ISO 21149

The ISO 21149 standard, which includes test methods for the enumeration and detection of aerobic mesophilic bacteria in cosmetic products, gives two different guidelines:

- by counting the colonies on agar medium after aerobic incubation or
- checking for the absence of bacterial growth after enrichment. by

General method under ISO 21149 standard; It involves counting colonies based on the presence or absence of bacterial growth on a non-selective agar medium or after enrichment.

Possible inhibition of microbial growth by the sample should be neutralized to allow detection of viable microorganism. In any case and whatever the methodology, the neutralization of the antimicrobial properties of the product must be checked and confirmed.

Another method is “Plate Counting”
Plate counting consists of the following steps:
⎯ Preparation of poured plates or smear plates using a specific culture medium and Inoculation of plates using a defined amount of initial suspension or dilution of the product.
⎯ Aerobic incubation of plates at 32.5 °C ± 2.5 °C for 72 hours ± 6 hours.
⎯ Number of colony forming units (CFU) counting and calculating the number of aerobic mesophilic bacteria per milliliter or gram of product.

Third method “Membrane Filtration”
Membrane filtration consists of the following steps:
⎯ Add an appropriate amount of the validated sample to a small amount of suitable sterile diluent. into the filtration apparatus moistened with water, drain immediately and wash according to the validated procedure (see 13.3.4). Transfer the membrane filter to the surface of the specified agar medium as specified in ISO 21148.
⎯ Aerobic incubation of membranes at 32.5 °C ± 2.5 °C for 72 hours ± 6 hours.
⎯ Colony forming units Counting the number of CFUs and calculating the number of aerobic mesophilic bacteria per milliliter or gram of product.

The fourth method is “Bacteria Detection by Enrichment”
The determination of bacteria by enrichment consists of the following steps:
⎯ 32.5 °C ± 2.5 Incubation of a defined amount of starting suspension for at least 20 hours at °C in a non-selective liquid medium containing suitable neutralizers and/or dispersing agents.
⎯ Transfer of a specified amount of the previous suspension onto non-selective solid agar medium.
⎯ Aerobic incubation for 48 hours to 72 hours at 32.5 °C ± 2.5 °C.
⎯ Detection of growth of aerobic mesophilic bacteria per S sample of product and expressing results as "presence/absence".