SIST EN ISO 6579:2003
(Main)Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp (ISO 6579:2002)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp (ISO 6579:2002)
This International Standard specifies a horizontal method for the detection of Salmonella, including Salmonella Typhi and Salmonella Paratyphi. Subject to the limitations discussed in the Introduction, this International Standard is applicable to products intended for human consumption and the feeding of animals.
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis von Salmonellen spp (ISO 6579:2002)
Diese Internationale Norm legt ein horizontales Verfahren zum Nachweis von Salmonellen fest, einschließlich Salmonella Typhi und Salmonella Paratyphi.
In Abhängigkeit von den in der Einleitung erörterten Einschränkungen gilt diese Internationale Norm für
3 Produkte, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind;
3 Umgebungsproben im Bereich der Herstellung von Lebensmitteln und beim Umgang mit Lebensmitteln.
WARNUNG - Es ist möglich, dass nach diesem Untersuchungsverfahren nicht alle Salmonella Typhi und Salmonella Paratyphi wachsen.
Microbiologie des aliments - Méthode horizontale pour la recherche des Salmonella spp (ISO 6579:2002)
La présente Norme internationale spécifie une méthode horizontale de recherche des Salmonella, incluant Salmonella Typhi et Salmonella Paratyphi.
La présente Norme internationale est applicable aux produits destinés à la consommation humaine ou à l'alimentation animale.
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti vrst Salmonella (ISO 6579:2002)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 6579:2003
01-april-2003
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti vrst
Salmonella (ISO 6579:2002)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection of
Salmonella spp (ISO 6579:2002)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum
Nachweis von Salmonellen spp (ISO 6579:2002)
Microbiologie des aliments - Méthode horizontale pour la recherche des Salmonella spp
(ISO 6579:2002)
Ta slovenski standard je istoveten z: EN ISO 6579:2002
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 6579:2003 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 6579:2003
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SIST EN ISO 6579:2003
EUROPEAN STANDARD
EN ISO 6579
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2002
ICS 07.100.30 Supersedes EN ISO 12824:1997
English version
Microbiology of food and animal feeding stuffs - Horizontal
method for the detection of Salmonella spp (ISO 6579:2002)
Microbiologie des aliments - Méthode horizontale pour la Mikrobiologie von Lebensmitteln und Futtermitteln -
recherche des Salmonella spp (ISO 6579:2002) Horizontales Verfahren zum Nachweis von Salmonellen
spp (ISO 6579:2002)
This European Standard was approved by CEN on 30 May 2002.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2002 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 6579:2002 E
worldwide for CEN national Members.
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SIST EN ISO 6579:2003
EN ISO 6579:2002 (E)
CORRECTED 2002-09-18
Foreword
This document (ISO 6579:2002) has been prepared by Technical Committee ISO/TC 34
"Agricultural food products" in collaboration with Technical Committee CEN/TC 275 "Food
analysis - Horizontal methods", the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication
of an identical text or by endorsement, at the latest by January 2003, and conflicting national
standards shall be withdrawn at the latest by January 2003.
This document supersedes EN 12824:1997.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the
United Kingdom.
Endorsement notice
The text of ISO 6579:2002 has been approved by CEN as EN ISO 6579:2002 without any
modifications.
NOTE Normative references to International Standards are listed in Annex ZA (normative).
2
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SIST EN ISO 6579:2003
EN ISO 6579:2002 (E)
Annex ZA
(normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions
of any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications,
indicated by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 6887-1 1999 Microbiology of food and animal feeding EN ISO 6887-1 1999
stuffs — Preparation of test samples, initial
suspension and decimal dilutions for
microbiological examination — Part 1:
General rules for the preparation of the
initial suspension and decimal dilutions
ISO 8261 2001 Milk and milk products - General guidance EN ISO 8261 2001
for the preparation of test samples, initial
suspensions and decimal dilutions for
microbiological examination
3
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SIST EN ISO 6579:2003
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SIST EN ISO 6579:2003
INTERNATIONAL ISO
STANDARD 6579
Fourth edition
2002-07-15
Microbiology of food and animal feeding
stuffs — Horizontal method for the
detection of Salmonella spp.
Microbiologie des aliments — Méthode horizontale pour la recherche des
Salmonella spp.
Reference number
ISO 6579:2002(E)
©
ISO 2002
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SIST EN ISO 6579:2003
ISO 6579:2002(E)
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ii © ISO 2002 – All rights reserved
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SIST EN ISO 6579:2003
ISO 6579:2002(E)
Contents Page
Foreword.iv
Introduction.v
1 Scope .1
2 Normative references.1
3 Terms and definitions .1
4 Principle.2
4.1 General.2
4.2 Pre-enrichment in non-selective liquid medium .2
4.3 Enrichment in selective liquid media .2
4.4 Plating out and identification .2
4.5 Confirmation of identity .2
5 Culture media, reagents and sera.3
5.1 General.3
5.2 Culture media and reagents .3
5.3 Sera .4
6 Apparatus and glassware .4
7 Sampling.5
8 Preparation of test sample.5
9 Procedure (see diagram in annex A).5
9.1 Test portion and initial suspension .5
9.2 Non-selective pre-enrichment .6
9.3 Selective enrichment.6
9.4 Plating out and identification .6
9.5 Confirmation .6
10 Expression of results .10
11 Test report .10
12 Quality assurance.11
Annex A (normative) Diagram of procedure .12
Annex B (normative) Composition and preparation of culture media and reagents.14
Annex C (informative) Results of interlaboratory trial .24
Bibliography.27
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SIST EN ISO 6579:2003
ISO 6579:2002(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 3.
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.
Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 6579 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology.
This fourth edition cancels and replaces the third edition (ISO 6579:1993), which has been technically revised.
Annexes A and B form a normative part of this Interntional Standard. Annex C is for information only.
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SIST EN ISO 6579:2003
ISO 6579:2002(E)
Introduction
Because of the large variety of food and feed products, this horizontal method may not be appropriate in every
detail for certain products. In this case, different methods, which are specific to these products, may be used if
absolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this
horizontal method as far as possible.
When this International Standard is next reviewed, account will be taken of all information then available regarding
the extent to which this horizontal method has been followed and the reasons for deviations from this method in the
case of particular products.
The harmonization of test methods cannot be immediate, and for certain groups of products International
Standards and/or national standards may already exist that do not comply with this horizontal method. It is hoped
that when such standards are reviewed they will be changed to comply with this International Standard so that
eventually the only remaining departures from this horizontal method will be those necessary for well-established
technical reasons.
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SIST EN ISO 6579:2003
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SIST EN ISO 6579:2003
INTERNATIONAL STANDARD ISO 6579:2002(E)
Microbiology of food and animal feeding stuffs — Horizontal
method for the detection of Salmonella spp.
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for detecting
Salmonella, and especially Salmonella Typhi and Salmonella Paratyphi, 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.
1 Scope
This International Standard specifies a horizontal method for the detection of Salmonella, including Salmonella
Typhi and Salmonella Paratyphi.
Subject to the limitations discussed in the Introduction, this International Standard is applicable to
products intended for human consumption and the feeding of animals;
environmental samples in the area of food production and food handling.
WARNING — The method may not recover all Salmonella Typhi and Paratyphi.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions
ISO 7218:1996, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations
ISO 8261, Milk and milk products — General guidance for the preparation of test samples, initial suspensions and
decimal dilutions for microbiological examination
3 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
3.1
Salmonella
microorganisms which form typical or less typical colonies on solid selective media and which display the
biochemical and serological characteristics described when tests are carried out in accordance with this
International Standard
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SIST EN ISO 6579:2003
ISO 6579:2002(E)
3.2
detection of Salmonella
determination of the presence or absence of Salmonella (3.1), in a particular mass or volume of product, when
tests are carried out in accordance with this International Standard
4 Principle
4.1 General
The detection of Salmonella necessitates four successive stages (see also annex A).
NOTE The Salmonella may be present in small numbers and are often accompanied by considerably larger numbers of
other Enterobacteriaceæ or other families. Furthermore, pre-enrichment is necessary to permit the detection of low numbers of
Salmonella or injured Salmonella.
4.2 Pre-enrichment in non-selective liquid medium
Buffered peptone water is inoculated at ambient temperature with the test portion, then incubated at 37 °C ± 1 °C
for 18 h ± 2 h.
For certain foodstuffs the use of other pre-enrichment procedures is necessary. See 9.1.2.
For large quantities, the buffered peptone water should be heated to 37 °C ± 1 °C before inoculation with the test
portion.
4.3 Enrichment in selective liquid media
Rappaport-Vassiliadis medium with soya (RVS broth) and Muller-Kauffmann tetrathionate/novobiocin broth (MKTTn
broth) are inoculated with the culture obtained in 4.2.
The RVS broth is incubated at 41,5 °C ± 1 °C for 24 h ± 3 h, and the MKTTn broth at 37 °C ± 1 °C for 24 h ± 3 h.
4.4 Plating out and identification
From the cultures obtained in 4.3, two selective solid media are inoculated:
xylose lysine deoxycholate agar (XLD agar);
any other solid selective medium complementary to XLD agar and especially appropriate for the isolation of
lactose-positive Salmonella and Salmonella Typhi and Salmonella Paratyphi strains; the laboratory may
choose which medium to use.
The XLD agar is incubated at 37 °C ± 1 °C and examined after 24 h ± 3 h. The second selective agar is incubated
according to the manufacturer's recommendations.
NOTE For information, Brilliant green agar (BGA), bismuth sulfite agar, etc., could be used as the second plating-out
medium.
4.5 Confirmation of identity
Colonies of presumptive Salmonella are subcultured, then plated out as described in 4.4, and their identity is
confirmed by means of appropriate biochemical and serological tests.
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ISO 6579:2002(E)
5 Culture media, reagents and sera
5.1 General
For current laboratory practice, see ISO 7218.
5.2 Culture media and reagents
NOTE Because of the large number of culture media and reagents, it is considered preferable, for clarity, to give their
compositions and preparations in annex B.
5.2.1 Non-selective pre-enrichment medium: Buffered peptone water
See B.1.
5.2.2 First selective enrichment medium: Rappaport-Vassiliadis medium with soya (RVS broth)
See B.2.
5.2.3 Second selective enrichment medium: Muller-Kauffmann tetrathionate novobiocin broth (MKTTn
broth)
See B.3.
5.2.4 Solid selective plating-out media
5.2.4.1 First medium: Xylose lysine deoxycholate agar (XLD agar)
See B.4.
5.2.4.2 Second medium
The choice of the second appropriate medium is left to the discretion of the testing laboratory. The manufacturer's
instructions should be precisely followed regarding its preparation for use.
5.2.5 Nutrient agar
See B.5.
5.2.6 Triple sugar/iron agar (TSI agar)
See B.6.
5.2.7 Urea agar (Christensen)
See B.7.
5.2.8 L-Lysine decarboxylation medium
See B.8.
5.2.9 Reagent for detection of β-galactosidase (or prepared paper discs used in accordance with the
manufacturer's instructions)
See B.9.
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ISO 6579:2002(E)
5.2.10 Reagents for Voges-Proskauer (VP) reaction
See B.10.
5.2.11 Reagents for indole reaction
See B.11.
5.2.12 Semi-solid nutrient agar
See B.12.
5.2.13 Physiological saline solution
See B.13.
5.3 Sera
Several types of agglutinating sera containing antibodies for one or several O-antigens are available commercially;
i.e. anti-sera containing one or more “O” groups (called monovalent or polyvalent anti-O sera), anti-Vi sera, and
anti-sera containing antibodies for one or several H-factors (called monovalent or polyvalent anti-H sera).
Every attempt should be made to ensure that the anti-sera used are adequate to provide for the detection of all
Salmonella serotypes. Assistance towards this objective may be obtained by using only anti-sera prepared by a
supplier recognized as competent (for example, by an appropriate government agency).
6 Apparatus and glassware
Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave)
See ISO 7218.
6.2 Drying cabinet or oven, ventilated by convection, capable of operating between 37 °C and 55 °C.
6.3 Incubator, capable of operating at 37 °C ± 1 °C.
6.4 Water bath, capable of operating at 41,5 °C ± 1 °C, or incubator, capable of operating at 41,5 °C ± 1 °C.
6.5 Water baths, capable of operating at 44 °C to 47 °C.
6.6 Water bath, capable of operating at 37 °C ± 1 °C.
It is recommended to use a water bath (6.4, 6.5 and 6.6) containing an antibacterial agent because of the low
infective dose of Salmonella.
6.7 Sterile loops, of diameter approximately 3 mm or 10 µl, or sterile pipettes.
6.8 pH-meter, having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C.
6.9 Test tubes or flasks, of appropriate capacity.
Bottles or flasks with non-toxic metallic or plastic screw-caps may be used.
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ISO 6579:2002(E)
6.10 Graduated pipettes or automatic pipettes, of nominal capacities 10 ml and 1 ml, graduated respectively in
0,5 ml and 0,1 ml divisions.
6.11 Petri dishes, of small size (diameter 90 mm to 100 mm) and/or large size (diameter 140 mm).
7 Sampling
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this International Standard. See the specific International Standard
dealing with the product concerned. If there is no specific International Standard, it is recommended that the parties
concerned come to an agreement on this subject.
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard dealing with the product concerned.
If there is no specific International Standard, it is recommended that the parties concerned come to an agreement
on this subject.
9 Procedure (see diagram in annex A)
9.1 Test portion and initial suspension
9.1.1 General
See ISO 6887-1 and the specific International Standard dealing with the product concerned. See ISO 8261 for milk
and milk products.
For preparation of the initial suspension, in the general case use as diluent the pre-enrichment medium specified in
5.2.1 and 4.2 (buffered peptone water).
If the specified mass of test portion is other than 25 g, use the necessary quantity of pre-enrichment medium to
yield a 1/10 dilution.
To reduce the examination workload when more than one 25 g test portion from a specified lot of food has to be
examined, and when evidence is available that compositing (pooling the test portions) does not affect the result for
that particular food, the test portions may be composited. For example, if 10 test portions of 25 g are to be
examined, combine the 10 units to form a composite test portion of 250 g and add 2,25 l of pre-enrichment broth.
Alternatively, the 0,1 ml (in 10 ml of RVS broth) and 1 ml (in 10 ml of MKTTn broth) portions of the pre-enrichment
broth from the 10 separate test portions (see 9.3.1) may be composited for enrichment in 100 ml of selective
enrichment media.
9.1.2 Specific preparations of the initial suspension for certain foodstuffs
NOTE The following specific preparations concern only the case of Salmonella. Specific preparations applicable for the
determination of any microorganisms are described in ISO 6887-2, ISO 6887-3, ISO 6887-4 and ISO 8261.
9.1.2.1 Cocoa and cocoa-containing products (e.g. more than 20 %)
Add to the buffered peptone water (5.2.1) preferably 50 g/l of casein (avoid the use of acid casein), or 100 g/l of
sterile skim milk powder and add, after about 2 h incubation, 0,018 g/l of Brilliant green if the foodstuff is likely to be
highly contaminated with Gram-positive flora.
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ISO 6579:2002(E)
9.1.2.2 Acidic and acidifying foodstuffs
Ensure that the pH does not fall to below 4,5 during pre-enrichment.
NOTE The pH of acidic and acidifying foodstuffs is more stable if double-strength buffered peptone water is used.
9.2 Non-selective pre-enrichment
Incubate the initial suspension (9.1) at 37 °C ± 1 °C for 18 h ± 2 h.
9.3 Selective enrichment
9.3.1 Transfer 0,1 ml of the culture obtained in 9.2 to a tube containing 10 ml of the RVS broth (5.2.2); transfer
1 ml of the culture obtained in 9.2 to a tube containing 10 ml of MKTTn broth (5.2.3).
9.3.2 Incubate the inoculated RVS broth (9.3.1) at 41,5 °C ± 1 °C for 24 h ± 3 h and the inoculated MKTTn broth
at 37 °C ± 1 °C for 24 h ± 3 h. Care should be taken that the maximum allowed incubation temperature (42,5 °C) is
not exceeded.
9.4 Plating out and identification
9.4.1 After incubation for 24 h ± 3 h, using the culture obtained in the RVS broth (9.3.2), inoculate by means of a
loop (6.7) the surface of one large-size Petri dish (6.11) containing the first selective plating-out medium (XLD agar,
see 5.2.4.1), so that well-isolated colonies will be obtained.
In the absence of large dishes, use two small dishes one after the other, using the same loop.
Proceed in the same way with the second selective plating-out medium (5.2.4.2) using a sterile loop and Petri
dishes as above.
9.4.2 After incubation for 24 h ± 3 h, using the culture obtained in the MKTTn broth (9.3.2), repeat the procedure
described in 9.4.1 with the two selective plating-out media.
9.4.3 Invert the dishes (9.4.1 and 9.4.2) so that the bottom is uppermost, and place them in the incubator (6.3)
set at 37 °C for the first plating-out medium (5.2.4.1). The manufacturer's instructions shall be followed for the
second plating-out medium (5.2.4.2).
9.4.4 After incubation for 24 h ± 3 h, examine the plates (9.4.3) for the presence of typical colonies of Salmonella
and atypical colonies that may be Salmonella (see Note). Mark their position on the bottom of the dish.
Typical colonies of Salmonella grown on XLD agar have a black centre and a lightly transparent zone of reddish
colour due to the colour change of the indicator.
NOTE Salmonella H S negative variants (e.g. S. Paratyphi A) grown on XLD agar are pink with a darker pink centre.
2
Lactose-positive Salmonella grown on XLD agar are yellow with or without blackening.
Incubate the second selective solid medium at the appropriate temperature and examine after the appropriate time
to check for the presence of colonies which, from their characteristics, are considered to be presumptive
Salmonella.
9.5 Confirmation
9.5.1 General
If shown to be reliable, commercially available identification kits for the biochemical examination of Salmonella may
be used. The use of identification kits concerns the biochemical confirmation of colonies. These kits should be used
following the manufacture
...
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