CEN/TS 17708:2022

Plant biostimulants - Preparation of sample for microbial analysis

This document defines general rules for the aerobic preparation of the initial suspension and of dilutions for microbiological examinations of microbial plant biostimulants.
This horizontal method might not be appropriate in very detail for certain products. In this case, different methods which are specific to these products can be used if necessary, for justified technical reasons.

Pflanzen-Biostimulanzien - Probenvorbereitung für die mikrobielle Analyse

Dieses Dokument legt allgemeine Regeln für die aerobe Herstellung von Erstverdünnungen und Verdünnungen für mikrobiologische Untersuchungen von mikrobiellen Pflanzen-Biostimulanzien fest.
Dieses horizontale Verfahren ist für bestimmte Produkte nicht unbedingt bis ins Detail geeignet. In diesem Fall können andere Verfahren, die speziell auf diese Produkte zutreffen, angewendet werden, wenn dies aus gerechtfertigten technischen Gründen notwendig ist.

Biostimulants des végétaux - Préparation de l’échantillon pour l’analyse microbienne

Le présent document définit les règles générales pour la préparation aérobie de la suspension mère et des dilutions en vue de l'examen microbiologique des biostimulants microbiens des végétaux.
Il se peut que cette méthode horizontale ne soit pas appropriée pour certains produits. Dans ce cas, des méthodes différentes, spécifiques à ces produits, peuvent être utilisées si cela s'avère nécessaire pour des raisons techniques justifiées.

Rastlinski biostimulanti - Priprava vzorcev za mikrobiološko analizo

Ta dokument določa splošna pravila za aerobno pripravo osnovne suspenzije in razredčin za mikrobiološke preiskave mikrobnih rastlinskih biostimulantov.
Ta vodoravna metoda v nekaterih podrobnostih morda ne bo primerna za nekatere izdelke. V tem primeru se lahko po potrebi iz upravičenih tehničnih razlogov uporabijo metode, ki so specifične za te izdelke.

General Information

• Status: Withdrawn
• Publication Date: 22-Mar-2022
• Withdrawal Date: 13-Apr-2025
• ICS: 65.080 - Fertilizers
• Technical Committee: CEN/TC 455 - Plant Biostimulants and Agricultural Micro-Organisms
• Drafting Committee: CEN/TC 455/WG 3 - Pathogenic and non-pathogenic microorganisms
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 27-Nov-2024
• Completion Date: 14-Apr-2025
• Directive: 2019/1009 - Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC) No 1107/2009 and repealing Regulation (EC) No 2003/2003
• Mandate: M/564 - Fertilising products in support of Regulation (EU) 2019/1009
• Ref Project: SIST-TS CEN/TS 17708:2023 - Plant biostimulants - Preparation of sample for microbial analysis

Relations

• Replaced By: EN 17708:2024 - Plant biostimulants - Preparation of sample for microbial analysis
• Effective Date: 15-Feb-2023

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2023
Rastlinski biostimulanti - Priprava vzorcev za mikrobiološko analizo
Plant biostimulants - Preparation of sample for microbial analysis
Pflanzen-Biostimulanzien - Probenvorbereitung für die mikrobielle Analyse
Biostimulants des végétaux - Préparation de l’échantillon pour l’analyse microbienne
Ta slovenski standard je istoveten z: CEN/TS 17708:2022
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 17708
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Preparation of sample for microbial
analysis
Biostimulants des végétaux - Préparation de Biostimulanzien für die pflanzliche Anwendung -
l'échantillon pour l'analyse microbienne Probenvorbereitung für die mikrobielle Analyse
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 NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

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 17708:2022 E
worldwide for CEN national Members.

CEN 17708:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
5 Diluents . 7
6 Apparatus . 7
7 Sampling and storage . 8
8 Preparation of samples and suspension . 8
8.1 Protocol for enumeration of beneficial microorganisms . 8
8.2 Protocol for enumeration of not-desirable microorganisms . 10
8.3 Protocol for the determination of anaerobic plate count . 11
8.4 Protocol for the detection of pathogenic microorganisms . 11
8.5 Further dilutions . 12
8.6 Duration of the procedure . 12
Annex A (informative) Plating procedure . 13
Annex B (informative) Diluents for general use . 15
Bibliography . 17

CEN 17708:2022 (E)
European foreword
This document (CEN/TS 17708:2022) 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.

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 [1]
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
This document defines the general rules for the preparation of samples, initial suspensions and
subsequent dilutions for microbiological examination of plant biostimulants.
Any special diluents or practices required in specific standard methods take priority over the
general rules listed in this document.
CEN 17708:2022 (E)
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 defines general rules for the aerobic preparation of the initial suspension and of
dilutions for microbiological examinations of microbial plant biostimulants.
This horizontal method might not be appropriate in very detail for certain products. In this case,
different methods which are specific to these products can be used if necessary, for justified
technical reasons.
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 17702-1, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
CEN/TS 17724, Plant biostimulants — Terminology
3 Terms and definitions
For the purposes of this document, the terms and definitions given in CEN/TS 17724 and the
following apply.
3.1
laboratory sample
sample sent to the laboratory and intended for inspection or testing
3.2
test sample
sample prepared from the laboratory sample (3.1) according to the procedure specified in the
method of test and from which test portions (3.3) are taken
3.3
test portion
measured (volume or mass) representative sample taken from the laboratory sample (3.1) for use
in the preparation of the initial suspension (3.4)
3.4
initial suspension
suspension, solution or emulsion obtained after a weighed or measured quantity of the product
under examination has been mixed with, normally, a nine-fold quantity of diluent, or other ratios
if required for specific purposes, allowing large particles, if present, to settle
3.5
further dilution
suspension or solution obtained by mixing a measured volume of the initial suspension (3.4) with
an x-fold volume of diluent and by repeating this operation with further dilutions until a dilution
series, suitable for the inoculation of culture media, is obtained
Note 1 to entry: Ten-fold dilutions are normally used to produce a decimal dilution series, but other ratios
can be required for specific purposes.
CEN 17708:2022 (E)
3.6
specific standard
standard or guidance document describing the examination of a specific product for the detection
or enumeration of a specific microorganism
4 Principle
Preparation of initial suspension (3.4) in such a way as obtain as uniform a distribution as possible
of the microorganisms contained in the test portion (3.3).
Preparation, if necessary, of further dilutions (3.5) in order to reduce the number of
microorganisms per unit volume to allow, after incubation, observation of their growth or not (in
case of tubes or bottles) or colony counting (in the case of plates).
5 Diluents
For the preparation of diluents follow the current laboratory practices and the manufacturer’s
instructions for commercial preparations.
In Annex B there are examples of general use diluents.
It is possible to add to diluents surface-active agents to improve the uniformity of the suspension
while avoiding the formation of clusters of microorganisms, and verifying that at the added
concentration does not alter cell viability.
6 Apparatus
Usual microbiological laboratory equipment as listed below:
6.1 Apparatus for dry sterilization (oven) and wet sterilization (autoclave).
6.2 Protective cabinets, for all work involving the handling of microbiological products.
6.3 Balances, capable of weighing to 1 %of the mass.
6.4 Incubator, capable of operating at the required temperatures of the test.
6.5 Total delivery pipettes, sterile, of nominal capacity 1 ml, and graduated in divisions of
0,1 ml.
6.6 Water bath, or similar apparatus, to maintain a specific temperature.
6.7 pH meter, accurate to 0,1 pH units at 25 °C.
6.8 Bottles, flasks and tubes, for boiling and storage of culture media, and for making of
dilutions.
6.9 Petri dishes, sterile, of glass or plastic, with a diameter 90 mm to 100 mm.
6.10 Optical Microscope, for bacteriological examinations, objectives with magnification from
x10 (dry lens) to about x100 (oil immersion).
6.11 Spreaders, made of glass or plastic (of diameter less than 2 mm and length 80 mm).
Diameter should not exceed 2 mm in order to minimize the amount of samples adhering to them
at the end of the spreading procedure.
6.12 Binocular magnifier, for discriminating and differentiation colonies/cells of yeasts and
moulds (magnification 6,5 to 50 times).
6.13 Refrigerator, chambers which allow maintenance of cold storage.
6.14 Centrifuge, to separate suspended particles from fluids.
For the preparation of the test sample, of the initial suspension or for the decimal dilutions series,
use one or more of the common apparatus reported below:
6.15 Peristaltic blender (stomacher) with sterile bags.
6.16 Rotatory homogenizer (blender), with a sterilizable glass or metals bowls equipped with
covers (speed between 8 000 r/min and 45 000 r/min).
6.17 Vibrational mixer (pulsifier) with sterile bags (usual operating time is 0,5 min to 1 min).
6.18 Ultraturrax, a dispersing tool and homogenizer instrumentation that produces
homogenous, uniform sample preparations.
6.19 Ultrasonic sonicator.
6.20 Mechanical stirrer.
6.21 Magnetic stirrer.
6.22 Vortex mixer, mainly used for liquid samples and decimal dilutions preparation.
6.23 Glass beads can be used for the preparation, by shaking, of the initial suspension of certain
viscous or thick products.
7 Sampling and storage
Carry out sampling in accordance with CEN/TS 17702-1. It is important that the laboratory
receives a sample which is representative and has not been damaged or changed during transport
or storage.
The samples should be delivered to the laboratory with the original storage conditions maintained
as nearly as possible.
The sample should be submitted in the original, unopened container.
Upon arrival, check the condition of the samples. Identify samples clearly and completely and
record the sample information.
T
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