kSIST-TS FprCEN/TS 17708:2021
(Main)Plant biostimulants - Preparation of sample for microbial analysis
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.
Biostimulanzien für die pflanzliche Anwendung - Probenvorbereitung für die mikrobielle Analyse
Dieses Dokument legt allgemeine Regeln für die aerobe Herstellung von Erstverdünnungen und Verdünnungen für mikrobiologische Untersuchungen von mikrobiellen Biostimulanzien für die pflanzliche Anwendung 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
General Information
Standards Content (sample)
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17708:2021
01-november-2021
Rastlinski biostimulansi - Priprava vzorcev za mikrobiološko analizo
Plant biostimulants - Preparation of sample for microbial analysis
Biostimulanzien für die pflanzliche Anwendung - Probenvorbereitung für die mikrobielle
AnalyseBiostimulants des végétaux - Préparation de l’échantillon pour l’analyse microbienne
Ta slovenski standard je istoveten z: FprCEN/TS 17708ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17708:2021 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 17708:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17708
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
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 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 17708:2021 E
worldwide for CEN national Members.---------------------- Page: 3 ----------------------
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FprCEN 17708:2021 (E)
Contents
European foreword ............................................................................................................................... 4
Introduction ............................................................................................................................................ 5
1 Scope ............................................................................................................................................ 7
2 Normative references ............................................................................................................ 7
3 Terms and definitions ........................................................................................................... 7
4 Principle ..................................................................................................................................... 8
5 Diluents ....................................................................................................................................... 8
6 Apparatus ................................................................................................................................... 8
7 Sampling and storage ............................................................................................................. 9
8 Preparation of samples and suspension ....................................................................... 10
8.1 Protocol for enumeration of beneficial microorganisms ....................................... 10
8.1.1 General ...................................................................................................................................... 10
8.1.2 Liquid - water based - formulations ............................................................................... 10
8.1.3 Liquid - oil based (emulsifiable concentrate - EC) formulations ......................... 10
8.1.4 Solid - wettable powder (WP) formulations ................................................................ 10
8.1.5 Solid - water dispersible granules (WDG) formulations ......................................... 10
8.1.6 Solid - pellets, granules, microgranules (slow release) formulations ............... 11
8.1.7 Solid - substrate ..................................................................................................................... 11
8.2 Protocol for enumeration of not-desirable microorganisms ................................ 11
8.2.1 General ...................................................................................................................................... 11
8.2.2 Liquid - water based - formulations ............................................................................... 11
8.2.3 Liquid - oil based (emulsifiable concentrate - EC) formulations ......................... 11
8.2.4 Solid - wettable powder (WP) formulations ................................................................ 11
8.2.5 Solid - water dispersible granules (WDG) formulations ......................................... 11
8.2.6 Solid - pellets, granules, microgranules (slow release) formulations ............... 12
8.2.7 Solid - substrate ..................................................................................................................... 12
8.3 Protocol for the determination of anaerobic plate count ....................................... 12
8.3.1 General ...................................................................................................................................... 12
8.3.2 Liquid - water based - formulations ............................................................................... 12
8.3.3 Liquid - oil based (emulsifiable concentrate - EC) formulations ......................... 12
8.3.4 Solid - wettable powder (WP) formulations ................................................................ 12
8.3.5 Solid - water dispersible granules (WDG) formulations ......................................... 12
8.3.6 Solid - pellets, granules, microgranules (slow release) formulations ............... 12
8.3.7 Solid - substrate ..................................................................................................................... 12
8.4 Protocol for the detection of pathogenic microorganisms .................................... 12
8.4.1 General ...................................................................................................................................... 12
8.4.2 Liquid formulations .............................................................................................................. 13
8.4.3 Solid formulations ................................................................................................................. 13
8.5 Further dilutions ................................................................................................................... 13
8.5.1 Decimal dilution series ....................................................................................................... 13
8.5.2 Other dilutions series .......................................................................................................... 13
8.6 Duration of the procedure ................................................................................................. 13
Annex A (informative) Plating procedure ................................................................................... 14
Annex B (informative) Diluents for general use ....................................................................... 16
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FprCEN 17708:2021 (E)
B.1 General ...................................................................................................................................... 16
B.2 Peptone salt solution ........................................................................................................... 16
B.2.1 Composition ............................................................................................................................ 16
B.2.2 Preparation ............................................................................................................................. 16
B.3 Buffered peptone water ...................................................................................................... 16
B.3.1 Composition ............................................................................................................................ 16
B.3.2 Preparation ............................................................................................................................. 16
B.4 Nacl 0,9 % ................................................................................................................................ 17
B.4.1 Composition ............................................................................................................................ 17
B.4.2 Preparation ............................................................................................................................. 17
B.5 PBS (Phosphate Buffered Saline) .................................................................................... 17
B.5.1 Composition ............................................................................................................................ 17
B.5.2 Preparation ............................................................................................................................. 17
Bibliography .......................................................................................................................................... 18
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FprCEN/TS 17708:2021 (E)
European foreword
This document (FprCEN/TS 17708:2021) 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 on TS.
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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FprCEN 17708:2021 (E)
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 [1]
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
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.---------------------- Page: 7 ----------------------
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WARNING — Person 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.---------------------- Page: 8 ----------------------
kSIST-TS FprCEN/TS 17708:2021
FprCEN 17708:2021 (E)
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.FprCEN/TS 17702-1, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
FprCEN/TS 17724, Plant biostimulants — Terminology3 Terms and definitions
For the purposes of this document, the terms and definitions 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
laboratory sample
sample sent to the laboratory and intended for inspection or testing
3.2
test sample
sample prepared from the laboratory sample (3.2) according to the procedure specified in the
method of test and from which test portions (3.4) are taken3.3
test portion
measured (volume or mass) representative sample taken from the laboratory sample (3.2) for use
in the preparation of the initial suspension (3.5)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
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3.5
further dilution
suspension or solution obtained by mixing a measured volume of the initial suspension (3.5) 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 obtainedNote 1 to entry: Ten-fold dilutions are normally used to produce a decimal dilution series, but other ratios
may be required for specific purposes.3.6
specific standard
standard or guidance document describing the examination of a specific product for the detection
or enumeration of a specific microorganism4 Principle
Preparation of initial suspension (3.5) in such a way as obtain as uniform a distribution as possible
of the microorganism contained in the test portion (3.4).Preparation, if necessary, of further dilutions (3.6) 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
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
avoiding the formation of cluster of microorganisms, verifying that at the added concentration
does not alter cell viability.6 Apparatus
Usual microbiological laboratory equipment is below listed:
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 Pet
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