EN ISO 21872-1:2017

SLOVENSKI STANDARD
01-september-2017
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje Vibrio
spp. - 1. del: Ugotavljanje potencialno enteropatogene Vibrio parahaemolyticus,
Vibrio cholerae in Vibrio vulnificus (ISO 21872-1:2017)
Microbiology of the food chain - Horizontal method for the determination of Vibrio spp. -
Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio
cholerae and Vibrio vulnificus (ISO 21872-1:2017)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum
Nachweis von potentiell enteropathogenen Vibrio spp. - Teil 1: Nachweis von vibrio
parahaemolyticus und vibrio cholerae (ISO 21872-1:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la détermination des
Vibrio spp. - Partie 1: Recherche des espèces de Vibrio parahaemolyticus, Vibrio
cholerae et Vibrio vulnificus potentiellement entéropathogènes (ISO 21872-1:2017)
Ta slovenski standard je istoveten z: EN ISO 21872-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 21872-1
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2017
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of food and animal feeding stuffs - Horizontal
method for the detection of potentially enteropathogenic
Vibrio spp. - Part 1: Detection of Vibrio parahaemolyticus
and Vibrio cholerae (ISO 21872-1:2017)
Microbiologie des aliments - Méthode horizontale pour Mikrobiologie von Lebensmitteln und Futtermitteln -
la recherche des Vibrio spp. potentiellement Horizontales Verfahren zum Nachweis von potentiell
entéropathogènes - Partie 1: Recherche de Vibrio enteropathogenen Vibrio spp. - Teil 1: Nachweis von
parahaemolyticus et Vibrio cholerae (ISO 21872- vibrio parahaemolyticus und vibrio cholerae (ISO
1:2017) 21872-1:2017)
This European Standard was approved by CEN on 14 May 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 21872-1:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 21872-1:2017) 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 January 2018 and conflicting national standards shall
be withdrawn at the latest by January 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
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 21872-1:2017 has been approved by CEN as EN ISO 21872-1:2017 without any
modification.
INTERNATIONAL ISO
STANDARD 21872-1
First edition
2017-06
Microbiology of the food chain —
Horizontal method for the
determination of Vibrio spp. —
Part 1:
Detection of potentially
enteropathogenic Vibrio
parahaemolyticus, Vibrio cholerae and
Vibrio vulnificus
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la détermination des Vibrio spp. —
Partie 1: Recherche des espèces de Vibrio parahaemolyticus, Vibrio
cholerae et Vibrio vulnificus potentiellement entéropathogènes
Reference number
ISO 21872-1:2017(E)
©
ISO 2017
ISO 21872-1:2017(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
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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 21872-1:2017(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Primary enrichment in a liquid selective medium . 2
4.3 Secondary enrichment in a liquid selective medium . 3
4.4 Isolation and identification . 3
4.5 Confirmation . 3
5 Culture media and reagents . 3
5.1 Enrichment medium: alkaline saline peptone water (ASPW). 4
5.2 Solid selective isolation media . 4
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS) . 4
5.2.2 Second medium. 4
5.3 Saline nutrient agar (SNA) . 4
5.4 Reagent for detection of oxidase . 4
5.5 Biochemical tests . 4
5.5.1 L-lysine decarboxylase saline medium (LDC) . 4
5.5.2 Arginine dihydrolase saline medium (ADH) . 4
5.5.3 Reagent for detection of β-galactosidase . 5
5.5.4 Saline medium for detection of indole . 5
5.5.5 Saline peptone waters . 5
5.5.6 Sodium chloride solution . 5
5.6 PCR . 5
5.6.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance
for the purpose) . 5
5.6.2 Mastermix . 5
5.6.3 Primers and probes . 5
5.6.4 Positive control material . 5
5.6.5 Negative extraction control . 6
6 Equipment and consumables . 6
7 Sampling . 6
8 Preparation of the test sample . 6
9 Procedure (See Figure A.1) . 7
9.1 Test portion and initial suspension . 7
9.2 Primary selective enrichment . 7
9.3 Secondary selective enrichment . 7
9.4 Isolation and identification . 8
9.5 Confirmation . 8
9.5.1 General. 8
9.5.2 Selection of colonies for confirmation and preparation of pure cultures . 9
9.5.3 Tests for presumptive identification . 9
9.5.4 Biochemical confirmation .10
9.5.5 PCR confirmation .12
9.5.6 DNA extraction .12
9.5.7 Conventional PCR .12
9.5.8 Real-time PCR .13
10 Expression of results .13
ISO 21872-1:2017(E)
11 Performance characteristics of the method .13
11.1 Interlaboratory study .13
11.2 Sensitivity .14
11.3 Specificity .14
11.4 LOD .14
12 Test report .14
Annex A (normative) Diagram of procedure .15
Annex B (normative) Composition and preparation of the culture media and reagents .17
Annex C (informative) Conventional PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin (tdh) and thermostable direct related haemolysin
(trh) genes, Vibrio cholerae and Vibrio vulnificus .23
Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin gene (tdh) and Vibrio vulnificus .27
Annex E (informative) Results of an interlaboratory study .29
Bibliography .32
iv © ISO 2017 – All rights reserved

ISO 21872-1:2017(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 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: www . i so .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 TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This first edition cancels and replaces ISO/TS 21872-1:2007, which has been technically revised. It also
incorporates ISO/TS 21872-1:2007/Cor.1:2008.
The main changes are as follows:
— introduction of optional molecular identification methods for major food borne Vibrio spp.
(V. parahaemolyticus, including potentially enteropathogenic strains, V. vulnificus and V. cholerae);
— performance characteristics of the method have been added in Annex E.
A list of all parts in the ISO 21872 series can be found on the ISO website.
ISO 21872-1:2017(E)
Introduction
Because of the large variety of food and feed products, the horizontal method described in this
document 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.
The main changes, listed in the foreword, introduced in this document compared to ISO/TS 21872-
1:2007 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
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 document so that eventually the only remaining departures from this horizontal method will
be those necessary for well-established technical reasons.
vi © ISO 2017 – All rights reserved

INTERNATIONAL STANDARD ISO 21872-1:2017(E)
Microbiology of the food chain — Horizontal method for
the determination of Vibrio spp. —
Part 1:
Detection of potentially enteropathogenic Vibrio
parahaemolyticus, Vibrio cholerae and Vibrio vulnificus
WARNING — In order to safeguard the health of laboratory personnel, it is essential that
tests for detection of Vibrio spp., and particularly toxigenic Vibrio cholerae, be conducted
only in laboratories equipped for this purpose and under the supervision of an experienced
microbiologist, and that great care is exercised in the disposal of contaminated material.
1 Scope
This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which
causes human illness in or via the intestinal tract. The species detectable by the methods specified
include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the following:
— products intended for human consumption and the feeding of animals;
— environmental samples in the area of food production and food handling.
NOTE 1 This method may not be appropriate in every detail for certain products (see Introduction).
NOTE 2 The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V.
vulnificus are the major food-borne Vibrio spp. However, the method in this document can also be appropriate for
[1]
the identification of other Vibrio spp. causing illness in humans.
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 6887-1:2017, Microbiology of the food chain — 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 6887-3, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 3: Specific rules for the preparation of fish and fishery
products
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
ISO 22118, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection and quantification of food-borne pathogens — Performance characteristics
ISO 21872-1:2017(E)
ISO 22119, Microbiology of food and animal feeding stuffs — Real-time polymerase chain reaction (PCR) for
the detection of food-borne pathogens — General requirements and definitions
ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — General requirements and definitions
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 .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
potentially enteropathogenic Vibrio spp.
microorganism which forms typical colonies on solid selective media and which possesses the described
biochemical or molecular characteristics when the test is performed in accordance with this document
Note 1 to entry: This document describes specific procedures for V. parahaemolyticus, V. cholerae and V. vulnificus.
3.2
detection of potentially enteropathogenic Vibrio spp.
determination of the presence or absence of potentially enteropathogenic Vibrio spp. (3.1)
(V. parahaemolyticus, V. cholerae and V. vulnificus) in a determined quantity of product, when the test is
performed in accordance with this document
4 Principle
4.1 General
The detection of potentially enteropathogenic Vibrio spp. (V. parahaemolyticus, V. cholerae and
V. vulnificus) requires four successive phases, as shown in the procedure diagram in Annex A.
Recovery of certain Vibrio spp. from foodstuffs may be improved by the use of different incubation
temperatures depending upon the target species or state of the food matrix. For example, recovery of
V. parahaemolyticus and V. cholerae in fresh products is enhanced by enrichment at 41,5 °C whereas
[2]
for V. vulnificus, and for V. parahaemolyticus and V. cholerae in deep frozen (<−18 °C), dried or salted
products, recovery is enhanced by enrichment at 37 °C. If detection of V. parahaemolyticus, V. cholerae
and V. vulnificus is required, all specified incubation temperatures should be used. If detection of
V. parahaemolyticus, V. cholerae and V. vulnificus together is not required, the specific procedure(s) may
be selected according to the species being sought. Such a selection should be clearly specified in the
test report.
NOTE V. parahaemolyticus, V. cholerae and V. vulnificus may be present in small numbers and are often
accompanied by a much larger number of other microorganisms belonging to the Vibrionaceae family or to other
families.
4.2 Primary enrichment in a liquid selective medium
Inoculation of the test portion in the primary enrichment medium alkaline saline peptone water
(ASPW) (5.1) at ambient temperature, followed by incubation at 41,5 °C for 6 h and/or 37 °C for 6 h.
The incubation conditions are determined by the target species and food product state.
For detection of all target species in deep frozen, dried or salted products, primary enrichment should
be at 37 °C.
2 © ISO 2017 – All rights reserved

ISO 21872-1:2017(E)
For detection of V. vulnificus in all products, primary enrichment should be at 37 °C.
For detection of V. parahaemolyticus and/or V. cholerae only, in fresh products, primary enrichment
should be at 41,5 °C.
4.3 Secondary enrichment in a liquid selective medium
Inoculation of the second enrichment medium (ASPW) with the cultures obtained in 4.2.
Incubation of inoculated enrichment medium at 41,5 °C for 18 h and/or 37 °C for 18 h.
For detection of V. vulnificus in all products, secondary enrichment should be at 37 °C.
For detection of V. parahaemolyticus and/or V. cholerae only, in all products, secondary enrichment
should be at 41,5 °C.
4.4 Isolation and identification
From the cultures obtained in 4.2 and in 4.3, inoculation of two solid selective media:
— thiosulfate citrate bile and sucrose agar (TCBS) medium (5.2.1);
— another appropriate solid selective medium (left to the choice of the laboratory), such as chromogenic
agar, complementary to the TCBS medium (5.2.2).
Incubation of the TCBS medium at 37 °C, then examination after 24 h. Incubation of the second selective
medium according to the manufacturer’s recommendations.
4.5 Confirmation
Presumptive colonies of V. parahaemolyticus, V. cholerae and V. vulnificus isolated in 4.4 are subcultured
and confirmed by means of appropriate biochemical and/or polymerase chain reaction (PCR) tests.
Biochemical and/or PCR confirmation of isolates may be used for species identification, however,
reliable detection of enteropathogenic V. parahaemolyticus as determined by presence of the direct
thermostable haemolysin (tdh) and/or direct related haemolysin (trh) genes can only be carried out
using PCR tests.
It has been demonstrated that by screening ASPW broths using conventional PCR, the absence of
[3]
amplification of Vp-toxR can indicate no detection of V. parahaemolyticus. To reduce the amount
of downstream testing and, if shown to be reliable by the user laboratory, screening of incubated
enrichment broths may be used. This approach is only recommended after secondary enrichment and
does not apply to molecular targets for other Vibrio spp.
NOTE 1 Validation data generated in the preparation of this document demonstrated that PCR based
identification can be achieved by conventional PCR for V. parahaemolyticus, V. vulnificus and V. cholerae or real-
time PCR for V. parahaemolyticus and V. vulnificus. The PCR methods used in the development of this document
are given in Annexes C and D.
NOTE 2 Validation data for screening of secondary enrichment broths for amplification of Vp-toxR were not
generated in the preparation of this document.
5 Culture media and reagents
For general laboratory practice, refer to ISO 7218.
For clarity of the text, details of the composition of culture media and reagents and their preparation
are described in Annex B.
ISO 21872-1:2017(E)
For performance testing of culture media, refer to ISO 11133.
NOTE Primers, probes and PCR running conditions used in the development of this document are given in
Annexes C and D.
5.1 Enrichment medium: alkaline saline peptone water (ASPW)
As specified in B.1.
5.2 Solid selective isolation media
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS)
As specified in B.2. See Table 1 for performance testing data.
Table 1 — Performance testing of thiosulphate, citrate, bile and sucrose agar medium (TCBS)
a e
Function Incubation Control strains WDCM Method of Criteria Characteristic
control reactions
b
Productivity 37 °C ± 1 °C for Vibrio parahaemolyticus 00185 Qualitative Good Green colonies
24 h ± 3 h growth (2) (sucrose negative)
b
37 °C ± 1 °C for Vibrio furnissii 00186 Qualitative Good Yellow colonies
24 h ± 3 h growth (2) (sucrose positive)
c d
Selectivity 37 °C ± 1 °C for Escherichia coli 00012, Qualitative Total
24 h ± 3 h 00013 or inhibition —
00090 (0)
a
World Data Centre for Microorganisms (WDCM) strain catalogue available at http:// refs .wdcm .org
b
Strain to be used as a minimum (see ISO 11133).
c
Some national restrictions and directions may require the use of a different E. coli serovar. Make reference to national
requirements relating to the choice of E. coli serovars.
d
Strain free of choice; one of the strains shall be used as a minimum (see ISO 11133).
e
Growth is categorized as 0: no growth, 1: weak growth (partial inhibition), and 2: good growth (see ISO 11133).
5.2.2 Second medium
The selection of the second medium is left to the choice of the test laboratory. Preparation of the medium
should be strictly according to the manufacturer’s instructions.
5.3 Saline nutrient agar (SNA)
As specified in B.3.
5.4 Reagent for detection of oxidase
As specified in B.4.
5.5 Biochemical tests
5.5.1 L-lysine decarboxylase saline medium (LDC)
As specified in B.5.
5.5.2 Arginine dihydrolase saline medium (ADH)
As specified in B.6.
4 © ISO 2017 – All rights reserved

ISO 21872-1:2017(E)
5.5.3 Reagent for detection of β-galactosidase
As specified in B.7.
5.5.4 Saline medium for detection of indole
As specified in B.8.
5.5.5 Saline peptone waters
As specified in B.9.
5.5.6 Sodium chloride solution
As specified in B.10.
5.6 PCR
5.6.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the
purpose)
As specified in B.11.
5.6.2 Mastermix
Reagents shall be added in quantities as specified by the manufacturer’s instructions. See Annexes C
and D for example details of master mixes used in the development of this document.
5.6.3 Primers and probes
Primer (and hydrolysis probe) sequences if required shall be published in a peer-reviewed journal and
be verified for use against a broad range of target Vibrio spp. and non-target strains. With advances
in whole genome sequencing of bacterial strains, more appropriate species-specific markers may be
identified in the future.
For V. parahaemolyticus the target region should be toxR.
For determination of pathogenic strains of V. parahaemolyticus genes encoding the thermostable direct
(TDH) and the thermostable direct related (TRH) haemolysins should be targeted.
For V. cholerae the target region for conventional PCR should be the 16S-23S rRNA intergenic spacer
region prVC.
For V. vulnificus the target region should be the V. vulnificus haemolysin region (vvha).
Target regions other than those specified above for the identification of V. parahaemolyticus, V. vulnificus
and V. cholerae can be used if they have been shown to demonstrate equivalent performance to those
used in the development of this document and described in Annexes C or D, are published in a peer-
reviewed journal and are verified against a broad range of target Vibrio spp. and non-target strains.
See Annex C for example details of primers used for conventional PCR and Annex D for primers and
hydrolysis probes for real-time PCR used in the development of this document.
5.6.4 Positive control material
Separate control material shall be used for each target Vibrio spp. See Annexes C and D for example
details of control strains used in the development of this document.
ISO 21872-1:2017(E)
5.6.5 Negative extraction control
Nuclease free water or sterile NaCl 0,85 % extracted according to 9.5.6.
6 Equipment and consumables
Disposable equipment is acceptable in the same way as reusable glassware, if the specifications are
similar.
Ordinary microbiology laboratory equipment as specified in ISO 7218, and in particular the following.
6.1 Refrigerator, adjustable to 5 °C ± 3,0 °C.
6.2 Incubator, adjustable to 37 °C ± 1,0 °C.
6.3 Incubator, adjustable to 41,5 °C ± 1,0 °C.
6.4 Freezer, adjustable to <−15 °C.
6.5 Micro-centrifuge tubes, with a capacity of 1,5 ml and 2,0 ml.
6.6 Micro-centrifuge, for reaction tubes with a capacity of 1,5 ml and 2,0 ml and capable of running at
10 000g.
6.7 Heating block capable of operating at 95 °C ± 2,0 °C or equivalent.
6.8 Vortex.
6.9 Graduated pipettes and pipette filter tips, for volumes between 1 µl and 1 000 µl.
6.10 Associated consumables for conventional or real-time PCR, e.g. optical plates and caps, optical
plate holder, suitable for use with the selected PCR machine.
6.11 Conventional or real-time PCR machine, gel electrophoresis and UV visualization equipment as
appropriate.
7 Sampling
It is important that the laboratory receives a truly representative sample which has not been damaged
or modified during transport and storage.
Sampling does not form part of the method specified in this document. See the International Standard
specific to the relevant product. If a specific document does not exist, it is recommended that the
relevant parties reach agreement on this subject.
8 Preparation of the test sample
Prepare the test sample in accordance with ISO 6887-1 and ISO 6887-3 and/or the document concerning
the product to be examined. If a specific document does not exist, it is recommended that the relevant
parties reach agreement on this subject.
6 © ISO 2017 – All rights reserved

ISO 21872-1:2017(E)
9 Procedure (See Figure A.1)
9.1 Test portion and initial suspension
This document has been validated for test portions of up to 25 g or 25 ml. A smaller test portion may be
used without the need for additional validation/verification provided that the same ratio between (pre-)
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 adverse effects on the detection
of Vibrio spp.
NOTE Validation can be conducted in accordance with the appropriate document 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.
For the preparation of the initial suspensions, use the first enrichment medium (ASPW) specified in 5.1.
Take test portions (25 g or 25 ml) and homogenize in 225 ml of enrichment medium.
In the case of large quantities (greater than 25 g or 25 ml), the ASPW should be warmed to 37 °C ± 1 °C
and/or 41,5 °C ± 1 °C (4.2) before inoculation with the test portion.
In order to reduce the amount of examination work, where more than one 25 g or 25 ml test portion
stemming from a determined batch of food is to be examined, and where proof is available indicating
that a mixture (gathering together the test portions) does not modify the results concerning this
product in particular, the test portions may be mixed. For example, if 10 test portions of 25 g or 25 ml
are to be examined, it is possible to combine these 10 units in order to obtain a composite sample of
250 g or 250 ml and to add 2,25 l of enrichment medium.
Cell counts of Vibrio spp. potentially decline significantly on storage at refrigeration temperatures.
Storage of samples and, to a lesser extent, of suspensions at such temperatures should be avoided where
possible and should otherwise be kept to a minimum.
9.2 Primary selective enrichment
Incubate the initial suspensions (9.1) at 41,5 °C ± 1 °C and/or 37 °C ± 1 °C for 6 h ± 1 h according to
Table 2.
Table 2 — Primary incubation and target species/product state
Target Vibrio spp. in fresh product
Vibrio
a
Incubation temperature Vibrio cholerae Vibrio vulnificus
parahaemolyticus
41,5 °C ± 1 °C  
37 °C ± 1 °C 
Target Vibrio spp. in deep frozen, dried or salted product (such as bacalhau, stockfish, boknafisk, katsuo-
bushi, obambo)
Vibrio
a
Incubation temperature Vibrio cholerae Vibrio vulnificus
parahaemolyticus
41,5 °C ± 1 °C
37 °C ± 1 °C   
a
Vibrio spp. other than those listed in Reference [1], such as V. alginolyticus, may be recovered at these incubation
temperatures.
9.3 Secondary selective enrichment
Transfer 1 ml of the culture obtained in 9.2 taken from the surface into a tube containing 10 ml of ASPW
(5.1). It is recommended that the sample is not agitated before taking the aliquot.
ISO 21872-1:2017(E)
Incubate the ASPW at 41,5 °C ± 1 °C and/or 37 °C ± 1 °C for 18 h ± 1 h according to Table 3.
NOTE Cultures and/or boiled broths obtained in 9.3 can be screened using PCR
Table 3 — Secondary incubation and target species/product state
Target Vibrio spp. in all product states
a
Incubation temperature Vibrio parahaemolyticus Vibrio cholerae Vibrio vulnificus
 
41,5 °C ± 1 °C
37 °C ± 1 °C 
a
Vibrio spp. other than those listed in Reference [1], such as V. alginolyticus, may be recovered at these incubation
temperatures.
9.4 Isolation and identification
From the cultures obtained in the ASPW (9.2 and 9.3), inoculate with a 1 μl sampling loop the surface of
a TCBS agar plate (5.2.1), so as to permit the development of well-isolated colonies.
Proceed likewise with the chosen second selective isolation medium (5.2.2) using a fresh sampling loop.
Invert the agar plates:
— for TCBS agar plates, incubate at 37 °C ± 1 °C for 24 h ± 3 h;
— for the second isolation medium, incubate according to the manufacturer’s instructions.
After incubation, examine the TCBS and second selective medium for the presence of typical colonies of
presumptive pathogenic Vibrio spp. Mark their position on the bottom of the plates.
On TCBS agar V. parahaemolyticus, V. vulnificus, and V. cholerae exhibit different typical colony
morphologies:
— typical colonies of V. parahaemolyticus and V. vulnificus are smooth, green (negative sucrose) and of
2 mm to 3 mm in diameter;
— typical colonies of V. cholerae are smooth, yellow (positive sucrose) and of 1 mm to 2 mm in diameter.
For the second selective medium, examine for the presence of colonies, which, according to their
characteristics, may be considered as possible isolates of V. parahaemolyticus, V. vulnificus, and/or V.
cholerae.
NOTE 1 Vibrio spp. other than those listed in Reference [1], such as V. alginolyticus, can be recovered on TCBS
and the second selective medium.
NOTE 2 Recognition of colonies of Vibrio spp. is largely a question of experience and their appearance can
sometimes vary, not only from one species to another, but also from one batch of culture medium to another.
NOTE 3 For enhanced recovery of V. vulnificus, medium containing derivatives of cellobiose-polymyxin
[4]
B-colistin and cellobiose-colistin has been shown to be effective.
9.5 Confirmation
9.5.1 General
Using this document, V. parahaemolyticus, V. vulnificus, and V. cholerae can be confirmed by molecular
PCR and/or biochemical approaches. Confirmation may be carried out at the end user laboratory or by a
specialist reference laboratory. Other Vibrio spp. may be isolated using this document.
Not all V. parahaemolyticus possess pathogenicity traits. In order to confirm the pathogenic character
of the strains, it is preferable to detect the presence of thermostable direct haemolysin (tdh) or TDH-
8 © ISO 2017 – All rights reserved

ISO 21872-1:2017(E)
related haemolysin (trh) genes. This should be carried out using PCR tests. PCR based confirmation
also may reduce the subjective in
...