SIST-TS CEN/TS 17716:2023

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17716:2021
01-november-2021
Rastlinski biostimulansi - Določevanje Escherichia coli
Plant biostimulants - Determination of Escherichia coli
Biostimulanzien für die pflanzliche Anwendung - Bestimmung von Escherichia coli
Ta slovenski standard je istoveten z: FprCEN/TS 17716
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17716:2021 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 17716:2021


FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17716
SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION

September 2021
ICS 65.080
English Version

Plant biostimulants - Determination of Escherichia coli
 Biostimulanzien für die pflanzliche Anwendung -
Bestimmung von Escherichia coli


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 17716:2021 E
worldwide for CEN national Members.

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Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
4.1 Qualitative method . 7
4.2 Quantitative method . 7
5 Diluent and culture media . 7
5.1 General . 7
5.2 Broth and culture media in the qualitative method . 8
5.2.1 Enrichment broth . 8
5.2.2 Selective culture media: Tryptone-bile-glucuronic medium (TBX) for isolation of
Escherichia coli . 8
5.3 Diluent and culture media in the quantitative method . 8
5.3.1 Diluent . 8
5.3.2 Culture media: Tryptone-bile-glucuronic medium (TBX) . 8
6 Apparatus and glassware . 8
7 Handling of plant biostimulants products and sampling . 8
8 Procedure . 8
8.1 General . 8
8.2 Qualitative method . 8
8.2.1 General . 8
8.2.2 Solid formulations: Wettable Powder (WP), Water dispersible granules (WDG),
Pellets, granules, microgranules (slow release) formulation . 9
8.2.3 Liquid formulations: water based formulation and oil based (emulsifiable
concentrate - EC) formulations . 9
8.2.4 Incubation of the inoculated enrichment broth . 9
8.2.5 Detection and identification of Escherichia coli . 9
8.3 Quantitative method . 10
8.3.1 Test portion and initial suspension . 10
8.3.2 Serial dilutions . 10
8.3.3 Inoculation (surface plate method) and incubation . 10
8.3.4 Counting the colony-forming units . 11
9 Expression of results . 11
9.1 Expression of results in the qualitative test . 11
9.2 Expression of results in the quantitative test . 11
10 Test report . 12
Annex A (informative) Enrichment broth in the qualitative method . 13
Annex B (informative) Selective agar medium in the qualitative and quantitative
method . 15
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Annex C (informative) Diluent in the quantitative method . 18
Annex D (informative) Neutralization of the antimicrobial properties of the product . 19
Bibliography . 21

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European foreword
This document (FprCEN/TS 17716:2021) 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 Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
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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 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 plants, 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.
Table 1 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
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 gives general guidelines for the detection and identification of the specified
microorganism Escherichia coli in technical and formulated biostimulant products, both in liquid and
solid state, and also the horizontal method for the enumeration of ß–glucuronidase-positive Escherichia
coli in plant biostimulants products (both in liquid and solid state).
The qualitative method described in this document is based on the detection of Escherichia coli in a non-
selective liquid medium (enrichment broth), followed by isolation on a selective agar medium. Other
methods can be appropriate, depending on the level of detection required.
NOTE For the detection of Escherichia coli, subcultures can be performed on non-selective culture media
followed by suitable identification steps (e.g. using identification kits).
The quantitative method described in this document uses a colony-count technique at 44 °C on a solid
medium containing a chromogenic ingredient for detection of the enzyme ß –glucuronidase.
WARNING — Strains of Escherichia coli which do not grow at 44 °C and, in particular, those that are ß -
glucuronidase negative, such as Escherichia coli O157, will not be detected.
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 17708, Plant Biostimulants – Preparation of sample for microbial analysis
1
EN ISO 11133:2014, Microbiology of food, animal feed and water - Preparation, production, storage and
performance testing of culture media (ISO 11133:2014)
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:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1
escherichia coli
gram-negative rod, motile, smooth colonies, member of Enterobacteriaceae
Note 1 to entry: The main characteristics for identification are catalase positive, oxidase negative, fermentation
of lactose, production of indole, growth on selective medium containing bile salts with characteristic colonies.
Note 2 to entry: Escherichia coli can be isolated from moist environmental sources (air, water, soil) and is a faecal
contamination indicator.

1
As amended EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020
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3.2
enrichment broth
non-selective liquid medium containing suitable neutralizers and/or dispersing agents and
demonstrated to be suitable for the product under test
3.3
ß-glucuronidase-positive Escherichia coli
bacteria which at 44 °C form typical blue colony on tryptone-bile-glucuronide medium (TBX) under the
conditions specified in the relative part of this document
3.4
enumeration of ß-glucuronidase-positive escherichia coli
determination of the number of colony-forming-unit (CFU) of ß-glucuronidase-positive Escherichia coli,
per millilitre or per grams of sample, when test and calculations are carried out with the relative part of
this document
4 Principle
4.1 Qualitative method
The first step of the qualitative procedure is to perform an enrichment by using a non-selective broth
medium to increase the number of microorganisms without the risk of inhibition by the selective
ingredients that are present in selective/differential growth media.
The second step of the test (isolation) is performed on a selective medium followed by identification
tests.
The presence or absence of Escherichia coli per gram or per millilitre of sample is calculated
(see Clause 9).
4.2 Quantitative method
In the quantitative method, duplicate plates of tryptone-bile-glucuronic medium (TBX) are inoculated
with the specified quantity of the test sample (if the product is liquid) or the initial suspension (if the
product is solid).
Under the same conditions, using decimal dilutions of the test sample or of the initial suspension, two
plates per dilution are inoculated.
The dishes are incubated for 18 h to 24 h at 44 °C ± 1 °C then examined to detect the presence of colonies
which, from their characteristics, are considered to be ß-glucuronidase-positive Escherichia coli.
The number of colony-forming units (CFU) of ß-glucuronidase-positive Escherichia coli per gram or per
millilitre of sample is calculated (see Clause 9).
5 Diluent and culture media
5.1 General
The following diluents and culture media are suitable for the detection of Escherichia coli and
enumeration of ß-glucuronidase-positive Escherichia coli according to the proper procedure. Other
diluents and culture media may be used if they have been demonstrated to be suitable for use.
Diluents and culture media may be prepared using the descriptions provided or from
reagents/dehydrated culture media, according to the instructions from the manufacturer. The
instructions provided by the supplier of the media/reagents should be followed for storage conditions,
expiry date and use.
NOTE Ready-to-use diluents and media can be used when their composition and/or growth yields are
comparable to those of the formulae given in the present document.
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5.2 Broth and culture media in the qualitative method
5.2.1 Enrichment broth
The enrichment broth is used in the qualitative method to disperse the sample and to increase the initial
microbial population. See Annex A for the list and recipes of the possible enrichment broth.
5.2.2 Selective culture media: Tryptone-bile-glucuronic medium (TBX) for isolation of
Escherichia coli
The selective agar medium is used in the qualitative method for the isolation and identification of
Escherichia coli See Annex B for the list and recipes of the selective agar medium.
5.3 Diluent and culture media in the quantitative method
5.3.1 Diluent
See Annex C for the list and recipes of the possible diluents to be use in the preparation of the initial
suspension and further decimal dilutions.
5.3.2 Culture media: Tryptone-bile-glucuronic medium (TBX)
See Annex B for the list and recipes of the culture medium to be use in the inoculation by plating
technique of the initial suspension and the further decimal dilutions.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware typical of microbiological laboratory according to
FprCEN/TS 17708 shall be used.
7 Handling of plant biostimulants products and sampling
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
Sampling is not part of the method specified in this document (FprCEN/TS 17716): refer to
FprCEN/TS 17702-1.
If necessary, the product to be tested may be equilibrated at room temperature before starting the
analysis.
8 Procedure
8.1 General
According to the aim of the analysis one of the following described methods may be performed.
The qualitative method (see 8.2) allows to evaluate the presence or absence of Escherichia coli in at least
1 g or 1 mL of the product under test.
The quantitative method (see 8.3) allows to determine the number of ß-glucuronidase-positive
Escherichia coli in terms of CFU per g or per mL of the product under test.
8.2 Qualitative method
8.2.1 General
Dispense 25 g or ml of sample in 225 ml of sterile enrichment broth. Note S, the exact weight or volume
of the sample.
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8.2.2 Solid formulations: Wettable Powder (WP), Water dispersible granules (WDG), Pellets,
granules, microgranules (slow release) formulation
The initial suspension (see 8.2) is processed in a stomacher for 2 min at highest speed. Soon after, 10 ml
of this suspension are incubated (see 8.2.3).
8.2.3 Liquid formulations: water based formulation and oil based (emulsifiable concentrate -
EC) formulations
10 ml of the well mixed initial suspension (see 8.2) are sampled and incubated (see 8.2.3).
8.2.4 Incubation of the inoculated enrichment broth
Incubate the initial suspension prepared in broth (see 8.2.2 for solid formulations or 8.2.3 for liquid
formulations) at 32,5 °C ± 2,5 °C for at least 20 h (maximum 72 h).
8.2.5 Detection and identification of Escherichia coli
8.2.5.1 Isolation
Using a sterile loop, streak an aliquot of the incubated enrichment broth (8.2.2) onto the surface of
Tryptone-bile-glucuronic medium to obtain isolated colonies.
Invert the Petri dish and then incubate at 44 °C for 18 h to 24 h. The total incubation time shall not be
longer than 24 h. Check for characteristic colonies (see Table 2).
WARNING — If the presence of stressed cells is suspected, incubate for an initial period of 4 h at 37 °C,
and then raise the incubation temperature to 44 °C for 18 h to 24 h. The incubation temperature shall
not exceed 45 °C.
Table 2 — Morphological characteristics of Escherichia coli on Tryptone-bile-glucuronic agar
medium
Selective medium Characteristic colonial morphology of Escherichia
coli
Tryptone-bile-glucuronic Blue to blue-green
medium
8.2.5.2 Identification of Escherichia coli
8.2.5.2.1 General
In case of doubts about the morphological characteristic grown colonies, proceed to the following tests
for these suspect colonies isolated on the selective agar medium. The presence of Escherichia coli may
be confirmed by other suitable, cultural and biochemical tests.
8.2.5.2.2 Gram’s stain
Perform the test specified in EN ISO 21148. Check for Gram-negative rods (bacilli).
8.2.5.2.3 Culture on levine eosin-methylene blue agar medium (EMB agar medium)
Inoculate the surface of the levine eosin-methylene blue agar medium (see Annex B for recipes) with
suspect isolated colonies grown on TBX agar medium, so that isolated colonies develop. Invert the Petri
dish and then incubate at 32,5 °C ± 2,5 °C for at least 24 h (maximum 48 h).
Check for characteristic colonies (see Annex B).
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8.3 Quantitative method
8.3.1 Test portion and initial suspension
8.3.1.1 General
A representative sample of the product is taken to prepare the initial suspension according to following
procedure which takes into consideration the different formulations of biostimulants based products.
8.3.1.2 Liquid - water based - formulations
Dispense 25 ml of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex C) maintained
at room temperature in a flask and shake for 10 min or more until the distribution is optimal, with a
magnetic stirrer at half speed.
8.3.1.3 Liquid – oil based (emulsifiable concentrate - EC) formulations
Dispense 25 ml or g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex C)
maintained at room temperature in a flask and shake for 10 min or more until the distribution is optimal,
with a magnetic stirrer at half speed.
8.3.1.4 Solid - Wettable Powder (WP) formulations
Dispense 25 g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex C) maintained
at room temperature in a flask and shake for 20 min or more until the distribution is optimal, with a
magnetic stirrer at half speed.
8.3.1.5 Solid - Water dispersible granules (WDG) formulations
Dispense 25 g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex C) maintained
at room temperature in a flask and shake for 40 min or more until the distribution is optimal, with a
magnetic stirrer at half speed. If required help the dispersion of the formulations with other apparatus
such as a stomacher after having sieved (100 mesh sieve) the particles and resuspend them in the same
suspension.
8.3.1.6 Solid – Pellets, granules, microgranules (slow release) formulations
Dispense 25 g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex C) maintained
at room temperature in a sterile bag and disperse them using a stomacher for maximum 2 min and then
repeat 3 times with 5 min interval where the bag is put in water with ice.
8.3.1.7 Solid - substrate
Dispense 25 g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex C) maintained
at room temperature in a flask and shake for 20 min or more until the distribution is optimal, with a
magnetic stirrer at half speed.
8.3.2 Serial dilutions
Additional serial dilutions (e.g. 1:10 dilution) may be performed from the initial suspension using the
same diluent (according to the expected level of contamination of the product). Mix the dilutions in
order to avoid sedimentation of microorganism-containing particles.
Refer to FprCEN/TS 17708 for general rules in the preparation of the decimal dilutions.
8.3.3 Inoculation (surface plate method) and incubation
8.3.3.1 Using a micropipette, transfer and spread over the surface of the medium a measured volume of
not less than 0,1 ml of the initial suspension and/or sample dilutions using a sterile spreader until the
liquid is completely absorbed into the medium.
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For liquid products is possible to inoculate 0,1 mL the undiluted product.
Repeat the procedure with the further decimal dilutions in order to have at least two successive
dilutions, if necessary.
−1
To facilitate enumeration of low populations, volumes of up to 1 ml of a 10 dilution of sample, or of
the test sample if liquid, can be spread over to three plates (dishes 85 mm to 100 mm) or over a single
plate of 140 mm.
It is recommended to perform the counting using at least two Petri dishes (preferably), but it is possible
to use only one Petri dish in case of routine testing, or if counts are performed on successive dilutions
of the same sample or according to previous results or as a confirmation of previously results for the
same sample.
8.3.3.2 Invert the inoculated dishes so that the bottom is uppermost and place them in an incubator set
at 44 °C for 18 h to 24 h. The total incubation time shall not be longer than 24 h.
WARNING — If the presence of stressed cells is suspected, incubate for an initial period of 4 h at 37 °C,
and then raise the incubation temperature to 44 °C for 18 h to 24 h. The incubation temperature shall
not exceed 45 °C.
8.3.4 Counting the colony-forming units
After the specified period of incubation count the typical CFU of -ß-glucuronidase-positive Escherichia
coli in each dish containing less than 150 typical CFU and less than 300 total (typical and non-typical)
CFU.
If they form part of the retained dishes, the dishes containing 0 typical CFU should be taken into
consideration in the different calculation methods defined in FprCEN/TS 17714.
9 Expression of results
9.1 Expression of results in the qualitative test
If the identification of the colonies confirms the presence of this species, express the result as:
“Presence of Escherichia coli in the sample S.”
If no growth after enrichment is observed and/or if the identification of the colonies does not confirm
the presence of this species, express the result as:
“Absence of Escherichia coli in the sample S.”
9.2 Expression of results in the quantitative test
Calculate the number, N, of Escherichia coli ß-glucuronidase-positive present in the sample, S, using:
— m, the arithmetic mean of the counts obtained from the duplicates [Formula (1)],
— c, the number of colonies counted on a single plate [Formula (2)], or
— the weighted mean of the counts obtained from two successive dilutions [Formula (3)], according
to the following formulae:
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m
N= (1)
Vd⋅
c
(2)
N=
Vd⋅
∑ c
N= (3)
V⋅+n1 0,·1nd2
( )
where
m is the arithmetic mean of the counts obtained from the duplicates;
V is the volume of inoculum applied to each dish, in millilitres;
d is the dilution of the initial suspension (see 8.3.1) or for the first counted dilution;
c is the number of colonies counted on a single plate;
is the sum of colonies counted on all the dishes retained from two successive dilution;
∑ c
n1 is the number of dishes counted for the initial suspension (or for the first counted dilution);
n2 is the number of dishes counted for the 1:10 dilution of the initial suspension (or for the
second counted dilution).
Round off the result calculated to two significant figures. For this, if the last figure is below 5, the
preceding figure is not modified; if the last figure is 5 or more, the preceding figure is increased by one
unit. Proceed stepwise until two significant figures are obtained. Note the number, N, obtained.
See FprCEN/TS 17714 for detailed explanation (low counts or counts after confirmation) of the
quantitative results as CFU of -ß-glucuronidase-positive Escherichia coli per g or per mL of product.
10 Test report
The test report shall specify the following information:
a) all information necessary for the complete identification of the plant biostimulants product;
b) the sampling method used, if known;
c) the test method used (qual
...