Here, information is given on preparation of specific products, on analytical methods for detection, enumeration and typing of Salmonella, and on reference materials.
Detection of Salmonella
EN-ISOInternational Standardisation Organisation
The internationally accepted horizontal method for the detection of Salmonella is described in EN-ISO 6579-1:2017 ‘Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp.’
The scope of this document includes:
• products intended for human consumption and the feeding of animals;
• environmental samples in the area of food production and food handling;
• samples from the primary production stage such as animal faeces, dust, and swabs.
In March 2020 Amendment 1 to EN-ISO 6579-1 was published, entitled:
EN ISO 6579-1:2017/A1:2020 Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp. AMENDMENT 1: Broader range of incubation temperatures, amendment to the status of Annex D, and correction of the composition of MSRV and SC.
The main amendments to EN ISO 6579-1 are the following:
• The temperature range for incubation of selective media has been extended from 37 °C ± 1 °C to 34 °C to 38 °C without further tolerance (like it already was for non-selective media). This amendment also resulted in an update of the flow schemes in Annex A.
• In Annex B (B.4) the composition of Modified semi-solid Rappaport-Vassiliadis (MSRV) agar was corrected when preparing it from individual ingredients. In the composition described in EN-ISO 6579-1:2017 the final concentration of MgCl2 in MSRV agar was not correct.
• The status of Annex D on detection of Salmonella Typhi and Salmonella Paratyphi was changed from normative to informative.
• A few corrections were included in Annex D, especially concerning the composition of selenite cystine medium (broth) in Annex D.3. In EN-ISO 6579-1:2017 it is indicated that 100 ml L-cystine solution (D.3.1.2) should be added to 1000 ml base medium, but this has to be only 10 ml.
Occasionally problems may occur when using the selective enrichment medium Modified semi-solid Rappaport Vassiliadis (MSRV) agar. Information on different causes of possible problems, as well as possible solutions have been published in the EURLEuropean Union Reference Laboratory -Salmonella Newsletter Vol. 24 No. 3 (September 2018), page 9-10: [link]
In many EUEuropese unie legislation, EN-ISO 6579-1 is prescribed as the reference method for detection of Salmonella. In this EU legislation it is also indicated that alternative methods may be used if validated against the reference method. In Regulation (EC) 2073/2005 ‘on microbiological criteria for foodstuffs’ the following information on the use of alternative methods is given in Article 5 (consolidated version 2019):
The use of alternative analytical methods is acceptable provided they are:
• validated against the specific reference method provided for in Annex I in accordance with the protocol set out in standard EN-ISO 16140-2, and
• validated for the food category specified in the relevant microbiological criterion set in Annex I the compliance with which is verified by the food business operator, or validated for a broad range of food as referred to in EN-ISO 16140-2.
Proprietary methods may be used as alternative analytical methods, provided they are:
• validated, in accordance with the protocol set out in standard EN-ISO 16140-2, against the specific reference method provided for verifying compliance with the microbiological criteria laid down in Annex I, as provided for in the third subparagraph, and
• certified by an independent certification body.
The certification of the proprietary method shall:
• be subject, at least every 5 years, to reassessment through renewal procedures,
• show that the production process assurance of the manufacturer was evaluated, and
• include a summary of or a reference to the validation results of the proprietary method and a statement on the quality management of the production process of the method.
In Europe, the following organisations perform (independent) validations following the procedure of EN-ISO 16140-2 (‘Microbiology of the food chain – Method validation - Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method’). Certificates of validated methods are available at the relevant websites:
AFNOR validation: https://nf-validation.afnor.org/en/
Enumeration of Salmonella
A procedure for the enumeration of Salmonella spp. is described in CENEuropean Committee for Standardisation
-ISOInternational Standardisation Organisation
/TS 6579-2:2012 ‘Microbiology of food and animal feed - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 2: Enumeration by a miniaturized most probable number technique’.
The procedure describes a miniaturized MPN method in 12-wells microtiter plates with Modified semi-solid Rappaport Vassiliadis (MSRV) agar as selective enrichment medium.
Serotyping of Salmonella
CENEuropean Committee for Standardisation
-ISOInternational Standardisation Organisation
Guidelines for serotyping of Salmonella are given in CEN-ISO/TR 6579-3:2014 ‘Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 3: Guidelines for serotyping of Salmonella spp.’
In this guidance document reference is made to the ‘White-Kauffmann-Le Minor scheme’, as described by Grimont and Weill (2007).
Grimont P .A.D., & W eill F.-X., 2007. Antigenic formulae of the Salmonella serovars, 9th edition. WHOWorld Health Organization Collaborating Centre for Reference and Research on Salmonella. Paris: Institut Pasteur. The scheme can be found at: https://www.pasteur.fr/sites/default/files/veng_0.pdf
Supplements to the White-Kauffmann-Le Minor scheme are published in Research in Microbiology, a publication of Institute Pasteur:
Guibourdenche, M., Roggentin, P. Mikoleit, M., Fields, P.I., Bockemühl, J., Grimont, P.A.D., Weill, F-X. Supplement 2003–2007 (No. 47) to the White–Kauffmann–Le Minor scheme. Research in Microbiology, 2010, 161, pp. 26–29.
Issenhuth-Jeanjean, S., Roggentin, P., Mikoleit, M., Guibourdenche, M., de Pinna, E., Nair, S., I. Fields, P.I., Weill, F-X. Supplement 2008-2010 (no. 48) to the White-Kauffmann-Le Minor scheme. Research in Microbiology, 2014, 165, pp. 526-530.
For the validation of alternative methods for serotyping of Salmonella, the protocol of EN-ISO 16140-6 (‘Microbiology of the food chain – Method validation - Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing procedures’) shall be followed. For the use of alternative serotyping methods, similar rules apply as indicated for alternative detection methods [link].
MLVA typing of Salmonella
The Standard Operating Procedure for Multiple-Locus Variable-number tandem repeat Analysis (MLVA) typing of Salmonella Typhimurium is described in Annex D of the following report:
Jacobs, W., Kuiling, S., and van der Zwaluw, K., 2014. Molecular typing of Salmonella strains isolated from food, feed and animals: state of play and standard operating procedures for pulsed field gel electrophoresis (PFGE) and Multiple-Locus Variable number tandem repeat Analysis (MLVA) typing, profiles interpretation and curation. EFSA supporting publication 2014: EN-703, 74 pp. http://www.efsa.europa.eu/en/supporting/pub/703e.htm
For the Standard Operating Procedure for MLVA typing of Salmonella Enteritidis, reference is made to the procedure published by ECDCEuropean Centre for Disease Prevention and Control :
European Centre for Disease Prevention and Control. Laboratory standard operating procedure for multiple-locus variable-number tandem repeat analysis of Salmonella enterica serotype Enteritidis. Stockholm: ECDC; 2016. doi 10.2900/973540 [link]
Next Generation Sequencing (NGS)
Inter-European Union Reference Laboratories Working Group on Next Generation Sequencing
The Inter-European Union Reference Laboratories (EURLs) Working Group (WG) has been established by the European Commission with the aim to promote the use of Next Generation Sequencing (NGS) across the EURLs' networks, build NGS capacity within the EUEuropese unie and ensure liaison with the work of the EURLs and the work of EFSA and ECDCEuropean Centre for Disease Prevention and Control on the NGS mandate sent by the Commission. The WG includes all the EURLs operating in the field of the microbiological contamination of food and feed and is coordinated by the EURLEuropean Union Reference Laboratory
for E. coli.
The documents are meant to provide guidance to the laboratories in the area of application of NGS and have been produced by the working group with the aim of being diffused to all the networks of NRLsNational Reference Laboratories . Links to these documents at the respective curators' websites will be added below as soon as the documents become available (expected by the end of 2020).
• Overview of conducted and planned PTs – curated by EURL-Antimicrobial Resistance
• Reference Whole Genome Sequencing collection – curated by EURL-Salmonella
• NGS laboratory procedures – curated by EURL-Parasites
• Bioinformatics tools for basic analysis of Next Generation Sequencing data – curated by EURL-E. coli
• Guidance document for cluster analysis of whole genome sequence data – curated by EURL-Campylobacter
• Guidance document for NGS-Benchmarking – curated by EURL-Listeria monocytogenes
• Inventory of training supports – curated by EURL-Coagulase-Positive Staphylococci
• Survey on the use of NGS across the NRLs networks – curated by EURL-E. coli
On 25 September 2020 the inter-EURLs WG on NGS organised a conference with the support of the Med-Vet-Net association, entitled:
‘Science meets Policy’ conference: Modern technologies to enable response to crises. Next Generation Sequencing to tackle food-borne diseases.
The presentations given at this conference can be found at the website of the EURL-E.coli, through this link: link to presentations 25 September 2020
Sampling whole melons to check for the presence of Salmonella
In 2012 a request was received from EC DG SANTE to give advice on the best way to sample whole melons to check for Salmonella. After consulting experts and literature the following document [link] (pdf, 97KB) was drafted.
Detection of Salmonella in shellfish
Background information on the detection of Salmonella in live bivalve molluscs (LBM), and especially on the transport and handling of the samples is given in the document as shown by this [link] (pdf, 234KB).
Definitions of reference materials (RM) and certified reference materials (CRM) are given in EN-ISOInternational Standardisation Organisation 17034:2016 ‘General requirements for the competence of reference material producers’ and in ISO Guide 30:2015 ‘Reference materials - Selected terms and definitions’.
Reference material (RM)
material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process
Note 1 to entry: Reference material is a generic term.
Note 2 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.
Note 3 to entry: Uses may include the calibration of a measurement system, assessment of a measurement procedure, assigning values to other materials, and quality control.
Note 4 to entry: ISO/IEC Guide 99:2007 has an analogous definition, but restricts the term “measurement” to apply to quantitative values. However, Note 3 of the definition in ISO/IEC Guide 99:2007 specifically includes qualitative properties, called “nominal properties”.
[SOURCE: EN-ISO 17034:2016]
Certified reference material (CRM)
reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a reference material certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability
Note 1 to entry: The concept of value includes a nominal property or a qualitative attribute such as identity or sequence. Uncertainties for such attributes may be expressed as probabilities or levels of confidence.
Note 2 to entry: Metrologically valid procedures for the production and certification of reference materials are given in, among others, ISO Guide 35.
Note 3 to entry: ISO Guide 31 gives guidance on the contents of reference material certificates.
Note 4 to entry: ISO/IEC Guide 99:2007 has an analogous definition.
[SOURCE: EN-ISO 17034:2016]
Development of (certified) reference materials
From 1986 up to 1995, the Microbiological Laboratory for Health Protection (MGB) of the Dutch National Institute for Public Health and the Environment (RIVMRijksinstituut voor Volksgezondheid en Milieu) led EUEuropese unie funded projects on the development and certification of microbiological reference materials. After their development, the non-certified reference materials were produced by the Foundation affiliated to the RIVM until 2003. The experiences and knowledge obtained from the development and production of these (C)RMs have been applied in preparing reference materials for use as standard samples in the Proficiency Tests of the EURLEuropean Union Reference Laboratory -Salmonella up to 2010.
Availability of microbiological (certified) reference materials
Microbiological reference materials (certified and non-certified) are produced in different formats by several national and international organisations. For example: reference strains from culture collections, reference materials containing a set number of one or more culturable organism(s) or reference materials containing genomic DNA of a microorganism.
For information, the details of some (C)RM producers are given below.
Mind that this list is not exhaustive and that the information is given for the convenience of the users and does not constitute an endorsement by the EURL-Salmonella.
World Data Centre for Microorganisms (WDCM)
Many national and international culture collections exist which preserve, authenticated microbial strains. These collections are developed, managed and maintained in accordance with internationally recognized quality standards.
Some cultures representing the same strain are available at different culture collections, obtaining a unique culture collection number at each culture collection. Some of these strains are listed in the catalogue of the World Data Centre for Microorganisms (WDCM), especially the reference strains listed in EN-ISO 11133:2014 (‘Microbiology of food, animal feed and water - Preparation, production, storage and performance testing of culture media’) for the quality control of culture media.
For example: In EN-ISO 11133:2014 and EN-ISO 6579-1:2017 for the performance testing of MSRV agar, the use of Salmonella Typhimurium WDCM 00031 or Salmonella Enteritidis WDCM 00030 is prescribed. In the WDCM Reference strain catalogue it can be found that Salmonella Typhimurium WDCM 00031 is available at 13 different culture collections and Salmonella Enteritidis WDCM 00030 is available at 9 different culture collections. The different culture collection numbers represent the same strain for one WDCM number. E.g. WDCM 00031 is available as ATCC 14028, but also as NCTC 12023, or CIP 104115. Whereas WDCM 00030 is available as ATCC 13076, but also as NCTC 12694.
More information on the World Data Centre for Microorganisms (WDCM) can be found at: http://www.wdcm.org/
Examples of culture collections
Belgian Co-ordinated Collections of Micro-organsims (BCCM)
Czech Collection of Microorganisms (CCM)
Masaryk University, Czech Republic
Collection de l’Institut Pasteur (CIP)
Institute Pasteur, France
German Collection of Microorganisms and Cell Cultures (DSMZ)
Leibniz Institute, Germany
The Netherlands Culture Collection of Bacteria (NCCB)
Westerdijk Fungal biodiversity Institute, The Netherlands
Spanish Type Culture Collection (CECT)
University of Valencia, Spain
National Collection of Type Cultures (NCTC) for bacteria
Public Health England, United Kingdom
American Type Culture Collection (ATCC)
LGC Standards, USA
Microbiological (certified) reference materials
EC-Joint Research Centre (JRC)
The European Commission’s Joint Research Centre (JRC) in Geel, Belgium develops and produces (certified) reference materials and most of the reference material projects are performed in collaboration with various external institutions. Since 2004, JRC is accredited according to ISO 17034 and ISO/IEC 17025. The catalogue of JRC also contains a (small) list of certified microbiological reference materials.
More information, as well as the online Certified Reference Materials catalogue can be found at: https://ec.europa.eu/jrc/en/reference-materials
Examples of producers of microbiological (certified) reference materials
BIOBALL® Reference Materials
Vitroids™ and LENTICULE® discs Reference Materials
Sigma-Aldrich/ Merck, Germany
Biosisto (Certified) Reference Materials
ielab Reference Materials
easi-tabTM Reference Materials
LGC Standards, United Kingdom
Validation and verification
The ISOInternational Standardisation Organisation
16140 series is dedicated to the validation and verification of microbiological methods. These International Standards are designed to help food and feed testing laboratories, test kit manufacturers, competent authorities, and food and feed business operators to implement microbiological methods.
Detailed information about the ISO 16140 series, and the necessary stages of validation and verification of methods before use, can be found on the website of ISO/TC 34/SC 9 Food products – Microbiology:
To facilitate the implementation of the ISO 16140 series and especially ISO 16140-3:2021 ‘Method verification’, the following supporting materials are available at the bottom of the ISO/TC34Technical Committee 34 on Feed and Food products
/SC9Subcommittee 9 on microbiology
• Presentation: Overview of the ISO 16140 series – standards for validation and verification of microbiology methods
• Presentation: Overview of ISO 16140-3 ‘Method verification’ – improving confidence in laboratory results
• Presentation: “Deep dive” into ISO 16140-3 ‘Method verification’ – an extended training for improving confidence in laboratory results
• Excel-calculation tool ISO 16140-3:2021 for assistance on statistics
• Recording of the Webinar on 2 March 2021 ‘Publication of ISO 16140-3 ‘Method verification’ – improving confidence in laboratory results’
Guidance Document for the organisation of Proficiency Tests by NRLsNational Reference Laboratories for national networks, including partial outsourcing
One of the tasks of a National Reference Laboratory (NRLNational Reference Laboratorie
) is to organize, where appropriate, Proficiency Tests (PTs) for the national network of designated official laboratories (OLs) it coordinates, according to Regulation (EUEuropese unie) 2017/625 (article 101) [link].
For different reasons (too small or too large size of the network, economic aspects, availability of human resources etc.), some NRLs outsource (parts of) these PTs.
The five EU Reference Laboratories (EURLs) on Campylobacter, Coagulase-Positive Staphylococci (CPS), Listeria monocytogenes (Lm), Salmonella and Shiga-toxin-producing Escherichia coli (STEC), in agreement with their respective NRL networks, identified the need to draft a common guidance document, defining:
• criteria for NRLs to organize PTs in their respective scope;
• if PTs are outsourced:
o criteria to outsource (parts of) PTs organised by NRLs for national networks, including PT steps that can be outsourced or not;
o criteria to select PT providers.
This document [link] gives only guidance, since organisation of PTs at national level is a Member State’s responsibility. The document is based upon an EURLEuropean Union Reference Laboratory -Listeria monocytogenes document on the same topic.