ISO 16140-4-2020 amd2-2025 PDF

Full title and description

ISO 16140-4:2020/Amd 2:2025 — Microbiology of the food chain — Method validation — Part 4: Protocol for method validation in a single laboratory — Amendment 2: Protocol for single‑laboratory validation of identification methods of microorganisms. This amendment adds a dedicated protocol and normative provisions for the single‑laboratory validation of methods that identify microorganisms (e.g., biochemical, molecular, MALDI‑TOF and other identification technologies) used in food, feed and related environmental testing within the food chain.

Abstract

This amendment updates ISO 16140‑4:2020 by defining a study design, performance criteria, test panels and reporting requirements specifically for single‑laboratory validation of identification methods for microorganisms. It clarifies inclusivity/exclusivity testing, choice and handling of reference strains and isolates, acceptance criteria for identification results, recommended numbers of isolates and replicates, data analysis approaches and documentation needed to demonstrate that an identification method is fit for use in the validating laboratory. The amendment is intended to harmonize in‑house validation practices for identification procedures so results are robust and auditable.

General information

• Status: Published.
• Publication date: 14 August 2025.
• Publisher: International Organization for Standardization (ISO).
• ICS / categories: 07.100.30 — Food microbiology.
• Edition / version: ISO 16140-4:2020/Amd 2:2025 (Amendment 2, Edition 1, 2025).
• Number of pages: 5.

Scope

This amendment applies to ISO 16140‑4:2020 and provides a protocol for single‑laboratory (in‑house) validation of identification methods for microorganisms used in the analysis of products and environments in the food chain. It is applicable only to methods that are fully specified (all parameters, tolerances and media defined) and already optimized. The amendment is specific to single‑laboratory validation and does not replace interlaboratory validation routes; it does not itself create a reference method.

Key topics and requirements

• Definition of objectives and scope for single‑laboratory identification validation studies.
• Study design guidance: selection and number of isolates, replicates, and sample matrices for inclusivity and exclusivity testing.
• Requirements for reference materials, strain panels and characterization of test isolates (reference identification).
• Performance characteristics and acceptance criteria (e.g., correct identification rates, specificity, sensitivity, repeatability within one laboratory).
• Recommended procedures for blind testing, randomization and avoidance of bias.
• Data analysis and reporting: how to present results, calculate key metrics and document deviations.
• Quality control, traceability, and documentation necessary for accreditation and audit purposes.
• Limitations, applicability statements and statements on the validated range (taxa, matrices, concentrations).

Typical use and users

Primary users are food and feed microbiology laboratories performing in‑house method validation for identification techniques, test kit and instrument manufacturers documenting method performance, method developers, accreditation bodies assessing laboratories (e.g., ISO/IEC 17025 assessors), and competent authorities evaluating laboratory competence. The amendment is used when a laboratory needs documented evidence that an identification method performs acceptably within its own operational conditions.

Related standards

This amendment is part of the ISO 16140 series on microbiological method validation (notably ISO 16140‑1, ISO 16140‑2, ISO 16140‑3, ISO 16140‑4, ISO 16140‑5 and ISO 16140‑6). It should be used alongside ISO 16140‑4:2020 (the base single‑laboratory protocol) and ISO 16140‑1 (vocabulary). Other relevant documents include ISO 17468 (guidance on standardized reference methods) and applicable parts of ISO/IEC 17025 for laboratory competence and quality management. National or regional adoptions of ISO 16140 parts may also provide complementary guidance.

Keywords

ISO 16140, amendment 2, single‑laboratory validation, identification methods, microbiology, food microbiology, inclusivity, exclusivity, performance criteria, method validation, in‑house validation.

FAQ

Q: What is this standard?

A: ISO 16140‑4:2020/Amd 2:2025 is Amendment 2 to Part 4 of the ISO 16140 series; it provides a protocol for single‑laboratory validation specifically for identification methods of microorganisms used in the food chain.

Q: What does it cover?

A: The amendment covers study design, selection of isolates and reference materials, inclusivity/exclusivity testing, acceptance criteria, data analysis, reporting and documentation requirements needed to demonstrate an identification method is fit for use in the validating laboratory.

Q: Who typically uses it?

A: Food and feed microbiology laboratories, method and instrument manufacturers, method developers, accreditation assessors and regulatory laboratories that need to validate identification methods in‑house.

Q: Is it current or superseded?

A: This document was published as an amendment on 14 August 2025 and is a current amendment to ISO 16140‑4:2020. It supplements (does not supersede) the base ISO 16140‑4:2020 standard; users should apply the amended protocol together with the 2020 base document.

Q: Is it part of a series?

A: Yes. It is part of the multipart ISO 16140 series (Method validation for microbiology of the food chain). Use it in conjunction with the other parts in the series (e.g., Parts 1, 2, 3, 4, 5 and 6) and related guidance documents where appropriate.

Q: What are the key keywords?

A: Single‑laboratory validation, identification methods, inclusivity, exclusivity, method validation, food microbiology, ISO 16140, performance criteria.