Ph. Eur. 2.6.12 – Microbiological examination of non-sterile products: microbial enumeration tests

Ph. Eur. 2.6.12 – Microbiological examination of non-sterile products: microbial enumeration tests provides harmonized, quantitative methods. Under ICH Q4B Annex 4A(R1), the Ph. Eur., USP <61>, and JP 4.05 chapters are considered interchangeable for regulatory purposes in ICH regions. Harmonization applies only if the chapter is followed in full, without selective mixing of methods across pharmacopoeias.

• Applies when a Ph. Eur. monograph requires microbial enumeration. Chapter 2.6.12 defines the test methods only; acceptance criteria are specified separately in individual monographs or in General Chapter 5.1.4.

• Core principle. Quantitative CFU enumeration: a prepared test portion is cultivated under aerobic conditions; colonies are counted and expressed as CFU/g or CFU/mL after accounting for dilutions.

• Key Requirements & Procedures

  • Method suitability (product-specific) requires demonstrating recovery of low inocula (~100 CFU) and showing that the product does not inhibit detection.

  • Media & incubation.

    • TAMC: Casein-soy (Soybean-Casein Digest) agar (SCDA/TSA), 30–35 °C for 3–5 days.

    • TYMC: Sabouraud-dextrose agar, 20–25 °C for 5–7 days (antibiotics may be added if needed to suppress bacteria).

    • Incubation times may be extended if growth is not readily visible, per Ph. Eur. guidance.

  • Enumeration methods (harmonized options).

    • Membrane filtration (preferred for filterable samples; typically 0.45 µm) with filters placed onto agar.

    • Plate-count methods: pour-plate or surface-spread for soluble/suspendable samples.

    • Most-Probable-Number (MPN): statistical alternative when low counts are expected or plate counts are unsuitable.

Membrane filtration is preferred; plate counts apply when filtration isn’t feasible; MPN is reserved for special cases with very low counts or interfering matrices.