SIST-TS CEN ISO/TS 6579-2:2013
(Main)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 (ISO/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 (ISO/TS 6579-2:2012)
This part of ISO 6579 gives a method for the enumeration of Salmonella spp. present in:
— products intended for human consumption and for the feeding of animals;
— environmental samples in the area of food production and food handling;
— animal faeces;
— environmental samples from the primary production stage; by calculation of the most probable number (MPN). The method is based on miniaturization of the dilution, pre-enrichment and selective enrichment steps. The selective enrichment medium, modified semi-solid Rappaport–Vassiliadis (MSRV), is intended for the detection of motile salmonellae and is not appropriate for the detection of non-motile salmonellae. It is possible that the method is less appropriate to enumerate Salmonella ser. Typhi and Salmonella ser. Paratyphi. The method is not appropriate for the enumeration of Salmonella spp. in (very) low contaminated samples (<1 cfu/g).
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis, zur Zählung und zur Serotypisierung von Salmonellen - Teil 2: Zählung unter Anwendung eines miniaturisierten Verfahrens der wahrscheinlichsten Keimzahl (ISO/TS 6579-2:2012)
Dieser Teil von ISO 6579 legt ein Verfahren zur Zählung von Salmonella spp. in
- Produkten, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind;
- Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln;
- Tierkot;
- Umgebungsproben im Bereich der Primärproduktion;
fest, und zwar durch Berechnung der wahrscheinlichsten Keimzahl (en: most probable number, MPN).
Das Verfahren beruht auf einer Miniaturisierung der Schritte der Verdünnung, Voranreicherung und selektiven Anreicherung. Das selektive Anreicherungsmedium, modifiziertes halbfestes Medium nach Rappaport-Vassiliadis (MSRV), ist für den Nachweis von beweglichen Salmonellen vorgesehen. Es ist nicht für den Nachweis von unbeweglichen Salmonellen geeignet.
Es ist möglich, dass dieses Verfahren weniger für Salmonella ser. Typhi und Samonella ser. Paratyphi geeignet ist.
Das Verfahren ist nicht für die Zählung von Salmonella spp. in (sehr) gering kontaminierten Proben ( 1 KBE/g) geeignet.
ANMERKUNG Bei den meisten der für dieses Verfahren relevanten Matrices ist im Allgemeinen die Anzahl der unbeweglichen Salmonellen gering. In dieser Anmerkung sind Beispiele für Proben aus der Primärproduktion genannt. Die unbeweglichen Salmonella-Biovaren von Salmonella Gallinarum (Salmonella Gallinarum biovar gallinarum und Salmonella Gallinarum biovar pullorum) scheinen in Umgebungsproben nicht lange zu überleben und werden deshalb in Kot- oder Umgebungsproben (wie z. B. Staubproben) selten nachgewiesen (unabhängig vom Verfahren). Die Anzahl von anderen unbeweglichen Salmonella-Serovare in Kotproben scheint gewöhnlich gering zu sein. Zum Beispiel waren in einer Studie [4], in der etwa 1 000 Kotproben aus Legehennenbeständen und etwa 900 Kotproben aus Jungmastgeflügelbeständen analysiert wurden, weniger als 1 % der Gesamtanzahl der Proben in einer selektiven Bouillon positiv und auf MSRV-Medium gleichzeitig negativ (und vermutlich unbeweglich). Ähnliche Ergebnisse wurden in einer niederländischen Studie mit etwa 3 200 Kotproben von Schweinen erhalten (unveröffentlichte Daten). Andererseits wären im Fall der Studie in Literaturhinweis [4] fast 40 % der positiven Proben nicht nachgewiesen worden (d. h. falsch-negative Proben), wenn nur eine selektive Bouillon (in diesem Fall nach Rappaport-Vassiliadis) anstelle eines halbfesten Mediums verwendet worden wäre.
Microbiologie des aliments - Méthode horizontale pour la recherche, le dénombrement et le sérotypage des salmonella - Partie 2: Dénombrement par une technique miniaturisée du nombre le plus probable (ISO/TS 6579-2:2012)
L'ISO/TS 6579-2:2012 fournit une méthode de dénombrement de Salmonella spp. présentes dans: les produits destinés à la consommation humaine ou à l'alimentation animale; les échantillons environnementaux dans le domaine de la production et de la manutention de produits alimentaires; les matières fécales des animaux; les échantillons environnementaux au stade de la production primaire; par le calcul du nombre le plus probable (NPP).
La méthode est fondée sur une miniaturisation des étapes de dilution, de préenrichissement et d'enrichissement sélectif. Le milieu d'enrichissement sélectif, le milieu semi-solide de Rappaport-Vassiliadis modifié (MSRV), est conçu pour la recherche des Salmonella mobiles et n'est pas adapté à la recherche des Salmonella immobiles.
Il est possible que la méthode soit moins appropriée au dénombrement de Salmonella ser. Typhi et Salmonella ser. Paratyphi.
La méthode n'est pas appropriée au dénombrement de Salmonella spp. présentes en (très) faibles concentrations dans des échantillons contaminés (< 1 ufc/g).
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti, števila in serotipov Salmonella - 2. del: Ugotavljanje števila z miniaturizirano metodo najbolj verjetnega števila (MPN) (ISO/TS 6579-2:2012)
Ta del standarda ISO 6579 podaja metodo za ugotavljanje števila Salmonella spp., prisotnih v:
– izdelkih, namenjenih za prehrano ljudi in krmo živali;
– okoljskih vzorcih na območju proizvodnje hrane in ravnanja s hrano;
– iztrebkih živali;
– okoljskih vzorcih iz faze primarne proizvodnje; z izračunom najbolj verjetnega števila (MPN). Metoda temelji na miniaturizaciji korakov redčenja, predbogatenja in selektivnega bogatenja. Medij selektivnega bogatenja, spremenjen poltrd Rappaport–Vassiliadis (MSRV), je namenjen za odkrivanje gibljivih salmonel in ni primeren za odkrivanje negibljivih salmonel. Možno je, da je metoda manj primerna za ugotavljanje števila Salmonella ser. Typhi in Salmonella ser. Paratyphi. Ta metoda ni primerna za ugotavljanje števila Salmonella spp. v (zelo) malo kontaminiranih vzorcih (< 1 cfu/g).
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 6579-2:2013
01-maj-2013
1DGRPHãþD
SIST EN ISO 6579:2003
SIST EN ISO 6579:2003/A1:2007
SIST EN ISO 6579:2003/AC:2004
SIST EN ISO 6579:2003/AC:2006
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti,
števila in serotipov Salmonella - 2. del: Ugotavljanje števila z miniaturizirano
metodo najbolj verjetnega števila (MPN) (ISO/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 (ISO/TS 6579-2:2012)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum
Nachweis, zur Zählung und zur Serotypisierung von Salmonellen - Teil 2: Zählung unter
Anwendung eines miniaturisierten Verfahrens der wahrscheinlichsten Keimzahl (ISO/TS
6579-2:2012)
Microbiologie des aliments - Méthode horizontale pour la recherche, le dénombrement et
le sérotypage des salmonella - Partie 2: Dénombrement par une technique miniaturisée
du nombre le plus probable (ISO/TS 6579-2:2012)
Ta slovenski standard je istoveten z: CEN ISO/TS 6579-2:2012
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST-TS CEN ISO/TS 6579-2:2013 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN ISO/TS 6579-2:2013
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SIST-TS CEN ISO/TS 6579-2:2013
TECHNICAL SPECIFICATION
CEN ISO/TS 6579-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
November 2012
ICS 07.100.30
English Version
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
(ISO/TS 6579-2:2012)
Microbiologie des aliments - Méthode horizontale pour la Mikrobiologie von Lebensmitteln und Futtermitteln -
recherche, le dénombrement et le sérotypage des Horizontales Verfahren zum Nachweis, zur Zählung und zur
salmonella - Partie 2: Dénombrement par une technique Serotypisierung von Salmonellen - Teil 2: Zählung unter
miniaturisée du nombre le plus probable (ISO/TS 6579- Anwendung eines miniaturisierten Verfahrens der
2:2012) wahrscheinlichsten Keimzahl (ISO/TS 6579-2:2012)
This Technical Specification (CEN/TS) was approved by CEN on 16 July 2012 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 6579-2:2012: E
worldwide for CEN national Members.
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Contents Page
Foreword .3
2
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CEN ISO/TS 6579-2:2012 (E)
Foreword
This document (CEN ISO/TS 6579-2:2012) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the
secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany,
Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO/TS 6579-2:2012 has been approved by CEN as a CEN/TS ISO 6579-2:2012 without any
modification.
3
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SIST-TS CEN ISO/TS 6579-2:2013
TECHNICAL ISO/TS
SPECIFICATION 6579-2
First edition
2012-11-01
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
Microbiologie des aliments — Méthode horizontale pour la recherche, le
dénombrement et le sérotypage des Salmonella —
Partie 2: Dénombrement par une technique miniaturisée du nombre le
plus probable
Reference number
ISO/TS 6579-2:2012(E)
©
ISO 2012
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SIST-TS CEN ISO/TS 6579-2:2013
ISO/TS 6579-2:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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ISO/TS 6579-2:2012(E)
Contents Page
Foreword .iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Pre-enrichment in non-selective liquid medium . 2
4.3 Enrichment on a selective semi-solid medium . 2
4.4 Selective plating and identification . 2
4.5 Confirmation . 2
4.6 Calculation of most probable number . 3
5 Culture media and sera . 3
5.1 General . 3
5.2 Culture media . 3
6 Apparatus and glassware . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure . 4
9.1 Test portion, initial suspension . 4
9.2 Dilution and pre-enrichment in non-selective liquid medium . 5
9.3 Selective enrichment on a semi-solid medium . 5
9.4 Selective plating . 5
9.5 Biochemical and serological confirmation . 6
10 Expression of results . 8
11 Test report . 8
Annex A (informative) Composition and preparation of culture media and reagents . 9
Annex B (informative) Diagram of procedure .17
Bibliography .18
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ISO/TS 6579-2:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
In other circumstances, particularly when there is an urgent market requirement for such documents, a technical
committee may decide to publish other types of document:
— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members
of the parent committee casting a vote;
— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee
casting a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further
three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed,
it is reviewed again after a further three years, at which time it must either be transformed into an International
Standard or be withdrawn.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO/TS 6579-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
ISO 6579 consists of the following part, under the general title 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 [Technical Specification]
Additional parts, dealing with a detection method and guidance for serotyping are planned. ISO 6579:2002 is
to be withdrawn at a later date.
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Introduction
The procedure described is based on the method reported in Reference [1].
The enumeration procedure described here concerns a miniaturized most probable number (MPN) technique.
For this mini-MSRV (modified semi-solid Rappaport–Vassiliadis) MPN technique, the volume of primary
dilution tested is less than the volume in the detection method specified in ISO 6579:2002 + Cor.1:2004 +
[5]
Amd.1:2007. For this reason, the sensitivity of the mini-MSRV technique is lower than in these detection
methods (Reference [1]). The detection limit of the mini-MSRV method is approximately 1 cfu/g, but can vary
according to Salmonella serovar and per matrix. For the previously mentioned detection methods, this is typically
1 cfu/25 g (0,04 cfu/g). For samples with (very) low numbers of Salmonella spp. (<1 cfu/g), it is possible that
the mini-MSRV procedure is not sufficiently sensitive. If a quantitative result is requested for samples likely to
contain such low numbers (and tested negative with this mini-MSRV technique, for example), it is advisable to
enumerate with a “conventional” MPN technique (not miniaturized). For other samples, the mini-MSRV method
can have an advantage over the conventional MPN technique because the performance of the miniaturized
MPN technique can take less time and need fewer resources (due to small amounts).
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SIST-TS CEN ISO/TS 6579-2:2013
TECHNICAL SPECIFICATION ISO/TS 6579-2:2012(E)
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
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for detecting Sal-
monella, are only undertaken in properly equipped laboratories, under the control of a skilled microbiologist, and
that great care is taken in the disposal of all incubated materials.
Persons using this International Standard should be familiar with normal laboratory practice. This standard does
not purport to address all of the safety aspects, if any, associated with its use. It is the responsibility of the user
to establish appropriate safety and health practices and to ensure compliance with any national regulatory condi-
tions.
1 Scope
This part of ISO 6579 gives a method for the enumeration of Salmonella spp. present in:
— products intended for human consumption and for the feeding of animals;
— environmental samples in the area of food production and food handling;
— animal faeces;
— environmental samples from the primary production stage;
by calculation of the most probable number (MPN).
The method is based on miniaturization of the dilution, pre-enrichment and selective enrichment steps. The
selective enrichment medium, modified semi-solid Rappaport–Vassiliadis (MSRV), is intended for the detection
of motile salmonellae and is not appropriate for the detection of non-motile salmonellae.
It is possible that the method is less appropriate to enumerate Salmonella ser. Typhi and Salmonella ser. Paratyphi.
The method is not appropriate for the enumeration of Salmonella spp. in (very) low contaminated samples (<1 cfu/g).
NOTE The number of non-motile salmonellae is generally low in most of the matrices relevant for this method. In
this note, examples are given for samples from primary production. The non-motile Salmonella biovars of Salmonella
Gallinarum (Salmonella Gallinarum biovar gallinarum and Salmonella Gallinarum biovar pullorum) do not seem to survive
long in environmental samples and are therefore rarely detected in faecal or environmental (such as dust) samples
(regardless of the method). The number of other non-motile Salmonella serovars in faecal samples seems to be generally
low. For example, in Reference [4] in which approximately 1 000 faecal samples of poultry layer flocks and approximately
900 faecal samples of broiler flocks were analysed, less than 1 % of the total number of samples were positive in a
selective broth and at the same time negative on MSRV medium (and likely to be non-motile). Similar results were found
in a Dutch study with ca 3 200 faecal samples of pigs (unpublished data). On the other hand, in the case of the study
reported in Reference [4], up to almost 40 % of positive samples would not have been detected (i.e. false negatives) if only
a selective broth (in this case Rappaport–Vassiliadis) had been used instead of a semi-solid medium.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced document
(including any amendments) applies.
ISO 6887 (all parts), Microbiology of food and animal feeding stuffs — Preparation of test samples, initial
suspension and decimal dilutions for microbiological examination
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ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO/TS 11133-1, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production of
culture media — Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory
ISO/TS 11133-2, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production
of culture media — Part 2: Practical guidelines on performance testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Salmonella
microorganism which forms typical or less typical colonies on solid selective media and which displays specific
biochemical and serological characteristics
NOTE Suitable tests for the specific biochemical and serological characteristics are specified in this part of ISO 6579.
3.2
count of Salmonella
number of Salmonella spp. found per millilitre or per gram of a test sample or per surface area or on an object
(e.g. bootsocks)
4 Principle
4.1 General
The enumeration of Salmonella spp. in the MPN format necessitates four successive stages (4.2 to 4.5).
4.2 Pre-enrichment in non-selective liquid medium
-1
Preparation of a 10 dilution of the sample in buffered peptone water (BPW) (initial suspension).
Addition of the initial suspension to the first (empty) row of three wells of a 12-well microtiter plate.
Inoculation of the second row of three wells containing non-selective pre-enrichment broth (BPW) with a
specified quantity, from the first row in a 12-well microtiter plate.
Inoculation of the third, fourth and if necessary more rows of three wells containing BPW.
Incubation of the 12-well microtiter plates at 37 °C for 18 h.
4.3 Enrichment on a selective semi-solid medium
Subculturing of each well obtained in 4.2 in a well containing a semi-solid agar (MSRV).
Incubation at 41,5 °C (MSRV) for 24 h. If MSRV is negative after 24 h, the plate is incubated for a further 24 h.
4.4 Selective plating and identification
From the (suspect) cultures (the highest dilutions) obtained in 4.3, a selective solid medium xylose–lysine–
deoxycholate (XLD) agar is inoculated and incubated at 37 °C for 24 h.
4.5 Confirmation
Colonies of presumptive Salmonella obtained in 4.4 are confirmed by means of appropriate biochemical and
serological tests.
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4.6 Calculation of most probable number
From the number of confirmed positive wells, the MPN of Salmonella spp. per millilitre or per gram of the test
sample is calculated.
5 Culture media and sera
5.1 General
For current laboratory practice, see ISO 7218.
For the performance testing of media, follow the recommendations of ISO/TS 11133-1, ISO/TS 11133-2 and
the information as given in A.8.
All media and reagents needed are described in Annex A. Alternatively, dehydrated complete media or diluents
can be used. In the latter case, follow the manufacturer’s instructions.
5.2 Culture media
5.2.1 Non-selective pre-enrichment medium: Buffered peptone water (BPW). See A.1.
5.2.2 Semi-solid selective enrichment agar: Modified semi-solid Rappaport–Vassiliadis medium
(MSRV). See A.2.
5.2.3 Xylose–lysine–deoxycholate (XLD) agar. See A.3.
5.2.4 Nutrient agar. See A.4.
5.2.5 Triple sugar–iron agar (TSI agar). See A.5.
As an alternative, a double sugar–iron agar (Kligler–Hajna) may be used.
5.2.6 Urea agar (Christensen). See A.6.
5.2.7 l-Lysine decarboxylation medium (LDC). See A.7.
5.2.8 Antisera. Several types of agglutinating sera containing antibodies for one or several O-antigens and
for one or several H-antigens are commercially available.
Every attempt should be made to ensure that the anti-sera used are adequate to provide for the detection of
all Salmonella serotypes.
6 Apparatus and glassware
Disposable supplies are an acceptable alternative to reusable glassware if it has similar specifications.
Usual microbiological laboratory equipment and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). See ISO 7218.
6.2 Drying cabinet or ventilated oven, capable of being maintained at between 25 °C and 50 °C or a
laminar air flow cabinet.
6.3 Incubators, capable of operating at 37 °C ± 1 °C and 41,5 °C ± 1 °C.
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6.4 Water bath, capable of operating at 47 °C to 50 °C.
6.5 Refrigerator (for storage of prepared media), capable of operating at 5 °C ± 3 °C.
6.6 pH-meter, having a resolution of 0,01 pH and accurate to within ± 0,1 pH units at 25 °C. See ISO 7218.
6.7 Sterile test tubes and flasks, of appropriate capacity. Flasks or bottles and test tubes with non-toxic
metallic or plastic (screw) caps may be used.
6.8 Sterile loops of 1 µl.
6.9 Sterile graduated pipettes or automatic pipettes, of nominal capacities 10 ml (with 0,5 ml division),
2 ml (with 0,1 ml division), 0,1 ml (with 0,01 ml division). Multi-channel pipettes of 0,5 ml and 0,02 ml nominal
capacity for pipetting three wells at a time.
6.10 Sterile Petri dishes, of approximately 90 mm diameter.
6.11 Sterile 12-well microtiter plates with wells of approximately 25 mm diameter and 20 mm deep (5 ml)
with a flat bottom with lid.
6.12 Homogenizer. See ISO 7218.
7 Sampling
Sampling is not part of the method specified in this part of ISO 6579. See the specific International Standard
dealing with the product concerned.
It is important that the laboratory receive a truly representative sample which has not been damaged or changed
during transport or storage.
If there is no specific International Standard dealing with sampling of the product concerned, it is recommended
that the parties concerned come to an agreement on the subject.
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard appropriate to the product concerned.
If there is no specific International Standard, it is recommended that the parties concerned come to an
agreement on the subject.
9 Procedure
9.1 Test portion, initial suspension
See ISO 6887 and any specific International Standard appropriate to the product concerned. Prepare an initial
suspension by diluting the test portion 10-fold in BPW (5.2.1). For example, add 25 g of sample to 225 ml of
BPW and homogenize, e.g. in a stomacher (6.12), for 1 min.
4 © ISO 2012 – All rights reserved
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SIST-TS CEN ISO/TS 6579-2:2013
ISO/TS 6579-2:2012(E)
9.2 Dilution and pre-enrichment in non-selective liquid medium
Take a 12-well microtiter plate (6.11) with empty wells in the first row of three wells and containing 2 ml BPW
(5.2.1) in the other wells (second, third and fourth row with each 3 wells; see Annex B).
NOTE 1 As a general case, the described procedure specifies dilutions for one 12-well microtiter plate. When a higher
number than 500 cfu/g of Salmonella is suspected, it is necessary to proceed with a second 12-well microtitre plate
containing 2 ml BPW (5.2.1) in each well. Prepare a sufficient number of plates (dilutions) to ensure that the final well in the
12-well microtiter plate yields a negative result.
Transfer to each well of the first (empty) row of three wells, using a pipette (6.9), 2,5 ml of the initial suspension (9.1).
Transfer 0,5 ml (e.g. by using a multi-channel pipette; 6.9) of each well from the first row into the 2 ml BPW in
-1
the successive wells in the second row (first 5 dilution).
Transfer 0,5 ml (e.g. by using a multi-channel pipette; 6.9, with new tips) of each well from the second row into
-1
the 2 ml BPW in the successive wells in the third row (second 5 dilution).
Before transferring the 0,5 ml of the second row into the third row, mix the suspensions in the wells by repeatedly
(carefully) sucking up and blowing out of the suspension in the pipette and in the wells.
Proceed in the same way for the other rows.
Incubate the 12-well microtiter plate at 37 °C (6.3) for 18 h ± 2 h.
NOTE 2 As the contamination level of the tested samples is generally unknown and often low, it can prove worthwhile to
check for the presence of Salmonella spp. in the sample by also culturing the initial suspension. For this purpose, incubate
(6.3) the initial suspension (9.1) at 37 °C for 18 h± 2 h. For the next culture steps, follow the procedures as described in
9.3 to 9.5. In 9.3: inoculate a Petri dish containing MSRV with 1–3 equally spaced spots with a total volume of 0,1 ml of the
incubated BPW culture of the initial suspension (9.1).
9.3 Selective enrichment on a semi-solid medium
Allow the MSRV (5.2.2) in the 12-well microtiter plates to equilibrate at room temperature if they were stored at
a lower temperature.
Inoculate each well containing 2 ml MSRV with 20 µl of the BPW culture (9.2), e.g. using a multi-channel pipette
(6.9). Use new tips for each row of three wells.
Place the 20 µl BPW culture at the margin of the well and on the surface of the medium (see Annex B).
When taking a subculture from BPW, try not to disturb particulate samples. Therefore, move the microtitre
plates carefully. Avoid pipetting particulate matter on to the MSRV plates.
Incubate the inoculated MSRV plates at 41,5 °C (6.3) for 24 h ± 3 h.
Do not invert the plates.
Suspect wells show a grey-white, turbid zone extending out from the inoculated drop. The turbid zone is
characterized by a white halo with a clearly defined edge.
If the wells are negative after 24 h, reincubate for a further 24 h ± 3 h.
9.4 Selective plating
Allow the XLD agar (5.2.3) plates to equilibrate at room temperature if they were stored at lower temperature.
If necessary, dry the surface of the plates before use.
Subculture sus
...
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