EN ISO 10273:2017

SLOVENSKI STANDARD
01-julij-2017
1DGRPHãþD
SIST EN ISO 10273:2003
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti patogene bakterije Yersinia enterocolitica (ISO 10273:2017)
Microbiology of the food chain - Horizontal method for the detection of pathogenic
Yersinia enterocolitica (ISO 10273:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis von
pathogenen Yersinia enterocolitica (ISO 10273:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche de
Yersinia enterocolitica pathogènes (ISO 10273:2017)
Ta slovenski standard je istoveten z: EN ISO 10273:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 10273
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 10273:2003
English Version
Microbiology of the food chain - Horizontal method for the
detection of pathogenic Yersinia enterocolitica (ISO
10273:2017)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche de Yersinia Verfahren zum Nachweis von pathogenen Yersinia
enterocolitica pathogènes (ISO 10273:2017) enterocolitica (ISO 10273:2017)
This European Standard was approved by CEN on 13 March 2017.

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 CEN-CENELEC 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 CEN-CENELEC Management
Centre has the same status as the official versions.

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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10273:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 10273:2017) 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.
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 October 2017, and conflicting national standards shall
be withdrawn at the latest by October 2017.
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.
This document supersedes EN ISO 10273:2003.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 10273:2017 has been approved by CEN as EN ISO 10273:2017 without any modification.

INTERNATIONAL ISO
STANDARD 10273
Third edition
2017-03
Microbiology of the food chain —
Horizontal method for the detection
of pathogenic Yersinia enterocolitica
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche de Yersinia enterocolitica pathogènes
Reference number
ISO 10273:2017(E)
©
ISO 2017
ISO 10273:2017(E)
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

ISO 10273:2017(E)
Contents Page
Foreword .v
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Principle . 2
5.1 General . 2
5.2 Direct plating from liquid enrichment medium . 2
5.3 Enrichment in liquid enrichment medium and selective liquid enrichment medium . 2
5.4 Plating out after enrichment and identification . 2
5.5 Confirmation . 3
6 Culture media and reagents . 3
7 Equipment and consumables . 3
8 Sampling . 3
9 Preparation of test sample . 4
Procedure (as shown in Annex A) . 4
10.1 Test portion and initial suspension . 4
10.2 Direct plating on selective agar . 4
10.3 Enrichment . 5
10.4 Plating out and incubation of plates . 5
10.4.1 Plating from PSB and ITC by KOH treatment on CIN agar. 5
10.4.2 Plating from PSB and ITC by KOH treatment on chromogenic agar (optional) . 5
10.5 Identification of characteristic colonies . 5
10.6 Confirmation . 6
10.6.1 General. 6
10.6.2 Selection of colonies for confirmation . 6
10.6.3 Determination of pathogenic Yersinia species . 6
10.6.4 Confirmation of pathogenic Y. enterocolitica . 8
10.6.5 Interpretation of confirmation tests for Y. enterocolitica.10
10.6.6 Interpretation of confirmation tests for pathogenic Y. enterocolitica .10
10.7 Biotyping of Y. enterocolitica (optional) .10
10.7.1 General.10
10.7.2 Fermentation of xylose .11
10.7.3 Tween-esterase test .11
10.7.4 Fermentation of salicin (optional) and trehalose .11
10.7.5 Indole formation .11
10.7.6 Interpretation of biotyping tests .11
11 Expression of results .12
12 Performance characteristics of the method .12
12.1 Interlaboratory study .12
12.2 Sensitivity .12
12.3 Specificity .12
12.4 LOD .12
13 Test report .12
14 Quality assurance .13
Annex A (normative) Diagrams of the procedures .14
ISO 10273:2017(E)
Annex B (normative) Composition and preparation of culture media and reagents .17
Annex C (informative) Method validation studies and performance characteristics .32
Annex D (informative) Procedure for cold enrichment .34
Bibliography .39
iv © ISO 2017 – All rights reserved

ISO 10273:2017(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with ISO Technical
Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the
agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This third edition cancels and replaces the second edition (ISO 10273:2003), which has been technically
revised with the following main changes.
— In confirmation of pathogenic Y. enterocolitica, tests related to pathogenicity have been added or
specified and relocated in the frontline. Accordingly, the word “presumptive” has been removed
from the title wording (pathogenic Y. enterocolitica) since standard contains mandatory tests
related to pathogenicity and allows separation of pathogenic and non-pathogenic Y. enterocolitica.
1)
TM
— Direct plating on cefsulodin, Irgasan and novobiocin (CIN) agar has been added.
— Incubation time for peptone, sorbitol and bile salts (PSB) enrichment broth and CIN agar has been
changed.
TM
— Inoculation and incubation time for Irgasan , ticarcillin and potassium chlorate (ITC) enrichment
broth has been changed and specified.
— Salmonella/shigella agar with sodium desoxycholate and calcium chloride (SSDC) has been replaced
by CIN agar and optional chromogenic medium.
— Inoculation of CIN agar without prior potassium hydroxide (KOH) treatment of enrichment broth
has been changed to optional procedure (in parallel to mandatory KOH treatment).
— The preparation (shelf life) of KOH and ammonium iron(II) sulfate solutions has been specified.
TM
1) Irgasan is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of this product.
ISO 10273:2017(E)
— Suspect colonies from primary culture are streaked (purified) on CIN agar and (optionally)
on chromogenic agar to facilitate better selection of characteristic colonies that need further
confirmation. The use of stereomicroscope in identification of characteristic colonies is emphasized.
— All biochemical confirmation tests, except for pyrazinamidase test, can be replaced by real-time
polymerase chain reaction (PCR) detection of ail-gene in accordance with ISO/TS 18867.
— Five confirmation tests (indole, trehalose, xylose, citrate, tween-esterase) have become optional.
Test for salicin has been added as an optional (biotyping) test. Test for calcium requirements at 37°C
has been replaced by congo red magnesium-oxalate (CR-MOX) test. Three tests (oxidase, Kligler’s
agar and ornithine decarboxylase) have been deleted.
— The procedure for cold-enrichment of Y. enterocolitica has been added as Annex D;
— Performance characteristics have been added to Annex C.
— Performance testing for the quality assurance of the culture media has been added to Annex B and
Annex D.
vi © ISO 2017 – All rights reserved

ISO 10273:2017(E)
Introduction
This document specifies a horizontal method for the detection of Yersinia enterocolitica associated with
human disease. Because of the large variety of food and feed products, this horizontal method may not
be appropriate in every detail for certain products, and for some other products it may be necessary to
use different methods. Nevertheless, it is hoped that in all cases every attempt will be made to apply
this horizontal method as far as possible and that deviations from this will only be made if absolutely
necessary for technical reasons.
The main changes, listed in the foreword, introduced in this document compared to ISO 10273:2003,
are considered as major (see ISO 17468).
When this document 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
in the case of particular products.
The harmonization of test methods cannot be immediate and, for certain group 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 document so that eventually the only remaining departures from this horizontal method will
be those necessary for well-established technical reasons.
INTERNATIONAL STANDARD ISO 10273:2017(E)
Microbiology of the food chain — Horizontal method for
the detection of pathogenic Yersinia enterocolitica
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests
for detecting pathogenic Yersinia enterocolitica 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 document should be familiar with normal
laboratory practice. This document 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 conditions.
1 Scope
This document specifies a horizontal method for the detection of Y. enterocolitica associated with
human disease. It 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 documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www.e lectropedia. org/
— ISO Online browsing platform: available at http:// www. iso. org/o bp
3.1
pathogenic Yersinia enterocolitica
psychrotrophic bacteria forming characteristic colonies on solid selective media and possessing the
biochemical and molecular properties meeting the pathogenicity criteria described when confirmation
tests are carried out in accordance with this document
ISO 10273:2017(E)
3.2
detection of pathogenic Yersinia enterocolitica
determination of the presence or absence of pathogenic Yersinia enterocolitica (3.1) in a given mass
or volume of product or a specified surface, when the tests are carried out in accordance with this
document
4 Abbreviated terms
For the purposes of this document, the following abbreviations apply.
CEB cold enrichment broth
TM
CIN Cefsulodin, Irgasan and Novobiocin
CR-MOX congo red magnesium-oxalate
TM
ITC Irgasan , Ticarcillin and potassium chlorate
KOH potassium hydroxide
MRB modified rappaport broth
PCR: polymerase chain reaction
PSB peptone, sorbitol and bile salts
TSB tryptic soy broth
WDCM World Data Centre for Microorganisms
5 Principle
5.1 General
Detection of pathogenic Y. enterocolitica involves four successive stages (see Annex A for a diagram
of procedure and confirmation). In addition to the general procedure, for example during outbreak
investigations, optional cold enrichment procedure as described in Annex D may be used.
5.2 Direct plating from liquid enrichment medium
The sample is homogenized into a liquid enrichment medium (PSB broth), after which a specified amount
[15]
is inoculated onto two to four CIN agar plates. Inoculated plates are incubated at 30 °C for 24 h.
NOTE Additional plates like chromogenic agar medium for detection of pathogenic Y. enterocolitica can also
[9,13,18]
be used.
5.3 Enrichment in liquid enrichment medium and selective liquid enrichment medium
A specified amount of inoculated PSB enrichment medium (5.2) is transferred into a selective liquid
[17]
enrichment medium ITC broth. The ITC broth and initial PSB suspension are incubated at 25 °C
for 44 h.
5.4 Plating out after enrichment and identification
Using the enrichments obtained in 5.3, surface plating on the CIN agar is performed by transferring first
a specified amount of enrichment (5.3, see Clause 10 for the procedure) into 0,5 % KOH solution and,
after mixing for specified amount of time (KOH treatment or alkaline treatment), inoculating onto a CIN
plate. Inoculated plates are incubated at 30 °C for 24 h. Colonies typical of pathogenic Y. enterocolitica are
2 © ISO 2017 – All rights reserved

ISO 10273:2017(E)
identified (see 10.5) and the colony morphology is verified as presumptive pathogenic Y. enterocolitica
by successive culturing onto selective plates (see 10.5).
NOTE Additional plates like chromogenic agar medium for detection of pathogenic Y. enterocolitica can also
[9,13,18]
be used.
5.5 Confirmation
On colonies identified as presumptive pathogenic Y. enterocolitica (5.2 and 5.4), confirmation of
pathogenic Y. enterocolitica is carried out by appropriate biochemical or/and molecular confirmation
tests (see 10.6 and Figure A.2).
6 Culture media and reagents
For current laboratory practice, see ISO 7218.
For performance testing of culture media see ISO 11133 and Annex B.
Composition of culture media and reagents and their preparation are described in Annex B.
Alternatively, dehydrated complete media, diluents or ready-to-use media may be used; follow the
manufacturer’s instructions.
7 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
7.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).
As specified in ISO 7218.
7.2 Incubators, in accordance with ISO 7218, capable of operating at 4 °C ± 2 °C, 25 °C ± 1 °C,
30 °C ± 1 °C and 37 °C ± 1 °C.
7.3 Sterile blender bags, test tubes, bottles and/or flasks, of appropriate capacity.
7.4 Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
7.5 Pipettes. Graduated pipettes or automatic pipettes, with a wide opening, and of nominal capacities
1 ml and 10 ml, graduated respectively in 0,1 ml and 0,5 ml divisions and Pasteur pipettes.
7.6 Loops and spreaders. Sterile loops, approximately 6 mm in diameter (10 µl volume), and
inoculation needle or wire. L-shaped or T-shaped single-use spreaders. Cotton buds (see optional
protocol in Annex D).
7.7 Stereomicroscope, equipped with dark field illumination or obliquely (45° angle) transmitted light.
7.8 Peristaltic blender.
8 Sampling
Sampling is not part of the method specified in this document. See the specific International Standard
dealing with the product concerned. If there is no specific International Standard dealing with the
ISO 10273:2017(E)
sampling of the product concerned, it is recommended that the parties concerned come to an agreement
on this subject.
Recommended sampling techniques are given in the following documents:
— ISO/TS 17728 for food and animal feed;
— ISO 13307 for primary production stage;
— ISO 17604 for carcasses;
— ISO 18593 for environmental samples.
It is important that the laboratory receives a sample that is representative and the sample should not
have been damaged or changed during transport or storage.
9 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.
10 Procedure (as shown in Annex A)
10.1 Test portion and initial suspension
10.1.1 See the relevant document of ISO 6887 (all parts) or any specific International Standard
appropriate to the product concerned.
10.1.2 For preparation of the initial suspension, in the general case, use as diluent the pre-enrichment
medium specified in B.2 (PSB broth). Pre-warm the PSB broth to room temperature before use.
In general, an amount of test portion (mass or volume) is added to a quantity of PSB (mass or volume) to
yield a tenfold dilution. For this, a 25 g test portion is mixed with 225 ml of PSB.
Homogenize the suspension, preferably by using a peristaltic blender (7.8) for 1 min.
This document has been validated for test portions of 25 g or ml. A smaller test portion may be
used, without the need for additional validation/verification, providing that the same ratio between
enrichment broth and test portion is maintained. A larger test portion than that initially validated may
be used, if a validation/verification study has shown that there are no negative effects on the detection
of pathogenic Y. enterocolitica.
NOTE Validation can be conducted in accordance with the appropriate documents of ISO 16140 (all parts).
Verification for pooling samples can be conducted in accordance with the protocol described in ISO 6887-1:2017,
Annex D (verification protocol for pooling samples for qualitative tests).
10.1.3 Prepare the selective enrichment ITC suspension by transferring 10 ml of PSB suspension
(10.1.2) into 90 ml of ITC broth (B.3) and mix.
10.2 Direct plating on selective agar
Using the initial PSB suspension obtained (10.1.2), divide a total volume of 1 ml onto two to four CIN
agar plates (B.6) and spread it over the plates with a spreader (7.6).
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ISO 10273:2017(E)
Invert the CIN plates and place them in the incubator set at 30 °C (7.2) for 24 h ± 2 h.
NOTE 1 Drying of CIN agar plates (e.g. in laminar airflow cabinet) before inoculation for half an hour can be
required for complete absorption of the inoculum in the agar.
NOTE 2 The number of CIN agar plates to use depends on the expected level of background microflora of the
samples.
10.3 Enrichment
Incubate the initial suspension in PSB (10.1.2) and selective enrichment broth ITC (10.1.3) at 25 °C (7.2)
for 44 h ± 4 h (without agitation).
10.4 Plating out and incubation of plates
10.4.1 Plating from PSB and ITC by KOH treatment on CIN agar
Using a sterile pipette (7.5), transfer 0,5 ml of the PSB enrichment (10.3) into 4,5 ml of KOH solution
[7]
(B.5) (prepared the day before use) and mix. After 20 s ± 5 s of the addition of the PSB enrichment
to the KOH solution, streak by means of a loop (7.6), the surface of a CIN agar plate (B.6) to obtain well-
separated colonies. Repeat the procedure for ITC enrichment (10.3).
NOTE 1 It is crucial for method performance to prepare KOH on the day before use, see Annexes B and C.
Invert the CIN plates and place them in the incubator set at 30 °C (7.2) for 24 h ± 2 h.
NOTE 2 Additionally, it can be advantageous to inoculate [by means of a loop (7.6)] CIN agar plates with
untreated (no KOH treatment) PSB and ITC.
NOTE 3 During KOH treatment the enrichment is diluted tenfold. Furthermore, this treatment can reduce the
number of pathogenic Y. enterocolitica in the solution. Consequently, it can be advantageous, in some cases, to
inoculate an additional CIN plate with 0,1 ml of inoculum.
10.4.2 Plating from PSB and ITC by KOH treatment on chromogenic agar (optional)
Repeat the procedure in 10.4.1 and inoculate, after KOH treatment, by means of a loop (7.6), the surface
[9,13,18]
of a chromogenic agar plate to obtain well-separated colonies.
Incubate the chromogenic plates according to the instructions of the manufacturer.
10.5 Identification of characteristic colonies
After incubation for 24 h ± 2 h, examine the CIN plates in order to detect the presence of characteristic
colonies of Y. enterocolitica. This should be done with the help of a stereomicroscope (7.7) equipped
with dark field illumination or obliquely transmitted light (45° angle).
On CIN agar, pathogenic Y. enterocolitica appears as small (approximately 1 mm or under), circular,
smooth colonies with entire edge. The colonies have a small, deep red sharp bordered centre (“bull’s
eye”). The surrounding rim is translucent or transparent and, when examined with obliquely
transmitted light, non-iridescent and finely granular.
NOTE 1 Dark field illumination or obliquely transmitted light helps to distinguish characteristic colonies of
[12]
Yersinia enterocolitica from very similar colonies of other Yersinia species and some non-Yersinia species.
NOTE 2 In case of dense growth of background flora on the CIN plates, the colony size of pathogenic
Y. enterocolitica can be smaller and the typical red centre can be unclear or absent.
ISO 10273:2017(E)
10.6 Confirmation
10.6.1 General
The use of control strains of Yersinia species is required especially in helping to distinguish between
pathogenic Y. enterocolitica from other Yersinia species on CIN agar. Appropriate positive and negative
control strains for each of the confirmation tests shall be used. Examples of suitable control strains are
given in chapters dealing with these tests. A flow-diagram of the confirmation is given in Figure A.2.
10.6.2 Selection of colonies for confirmation
For confirmation, take from each plate of each selective medium (see 10.3) five colonies considered to
be typical for pathogenic Y. enterocolitica if available (see 10.5).
Streak the selected colonies onto the surface of CIN agar plates (B.6) in order to allow well separated
colonies to develop. Streak also control strains of Y. enterocolitica bioserotype 4/O:3, 2/O:9, and biotype
1A and other Yersinia species for comparison of the colony morphology.
Additionally, it is advantageous to streak typical colonies for confirmation and appropriate control
strains on chromogenic agar, in parallel to CIN agar plating. For identification of characteristic colonies
on chromogenic agar, follow the manufacturer’s instructions on evaluation of typical morphology of the
colonies.
EXAMPLE Suitable Y. enterocolitica control strains are WDCM 00216 (bioserotype 4/O:3), WDCM 00215
(bioserotype 2/O:9), and WDCM 00160 (bioserotype 1B/O:8).
Invert the inoculated plates and place them in the incubator set at 30 °C (7.2) for 24 h ± 2 h.
Examine the incubated plates for characteristic colonies (see 10.5) and purity of culture. This should be
done with the help of a stereomicroscope (7.7). Compare the morphology of suspect colonies to colonies
of control strains for better distinction between typical and atypical colonies. Discard plates with
atypical colonies. If mixed cultures with typical colonies are present, subculture typical colonies onto
CIN agar plates (B.6) and incubate as above.
Proceed with one pure culture representing initial typical colonies on the primary plate. Retain the
other typical pure cultures (up to five, if available) for confirmation in case the first culture does not
confirm. Streak the selected colonies onto the surface of non-selective agar (for example, nutrient
agar (B.7), blood agar, or tryptone soya agar) in a manner which will allow well-separated colonies to
develop.
Invert the inoculated plates and place them in the incubator set at 30 °C (7.2) for 18 h to 24 h or until
growth is satisfactory.
Use pure cultures for the biochemical confirmations and pathogenicity tests.
NOTE 1 It is not necessary to proceed to confirmation from all successive enrichment steps if pathogenic Y.
enterocolitica from earlier step has been confirmed.
NOTE 2 For epidemiological purposes or during outbreak investigations, confirmation of additional colonies,
e.g. five typical or suspect colonies from each selective enrichment/isolation medium combination, can be
beneficial.
10.6.3 Determination of pathogenic Yersinia species
10.6.3.1 Detection of urease
Streak bacteria onto the slant surface of the agar (B.10). Close the caps of the tubes loosely so that air
can enter and aerobic growth conditions prevail.
Incubate at 30 °C (7.2) for 24 h ± 2 h.
6 © ISO 2017 – All rights reserved

ISO 10273:2017(E)
Pink-violet or red-pink colours indicate a positive urease reaction.
EXAMPLE Suitable positive control strain is WDCM 00216 (Y. enterocolitica, bioserotype 4/O:3) or WDCM
00160 (Y. enterocolitica, bioserotype 1B/O:8).
An orange-yellow colour indicates a negative urease reaction.
Retain for further confirmation all urease positive colonies with typical colony morphology.
NOTE 1 Pathogenic Y. enterocolitica strains inoculated on some types of commercially available urea agars
can need more time (up to 7 days) for positive reaction to develop.
NOTE 2 Pathogenic urease-negative strains of Y. enterocolitica do exist, but they are extremely rare (0,01 %).
10.6.3.2 Hydrolysis of esculin
Streak bacteria onto the slant surface (B.12) of the agar.
Incubate at 30 °C (7.2) for 24 h ± 2 h.
A black halo around the colonies indicates a positive reaction.
EXAMPLE Suitable negative control strain is WDCM 00216 (Y. enterocolitica, bioserotype 4/O:3) or
WDCM 000160 (Y. enterocolitica, bioserotype 1B/O:8) and positive control strain is any Y. enterocolitica strain
representing biotype 1A or Y. intermedia WDCM 00217.
NOTE This test for hydrolysis of esculin is equivalent to the test for fermentation of salicin in determining
pathogenicity.
10.6.3.3 Detection of virulence plasmid (pYV) by CR-MOX agar test
Congo red binding and formation of pin-point colonies at 37 °C are typical features of pathogenic
Y. enterocolitica. The virulence plasmid (pYV) determines traits related to the pathogenicity of Yersinia,
and many of them, including calcium dependent growth are switched on only at 37 °C.
The virulence plasmid (pYV) can be spontaneously lost in the laboratory during storage, lengthy
culture and repeated passages. Therefore, the test for virulence plasmid (CR-MOX test) shall be carried
out at an early stage of confirmation.
Using a loop (7.6), touch several colonies of the pure culture of the strain selected for further
confirmation (urease positive, typical colony morphology). Inoculate the surface of CR-MOX agar (B.11)
to obtain well-separated colonies.
Incubate at 37 °C (7.2) for 24 h to 48 h.
If needed, examine the plates for positive, pYV containing colonies after 24 h and continue incubation
for further 24 h if positive colonies are not present.
A plate giving a positive reaction contains sharp orange-red (congo red binding) pinpoint colonies
(calcium dependent growth at 37 °C) and possibly colourless larger colonies. A plate giving a negative
reaction contains only colourless colonies.
NOTE 1 In a pure culture, it is normal that some of the colonies contain cells with plasmid pYV while other
colonies in the same culture contain plasmid-free cells. When preparing inoculum for this test, collecting
material with a loop from several colonies helps to avoid choosing for plasmid-free bacterial cells.
NOTE 2 For better distinction between positive and negative reactions, it is advantageous to inoculate two
parallel CR-MOX plates from the same inoculant of the strain tested and incubate one plate at 37 °C (7.2) and the
other at 25 °C (7.2). The plate incubated at 25 °C (7.2) always gives a negative reaction (even if the strain contains
pYV). Therefore, the difference between possible positive result at 37 °C (7.2) and negative reaction at 25 °C (7.2)
is better visualized.
ISO 10273:2017(E)
A suitable positive control is any pathogenic Y. enterocolitica strain that has been verified to possess the
virulence plasmid, before use.
EXAMPLE Suitable positive control strain is WDCM 00216 (Y. enterocolitica, bioserotype 4/O:3) and
negative control strain is WDCM 00160 (Y. enterocolitica, bioserotype 1B/O:8, plasmid-free) or WDCM 00217
(Y. intermedia).
Since the virulence plasmid (pYV) can be lost during subculturing in the laboratory, continue
confirmation also with strains giving a negative reaction in the test.
Additionally, preserve positive strains as frozen stock cultures at an early stage of confirmation
[preferably after positive reactions in urease (10.6.3.1) and CR-MOX tests (10.6.3.3)].
An example of preserving strains is given below (other suitable means of preserving cultures can also
be used):
— immediately subculture each pure culture into TSB (B.8);
— incubate at 30 °C (7.2) for 24 h ± 2 h;
— add an equal volume of 40 % sterile glycerol (B.9) to get a final glycerol concentration of 20 %;
— mix well and freeze, preferably at −70 °C.
10.6.3.4 Detection of pyrazinamidase
Inoculate a large area of the slant surface (B.13) of the medium with generous loopful (7.6) of bacteria.
Incubate at 30 °C (7.2) for 48 h ± 4 h.
Add 1 ml of freshly prepared (on the day of use) 1 % ammonium iron(II) sulfate solution (B.14).
If the reaction is positive, a pinkish-brown colour develops within 15 min indicating presence of
pyrazinoic acid formed by pyrazinamidase enzyme.
EXAMPLE Suitable negative control strain is WDCM 00216 (Y. enterocolitica, bioserotype 4/O:3) or
WDCM 000160 (Y. enterocolitica, bioserotype 1B/O:8) and positive control strain is any Y. enterocolitica strain
representing biotype 1A or Y. intermedia WDCM 00217.
NOTE The possibility of obtaining false positive reactions increases if old 1 % ammonium iron(II) sulfate
solution is used.
10.6.3.5 Interpretation of pathogenicity tests
The strain is considered to be a pathogenic Yersinia if it is urease positive, an
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