prEN 17710
(Main)Plant biostimulants - Detection of Listeria monocytogenes
Plant biostimulants - Detection of Listeria monocytogenes
This document provides a method for the detection of Listeria monocytogenes in microbial plant biostimulants for verifying that the content of this human pathogen agrees with the respective limits outlined in the EU Regulation on Fertilising Products [1].
Pflanzen-Biostimulanzien - Nachweis von Listeria monocytogenes
Dieses Dokument enthält ein Verfahren zum Nachweis von Listeria monocytogenes in mikrobiellen Pflanzen-Biostimulanzien, um zu verifizieren, dass der Gehalt dieses Humanpathogens die entsprechenden Grenzwerte der EU Düngeprodukteverordnung[1] einhält.
Biostimulants des végétaux - Détection de Listeria monocytogenes
Le présent document fournit une méthode pour la détection de Listeria monocytogenes dans les biostimulants microbiens des végétaux pour vérifier que la teneur de ce pathogène pour l’homme est conforme aux limites correspondantes énoncées dans le Règlement UE sur les fertilisants [1].
Rastlinski biostimulanti - Ugotavljanje prisotnosti Listeria monocytogenes
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17710:2023
01-maj-2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti Listeria monocytogenes
Plant biostimulants - Detection of Listeria monocytogenes
Pflanzen-Biostimulanzien - Nachweis von Listeria monocytogenes
Biostimulants des végétaux - Détection de Listeria monocytogenes
Ta slovenski standard je istoveten z: prEN 17710
ICS:
65.080 Gnojila Fertilizers
oSIST prEN 17710:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17710:2023
DRAFT
EUROPEAN STANDARD
prEN 17710
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2023
ICS 65.080 Will supersede CEN/TS 17710:2022
English Version
Plant biostimulants - Detection of Listeria monocytogenes
Biostimulants des végétaux - Détection de Listeria Pflanzen-Biostimulanzien - Nachweis von Listeria
monocytogenes monocytogenes
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 455.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
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
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17710:2023 E
worldwide for CEN national Members.
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Contents Page
European foreword . 5
Introduction . 6
1 Scope . 8
2 Normative references . 8
3 Terms and definitions . 8
4 Principle . 9
4.1 General . 9
4.2 Pre-enrichment in selective liquid medium . 9
4.3 Enrichment in/on selective media . 9
4.4 Plating out on selective solid media . 9
4.5 Confirmation . 9
5 Culture media, reagents, antisera . 9
6 Equipment and consumables . 10
7 Sampling . 10
8 Preparation of test sample . 10
9 Preparation of test sample . 10
9.1 Test portion and initial suspension . 10
9.1.1 General . 10
9.1.2 Liquid - water based- formulations . 11
9.1.3 Liquid - oil based (emulsifiable concentrate - EC) formulations . 11
9.1.4 Solid - Wettable Powder (WP) formulations . 11
9.1.5 Solid - Water dispersible granules (WDG) formulations . 11
9.1.6 Solid – Pellets, granules, microgranules (slow release) formulations . 11
9.1.7 Solid substrates . 11
9.2 Non-selective pre-enrichment . 12
9.3 Selective enrichment. 12
9.4 Plating out . 12
9.4.1 General . 12
9.4.2 Agar Listeria according to Ottaviani and Agosti (B.3) . 12
9.4.3 Second selective medium . 13
9.5 Confirmation of L. monocytogenes . 13
9.5.1 General . 13
9.5.2 Selection of colonies for confirmation . 13
9.5.3 Confirmation tests for L. monocytogenes . 13
9.6 Interpretation of morphological and physiological properties and of the biochemical
reactions . 16
9.7 Additional characterization of isolated strains (optional) . 16
10 Expression of results . 16
11 Performance characteristics of the method . 16
11.1 Interlaboratory studies . 16
11.2 Sensitivity . 16
11.3 Specificity . 16
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11.4 Predictive positive value (PPV) . 16
11.5 Predictive negative value (NPV) . 17
12 Test report . 17
13 Quality assurance . 17
Annex A (normative) Diagram of the procedures . 18
Annex B (normative) Composition and preparation of culture media and reagents . 19
B.1 Selective primary enrichment medium: half-Fraser broth . 19
B.1.1 Base . 19
B.1.2 Lithium chloride solution . 19
B.1.3 Solution of sodium salt of nalidixic acid . 20
B.1.4 Acriflavine hydrochloride solution . 20
B.1.5 Ammonium iron(III) citrate solution . 20
B.1.6 Complete medium . 21
B.2 Selective secondary enrichment medium: Fraser broth . 21
B.2.1 Base . 21
B.2.2 Acriflavine hydrochloride solution . 21
B.2.3 Ammonium iron(III) citrate solution . 22
B.2.4 Complete medium . 22
B.3 Agar Listeria according to Ottaviani and Agosti (ALOA) . 22
B.3.1 Base medium . 22
B.3.2 Nalidixic acid solution . 22
B.3.3 Ceftazidime solution . 23
B.3.4 Polymyxin B solution . 23
B.3.5 Antibiotic supplement . 23
B.3.6 Supplement . 24
B.3.7 Complete medium . 24
B.4 Second selective solid plating-out medium . 25
B.5 Performance testing for the quality assurance of the culture media . 25
B.6 Hydrogen peroxide solution . 25
B.7 Motility agar . 25
B.7.1 Composition . 25
B.7.2 Preparation . 25
B.8 Blood agar . 25
B.8.1 Base . 25
B.8.2 Defibrinated blood (sheep, calf or bovine) . 26
B.8.3 Complete medium . 26
B.9 Phosphate-buffered saline (PBS). 26
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B.9.1 Composition . 26
B.9.2 Preparation . 26
B.10 Red blood corpuscle suspension . 26
B.11 CAMP (Christie, Atkins, Munch-Petersen) medium and test strains . 27
B.11.1 General . 27
B.11.2 Base . 27
B.11.3 Blood medium . 27
B.11.4 Complete medium . 27
B.12 Carbohydrate utilization broth (L-Rhamnose and D-Xylose) . 27
B.12.1 Base . 27
B.12.2 Carbohydrate solutions . 28
B.12.3 Complete medium . 28
B.13 Tryptone soya yeast extract agar (TSYEA) . 28
B.13.1 Composition . 28
B.13.2 Preparation . 28
Annex C (informative) Results of the interlaboratory study for detection of Listeria
monocytogenes . 29
C.1 Results of the interlaboratory study . 29
C.2 Statistical analysis . 30
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 . 31
Bibliography . 32
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European foreword
This document (prEN 17710:2023) has been prepared by Technical Committee CEN/TC 455 “Plant
Biostimulants”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
This document will supersede CEN/TS 17710:2022.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of EU
Directive(s) / Regulation(s).
For relationship with EU Directive(s) / Regulation(s), see informative Annex ZA, which is an integral
part of this document.
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Introduction
This document was prepared by the experts of CEN/TC 455 “Plant Biostimulants”. 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 laying down rules on the making available on the market of
EU fertilizing products (“FPR” or “Fertilising Products Regulation”).
This request, presented as SR M/564 and M/564/Amd1, also contributes to the Communication on
“Innovating for Sustainable Growth: A Bio economy for Europe”. The Working Group 5 “Labelling and
denominations”, was created to develop a work program as part of this Request. The technical
committee CEN/TC 455 “Plant Biostimulants” was established to carry out the work program that will
prepare a series of standards. The interest in 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.
Biostimulants used in agriculture can be applied in multiple ways: on soil, on plant, as seed treatment,
etc. A microbial plant biostimulant consists of a microorganism or a consortium of microorganisms, as
referred to in Component Material Category 7 of Annex II of the EU Fertilising Products Regulation.
This document is applicable to all microbial biostimulants in agriculture.
The Table 1 below summarizes many of the agro-ecological principles and the role played by
biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant-microorganism interactions
- symbiotic exchanges i.e. mycorrhize
- symbiotic exchanges i.e. rhizobiaciae/fava
- secretions mimicking plant hormones (i.e. trichoderma)
By regulating plant physiological processes
- for ex growth, metabolism, plant development…
Improve biogeochemical cycles
- improve absorption of nutritional elements
- improve bioavailability of nutritional elements in the soil
- stimulate degradation of organic matter
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.
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IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably trained staff.
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1 Scope
This document provides a method for the detection of Listeria monocytogenes in microbial plant
biostimulants for verifying that the content of this human pathogen agrees with the respective limits
outlined in the EU Regulation on Fertilising Products [1].
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.
1
prEN 17724:— , Plant biostimulants — Terminology
1
prEN 17708:— , Plant biostimulants — Preparation of sample for microbial analysis
EN ISO 11290-1:2017, Microbiology of the food chain — Horizontal method for the detection and
enumeration of Listeria monocytogenes and of Listeria spp. — Part 1: Detection method
(ISO 11290-1:2017)
1
EN ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
EN ISO 7218:2007, Microbiology of food and animal feeding stuffs — General requirements and guidance
for microbiological examinations (ISO 7218:2007)
EN 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 6887-1:2017)
3 Terms and definitions
2
For the purposes of this document, the terms and definitions are given in prEN 17724:— 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
Listeria monocytogenes
bacterium which forms colonies fitting the description of the species on the specified selective medium
after incubation of 24 h at a temperature of 37 °C under aerobic conditions.
Note 1 to entry: L. monocytogenes is a Gram-positive, non-spore forming, rod-shaped bacterium that belongs to
the genus Listeria, phylum Firmicutes.
Note 2 to entry: L. monocytogenes is a ubiquitous bacterial pathogen that causes serious localized and generalized
infections in humans.
1
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020.
2
Under preparation.
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Note 3 to entry: L. monocytogenes are catalase positive and oxidase negative. They produce flagella when grown
at a temperature between 20 °C and 25 °C but not at 37 °C. They produce a β-hemolysin on blood agar plates, which
is part of the CAMP (Christie, Atkins, and Munch-Petersen) diagnostic test.
Note 4 to entry: L. monocytogenes can grow at temperatures between −0,4 °C and 50 °C, with an optimum
temperature of 30–37 °C. They can withstand freezing, but they are inactivated by heating at 60 °C for 30 min.
Note 5 to entry: L. monocytogenes are facultative anaerobes that utilize glucose, lactose, and rhamnose under
aerobic conditions and can ferment several hexoses and pentoses under anaerobic conditions.
Note 6 to entry: L. monocytogenes can grow over a pH range of 4–9,5 and a water activity of from 0,90 to 0,97.
They can also grow in 10 % sodium chloride.
[SOURCE: EN ISO 11290-1:2017, 3.1]
4 Principle
4.1 General
The detection of Listeria monocytogenes requires four successive stages as specified in Annex A.
NOTE L. monocytogenes can be present in small numbers and is often accompanied by considerably larger
numbers of bacteria belonging to different taxonomic groups or different Listeria species. Pre-enrichment is used
to permit the detection of low numbers of L. monocytogenes or injured L. monocytogenes.
4.2 Pre-enrichment in selective liquid medium
Half-Fraser broth (225 ml) at ambient temperature is inoculated with the test portion sample (25 g or
25 ml), then incubated at 30 °C ± 1 °C for 24 h to 26 h.
For large quantities (e.g. 1-L or more), it is recommended to pre-warm the broth to 30 °C before mixing
it with the test portion.
4.3 Enrichment in/on selective media
Fraser broth is inoculated at 37°C (0,1 ml of culture in 10 ml of Fraser broth) and incubated at 37°C ± 1°C
for 24 h ± 2 h.
4.4 Plating out on selective solid media
Streak both primary AND secondary enrichments onto:
— Agar Listeria according to Ottaviani and Agosti (ALOA) [3];
— A second selective agar of choice, e.g. PALCAM agar, Oxford agar.
The agar prepared according to Ottaviani and Agosti is incubated 24 h ± 2 h, 37 °C ± 1 °C and
additionally 24 h ± 2 h, 37 °C ± 1 °C, then examined. The second selective agar is incubated as specified
by the manufacturer.
4.5 Confirmation
Colonies of presumptive L. monocytogenes are subcultured and their identity is confirmed by means of
appropriate morphological and biochemical tests.
5 Culture media, reagents, antisera
For current laboratory practices prEN 17708:—2 and EN ISO 11133:20141 shall be used.
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Composition of culture media and reagents and their preparation are described in Annex B.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment according to EN ISO 7218:2007 shall be used and, in
particular, the following.
6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), as specified in
EN ISO 7218:2007.
6.2 Drying cabinet or incubator, capable of operating between 25 °C and 50 °C.
6.3 Incubators, capable of operating at 30 °C ± 1 °C, 37 °C ± 1 °C, and at 25 °C ± 1 °C (optional).
6.4 Water bath, capable of operating at 47 °C to 50 °C.
6.5 Sterile loops, approximately 3 mm in diameter or 10 μl, and inoculating needle or wire.
6.6 pH-meter, having an accuracy of calibration of ± 0,1 pH unit at 25 °C.
6.7 Sterile graduated pipettes or automatic pipettes of nominal capacities of 1 ml, and 10 ml.
6.8 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
6.9 Microscope, preferably with phase-contrast, and with slides and cover slips.
6.10 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.11 Peristaltic blender (stomacher) with 400 ml sterile bags.
6.12 Blender motor and jars or vortex.
7 Sampling
Sampling is not part of the method specified in this document (see the specific European Standard
dealing with the product concerned). If there is no specific International or European Standard, it is
recommended that the parties concerned come to an agreement on this subject.
It is important that the laboratory receives a sample which is representative and has not been damaged
or changed during transport or storage.
8 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International or
European Standard
...
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