SIST EN 17714:2025

SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17714:2023
Rastlinski biostimulanti - Določanje koncentracije mikroorganizmov
Plant biostimulants - Determination of microorganisms' concentration
Pflanzen-Biostimulanzien - Bestimmung der Konzentration von Mikroorganismen
Biostimulants des végétaux - Détermination de la concentration en microorganismes
Ta slovenski standard je istoveten z: EN 17714:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17714
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17714:2022
English Version
Plant biostimulants - Determination of microorganisms'
concentration
Biostimulants des végétaux - Détermination de la Pflanzen-Biostimulanzien - Bestimmung der
concentration en microorganismes Konzentration von Mikroorganismen
This European Standard was approved by CEN on 26 August 2024.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17714:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle of the method . 6
5 Sampling . 7
6 Preparation of sample for microbial analysis . 7
7 Method for the enumeration of microorganisms . 7
7.1 General. 7
7.2 Enumeration using solid media . 7
7.2.1 General. 7
7.2.2 Number of Petri dishes per dilution . 7
7.2.3 Pour plate techniques . 8
7.2.4 Surface inoculation . 8
7.2.5 Incubation. 9
7.2.6 Calculation and expression of results obtained with solid media . 9
7.3 Enumeration and quantification using a liquid medium . 15
7.3.1 Principle . 15
7.3.2 Inoculation . 16
7.3.3 Choice of inoculation system . 16
8 Expression of results . 17
9 Test report . 18
10 Quality assurance . 18
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 . 19
Bibliography . 20
European foreword
This document (EN 17714:2024) has been prepared by Technical Committee CEN/TC 455 “Plant
biostimulants”, the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes CEN/TS 17714:2022.
— the European foreword and the Introduction have been updated;
— normative references have been updated;
— Annex ZA has been added.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
For the relationship with EU Legislation, see informative Annex ZA, which is an integral part of this
document.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
Introduction
The European Committee for Standardization (CEN) was requested by the European Commission (EC) to
draft European Standards or European Standardization deliverables to support the implementation of
Regulation (EU) 2019/1009 of 5 June 2019 [1] laying down rules on the making available on the market
of EU fertilising products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as SR M/564 and relevant amendments, also contributes to the
Communication on “Innovating for Sustainable Growth: A Bio economy for Europe”. The interest in plant
biostimulants has increased significantly in Europe as a valuable tool to use in agriculture.
Standardization was identified as having an important role in order to promote the use of biostimulants.
The work of CEN/TC 455 seeks to improve the reliability of the supply chain, thereby improving the
confidence of farmers, industry, and consumers in biostimulants, and will promote and support
commercialisation of the European biostimulant industry.
WARNING — Persons using this document should be familiar with normal laboratory practice. This
document does not purport to address all of the safety problems, if any, associated with its use. It is the
responsibility of the user to establish appropriate safety and health practices and to ensure compliance
with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably trained staff.
1 Scope
This document specifies the general rules to determine the concentration of microorganisms present in
plant biostimulants expressed as the number of active units per volume or weight, or in any other manner
that is relevant to the microorganism, e.g. colony forming units per gram (cfu/g).
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of
the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers,
Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest
% in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not
the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend
of fertilizing products is composed of components in equal quantity or in case the component EU
fertilising products used for the blend have identical formulations , the user decides which standard to
apply.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 17708:2024, Plant biostimulants — Preparation of sample for microbial analysis
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp;
— IEC Electropedia: available at https://www.electropedia.org/
3.1
plant biostimulant
product stimulating plant nutrition processes independently of the product’s nutrient content with the
sole aim of improving the nutrient use efficiency, the tolerance to abiotic stress, the quality traits of the
plant or the plant rhizosphere or the availability of confined nutrient in soil or rhizosphere
[SOURCE: EN 17724:2024, 3.1.1.8 [3], modified – editorial changes]
3.2
microorganism
any microbiological entity, including lower fungi, bacteria and viruses, cellular or non-cellular, capable of
replication or of transferring genetic material, including dead or empty cells, microorganisms and non-
harmful elements of the media on which they were produced
[SOURCE: EN 17724:2024, 3.2.2 [3], modified – “including dead or empty cells, microorganisms and non-
harmful elements of the media on which they were produced” added]

An example of such a blend is a product with 2 claimed functions consisting of a non-microbial plant biostimulant
and an organic fertilizer composed of 1kg/kg of plant biostimulant from seaweed.
3.3
colony
localized visible accumulation of microbial mass (such as prokaryotes, bacteria, micromycetes, yeasts and
fungi) or organisms (such as Dreissena species) developed on or in a solid nutrient medium from a viable
particle or organism
Note 1 to entry: Frequently, microcolonies from nearby viable particles, before becoming visible, fuse into one
macrocolony. The number of visible colonies is, therefore, usually an underestimate of the number of viable
particles.
[SOURCE: ISO 6107:2021, 3.119 [4]]
3.4
product
portion of an identified plant biostimulant received in the laboratory for testing
3.5
laboratory sample

final sample intended for laboratory testing and in relation to microbiological testing, each separate
segment sample intended for laboratory testing
3.6
initial suspension
suspension (or solution) of the laboratory sample (3.5) in a defined volume of an appropriate diluent
3.7
Unit Potential Mycorrhizal
UPM
unit of counting for mycorrhiza
where
U is unit, spore or propagule of any type able to initiate mycorrhiza formation in a host plant’s root;
P is potential, since the development of the symbiosis depend on different factors (soil, plant,
agriculture practises, competition with other soil borne microorganisms, etc.);
M is mycorrhizal, since the inoculum is able to synthesize new mycorrhizae in association with plant
roots depending on factors previously cited
EXAMPLE UPM per gram (% spores, % propagules) (in vivo, in vitro).
4 Principle of the method
The method for determining the concentration of microorganisms has been developed to provide a
general method for the enumeration of the microorganisms present in the plant biostimulant. The results
are expressed as the number of active units by volume or mass, or in any other way relevant to the
microorganism, for example colony forming units per gram or the UPM for Mycorrhizae.
5 Sampling
Sampling is not part of the method specified in this document (see EN 17702-1:2024 [2]). 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.
6 Preparation of sample for microbial analysis
The test sample shall be prepared from the laboratory sample in accordance with EN 17708:2024. If there
is no specific International or European Standard, it is recommended that the parties concerned come to
an agreement on this subject.
7 Method for the enumeration of microorganisms
7.1 General
When assessing the microbiological quality of plant biostimulants, it is often not enough to know only
which microorganisms are present. In most cases, the quantitative aspect is equally important, which
brings about the need to enumerate microorganisms [5]. This may be achieved in various ways: through
direct examination (microscopy), by inoculating solid or liquid media. However, this document only
covers enumeration using solid and liquid media.
Enumeration on solid media is based on the capacity of many microorganisms to produce colonies in or
on agar media that can be recognized as such with the naked eye or with the aid of a simple magnifying
glass.
If the level of bacteria is expected to be very low (less than 10 colonies in or on a plate at the lowest
dilution), enumeration using liquid media is recommended (e.g. Most Probal Number (MPN)) to improve
the statistical reliability of the results.
7.2 Enumeration using solid media
7.2.1 General
The different methods for enumeration using solid media (number of Petri dishes/dilutions, pour plate
techniques, surface inoculation, incubation) are described in the different chapters below and/or in
specific standards for microorganism detection/determination.
Petri dishes should be labelled with the sample number, dilution, date and any other desired information.
Dilutions should be selected to ensure that plates containing the appropriate number of colonies are
obtained (see 7.2.6.1) and to overcome any possible inhibitory properties.
Use a separate sterile pipette for transfers from each dilution, except if working from the highest dilution
to the lowest dilution.
7.2.2 Number of Petri dishes per dilution
For enumeration techniques in plant biostimulant(s), one plate per dilution shall be used with at least
two successive dilutions. Two plates per dilution may also be used to improve reliability.
If only one dilution is used, then two plates of this dilution shall be used to improve reliability of the
results.
For laboratories that do not operate under quality assurance principles, two plates per dilution shall be
used to improve reliability of the results.
7.2.3 Pour plate techniques
Withdraw the defined volumes of the dilution to be examined, touching the tip of the pipette against the
side of the tube to remove excess liquid adhering to the outside. Lift the sterile Petri dish lid just high
enough to insert the pipette, then dispense the contents.
After removing the tempered agar medium from the water bath, blot the bottle dry with a clean towel to
prevent water from contaminating the plates. Avoid spilling the medium on the outside of the container
or on the inside of the plate lid when pouring. To avoid contamination of the medium, hold the bottle in
a near horizontal position.
Also avoid setting down the bottle between pouring steps. Pour the agar medium at (44 ± 1) °C to
(47 ± 1) °C into each Petri dish (generally 18 ml to 20 ml of agar in 90 mm Petri dishes and 45 ml to 50 ml
in 140 mm Petri dishes, to obtain at least 3 mm of thickness) within 15 min of inoculation (to avoid
aggregation of colonies). Avoid pouring the molten medium directly on the inoculum. Immediately mix
the molten medium and the inoculum carefully so as to obtain a homogeneous distribution of the
microorganisms within the medium, e.g. by gently moving the dish backwards and forwards, from side
to side and in a circular direction. Allow to cool and solidify by placing the Petri dish on a cool horizontal
surface (the solidification time of the agar shall not exceed 10 min).
7.2.4 Surface inoculation
7.2.4.1 General
Methods of plating designed to produce only surface colonies on agar plates have certain advantages. The
morphology of surface colonies is easily observed, improving the analyst’s ability to distinguish between
different types of colonies.
Use pre-poured plates, of at least 3 mm of thickness of the agar medium, that are level and free from air
bubbles and surface moisture.
To facilitate uniform spreading, the surface of solidified agar should be dried in accordance with
EN ISO 11133:2014 [5] or as specified in the relevant International Standard so that the inoculum is
absorbed within 15 min.
7.2.4.2 Spreading-spatula method
Using a sterile pipette, transfer the inoculum (usually 0,1 ml or 0,5 ml) of the liquid test sample, or of the
initial suspension in the case of other samples, to the agar plate (90 mm or 140 mm in diameter,
respectively). Repeat this step for the next decimal dilution (the colonies to be counted will then be
−1 −2
present in a dilution step of 10 in the case of liquid sample materials and 10 in the case of other sample
materials) and, if necessary, repeat for further decimal dilutions.
The limit of enumeration can be lowered by a factor of 10 by inoculating 1,0 ml of the test sample if liquid,
or 1,0 ml of the initial suspension for other products, either on the surface of one large agar plate
(140 mm) or on the surface of three small agar plates (90 mm). In both cases, if only one dilution is used,
prepare duplicates by using two large plates or six small ones.
Using a spreading spatula made of glass, plastic or steel (for example made from a glass rod and shaped
like a hockey stick of about 3,5 mm in diameter and 20 cm long, bent at right angles at about 3 cm from
one end and flattened at the ends by heating), spread the inoculum as quickly as possible evenly over the
agar surface without touching the side walls of the Petri dish. Allow the inoculum to absorb with the lids
in place for about 15 min at room temperature.
In certain cases (as stated in the relevant International Standard), the inoculum may be deposited on a
membrane then spread as described previously.

As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020
7.2.5 Incubation
Unless otherwise stated in specific standards, invert dishes once they have been inoculated, and place
them quickly in the incubator set at the appropriate temperature. If excessive dehydration occurs (e.g. at
(55 ± 1) °C or in the event of strong air circulation), wrap the dishes loosely in plastic bags prior to
incubation or use any similar system of equivalent efficiency.
During the incubation period, minor variations in the incubation temperature may be unavoidable and
acceptable, for example during the usual operations of loading or unloading the incubator, but it is
important that these periods are kept to a minimum. The duration of these variations should be
monitored to ensure that they do not have a significant effect on the result.
It may sometimes be useful to laboratory operations to refrigerate inoculated dishes before incubation
for no more than 24 h. If this is done, the laboratory shall ensure that this practice does not affect the
resulting counts.
Generally, Petri dishes should be stacked no more than six heights for aerobic incubation and the stacks
should be separated from each other and from the incubator walls by at least 25 mm. However, higher
stacks with less spacing may be acceptable in incubators fitted with air circulation systems; in this case,
the temperature distribution should be verified. After incubation, the dishes should normally be
examined immediately. They may, however, be stored, unless otherwise specified in specific standards,
for up to 48 h in a refrigerator. Refrigerated storage is only acceptable if it has been shown to have no
effect on the numbers, appearance or the subsequent confirmation of the colonies. With certain media
containing indicator dyes, refrigerated plates should be allowed to equilibrate at room temperature
before examining, to ensure that the correct colour is regained.
7.2.6 Calculation and expression of results obtained with solid media
7.2.6.1 Counting of colonies
Following the period of incubation stated in the specific standard, count the colonies (total colonies,
typical colonies or presumed suspect colonies) for each dish containing up to and including 300 colonies
(or any other number stated in the specific standard).
In this subclause, the cases dealt with correspond to the following general cases:
— inoculation of one 90 mm-diameter Petri dish per dilution, and at least two successive dilutions are
performed;
— maximum countable number for the total colonies present: 300 per dish;
— maximum total number of colonies (typical and atypical) present on a dish when counting typical or
presumptive colonies: preferably 300 per dish;
— maximum countable number for typical or presumptive colonies: 150 per dish;
— number of presumptive colonies inoculated for identification or confirmation in each dish retained:
in general 5.
These figures shall be defined in the specific standards.
The methods of calculation defined in the following are for the cases which occur most frequently when
tests are carried out in accordance with good laboratory practice. Special cases can occasionally occur
(e.g. the number of colonies in two dishes with the same dilution may show significant discrepancy or the
ratio of the dilution factors used for two successive dilutions may be very different) and it is therefore
necessary for the counting results obtained to be examined and interpreted by a qualified microbiologist
and, if necessary, rejected.
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