SIST-TS CEN/TS 17714:2023

SLOVENSKI STANDARD
SIST-TS CEN/TS 17714:2023
01-februar-2023
Rastlinski biostimulanti - Določevanje 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: CEN/TS 17714:2022
ICS:
65.080 Gnojila Fertilizers
SIST-TS CEN/TS 17714:2023 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 17714:2023


CEN/TS 17714
TECHNICAL SPECIFICATION

SPÉCIFICATION TECHNIQUE

March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
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 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 17714:2022 E
worldwide for CEN national Members.

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Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principe of the method . 7
5 Sampling . 7
6 Preparation of sample for microbial analysis . 7
7 Method for enumeration of microorganism . 7
7.1 General. 7
7.2 Enumeration using a solid media. 8
7.3 Enumeration and quantification using a liquid medium . 16
8 Expression of results . 18
9 Test report . 18
10 Quality assurance . 18
Bibliography . 19

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European foreword
This document (CEN/TS 17714:2022) 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 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
- 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 determination of microorganism concentration in a
sample of biostimulant product.
The specific concentrations of microorganisms required in specific standard methods take precedence
over the general rules listed in this document.

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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 specifies general rules to determine the concentration of microorganisms present in plant
biostimulant products.
The method is applicable to microbial plant biostimulants for verifying that the concentration of
microorganisms does not exceed the respective limits outlined in the EU Regulation on Fertilising
Products [1].
This horizontal method might not be appropriate in every detail for certain products. In this case, it is
necessary to refer to the methodology of specific determination and quantification of the
microorganisms.
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 17708, Plant biostimulants — Preparation of sample for microbial analysis
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
plant biostimulant
product stimulating plant nutrition processes independently of the product's nutrient content with the
sole aim of improving one or more of the following characteristics of the plant or the plant rhizosphere:
a) nutrient use efficiency;
b) tolerance to abiotic stress;
c) crop quality traits;
d) availability of confined nutrients in soil or rhizosphere
3.2
microorganism
entity of microscopic size, encompassing bacteria, fungi, protozoa and viruses
[SOURCE: EN ISO 11139:2018, 3.176]
3.3
colony
localized visible accumulation of microbial mass (such as prokaryotes, bacteria, micromycetes, yeast 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, micro colonies from nearby viable particles, before becoming visible, fuse into one
macro colony. The number of visible colonies is, therefore, usually and underestimate of the number of viable
particles.
[SOURCE: ISO 6107-6:2021, [5], modified]
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3.4
product
portion of an identified plant biostimulant product received in the laboratory for testing
3.5
sample
portion of the product (3.4) (at least 1 g or 1 ml) that is used in the test to prepare the initial suspension
3.6
initial suspension
suspension (or solution) of the sample (3.5) in a defined volume of an appropriate diluent
4 Principe of the method
The method of determining the concentration of microorganisms has been developed to provide a
general method for the enumeration of the microorganisms present in the plant biostimulant product.
The results are expressed as the number of active units by volume or weight, 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 CEN/TS 17702-1). 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
Prepare the test sample from the laboratory sample in accordance with CEN/TS 17708. 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 enumeration of microorganisms
7.1 General
When assessing the microbiological quality of biostimulants products, 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. 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. MPN) to improve the statistical reliability
of the results.
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7.2 Enumeration using a solid media
7.2.1 General
The different steps for enumeration using a solid media (number of petri dishes/dilution, Pour plate
techniques, surface inoculation, Incubation) are described in the different chapters below and/or specific
standard 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 biostimulant(s) product, 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 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 media, hold the bottle in a
near horizontal position.
Also avoid setting down the bottle between pouring steps. Pour molten agar medium at 44 °C to 47 °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 thickness) within 15 min of inoculation (to avoid aggregation of
colonies). Avoid pouring the molten medium directly on to 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 colony.
Use pre-poured plates, of at least 3 mm thickness of the agar medium, that are level and free from air
bubbles and surface moisture.
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To facilitate uniform spreading, the surface of solidified agar should be dried in accordance with
EN ISO 11133:2014 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 material and 10 in the case of other sample
material) 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 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.
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 °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 high for aerobic incubation and 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.
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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 may 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.
7.2.6.2 Method of calculation: general case (counting of total colonies or typical colonies)
For a result to be valid, it is generally considered necessary to count the colonies on at least one dish
containing at least 10 colonies [total colonies, typical colonies or colonies complying with identification
or confirmation criteria (see 7.2.6.3).
Calculate the number N of microorganisms present in the test sample as a weighted mean from two
successive dilutions using Formula (1):
C
∑
(1)
N=
Vd ××1,1
where
ƩC is the sum of the colonies counted on the two dishes retained from two successive dilutions, at
least one of which contains a minimum of 10 colonies;
V is the volume of inoculum placed in each dish, in millilitres;
d is the dilution corresponding to the first dilution retained [d = 1 when the undiluted liquid
product (test sample) is retained].

If more than one dilution is used, the ratio between the colony count of dilution d and the colony count
2
of dilution d is expected to be 10 %. The upper and lower limits should be specified by the laboratory
1
for the colony count of dilution d
2.
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EXAMPLE If colony count of dilution d1 is 250, the colony count of dilution d2 should not be less than 13
(5,2 %) and not be more than 39 (15,6 %).
Round off the calculated result to two significant figures. When doing this, if the third figure is less than
5, do not modify the preceding figure; if the third figure is greater than or equal to 5, increase the
preceding figure by one unit.
Express the result preferably as a number between 1,0 and 9,9 multiplied by the appropriate power of
10, or a whole number with two significant figures.
Report the result as the number N of microorganisms per millilitre (liquid products) or per gram (other
products).
7.2.6.3 Method of calculation: Case after identification or confirmation
7.2.6.3.1 General
When the method used requires identification or confirmation, a given number A (generally 5) of
presumptive colonies is identified from each of the dishes retained for the colony counting. After
identification or confirmation, calculate, for each of the dishes, the number a of colonies complying with
identification or confirmation criteria, using Formula (2):
b
a= xC
(2)
A
where
b is the number of colonies complying with identification or confirmation criteria among the
identified colonies A;
C is the total number of presumptive colonies counted on the dish.
Round off the calculated result to the nearest whole number. When doing this, if the first figure after the
decimal point is less than 5, do not modify the preceding figure; if the first figure after the decimal point
is greater than or equal to 5, increase the preceding figure by one unit.
Calculate the number N, N or N ' of identified or confirmed microorganisms present in the test sample
E
by replacing C by a in the formula given in 7.2.6.2 or by replacing C by a in the formula given in
∑ ∑
7.2.6.4.1 and 7.2.6.5.3. Round off the result as specified in 7.2.6.2.
Express the result as specified in 7.2.6.2, 7.2.6.4.1 and 7.2.6.5.3, respectively.
7.2.6.3.2 Example
Counting has produced the following results:
−3
— at the first dilution retained (10 ): 66 colonies;
−4
— at the second dilution retained (10 ): 4 colonies.
Testing of selected colonies was carried out:
— of the 66 colonies, 8 colonies were tested, 6 of which complied with the criteria, hence a = 50;
— of the 4 colonies, all 4 complied with the criteria; hence a = 4.
∑ a 50+ 4 54

N  49 090
−−33
V x 1,1 xd
11 xx ,11 0 1,11 x 0
11
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4
Rounding off the result as specified in 7.2.6.2, the number of microorganisms is 49 000 or 4,9 × 10 per
millilitre or per gram of product.
7.2.6.4 Method of calculation: estimated counts
7.2.6.4.1 Case of two dishes (test sample or initial suspension or first dilution) contains less than
10 colonies
Requirements and recommendations of 7.2.6.2 concerning the lower limit of determination are
applicable.
If each of the two dishes from the test sample (liquid products), or from the initial suspension (other
products), or from the first dilution inoculated or retained, contains less than 10 colonies (total colonies,
typical colonies or colonies complying with identification or confirmation criteria) and the set of the two
dishes contains at least 4 colonies, calculate the estimated number NE of microorganisms present in the
test sample as an arithmetical mean of the colonies counted on the two dishes using Formula (3):
∑ C
N= (3)
V
nd
where
ΣC is the sum of the colonies counted on the two dishes;
V is the volume of inoculum applied to each dish, in millilitres;
n is the number of dishes retained (in this case, n = 2);
d is the dilution factor of the initial suspension or of the first dilution inoculated or retained
[d = 1 when the undiluted liquid product (test sample) is used].
Round off the result as recommended in 7.2.6.2.
Express the result as follows:
— estimated number N of microorganisms per millilitre (liquid products) or per gram (other
E
products).
7.2.6.4.2 Example
Counting has produced the following results:
−2
— at the first dilution (10 ) retained, 8 and 9 colonies were counted
8+ 9 17
N 850
−2
0,02
1 xx 2 10
By rounding off the result as recommended in 7.2.6.2, the estimated number N of microorganisms is 850
E
2
or 8,5 × 10 per millilitre or per gram of product.
7.2.6.4.3 Case of two dishes (test sample or initial suspension or first dilution) contains no
colonies
If the two dishes from the test sample (liquid products) or from the initial suspension (other products)
or from the first dilution inoculated or retained, do not contain any colonies, express the result as follows:
“less than 1/Vd of microorganisms per millilitre” (liquid products)
“less than 1/Vd microorganisms per gram” (other products)
12
== =

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where
d is the dilution factor of the initial suspension or of the first dilution inoculated or retained
(d = 100 = 1 where the directly inoculated test sample of liquid product is retained);
V is the volume of the inoculum used in each dish, in millilitres.
7.2.6.4.4 Special cases (Counting of typical or presumed suspect colonies)
7.2.6.4.4.1 Case 1
If the number of typical and atypical colonies for th
...