SIST EN ISO 10273:2003
(Main)Microbiology of food and animal feedings stuffs - Horizontal method for the detection of presumptive pathogenic Yersinia enterocolitica (ISO 10273:2003)
Microbiology of food and animal feedings stuffs - Horizontal method for the detection of presumptive pathogenic Yersinia enterocolitica (ISO 10273:2003)
This method describes the detection of Yersinia enterocolitica.
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis von präsumtiv pathogenen Yersenia enterocolitica (ISO 10273:2003)
Diese Internationale Norm legt ein horizontales Verfahren zum Nachweis von präsumtiv pathogenen Yersinia enterocolitica fest. Diese Internationale Norm gilt für
Erzeugnisse, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind,
Umgebungsproben im Bereich der Herstellung und der Behandlung von Lebensmitteln.
Microbiologie des aliments - Méthode horizontale pour la recherche de Yersinia enterocolitica présumées pathogenes (ISO 10273:2003)
L'ISO 10273:2003 spécifie une méthode horizontale pour la recherche des Yersinia enterocolitica présumées pathogènes chez l'homme. Elle est applicable aux produits pour l'alimentation humaine et animale, ainsi qu'aux échantillons d'environnement pour la production et la distribution des aliments.
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti domnevno patogene Yersinie enterocolitice (ISO 10273:2003)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 10273:2003
01-november-2003
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti
domnevno patogene Yersinie enterocolitice (ISO 10273:2003)
Microbiology of food and animal feedings stuffs - Horizontal method for the detection of
presumptive pathogenic Yersinia enterocolitica (ISO 10273:2003)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum
Nachweis von präsumtiv pathogenen Yersenia enterocolitica (ISO 10273:2003)
Microbiologie des aliments - Méthode horizontale pour la recherche de Yersinia
enterocolitica présumées pathogenes (ISO 10273:2003)
Ta slovenski standard je istoveten z: EN ISO 10273:2003
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 10273:2003 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10273:2003
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SIST EN ISO 10273:2003
EUROPEAN STANDARD
EN ISO 10273
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2003
ICS 07.100.30
English version
Microbiology of food and animal feedings stuffs - Horizontal
method for the detection of presumptive pathogenic Yersinia
enterocolitica (ISO 10273:2003)
Microbiologie des aliments - Méthode horizontale pour la Mikrobiologie von Lebensmitteln und Futtermitteln -
recherche de Yersinia enterocolitica présumées Horizontales Verfahren zum Nachweis von präsumtiv
pathogènes (ISO 10273:2003) pathogenen Yersenia enterocolitica (ISO 10273:2003)
This European Standard was approved by CEN on 20 May 2003.
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,
Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, 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
© 2003 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10273:2003 E
worldwide for CEN national Members.
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SIST EN ISO 10273:2003
EN ISO 10273:2003 (E)
CORRECTED 2003-09-24
Foreword
This document (EN ISO 10273:2003) 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 December 2003, and conflicting national
standards shall be withdrawn at the latest by December 2003.
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, Hungary, Iceland, Ireland, Italy,
Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and
the United Kingdom.
Endorsement notice
The text of ISO 10273:2003 has been approved by CEN as EN ISO 10273:2003 without any
modifications.
NOTE Normative references to International Standards are listed in Annex ZA (normative).
2
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SIST EN ISO 10273:2003
EN ISO 10273:2003 (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 EN ISO 6887-1 1999
feeding stuffs - Preparation of test
samples, initial suspension and
decimal dilutions f or
microbiological examination - Part
1: General rules for the
preparation of the initial
suspension and decimal dilutions
ISO 8261 2001 Milk and milk products - General EN ISO 8261 2001
guidance for the preparation of
test samples, initial suspensions
and decimal dilutions for
microbiological examination
ISO/TS 11133-1 2000 Microbiology of food and animal ENV ISO 11133-1 2000
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
3
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SIST EN ISO 10273:2003
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SIST EN ISO 10273:2003
INTERNATIONAL ISO
STANDARD 10273
Second edition
2003-06-15
Microbiology of food and animal feeding
stuffs — Horizontal method for the
detection of presumptive pathogenic
Yersinia enterocolitica
Microbiologie des aliments — Méthode horizontale pour la recherche de
Yersinia enterocolitica présumées pathogènes
Reference number
ISO 10273:2003(E)
©
ISO 2003
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SIST EN ISO 10273:2003
ISO 10273:2003(E)
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ii © ISO 2003 — All rights reserved
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SIST EN ISO 10273:2003
ISO 10273:2003(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 Enrichment in selective liquid media. 2
4.3 Plating out and identification. 2
4.4 Confirmation. 2
5 Reagents and media . 2
6 Apparatus and glassware. 4
7 Sampling . 5
8 Preparation of test sample. 5
9 Procedure (see Annex A). 5
9.1 Test portion and initial suspension . 5
9.2 Enrichment. 6
9.3 Plating out and identification. 6
9.4 Confirmation. 6
10 Test report. 12
Annex A (normative) Diagram of procedure. 13
Annex B (normative) Composition and preparation of culture media and reagents . 14
Annex C (informative) Biochemical characteristics at 30 °C of Yersinia pseudotuberculosis,
Yersinia enterocolitica and biochemically related species . 29
Annex D (informative) Biovars (biotyping) of Yersinia enterocolitica . 30
Bibliography . 31
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SIST EN ISO 10273:2003
ISO 10273:2003(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.
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 10273 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
This second edition cancels and replaces the first edition (ISO 10273:1994), Subclause 9.4 of which has been
technically revised.
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SIST EN ISO 10273:2003
ISO 10273:2003(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 will 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 10273:2003
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SIST EN ISO 10273:2003
INTERNATIONAL STANDARD ISO 10273:2003(E)
Microbiology of food and animal feeding stuffs — Horizontal
method for the detection of presumptive pathogenic Yersinia
enterocolitica
WARNING — The use of this standard may involve hazardous materials, operations and equipment. It
is the responsibility of the user of this standard to establish appropriate safety and health practices
and to determine the applicability of regulatory limitations prior the use.
1 Scope
This International Standard specifies a horizontal method for the detection of Yersinia enterocolitica presumed
to be pathogenic to human subjects. This International Standard is applicable to
products intended for human consumption and the feeding of animals, and
environmental samples in the area of food production and food handling.
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
ISO 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations,
and Amd.1:2001
ISO 8261, Milk and milk products — General guidance for the preparation of test samples, initial suspensions
and decimal dilutions for microbiological examination
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
presumptive pathogenic Yersinia enterocolitica
psychrotrophic bacteria forming characteristic colonies on solid selective media and having the biochemical
properties meeting the pathogenicity criteria described when the test is carried out in accordance with this
International Standard
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SIST EN ISO 10273:2003
ISO 10273:2003(E)
3.2
detection of presumptive pathogenic Yersinia enterocolitica
determination of the presence or absence of these bacteria in a predetermined quantity of product, when the
test is carried out in accordance with this International Standard
4 Principle
4.1 General
Presumptive pathogenic Yersinia enterocolitica are detected by the following three successive stages.
4.2 Enrichment in selective liquid media
The test portion is inoculated into two enrichment media
peptone, sorbitol and bile salts (PSB) broth, and
irgasan, ticarcillin and potassium chlorate (ITC) broth.
The ITC broth is incubated at 25 °C for 48 h and the PSB broth for 3 to 5 days.
NOTE Enrichment in ITC broth has been proposed (see reference [1]) for the isolation of Yersinia enterocolitica
biovar 4/serovar O:3 but not for biovar 1B serovar O:8, biovar 2/serovar O:9 (see reference [2]), or biovar 2 serovar
O:5,27. Isolation of Yersinia enterocolitica biovar 2/serovar O:9 needs the use of an ITC medium without chlorate and
which contains 80 % of the original concentration of magnesium chloride and malachite green (see reference [3]).
4.3 Plating out and identification
Using the cultures obtained in 4.2, surface plating of the following two solid selective culture media is carried
out:
agar with cefsulodin, irgasan and novobiocin (CIN) (see reference [7]);
Salmonella/Shigella agar, with sodium desoxycholate and calcium chloride (SSDC).
The media are incubated at 30 °C, then examined after 24 h and, if necessary, after 48 h depending on the
medium, to check if any characteristic colonies of Yersinia enterocolitica are present.
4.4 Confirmation
On plated-out colonies, tests for presumptive Yersinia enterocolitica are carried out, followed by biochemical
confirmation tests, biotyping tests, tests to establish pathogenic criteria, and possibly serological tests.
5 Reagents and media
For current laboratory practice, see ISO 7218.
See ISO/TS 11133-1 for specific requirements about quality assurance and performance of media.
NOTE ISO/TS 11133-2 on practical guidelines on performance testing of culture media is under preparation.
In view of the large number of culture media and reagents and for the clarity of the text, their compositions are
given in Annex B, which also includes details of dispensing, storage, etc.
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SIST EN ISO 10273:2003
ISO 10273:2003(E)
5.1 Enrichment media
5.1.1 Peptone, sorbitol and bile salts (PSB) broth
See B.1.
5.1.2 Irgasan, ticarcillin and potassium chlorate (ITC) broth
See B.2.
5.2 Plating out media
5.2.1 Cefsulodin, Irgasan and novobiocin (CIN) agar (see reference [7])
See B.3.
5.2.2 Salmonella/Shigella agar, with sodium desoxycholate and calcium chloride (SSDC)
See B.4.
5.2.3 Nutrient agar
See B.5.
5.3 Identification media and reagents
5.3.1 Urea indole medium
See B.6.
5.3.2 Reagent for indole detection
See B.7.
5.3.3 Kligler’s agar
See B.8.
5.3.4 Reagent for detection of oxidase
See B.9.
5.3.5 Decarboxylation media
5.3.5.1 Lysine decarboxylation medium
See B.10.
5.3.5.2 Ornithine decarboxylation medium
See B.11.
5.3.6 Media for fermentation of carbohydrates (sucrose, rhamnose, trehalose and xylose)
See B.12.
5.3.7 Simmons’ citrate medium
See B.13.
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SIST EN ISO 10273:2003
ISO 10273:2003(E)
5.3.8 Tween-esterase medium
See B.14.
5.3.9 Bile and aesculin agar
See B.15.
5.3.10 Casein soya agar
See B.16.
5.3.11 Casein soya agar, for detection of pyrazinamidase.
See B.17.
5.3.12 Ammonium iron(II) sulfate solution, for detection of pyrazinamidase.
See B.18.
5.3.13 Casein-soya agar, with magnesium and oxalate.
See B.19.
5.4 Saline solution
See B.20.
5.5 Potassium hydroxide in saline solution
See B.21.
5.6 Veal infusion broth
See B.22.
5.7 Sterile glycerol
See B.23.
6 Apparatus and glassware
Disposable apparatus is an acceptable alternative to reusable glassware, if it has suitable specifications.
Usual microbiology laboratory equipment and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave)
See ISO 7218.
6.2 Incubators, capable of operating at 22 °C ± 1 °C, 25 °C ± 1 °C, 30 °C ± 1 °C and 37 °C ± 1 °C.
6.3 Drying cabinet or oven, with ventilation by convection, capable of operating between 37 °C ± 1 °C and
50 °C ± 1 °C.
6.4 Water baths or incubators, capable of operating between 22 °C ± 1 °C, 24 °C ± 2 °C and 25 °C ± 1 °C,
preferably with a suitable agitation device.
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SIST EN ISO 10273:2003
ISO 10273:2003(E)
6.5 Water bath, capable of operating at 44 °C to 47 °C.
6.6 Test tubes, of dimensions 18 mm × 180 mm, 9 mm × 180 mm, and 12 mm × 50 mm.
6.7 Bottles and/or flasks, of suitable capacity.
6.8 Petri dishes, made of glass or plastics, of diameter 90 mm to 100 mm.
6.9 Total-delivery pipettes, of nominal capacities 10 ml and 1 ml, with large opening and 0,1 ml
graduations.
6.10 Rubber teats, or other microbiologically safe pipetting systems.
6.11 Loop, of approximately 3 mm diameter, straight wires of platinum/iridium and/or nickel/chromium,
glass rods and Pasteur pipettes.
Sterile plastic disposable loops or needles may be used. Nickel chromium is not suitable for the oxidase test
(see 9.4.3.5).
6.12 pH-meter, accurate to within ± 0,1 pH units at 25 °C.
6.13 Lighting, appropriate for oblique illumination.
6.14 Magnifying glass or stereomicroscope.
6.15 Peristaltic blender.
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.
Freezing of samples before analysis is not recommended, despite Yersinia spp. being recovered from frozen
products.
Sampling is not part of the method specified in this International Standard. 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 this 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 available, it is recommended that the parties
concerned come to an agreement on this subject.
9 Procedure (see Annex A)
9.1 Test portion and initial suspension
9.1.1 See the relevant part of ISO 6887, or ISO 8261, or any specific International Standard appropriate to
the product concerned.
9.1.2 In general, for preparing the initial suspension, place a quantity (x) of the test portion (of known mass
or volume) in a known volume of the PSB broth (B.1), to give a 1/10 dilution (by mass/volume or
volume/volume). Homogenize the suspension using a peristaltic blender (6.15) for 2 min.
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9.1.3 Prepare the second initial suspension in the same way with the ITC broth (B.2), so as to obtain a test
portion/enrichment medium dilution of 1/100 (mass/volume or volume/volume).
9.2 Enrichment
Incubate the two initial suspensions (9.1.2 and 9.1.3) as follows:
a) PSB medium at 22 °C to 25 °C for 48 h to 72 h with agitation, or for 5 days without agitation;
b) ITC medium at 25 °C for 48 h.
9.3 Plating out and identification
9.3.1 After incubation of the enrichment media (9.2), proceed as follows.
9.3.2 Using the PSB culture (9.2), inoculate, by means of a loop (6.11), the surface of a CIN agar plate
(B.3) to obtain well-separated colonies.
9.3.3 Using a sterile pipette (6.9), transfer 0,5 ml of the PSB culture (9.2) into 4,5 ml of potassium hydroxide
solution (B.21) and mix (see reference [5]). After 20 s ± 5 s, immediately inoculate, by means of a loop (6.11),
the surface of a CIN agar plate (B.3) to obtain well-separated colonies.
9.3.4 Using the ITC culture (9.2), inoculate, by means of a loop (6.11), the surface of an SSDC agar plate
(B.4) to obtain well-separated colonies.
9.3.5 Invert the dishes (9.3.2 to 9.3.4) and place them in the incubator (6.2) set at 30 °C.
9.3.6 After incubation for 24 h, examine the dishes with a magnifying glass (6.14) preferably equipped with
an obliquely transmitted light (6.13) in order to detect the presence of characteristic colonies of Yersinia
enterocolitica as follows.
a) On CIN agar, characteristic colonies of Yersinia enterocolitica are small (u 1 mm) and smooth with a red
centre and translucent rim and, when examined with obliquely transmitted light (6.13), are non-iridescent
and finely granular.
b) On SSDC agar, characteristic colonies of Yersinia enterocolitica are small (u 1 mm) and grey with an
indistinct rim, non-iridescent and very finely granular when examined with obliquely transmitted light.
NOTE Obliquely transmitted light helps to distinguish characteristic colonies of Yersinia enterocolitica from very
similar colonies of Pseudomonas.
9.3.7 If the development of colonies is slow, if coloration is weak, or if there are no characteristic colonies,
continue incubation of the plates for up to 48 h, then re-examine them.
9.4 Confirmation
9.4.1 General
Miniaturized biochemical identification kits, currently available commercially and permitting the identification of
Yersinia enterocolitica, may be used. Some miniaturized biochemical identification kits do not identify with
accuracy Yersinia species such as Yersinia mollaretii and Yersinia bercovieri (previous biovars of Yersinia
enterocolitica 3A and 3B) and Yersinia intermedia which are identified as Yersinia enterocolitica. In this last
case, the Mucate test shall be performed to discriminate between these species. An improvement of the
discriminatory powers of these miniaturized biochemical identification kits has been proposed (see references
[6] and [7]).
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9.4.2 Selection of colonies for confirmation
9.4.2.1 For confirmation, take from each dish of each selective medium (see 9.3.2 to 9.3.4), five colonies
considered to be characteristic or suspect.
If on one dish there are fewer than five characteristic or suspect colonies, take for confirmation all the
characteristic or suspect colonies.
Streak the selected colonies onto the surface of nutrient agar plates (B.5), in a manner which will allow well-
isolated colonies to develop.
Incubate the inoculated plates at 30 °C for 24 h.
Examine the incubated plates for purity of culture. If mixed cultures are present, subculture each individual
colony type onto further nutrient agar plates and incubate as above.
Use pure cultures for the biochemical confirmations and pathogenicity tests.
9.4.2.2 Plasmids that determine traits related to the pathogenicity of Yersinia can be spontaneously lost
during culture above 30 °C or with lengthy culture and passage below 30 °C in the laboratory.
Therefore preserve these as a frozen culture by immediately subculturing each pure culture into veal infusion
broth (B.22).
Incubate at 22 °C to 25 °C for 24 h to 48 h.
Add 10 % sterile glycerol (B.23), mix well and freeze, preferably at −70 °C.
9.4.3 Presumptive tests
9.4.3.1 General
By means of a wire (6.10), inoculate the media specified in 9.4.3.2 to 9.4.3.4 and perform the detection of
oxidase as described in 9.4.3.5 with each of the cultures obtained from the colonies selected in 9.4.2.
9.4.3.2 Detection of urease
Use a heavy inoculum to inoculate the broth just below the surface of the urease/indole medium (B.6).
Incubate at 30 °C for 24 h, preferably in a water bath.
Pink-violet or red-pink colours indicate a positive urease reaction. Yersinia enterocolitica mostly gives positive
urease reaction within 1 min to 5 min. Recording of the speed of a positive reaction can be of diagnostic value.
An orange-yellow colour indicates a negative urease reaction. Possible false negative reactions can occur if
the medium is not inoculated with sufficient microorganisms.
9.4.3.3 Detection of indole
Add 0,1 ml to 0,2 ml of the reagent (B.7) to the tubes (9.4.3.2) for the detection of indole.
A red ring at the surface of the culture indicates a positive reaction in 15 min.
9.4.3.4 Kligler’s agar
Stab the butt to the bottom of the agar and streak over the slant surface (B.7.2).
Incubate at 30 °C for 24 h to 48 h.
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