EN 17717:2024

SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17717:2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti salmonele (Salmonella spp.)
Plant biostimulants - Detection of Salmonella spp.
Pflanzen-Biostimulanzien - Nachweis von Salmonella spp.
Biostimulants des végétaux - Recherche des Salmonella spp.
Ta slovenski standard je istoveten z: EN 17717:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17717
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17717:2022
English Version
Plant biostimulants - Detection of Salmonella spp.
Biostimulants des végétaux - Recherche des Pflanzen-Biostimulanzien - Nachweis von
Salmonella spp. Salmonella 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

E U R OP ÄI S C HES 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 17717:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
4.1 General . 7
4.2 Enrichment in selective liquid medium . 7
4.3 Plating out on selective solid media . 7
4.4 Confirmation . 7
5 Culture media, reagents, antisera . 8
5.1 General . 8
5.2 Isolation chromogenic agar . 8
5.3 Non-selective agar . 8
5.4 Confirmation selective agar . 8
6 Equipment and consumables . 8
7 Sampling . 9
8 Preparation of test sample . 9
9 Procedure . 9
9.1 Test portion and initial suspension . 9
9.2 Selective enrichment . 10
9.3 Isolation . 10
9.4 Confirmation . 10
10 Expression of results . 12
11 Performance characteristics of the method . 12
11.1 Interlaboratory studies. 12
11.2 Sensitivity . 12
11.3 Specificity . 13
11.4 Positive predictive value (PPV) . 13
11.5 Negative predictive value (NPV) . 13
12 Test report . 13
Annex A (normative) Diagram of the procedures . 14
Annex B (normative) Culture media and reagents . 15
Annex C (informative) Examples of selective plating-out media . 19
Annex D (informative) Method validation study and performance characteristics . 21
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 . 24
Bibliography . 25

European foreword
This document (EN 17717:2024) 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 17717:2022.
CEN/TS 17717:2022:
— the European foreword and introduction have been updated;
— in Table 1, errors in the interpretation of the serological reactions have been corrected;
— in Annex B, the layout of Table B.1 has been improved;
— in Annex C, the footnotes have been revised;
— Annex D (method validation study) has been added;
— Annex ZA has been added.
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 method for the detection of Salmonella spp. in biostimulants.
This document is applicable to all microbial biostimulants in agriculture.
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 17708:2024, Plant biostimulants — Preparation of sample for microbial analysis
EN 17724:2024, Plant Biostimulants — Terminology
EN ISO 7218:2024, Microbiology of food and animal feeding stuffs — General requirements and
guidance for microbiological examinations (ISO 7218:2024)
EN 17702-1:2024, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
EN ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production,
storage and performance testing of culture media (ISO 11133:2014)
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 terminology databases for use in standardization at the following addresses:
• ISO Online browsing platform: available at https://www.iso.org/obp
• IEC Electropedia: available at https://www.electropedia.org/
3.1
Salmonella spp.
microorganisms which form typical colonies on solid selective media and which display the
morphological, physiological and biochemical characteristics described when the analysis is
carried out in accordance with this document

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 1 kg/kg of plant biostimulant from seaweed
As amended by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020
3.2
detection of Salmonella spp.
determination of the detection or not detection of Salmonella spp. (3.1), in 25 g or 25 ml of
product, when tests are carried out in accordance with this document
3.3
laboratory sample
sample intended for laboratory inspection or testing
3.4
test sample
sample prepared from the laboratory sample and from which test portions will be taken
3.5
test portion
quantity of material taken from the test sample (or if both are the same, from the laboratory
sample) and on which the test or observation is carried out
4 Principle
4.1 General
The detection of Salmonella spp. requires three successive steps as specified in Annex A. The three
steps are the selective enrichment, the isolation on a chromogenic agar, and the confirmation with
a serological test (and if required, a selective media).
NOTE Salmonella spp. can be present in small numbers and are often accompanied by considerably
larger numbers of other bacteria, such as Enterobacteriaceae or of other families. Enrichment is used to
allow the detection of low numbers of Salmonella spp. or stressed Salmonella spp.
Stressed microorganisms are defined here as those present in the environment that can be injured
or that can have developed in harsh environments. Such organisms can be difficult to detect
because they struggle to grow on selective media. However, under suitable conditions, they can
repair the cellular damages and recover their normal properties.
4.2 Enrichment in selective liquid medium
Buffered Peptone Water (BPW) containing 10 mg/l Novobiocin at room temperature is inoculated
with the test portion, then incubated from 34 °C to 38 °C for 18 h ± 2 h.
For large quantities (e.g. 1 l or more), pre-warm the BPW to between 34 °C and 38 °C before
mixing it with the test portion.
4.3 Plating out on selective solid media
From the enrichment obtained in 4.2, the chromogenic solid media (5.2) is inoculated.
This selective agar is incubated from 34 °C to 38 °C for 24 h ± 3 h (or according to the
manufacturer’s instructions if explicitly recommended).
4.4 Confirmation
Colonies of presumptive Salmonella spp. are confirmed by means of appropriate serological
testing. If the serological test gives a negative result, the inoculation of a selective agar (B.5) is
required.
If the test gives a negative result, up to 4 other presumptive colonies will be tested (if possible and
up to 5 colonies in total).
5 Culture media, reagents, antisera
5.1 General
For current laboratory practice, EN ISO 7218:2024 and EN ISO 11133:2014 shall be used.
Composition of culture media and reagents and their preparation are described in Annex B.
5.2 Isolation chromogenic agar
This isolation medium is chosen by the testing laboratory and shall highlight the C8-esterase
enzymatic activity. For examples of isolation media, see Annex C, Table C.1.
5.3 Non-selective agar
General purpose agar supporting the growth of a wide range of non-fastidious strains. See B.4.
5.4 Confirmation selective agar
This isolation medium is chosen by the testing laboratory and shall highlight the production of
hydrogen sulphide (H S) by the strains (see B.5). For examples of isolation media, see Annex C,
Table C.2.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable
specifications.
Usual microbiological laboratory equipment as described in EN ISO 7218:2024 shall be used and,
in particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave)
6.2 Drying cabinet or oven, capable of operating between 25 °C and 50 °C.
6.3 Incubator(s), capable of operating from 34 °C to 38 °C and at 37 °C ± 1 °C.
6.4 Water bath, capable of operating from 47 °C to 50 °C.
6.5 Water bath, capable of operating at 37 °C ± 1 °C.
It is recommended to use a water bath (6.4 and 6.5) containing an antibacterial agent because of
the low infective dose of Salmonella spp.
6.6 Cooling unit, adjustable at 5 °C ± 3 °C.
6.7 Freezer, capable of operating at −20 °C ± 5 °C.
6.8 Sterile loops with a diameter of approximately 3 mm (10 μl volume).
6.9 pH-meter having an accuracy of calibration of ± 0,1 pH unit from 20 °C to 25 °C.
6.10 Sterile tubes, bottles, or flasks with caps, of appropriate capacity.
6.11 Sterile graduated pipettes or automatic pipettes of nominal capacities of 25 ml, 10 ml,
1 ml, and 0,1 ml.
6.12 Sterile Petri dishes with a diameter of approximately 90 mm and (optional) large size
(diameter approximately 140 mm).
® 3
6.13 Peristaltic blender (Stomacher ) with sterile bags.
6.14 Sterile filter with a 0,2 µm porosity.
7 Sampling
The laboratory shall receive a truly representative laboratory sample (3.3) which has not been
damaged or modified during transport or storage; for the sampling EN 17702-1:2024 shall be
applied.
Sampling is not part of the method specified in this document.
8 Preparation of test sample
To prepare the test sample (3.4), EN 17702-1:2024 shall be applied and/or the document
concerning the product to be examined.
For the microbiological examination, follow a specific standard appropriate to the product
concerned if no specific method is provided by the manufacturer. If necessary, use one or more of
the apparatus on the basis of the nature of the product.
All the operations, before and after opening the products, shall be carried out aseptically to avoid
external contamination.
Sterile material and equipment shall be used.
Frozen products may be defrosted before testing, standing at 18 °C to 27 °C (laboratory ambient
temperature) for a maximum of 3 h, or at 5 °C ± 3 °C for a maximum of 24 h. After this, samples
shall be tested as quickly as possible.
Solid (powdered and granulated) products shall be thoroughly mixed in their container and
weighed out using aseptic techniques, taking the required test portion at random in small
increments with a spatula.
For dehydrated and other low-moisture products, it is important to weigh the diluent and then
add it gradually onto the test portion to reduce osmotic shock on any microorganism present.
For liquid products, before taking the test portion, the laboratory sample should be shaken by
hand in order to ensure that the microorganisms are uniformly distributed.
9 Procedure
9.1 Test portion and initial suspension
For the preparation of the initial suspension, use as diluent the enrichment medium specified in
B.2 (buffered peptone water). Pre-warm the BPW supplemented with Novobiocin (nBPW, see B.3)
to room temperature before use.
An amount of test portion (3.5) of 25 g or 25 ml shall be weighed and 225 ml of nBPW shall be
added to yield a tenfold dilution, according to EN 17708:2024
Relating solid formulations: soon after, take the entire suspension and process it in a stomacher
(6.13) for 2 min at highest speed. Proceed then with the incubation.
Relating liquid formulations: soon after, take the entire suspension and proceed then with the
incubation.
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.
9.2 Selective enrichment
Incubate the initial suspension (9.1) from 34 °C to 38 °C (6.3) for 18 h ± 2 h.
It is permissible to store the enriched sample after incubation at 5 °C (6.6) for a maximum of 72 h
(see references [2], [3], [4], [5]).
9.3 Isolation
Allow the plates of chromogenic isolation agar (5.2) to equilibrate at room temperature if they
were stored at a lower temperature. If necessary, the surface of the plates shall be dried before
use according to EN ISO 11133:2014 .
From the selective enrichment (9.2), inoculate the surface of the chromogenic isolation agar (5.2)
by means of a 10 µl loop (6.8) so that well-isolated colonies will be obtained.
Incubate the selective plating-out medium from 34 °C to 38 °C (6.3) for 24 h ± 3 h (or according to
the manufacturer’s instructions if explicitly recommended).
Typical colonies exhibiting the C8-esterase activity are smooth (or rarely rough), round and
magenta.
After incubation, check the chromogenic medium for the presence of colonies which, from their
characteristics, are considered to be suspect colonies.
9.4 Confirmation
9.4.1 General
The combination of enzymatic activity and serological test results indicate whether an isolate
belongs to the genus Salmonella or not. For the characterization of Salmonella strains, full
serotyping is needed.
For a clear distinction between positive and negative serological reactions, it is helpful to verify
the reactions of the media of each biochemical test with well-characterized positive and negative
control strains.
9.4.2 Selection of colonies for confirmation
Mark suspect colonies on each plate (9.3). Select one suspect colony for
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