SIST-TS CEN/TS 17710:2023
(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 concentration of this human pathogen does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].
Pflanzen-Biostimulanzien - Nachweis von Listeria monocytogenes
Dieses Dokument stellt ein Verfahren zum Nachweis von Listeria monocytogenes in mikrobiellen Bio-stimulanzien für die pflanzliche Anwendung zur Verfügung, und zwar um zu verifizieren, dass die Konzentration dieses Humanpathogens die entsprechenden Grenzwerte der EU Düngeprodukte-verordnung [1] nicht überschreitet.
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 concentration de ce pathogène pour l’homme ne dépasse pas les limites correspondantes indiquées dans le Règlement UE sur les fertilisants [1].
Rastlinski biostimulanti - Ugotavljanje prisotnosti Listeria monocytogenes
Ta dokument zagotavlja metodo za ugotavljanje prisotnosti bakterije Listeria monocytogenes v mikrobnih rastlinskih biostimulantih za preverjanje, ali koncentracija tega človeškega patogena ne presega ustreznih mejnih vrednosti, določenih v Uredbi EU o sredstvih za gnojenje [1].
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 17710:2023
01-februar-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: CEN/TS 17710:2022
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 17710:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 17710:2023
CEN/TS 17710
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Detection of Listeria monocytogenes
Biostimulants des végétaux - Détection de Listeria Pflanzen-Biostimulanzien - Nachweis von Listeria
monocytogenes monocytogenes
This Technical Specification (CEN/TS) was approved by CEN on 3 January 2022 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.
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, Turkey 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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17710:2022 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
4.1 General . 5
4.2 Pre-enrichment in non-selective liquid medium . 5
4.3 Enrichment in/on selective media . 6
4.4 Plating out on selective solid media . 6
4.5 Confirmation . 6
5 Culture media, reagents, antisera . 6
6 Equipment and consumables . 6
7 Sampling . 7
8 Preparation of test sample . 7
9 Preparation procedure . 7
9.1 Test portion and initial suspension . 7
9.2 Non-selective pre-enrichment . 8
9.3 Selective enrichment. 8
9.4 Plating out . 8
9.5 Confirmation of L. monocytogenes . 9
9.6 Interpretation of morphological and physiological properties and of the biochemical
reactions . 12
9.7 Additional characterization of isolated strains (optional) . 12
10 Expression of results . 12
11 Performance characteristics of the method . 13
11.1 Interlaboratory studies . 13
11.2 Sensitivity . 13
11.3 Specificity . 13
11.4 LOD . 13
50
12 Test report . 13
13 Quality assurance . 13
Annex A (normative) Diagram of the procedures . 14
Annex B (normative) Composition and preparation of culture media and reagents . 15
Bibliography . 25
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European foreword
This document (CEN/TS 17710:2022) has been prepared by Technical Committee CEN/TC 455 “Plant
Biostimulants”, the secretariat of which is held by AFNOR.
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 Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
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 announce this Technical Specification: 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, Turkey and the
United Kingdom.
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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 standardization request, presented as M/564, 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. Mycorrhizae
- symbiotic exchanges i.e. Rhizobiaceae/Faba
- secretions mimicking plant hormones (i.e. Trichoderma)
By regulating plant physiological processes
- for e.g. 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.
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 concentration of this human pathogen does not exceed 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.
CEN/TS 17724, Plant biostimulants — Terminology
CEN/TS 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, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
EN ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations (ISO 7218)
3 Terms and definitions
For the purposes of this document, the terms and definitions are given in CEN/TS 17724 and the
following apply.
3.1
Listeria monocytogenes
microorganisms which form typical colonies on solid selective media described and which display the
morphological, physiological and biochemical characteristics described when the analysis is carried out
in accordance with this document
[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 non-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.
1
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020.
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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 (4.2) 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];
2
— 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 CEN/TS 17708 and EN ISO 11133 shall be used.
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 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.
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.
2
PALCAM agar and Oxford agar are examples of a suitable products available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of these products.
https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam
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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 dealing with the product concerned (see EN ISO 6887-1).
9 Preparation procedure
9.1 Test portion and initial suspension
9.1.1 General
To ensure a truly representative analytical unit, agitate liquids or free flowing materials until the
contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion
from several locations within the sample unit. To reduce the workload, the analytical units may be
combined for analysis. It is recommended that a composite contains no more than five analytical units.
General rules for the preparation of the initial suspension for microbiological examination are described
in CEN/TS 17708. The initial suspension of the microbial biostimulant product samples can be prepared
according to EN ISO 11290-1:2017 using, as dilution fluid, the selective primary enrichment medium,
half-Fraser broth, described in B.1.
A representative sample of the product should be prepared taking into consideration the different
formulations of the biostimulant-based products.
Add 25 g or 25 ml of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml
stomacher bag (6.11) or a blender jar (6.12). For composite samples, analytical units may be combined
up to 125 g or ml (e.g. 125 g or ml of food to 1 125 ml of half-Fraser broth). Pre-warm the half-Fraser
broth to room temperature before use.
If alternate analytical units are required, maintain a ratio of 1 part sample material to 9 parts half-Fraser
broth.
9.1.2 Liquid – water based– formulations
Aseptically add 25 ml of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml
sterile stomacher bag (6.11) or a blender jar (6.12). Pre-warm the half-Fraser broth to room
temperature before use. Blend, stomach or vortex as required for thorough mixing.
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9.1.3 Liquid – oil based (emulsifiable concentrate – EC) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11) or a blender jar (6.12). Pre-warm the half-Fraser broth to room temperature
before use. Blend, stomach or vortex as required for thorough mixing.
9.1.4 Solid – Wettable Powder (WP) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture 2 min at higher speed with a stomacher (6.11).
9.1.5 Solid – Water dispersible granules (WDG) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture 2 min at higher speed with a stomacher (6.11).
9.1.6 Solid – Pellets, granules, microgranules (slow release) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture for 2 min at higher speed with a stomacher (6.11).
9.1.7 Solid substrates
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture 2 min at higher speed with a stomacher (6.11).
9.2 Non-selective pre-enrichment
Prepare the test portion sample (25 g or 25 ml) in half-Fraser broth (225 ml). Incubate for 24 h to 26 h
at 30 °C.
NOTE 1 A black coloration can develop during incubation.
NOTE 2 It is possible to store at 5 °C (6.10) the pre-enriched sample after incubation before transfer to Fraser
broth for a maximum of 72 h.
9.3 Selective enrichment
9.3.1 After incubation of the initial suspension (primary enrichment in half-Fraser broth) for 24 h to
26 h (9.2), transfer 0,1 ml of the culture obtained in 9.2 to a tube or bottle containing 10 ml of secondary
enrichment medium (Fraser Broth) (described in B.2).
9.3.2 Incubate the inoculated medium (9.3.1) for 24 h ± 2 h at 37 °C (6.3).
Half-Fraser broth and Fraser broth may be refrigerated before transfer or isolation on selective agar for
a maximum of 72 h. Refrigeration provides for greater laboratory productivity and analytical flexibility.
Following the period of refrigeration, the secondary enrichment broth is resuspended before transfer
or plating onto agar media.
9.4 Plating out
9.4.1 General
9.4.1.1 From the primary enrichment culture (9.2) incubated for 25 h ± 1 h at 30 °C (6.3), inoculate,
by means of a loop (6.5), the surface of the first selective plating medium, Agar Listeria according to
Ottaviani and Agosti (ALOA) (described in B.3), to obtain well-separated colonies.
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Proceed in the same way with the second selective plating-out medium of choice (B.4).
NOTE Half-Fraser broth and Fraser broth can be refrigerated at 5 °C (6.10) before isolation on selective agar
for a maximum of 72 h [4].
9.4.1.2 From the secondary enrichment medium incubated for 24 h ± 2 h at 37 °C (6.3) (9.3.2),
repeat the procedure described in 9.4.1.1 with the two selective plating-out media.
9.4.1.3 Invert the Petri dishes obtained in 9.4.1.1 and 9.4.1.2 and place them in an incubator set at
37 °C (6.3) for Agar Listeria according to Ottaviani and Agosti (B.3). For the second selective medium
(B.4), follow the manufacturer’s instructions.
9.4.1.4 For Agar Listeria according to Ottaviani and Agosti (B.3), incubate for a total of 48 h ± 2 h. If
colonies of presumptive L. monocytogenes are evident at 24 h ± 2 h the incubation may be stopped at
this stage. For second selective agar incubate for the appropriate time. Examine the dishes (9.4.1.3) for
the presence of presumptive colonies of L. monocytogenes.
NOTE After incubation plates can be refrigerated at 5 °C (6.10) for a maximum of 48 h before reading.
9.4.2 Agar Listeria according to Ottaviani and Agosti (B.3)
Consider as presumptive L. monocytogenes the blue-green colonies surrounded by an opaque halo
(typical colonies). Colonies of Listeria ivanovii are also blue-green and surrounded by an opaque halo.
NOTE 1 Some strains of L. monocytogenes exposed to stress conditions, particularly acid stress, can show a very
weak halo (or even no halo).
NOTE 2 Some rare L. monocytogenes are characterized by a slow PIPLC (phosphatidyl inositol phospholipase
C) activity. Such bacteria are detected when the total duration of incubation is more than, for example, four days.
Some of these strains could be pathogenic [5]. No L. monocytogenes strains have been described as PIPLC negative.
NOTE 3 Some organisms other than Listeria spp. can produce blue colonies on this medium [6].
9.4.3 Second selective medium
After the appropriate time, examine the plates for the presence of colonies which are considered to be
presumptive L. monocytogenes, based on their characteristics for the type of medium used (B.4).
9.5 Confirmation of L. monocytogenes
9.5.1 General
Confirmation tests are to be conducted in accordance with the standard EN ISO 11290-1:2017; in the
recent revision and validation of this standard, catalase test and Camp-test became optional for L.
monocytogenes. The microscopic aspect of confirmation remains mandatory, with an agar allowing
distinction of pathogenic Listeria spp. used. For the haemolysis test or CAMP test, blood agar is now
extended from defibrinated sheep blood only, to calf or bovine blood. For the haemolysis test, blood agar
is to be inoculated by stabbing or by streaking (only if positive at purification step) [2].
Appropriate positive and negative control strains for each of the confirmatory tests shall be used.
9.5.2 Selection of colonies for confirmation
For confirmation of presumptive L. monocytogenes, take at least one colony presumed to be L.
monocytogenes (see 9.4.2 and 9.4.3). One confirmed isolate per sample is sufficient. If the first colony is
negative take further colonies presumed to be L. monocytogenes from selective medium (up to a
maximum of five colonies from each plate of each selective medium).
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Streak the selected colonies onto the surface of pre-dried plates of a non-selective agar, for example
blood agar, nutrient agar, tryptone soya yeast extract agar (TSYEA) (B.13), in a manner which will allow
isolated colonies to develop.
Use of blood agar for pure culture enables interpretation of haemolysis, when positive, already at that
stage. If streaking on blood agar does not show haemolysis, then the haemolysis test shall be done by
stabbing or in liquid medium.
Place the plates in the incubator set at 37 °C (6.3) for 18 h to 24 h or until growth is satisfactory.
If the colonies are not isolated, pick a typical L. monocytogenes colony onto another non-selective agar
plate. Carry out the following tests (9.5.2) from colonies of a pure culture on the non-selective agar.
9.5.3 Confirmation tests for L. monocytogenes
9.5.3.1 General
The mandatory and optional tests to be performed for confirmation of presumptive L. monocytogenes
are described in EN ISO 11290-1:2017 and reported in Table 2.
Table 2 — Confirmation tests for L. monocytogenes
L. monocytogenes confirmation
Tests Results
tests
Microscopic aspect Slim short rods or coccobacilli
Beta-haemolysis +
Mandatory
L-Rhamnose +
D-Xylose -
Catalase +
Optional Motility at 25 °C +
CAMP test +
An alternative procedure as mentioned in EN ISO 7218 can be used to confirm the isolate as Listeria
monocytogenes, providing the suitability of the relevant procedure is verified. If shown to be reliable,
miniaturized galleries for the biochemical identification of L. monocytogenes may be used (see
EN ISO 7218). Rare strains of L. monocytogenes do not show beta-haemolysis or a positive reaction to
the CAMP test under the conditions described in this document. If typical colonies on Agar Listeria
according to Ottaviani and Agosti with PIPLC activity even if it is low are negative for haemolysis, it is
recommended to perform additional tests (e.g. Gram-stain, catalase, motility, CAMP test, PCR), in order
to determine whether this isolate is a non-haemolytic L. monocytegenes.
9.5.3.2 Catalase reaction (optional)
Take an isolated colony obtained in 9.5.2 and suspend it in a drop of hydrogen peroxide solution (B.6)
on a slide. The immediate formation of gas bubbles indicates a positive reaction.
NOTE A catalase reaction performed from a colony originating from a blood agar can sometimes lead to false-
positive results.
9.5.3.3 Motility test (optional)
Take an isolated colony obtained in 9.5.2 and suspend it in a tube containing a non-selective nutrient
liquid medium.
Incubate in the incubator (6.3) set at 25 °C for 8 h to 24 h until the medium turns cloudy.
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Take
...
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17710:2021
01-november-2021
[Not translated]
Plant biostimulants - Detection of Listeria monocytogenes
Biostimulanzien für die pflanzliche Anwendung - Nachweis von Listeria monocytogenes
Ta slovenski standard je istoveten z: FprCEN/TS 17710
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17710:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN/TS 17710:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17710
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
August 2021
ICS 65.080
English Version
Plant biostimulants - Detection of Listeria monocytogenes
Biostimulanzien für die pflanzliche Anwendung -
Nachweis von Listeria monocytogenes
This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 455.
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, Turkey 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 Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17710:2021 E
worldwide for CEN national Members.
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Contents Page
European foreword . 5
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Principle . 8
4.1 General . 8
4.2 Pre-enrichment in non-selective liquid medium . 8
4.3 Enrichment in/on selective media . 8
4.4 Plating out on selective solid media . 8
4.5 Confirmation . 8
5 Culture media, reagents, antisera . 8
6 Equipment and consumables . 8
7 Sampling . 9
8 Preparation of test sample . 9
9 Preparation of test sample . 9
9.1 Test portion and initial suspension . 9
9.1.1 Liquid - water based- formulations . 10
9.1.2 Liquid - oil based (emulsifiable concentrate - EC) formulations . 10
9.1.3 Solid - Wettable Powder (WP) formulations . 10
9.1.4 Solid - Water dispersible granules (WDG) formulations . 10
9.1.5 Solid – Pellets, granules, microgranules (slow release) formulations . 10
9.1.6 Solid substrates . 10
9.2 Non-selective pre-enrichment . 10
9.3 Selective enrichment. 10
9.4 Plating out . 11
9.4.1 General . 11
9.4.2 Agar Listeria according to Ottaviani and Agosti (B.3) . 11
9.4.3 Second selective medium . 11
9.5 Confirmation of L. monocytogenes . 11
9.5.1 General . 11
9.5.2 Selection of colonies for confirmation . 12
9.5.3 Confirmation tests for L. monocytogenes . 12
9.6 Interpretation of morphological and physiological properties and of the biochemical
reactions . 14
9.7 Additional characterization of isolated strains (optional) . 14
10 Expression of results . 14
11 Performance characteristics of the method . 15
11.1 Interlaboratory studies . 15
11.2 Sensitivity . 15
11.3 Specificity . 15
11.4 LOD . 15
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12 Test report . 15
13 Quality assurance . 15
Annex A (normative) Diagram of the procedures . 16
Annex B (normative) Composition and preparation of culture media and reagents . 17
B.1 Selective primary enrichment medium: half-Fraser broth . 17
B.1.1 Base . 17
B.1.2 Lithium chloride solution . 17
B.1.3 Solution of sodium salt of nalidixic acid . 18
B.1.4 Acriflavine hydrochloride solution . 18
B.1.5 Ammonium iron(III) citrate solution . 18
B.1.6 Complete medium . 19
B.2 Selective secondary enrichment medium: Fraser broth . 19
B.2.1 Base . 19
B.2.2 Acriflavine hydrochloride solution . 19
B.2.3 Ammonium iron(III) citrate solution . 20
B.2.4 Complete medium . 20
B.3 Agar Listeria according to Ottaviani and Agosti (ALOA) . 20
B.3.1 Base medium . 20
B.3.2 Nalidixic acid solution . 20
B.3.3 Ceftazidime solution . 21
B.3.4 Polymyxin B solution . 21
B.3.5 Antibiotic supplement . 21
B.3.6 Supplement . 22
B.3.7 Complete medium . 22
B.4 Second selective solid plating-out medium . 23
B.5 Performance testing for the quality assurance of the culture media . 23
B.6 Hydrogen peroxide solution . 23
B.7 Motility agar . 23
B.7.1 Composition . 23
B.7.2 Preparation . 23
B.8 Blood agar . 23
B.8.1 Base . 23
B.8.2 Defibrinated blood (sheep, calf or bovine) . 24
B.8.3 Complete medium . 24
B.9 Phosphate-buffered saline (PBS). 24
B.9.1 Composition . 24
B.9.2 Preparation . 24
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B.10 Red blood corpuscle suspension . 24
B.11 CAMP (Christie, Atkins, Munch-Petersen) medium and test strains . 25
B.11.1 General . 25
B.11.2 Base . 25
B.11.3 Blood medium . 25
B.11.4 Complete medium . 25
B.12 Carbohydrate utilization broth (L-Rhamnose and D-Xylose) . 25
B.12.1 Base . 25
B.12.2 Carbohydrate solutions . 26
B.12.3 Complete medium . 26
B.13 Tryptone soya yeast extract agar (TSYEA) . 26
B.13.1 Composition . 26
B.13.2 Preparation . 26
Bibliography . 27
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European foreword
This document (FprCEN/TS 17710:2021) 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 vote.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
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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 fertilising products (“FPR” or “Fertilising Products Regulation”). This request, presented as SR
M/564, 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 Fertilizing 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.
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 concentration of this human pathogen does not exceed 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.
FprCEN/TS 17724, Plant biostimulants — Terminology
FprCEN/TS 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
For the purposes of this document, the terms and definitions are given in FprCEN/TS 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
microorganisms which form typical colonies on solid selective media described and which display the
morphological, physiological and biochemical characteristics described when the analysis is carried out
in accordance with this document
[SOURCE: EN ISO 11290-1:2017, 3.1]
1
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020.
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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 non-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 24-26 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 FprCEN/TS 17708 and EN ISO 11133:2014 shall be used.
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).
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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 EN 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 EN
Standard dealing with the product concerned [see EN ISO 6887-1:2017].
9 Preparation of test sample
9.1 Test portion and initial suspension
To ensure a truly representative analytical unit, agitate liquids or free flowing materials until the
contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion
from several locations within the sample unit. To reduce the workload, the analytical units may be
combined for analysis. It is recommended that a composite contains no more than five analytical units.
General rules for the preparation of the initial suspension for microbiological examination are described
in FprCEN/TS 17708. The initial suspension of the microbial biostimulant product samples can be
prepared according to EN ISO 11290-1:2017 using, as dilution fluid, the selective primary enrichment
medium, half-Fraser broth, described in B.1.
A representative sample of the product should be prepared taking into consideration the different
formulations of the biostimulant-based products.
Add 25 g or 25 ml of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml
stomacher bag (6.11) or a blender jar (6.12). For composite samples, analytical units may be combined
up to 125 g or ml (e.g. 125 g or ml of food to 1 125 ml of half-Fraser broth). Pre-warm the half-Fraser
broth to room temperature before use.
If alternate analytical units are required, maintain a ratio of 1 part sample material to 9 parts half-Fraser
broth.
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9.1.1 Liquid - water based- formulations
Aseptically add 25 ml of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml
sterile stomacher bag (6.11) or a blender jar (6.12). Pre-warm the half-Fraser broth to room
temperature before use. Blend, stomach or vortex as required for thorough mixing.
9.1.2 Liquid - oil based (emulsifiable concentrate - EC) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11) or a blender jar (6.12). Pre-warm the half-Fraser broth to room temperature
before use. Blend, stomach or vortex as required for thorough mixing.
9.1.3 Solid - Wettable Powder (WP) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture 2 min at higher speed with a stomacher (6.11).
9.1.4 Solid - Water dispersible granules (WDG) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture 2 min at higher speed with a stomacher (6.11).
9.1.5 Solid – Pellets, granules, microgranules (slow release) formulations
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture for 2 min at higher speed with a stomacher (6.11).
9.1.6 Solid substrates
Aseptically add 25 g of the product (the analytical unit) to 225 ml of half-Fraser broth in a 400 ml sterile
stomacher bag (6.11). Pre-warm the half-Fraser broth to room temperature before use. Homogenize the
mixture 2 min at higher speed with a stomacher (6.11).
9.2 Non-selective pre-enrichment
Prepare the test portion sample (25 g or 25 ml) in half-Fraser broth (225 ml). Incubate for 24 – 26 h at
30 °C.
NOTE 1 A black coloration can develop during incubation.
NOTE 2 It is possible to store at 5 °C (6.10) the pre-enriched sample after incubation before transfer to Fraser
broth for a maximum of 72 h.
9.3 Selective enrichment
9.3.1 After incubation of the initial suspension (primary enrichment in half-Fraser broth) for 24-26 h
(9.2), transfer 0,1 ml of the culture obtained in 9.2 to a tub
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