard) | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Tryptone sodium chloride solution | — | diluent for the bacterial suspension (reference strain) |
| Soybean-casein digest agar | — | suitability test of the method (neutralisation) |
| Eugon LT 100 broth | — | non-selective enrichment with neutralisation of the preservatives |
| Cetrimide agar (cetrimide 0.3 g/l, glycerol 10 ml/l) | — | selective isolation of P. aeruginosa |
| Pseudomonas agar P (glycerol 10 ml/l) | — | confirmation — production of pyocyanin |
Health and safety (OHS)
- P. aeruginosa is an opportunistic pathogen (infections of the eyes and of wounds) — work under the conditions of a microbiological laboratory, autoclaving of the cultures
- Cetrimide (a quaternary ammonium salt) — irritant; gloves and goggles when weighing it out
- Hot media after boiling and autoclaving — risk of burns
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 22717:2016-01 + A1:2023-03 (ISO 22717:2015 + Amd 1:2022) — “Cosmetics — Microbiology — Detection of Pseudomonas aeruginosa”.
How does this method work?
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, and the neutralisation of the antibacterial properties of the product must be checked and demonstrated (clause 11 of the standard, the suitability test on soybean-casein digest agar).
What is the measuring range and accuracy?
Sample: at least 1 g or 1 ml of product; Stages: non-selective enrichment → selective isolation → identification; Enrichment broth: Eugon LT 100 (or another in accordance with the standard); Isolation agar: cetrimide agar: cetrimide 0.3 g/l, glycerol 10 ml/l.
How long does the test take?
The procedure comprises 6 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Autoclave 121 °C, Incubator for the incubation of the cultures, Water bath / hot plate, Petri dishes, flasks, sterile pipettes, loop, Identification kits.
Where is this test used?
Control of the microbiological purity of cosmetics — detection of P. aeruginosa as a specified micro-organism; Water-based products of high risk (creams, gels, lotions, eye products) — batch release; Testing of raw materials, of process water and of products during work on preservation; Contract and in-house laboratories of the cosmetics industry (the set PN-EN ISO 21149/16212/21150/22717/22718/18416).
What safety precautions apply?
P. aeruginosa is an opportunistic pathogen (infections of the eyes and of wounds) — work under the conditions of a microbiological laboratory, autoclaving of the cultures; Cetrimide (a quaternary ammonium salt) — irritant; gloves and goggles when weighing it out; Hot media after boiling and autoclaving — risk of burns.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 22717.