EN 17711:2024

SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17711:2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti Vibrio spp.
Plant biostimulants - Detection of Vibrio spp.
Pflanzen-Biostimulanzien - Nachweis von Vibrio spp.
Biostimulants des végétaux - Détection de Vibrio spp.
Ta slovenski standard je istoveten z: EN 17711:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17711
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17711:2022
English Version
Plant biostimulants - Detection of Vibrio spp.
Biostimulants des végétaux - Détection de Vibrio spp. Pflanzen-Biostimulanzien - Nachweis von Vibrio spp.
This European Standard was approved by CEN on 26 August 2024.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17711:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 5
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 8
4 Principle . 8
4.1 General . 8
4.2 Primary enrichment in a liquid selective medium . 8
4.3 Secondary enrichment in a liquid selective medium . 9
4.4 Isolation and identification . 9
4.5 Confirmation . 9
5 Culture media and reagents . 9
5.1 Enrichment medium: alkaline saline peptone water (ASPW) . 9
5.2 Solid selective isolation media . 9
5.2.1 First medium: thiosulphate citrate bile and sucrose agar (TCBS) medium . 9
5.2.2 Second medium .10
5.3 Saline nutrient agar (SNA) .10
5.4 Reagent for detection of oxidase .10
5.5 Reagent for biochemical tests.10
5.5.1 L-lysine decarboxylase saline medium (LDC) .10
5.5.2 Arginine dihydrolase saline medium (ADH) .10
5.5.3 Reagent for detection of β-galactosidase .10
5.5.4 Saline medium for detection of indole .10
5.5.5 Saline peptone water .10
5.5.6 Sodium chloride solution .10
5.5.7 Toluene, p.a. .11
5.5.8 Sterile mineral oil, p.a. .11
6 Equipment and consumables .11
7 Sampling .11
8 Preparation of the test sample .11
9 Procedure (see Figure A.1) .12
9.1 Test portion and initial suspension .12
9.2 Primary selective enrichment .12
9.3 Secondary selective enrichment .13
9.4 Isolation and identification .13
9.5 Confirmation .14
9.5.1 General .14
9.5.2 Selection of colonies for confirmation and preparation of pure cultures .14
9.5.3 Tests for presumptive identification .14
9.5.4 Biochemical confirmation .15
10 Expression of results .17
11 Performance characteristics of the method . 17
11.1 Sensitivity . 17
11.2 Specificity . 17
11.3 LOD50 . 17
11.4 Precision . 17
12 Test report . 17
Annex A (normative) Diagram of procedure . 19
Annex B (normative) Composition and preparation of culture media and reagents . 20
B.1 General . 20
B.2 Water . 20
B.3 Alkaline saline peptone water (ASPW) . 20
B.4 Thiosulfate citrate bile and sucrose agar (TCBS) . 21
B.5 Saline nutrient agar (SNA) . 21
B.6 Reagent for detection of oxidase . 22
B.7 L-lysine decarboxylase saline medium (LDC) . 22
B.8 Arginine dihydrolase saline medium (ADH) . 23
B.9 Detection of β-galactosidase . 23
B.10 Saline medium for detection of indole . 24
B.11 Saline peptone water . 25
B.12 Sodium chloride solution . 25
B.13 Tris acetate EDTA (TAE) buffer . 25
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 . 26
C.1 General . 26
C.2 Equipment . 26
C.3 DNA extraction . 27
C.4 Procedure conventional PCR . 27
C.5 Primers and probes . 28
C.6 Control material — conventional PCR . 30
Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin gene (tdh) and Vibrio vulnificus . 32
D.1 General . 32
D.2 Equipment . 32
D.3 DNA extraction . 32
D.4 Real-time PCR. 33
D.5 Vibrio parahaemolyticus — primers and hydrolysis probes . 33
D.6 Thermostable direct haemolysin (tdh) gene Vibrio parahaemolyticus — primers and
hydrolysis probes . 33
D.7 Vibrio vulnificus — primers and hydrolysis probes .34
D.8 Cycling parameters .34
D.9 Control material — real-time PCR .34
Annex E (informative) Repeatability and reproducibility data .35
E.1 General .35
E.2 Samples .35
E.3 Procedure.36
E.4 Replicate determination .36
E.5 Results .36
Annex ZA (informative) Relationship of this European Standard and the essential
requirements of Regulation (EU) 2019/1009 making available on the market of EU
fertilising products aimed to be covered.38
Bibliography .39
European foreword
This document (EN 17711:2024) has been prepared by Technical Committee CEN/TC 455 “Plant
biostimulants”, the secretariat of which is held by AFNOR.
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 May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
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 supersedes CEN/TS 17711:2022.
— the European foreword and Introduction have been updated;
— normative references have been updated;
— Table 1 has been updated;
— in 5.5, reagents list for biochemical tests has been revised;
— Clause 7 and Clause 8 have been updated;
— Table 2 has been revised;
— 9.5.4.1 and 9.5.4.6 have been revised;
— Table 4 has been revised;
— Clause 10 has been revised;
— Annex ZA has been added;
— the Bibliography has been updated.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
For the relationship with EU Legislation, see informative Annex ZA, which is an integral part of this
document.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
Introduction
The European Committee for Standardization (CEN) was requested by the European Commission (EC) to
draft European Standards or European Standardization deliverables to support the implementation of
Regulation (EU) 2019/1009 of 5 June 2019 [1] laying down rules on the making available on the market
of EU fertilising products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as SR M/564 and relevant amendments, also contributes to the
Communication on “Innovating for Sustainable Growth: A Bio economy for Europe”. The interest in plant
biostimulants has increased significantly in Europe as a valuable tool to use in agriculture.
Standardization was identified as having an important role in order to promote the use of biostimulants.
The work of CEN/TC 455 seeks to improve the reliability of the supply chain, thereby improving the
confidence of farmers, industry, and consumers in biostimulants, and will promote and support
commercialisation of the European biostimulant industry.
WARNING — Persons using this document should be familiar with normal laboratory practice. This
document does not purport to address all of the safety problems, 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.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably trained staff.
1 Scope
This document specifies a horizontal method for the detection of enteropathogenic Vibrio species (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 microbial plant biostimulants.
NOTE 1 The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V.
vulnificus are the major contaminants of Vibrio spp. [2].
NOTE 2 For confirmation, it is possible to use PCR (Polymerase Chain Reaction) tests; in this case a validation is
carried out by the laboratory for the procedure and data generated.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of
the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers,
Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest
% in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not
the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend
of fertilizing products is composed of components in equal quantity or in case the component EU
fertilising products used for the blend have identical formulations , the user decides which standard to
apply.
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.
EN 17702-1:2024, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
EN 17724:2024, Plant biostimulants — Terminology
EN ISO 7218:2024, Microbiology of the food chain — General requirements and guidance for
microbiological examinations (ISO 7218:2024)
EN ISO 11133:2014 , Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media (ISO 11133:2014)
EN ISO 3696:1995, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987)

An example of such a blend is a product with 2 claimed functions consisting of a non-microbial plant biostimulant
and an organic fertilizer composed of 1kg/kg of plant biostimulant from seaweed.
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 17724:2024 and the following
apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://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. is improved by the use of different incubation temperatures depending upon the
target species or state of the matrix. In liquid products, recovery of V. parahaemolyticus and V. cholerae is
enhanced by enrichment at 41,5 °C and recovery of V. vulnificus is enhanced by enrichment at 37 °C.
Whereas in solid products, for V. vulnificus, V. parahaemolyticus and V. cholerae recovery is enhanced by
enrichment at 37 °C.
If detection of V. parahaemolyticus, V. cholerae and V. vulnificus is required, all specified incubation
temperatures shall be used. If detection of V. parahaemolyticus, V. cholerae and V. vulnificus together is
not required, the specific procedure(s) is selected according to the species being sought. Such a selection
shall be clearly specified in the test report.
V. parahaemolyticus, V. cholerae and V. vulnificus can 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 shall be 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 product state.
For detection of all target species in solid products, primary enrichment shall be at 37 °C.
For detection of V. vulnificus in all products, primary enrichment shall be at 37 °C.
For detection of V. parahaemolyticus and/or V. cholerae only, in liquid products, primary enrichment shall
be at 41,5 °C.
4.3 Secondary enrichment in a liquid selective medium
The second enrichment medium (ASPW) shall be inoculated with the cultures obtained in 4.2. Incubation
of inoculated enrichment medium shall be at 41,5 °C for 18 h and/or 37 °C for 18 h.
For detection of V. vulnificus in all products, secondary enrichment shall be at 37 °C.
For detection of V. parahaemolyticus and/or V. cholerae only, in all products, secondary enrichment shall
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 shall be done:
— 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 shall be at 37 °C, then examination after 24 h. Incubation of the second
selective medium shall follow the manufacturer’s recommendations.
4.5 Confirmation
Presumptive colonies of V. parahaemolyticus, V. cholerae and V. vulnificus isolated in 4.4 shall be
subcultured and confirmed by means of an appropriate biochemical test. The PCR test is also possible to
use for confirmation; the PCR methods are suggested in Annexes C and D, but the laboratory must validate
the procedure and the data generated.
5 Culture media and reagents
For general laboratory practice, EN ISO 7218:2024 shall be used as reference.
For clarity of the text, details of the composition of culture media and reagents and their preparation are
described in Annex B.
For performance testing of culture media, EN ISO 11133:2014 shall be used as reference.
5.1 Enrichment medium: alkaline saline peptone water (ASPW)
As specified in B.3.
5.2 Solid selective isolation media
5.2.1 First medium: thiosulphate citrate bile and sucrose agar (TCBS) medium
As specified in B.4. According to Table 1 for performance testing data.
Table 1 — Performance testing of thiosulphate citrate bile and sucrose agar (TCBS) medium
a b
Function Incubation Control strains WDCM Method of Criteria Characteristic
control reactions
c
Productivity 37 °C ± 1 °C Vibrio 00185 Qualitative Good Green colonies
for 24 h ± 3 h parahaemolyticus growth (2) (sucrose
negative)
c
37 °C ± 1 °C Vibrio furnissii 00186 Qualitative Good Yellow colonies
for 24 h ± 3 h growth (2) (sucrose positive)
d e
Selectivity 37 °C ± 1 °C Escherichia coli 00012, Qualitative Total —
for 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
Growth is categorized as 0: no growth, 1: weak growth (partial inhibition), and 2: good growth (see EN ISO 11133:2014).
c
Strain to be used as a minimum (see EN ISO 11133:2014).
d
Strain free of choice; one of the strains shall be used as a minimum (see EN ISO 11133:2014).
e
Some national restrictions and directions can require the use of a different E. coli serovar. National requirements relating
to the choice of E. coli serovars shall be followed.
5.2.2 Second medium
The selection of the second medium is left to the choice of the test laboratory. The medium shall be
prepared strictly according to the manufacturer’s instructions.
5.3 Saline nutrient agar (SNA)
As specified in B.5.
5.4 Reagent for detection of oxidase
As specified in B.6.
5.5 Reagent for biochemical tests
5.5.1 L-lysine decarboxylase saline medium (LDC)
As specified in B.7.
5.5.2 Arginine dihydrolase saline medium (ADH)
As specified in B.8.
5.5.3 Reagent for detection of β-galactosidase
As specified in B.9.
5.5.4 Saline medium for detection of indole
As specified in B.10.
5.5.5 Saline peptone water
As specified in B.11.
5.5.6 Sodium chloride solution
As specified in B.12.
5.5.7 Toluene, p.a.
5.5.8 Sterile mineral oil, p.a.
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 EN ISO 7218:2024, 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
10 000 g.
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 Magnetic stirrer with a magnetic stirring bar.
® 3
6.11 Stomacher apparatus.
7 Sampling
The laboratory shall receive a truly representative sample which has not been damaged or modified
during transport and storage; for the sampling EN 17702-1:2024 shall be applied.
Sampling does not form part of the method specified in this document.
8 Preparation of the test sample
To prepare the test sample, EN 17702-1:2024 shall be applied and/or the document concerning the
product to be examined.
3 ®
Stomacher 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 CEN of this product.
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 but the same ratio between (pre-)
enrichment broth and test portion shall be 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 the EN ISO 16140 series
[13]. Verification for pooling samples can be conducted in accordance with the protocol described in
EN ISO 6887-1:2017 [14], Annex C.
For the preparation of the initial suspensions, the first enrichment medium (ASPW) shall be used, as
specified in 5.1.
The initial suspension shall be prepared with a representative test portion in accordance with the
following procedure taking into consideration the different formulations of biostimulant-based products.
a) Liquid – water-based and oil-based formulations:
test portions (25 g or 25 ml) shall be taken and homogenized in 225 ml of enrichment medium at
room temperature in a flask with a magnetic stirrer (6.10) at half speed for 10 min or more, until the
distribution is optimal.
b) Solid – wettable powder or water-dispersible granules formulations:
test portions (25 g or 25 ml) shall be taken and homogenized in 225 ml of enrichment medium at ®
room temperature in a sterile bag using a Stomacher apparatus (6.11) for 2 min at highest speed.
c) Solid – pellets, granules, microgranules (slow release) formulations:
test portions (25 g or 25 ml) shall be taken and homogenized in 225 ml of enrichment medium at ®
apparatus (6.11) for 2 min at highest speed,
room temperature in a sterile bag with a Stomacher
until the dispersion is optimal. If required, repeat this operation twice.
In the case of large quantities (greater than 25 g or 25 ml), the ASPW shall 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 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 shall be avoided where
possible and shall otherwise be kept to a minimum.
9.2 Primary selective enrichment
The initial suspensions (9.1) shall be incubated 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 liquid product formulations
Vibrio
Incubation temperature Vibrio cholerae Vibrio vulnificus
parahaemolyticus
-
41,5 °C ± 1 °C
✓ ✓
37 °C ± 1 °C - -
✓
Target Vibrio spp. in solid dried product formulations
Vibrio
Incubation temperature Vibrio cholerae Vibrio vulnificus
parahaemolyticus
41,5 °C ± 1 °C - - -
37 °C ± 1 °C ✓ ✓ ✓
9.3 Secondary selective enrichment
1 ml of the culture obtained in 9.2 taken from the surface shall be transferred into a tube containing 10 ml
of ASPW (5.1). The sample shall not be agitated before taking the aliquot.
The ASPW shall be incubated 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 also be screened using PCR.
Table 3 — Secondary incubation and target species/product state
Target Vibrio spp. in all product states
Incubation Vibrio Vibrio Vibrio
temperature parahaemolyticus cholerae vulnificus
-
41,5 °C ± 1 °C ✓ ✓
- -
37 °C ± 1 °C
✓
9.4 Isolation and identification
From the cultures obtained in the ASPW (9.2 and 9.3), the surface of a TCBS agar plate (5.2.1) shall be
inoculated with a 1 μl sampling loop, 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, the incubation temperature shall be at 37 °C ± 1 °C for 24 h ± 3 h;
— for the second isolation medium, the incubation temperature shall be according to the manufacturer’s
instructions.
After incubation, the TCBS and second selective medium shall be examined for the presence of typical
colonies of presumptive pathogenic Vibrio spp. Their positions shall be marked 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, the presence of colonies, which according to their characteristics may
be considered as possible isolates of V. parahaemolyticus, V. vulnificus, and/or V. cholerae, shall be
examined.
NOTE 1 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 2 For enhanced recovery of V. vulnificus, medium containing derivatives of cellobiose-polymyxin B-colistin
and cellobiose-colistin has been shown to be effective [4].
9.5 Confirmation
9.5.1 General
Using this document, V. parahaemolyticus, V. vulnificus, and V. cholerae can be confirmed by 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. For confirmation using PCR tests, the
laboratory must validate the procedure and data generated; see Annexes C and D.
If shown to be reliable, commercially available biochemical test kits may be used to identify Vibrio at the
species level, provided they are inoculated with a suspension of the bacteria to be identified in a
sufficiently saline medium or dilution fluid, and as long as the database or identification table for the
product has been based on reactions obtained using similar media to those described in this document.
These kits shall be used in accordance with the manufacturer’s instructions.
If shown to be reliable, commercially available molecular detection kits may be used to identify Vibrio at
the species level. These kits shall be used in accordance with the manufacturer’s instructions.
9.5.2 Selection of colonies for confirmation and preparation of pure cultures
For confirmation, at least one well-isolated colony from each selective medium (9.4), that is considered
to be typical or similar to each of the potentially pathogenic Vibrio spp. sought shall be subcultured. If the
result of the first isolated colony tested is negative, a further four well-isolated colonies shall be tested.
In samples where it is important to optimize the detection of potentially pathogenic V. parahaemolyticus,
at least five and, where possible, all colonies exhibiting typical V. parahaemolyticus colony morphology,
shall be subcultured for downstream testing.
The colonies selected shall be inoculated onto the surface of plates of saline nutrient agar (SNA) (5.3) or
suitable medium of the laboratory’s choice to obtain isolated colonies. It shall be incubated at 37 °C ± 1 °C
for 24 h ± 3 h.
9.5.3 Tests for presumptive identification
9.5.3.1 Oxidase test
Using a sampling loop, platinum iridium straight wire or glass rod, a portion of the pure culture shall be
taken from the saline nutrient agar (9.5.2) and streaked onto the filter paper moistened with oxidase
reagent (5.4); if a commercially available test is used, the manufacturer’s instructions shall be followed.
WARNING — Neither a nickel-chromium sampling loop nor a metallic wire shall be used. Nickel or
chrome wire sampling loops can give false-positive results and should be avoided.
9.5.3.2 Microscopic examination (optional)
For each pure culture obtained in 9.4, test according to a) and b).
a) A film for Gram staining shall be prepared; EN ISO 7218:2024 shall be used as reference. After
staining, using a microscope the morphology and the Gram reaction shall be examined and their
results shall be recorded.
b) A tube of ASPW (5.1) shall be inoculated and incubated at 37 °C ± 1 °C for 1 h to 6 h. A drop of the
culture shall be deposited onto a clean slide, covered with a cover slip and examined for the motility
under the microscope. The cultures showing a positive result for motility shall be noted.
9.5.3.3 Selection of the cultures
For confirmation, the oxidase-positive colonies shall be retained.
For confirmation, also Gram-negative colonies which give a positive result in the motility test (if
examined) shall be retained.
9.5.4 Biochemical confirmation
9.5.4.1 General
Using an inoculation loop, the media indicated in 9.5.4.2 to 9.5.4.5 shall be inoculated with each of the
cultures obtained from the colonies retained in 9.5.2.
The tests mentioned in 9.5.4.2 to 9.5.4.5 can also be carried out in miniaturised strips by the laboratory.
9.5.4.2 L-lysine decarboxylase saline medium (5.5.1)
The liquid medium shall be inoculated just below the surface. About 1 ml of sterile mineral oil shall be
added on the top of the medium.
It shall be incubated at 37 °C ± 1 °C for 24 h ± 3 h.
Turbidity and a violet colour after incubation indicate a positive reaction (bacterial growth and
decarboxylation of the L-lysine). A yellow colour indicates a negative reaction.
9.5.4.3 Arginine dihydrolase saline medium (5.5.2)
The liquid medium shall be inoculated just below the surface. About 1 ml of sterile mineral oil shall be
added on the top of the medium.
It shall be incubated at 37 °C ± 1 °C for 24 h ± 3 h.
Turbidity and a violet colour after incubation indicate a positive reaction (bacterial growth and
dihydrolation of arginine). A yellow colour indicates a negative reaction.
9.5.4.4 Detection of β-galactosidase (5.5.3)
The presumptive colony shall be inoculated into a tube containing 0,25 ml of sodium chloride solution
(5.5.6).
One drop of toluene shall be added and the tube shaked.
The tube shall be placed in the incubator (6.2) set at 37 °C ± 1 °C and left to stand for approximately 5 min.
For the detection of β-galactosidase, 0,25 ml of the reagent shall be added and mixed.
The tube shall be replaced in the water bath set at 37 °C ± 1 °C, left to stand for 24 h ± 3 h, examining it
during this time.
A yellow colour indicates a positive reaction (presence of β-galactosidase). The reaction is often visible
after 20 min. Absence of colouring after 24 h indicates a negative reaction.
If ready-to-use paper disks (5.5.3) are used, the manufacturer’s instructions shall be followed.
9.5.4.5 Detection of indole (5.5.4)
A tube containing 5 ml of the tryptophan saline medium shall be inoculated with the presumptive colony.
It shall be incubated at 37 °C ± 1 °C for 24 h ± 3 h. After incubation, 1 ml of Kovacs reagent (5.5.4) shall be
added.
The formation of a red ring indicates a positive reaction (formation of indole). A yellow-brown ring
indicates a negative reaction.
9.5.4.6 Halotolerance test (5.5.5)
If identifying the species is needed, a halotolerance test shall be performed.
A series of tubes of saline peptone water with increasing salt (NaCl) concentration: 0 %, 6 % and 10 %
(% w/v) shall be produced, inoculated with the presumptive colony and incubated at 37 °C ± 1 °C for
24 h ± 3 h.
Observation of turbidity indicates that the bacterium can grow at the concentration of sodium chloride
present in the tube of saline peptone water.
9.5.4.7 Interpretation of biochemical tests
V. parahaemolyticus, V. cholerae and V. vulnificus generally give the reactions indicated in Table 4.
Table 4 — Interpretation of biochemical tests
a
Vibrio Vibrio parahaemolyticus Vibrio
Test
a a
cholerae vulnificus
Oxidase + + +
LDC + + +
ADH – – –
β-galactosidase + – +
Production of
+ + +
indole
Growth in peptone water with (% w/v):
0 % NaCl + – –
6 % NaCl – + +
10 % NaCl – – –
a
The sign + denotes 76 % to 89 % positive reactions.
NOTE 1 The reactions given in Table 4 are a guide to the identification of the listed species. Additional phenotypic
tests can be required to fully distinguish these species from each other and from non-pathogenic Vibrio spp.
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