🦠 Candida albicans in cosmetics — detection after enrichment in Eugon LT 100 broth and isolation on Sabouraud agar with chloramphenicol

Microbiology PN-EN ISO 18416

Overview

ISO 18416:2015 gives general guidelines for the detection and identification of Candida albicans — a "specified" micro-organism in cosmetic products; the list of specified micro-organisms may differ between countries. The method is based on the detection of C. albicans 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.

In Poland it is in force as PN-EN ISO 18416:2016-01 with amendment A1:2023-03 (it replaced PN-EN ISO 18416:2009). It belongs to the set of PN-EN ISO standards used in laboratories for testing the microbiological purity of cosmetics (alongside 21149, 16212, 21150, 22717, 22718); the strain Candida albicans ATCC 10231 is also one of the test organisms in the testing of the effectiveness of preservation.

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 antimicrobial properties of the product must be checked and demonstrated (clause 11 of the standard). 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 yeast suspension, Sabouraud dextrose agar (SDA) for the suitability test (other agars are permitted), Eugon LT 100 broth for the enrichment (other broths are permitted), Sabouraud dextrose agar with chloramphenicol for the isolation of C. albicans (after sterilisation at 121 °C for 15 min the pH is 5.6 ± 0.2; other selective agars are permitted) and corn meal agar with 1 % polysorbate 80 for the identification.

Applications

• Control of the microbiological purity of cosmetics — detection of C. albicans as a specified micro-organism
• Water-based products of high microbiological risk (creams, lotions, gels) — batch release
• Testing of raw materials 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

Standard

Standard number

PN-EN ISO 18416:2016-01 + A1:2023-03 (ISO 18416:2015 + Amd 1:2022)

Title (PL)

Kosmetyki — Mikrobiologia — Wykrywanie obecności Candida albicans

Title (EN)

Cosmetics — Microbiology — Detection of Candida albicans

Step-by-step procedure

1. 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.

2. Suitability test

   Demonstrate the neutralisation of the antimicrobial properties of the product (clause 11) using a suspension of the reference strain in tryptone sodium chloride solution and SDA agar.

3. Enrichment

   Incubate the initial suspension in non-selective Eugon LT 100 broth under the conditions given in the standard.

4. Isolation

   From the culture inoculate Sabouraud dextrose agar with chloramphenicol and incubate it; assess the growth of yeast-like colonies.

5. Identification

   Identify the suspect colonies by identification tests, among other things by inoculation onto corn meal agar with 1 % polysorbate 80 and microscopic assessment.

6. Result

   Report the presence or absence of C. albicans 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)

• Candida albicans is an opportunistic pathogen — work under the conditions of a microbiological laboratory, autoclaving of the cultures
• Chloramphenicol in the medium — avoid inhaling the powder and contact with the skin when weighing it out
• Hot media after autoclaving — risk of burns

Frequently asked questions