oSIST prEN 17707:2023
(Main)Plant biostimulants - Determination of the yeast and mould content
Plant biostimulants - Determination of the yeast and mould content
This document specifies a horizontal method for the enumeration of yeasts and moulds present in plant biostimulants intended for use in agriculture, by means of the colony count technique after aerobic incubation at 25 °C ± 2,5 °C.
This document allows the enumeration of yeasts and moulds, in technical and formulated plant biostimulant, both in liquid and solid state. The method is applicable to microbial plant biostimulant except those composed of fungi or yeast to verify that the concentration of yeast and moulds does not exceed the respective limits described in the EU Fertilisers Regulation [1].
If necessary, yeast and mould enumerated can be identified using suitable identification tests.
Pflanzen-Biostimulanzien - Bestimmung des Gehalts an Hefen und Schimmelpilzen
Dieses Dokument legt ein horizontales Verfahren zur Zählung von Hefen und Schimmelpilzen in Pflanzen-Biostimulanzien, die zur Verwendung in der Landwirtschaft bestimmt sind, mithilfe des Koloniezähl-verfahrens nach aerober Bebrütung bei (25 ± 2,5) °C fest.
Dieses Dokument hat die Zählung von Hefen und Schimmelpilzen in technischen und formulierten Pflanzen-Biostimulanzien, die im flüssigen oder im festen Zustand vorliegen können, zum Gegenstand. Das Verfahren ist auf mikrobielle Pflanzen-Biostimulanzien anwendbar, ausgenommen jene, die aus Pilzen oder Hefe bestehen, um zu überprüfen, dass die Konzentration von Hefen und Schimmelpilzen die jeweiligen Grenzwerte, die in der EU Düngeprodukteverordnung [1] angegeben sind, nicht überschreitet.
Gegebenenfalls können die gezählten Hefen und Schimmelpilze durch geeignete Identifizierungsprüfungen identifiziert werden.
Biostimulants des végétaux - Détermination de la teneur en levures et en moisissures
Le présent document spécifie une méthode horizontale pour le dénombrement des levures et des moisissures présentes dans les biostimulants des végétaux destinés à être utilisés en agriculture, par la technique de comptage des colonies après incubation aérobie à 25 °C ± 2,5 °C.
Le présent document permet le dénombrement des levures et des moisissures, dans les biostimulants des végétaux de qualité technique, à la fois à l’état liquide et à l’état solide. La méthode est applicable aux biostimulants microbiens des végétaux, excepté ceux composés de champignons ou de levures, pour vérifier que la concentration des levures et des moisissures ne dépasse pas les limites correspondantes indiquées dans le Règlement UE sur les fertilisants [1].
Si nécessaire, les levures et les moisissures dénombrées peuvent être identifiées par des essais d’identification appropriés.
Rastlinski biostimulanti - Ugotavljanje prisotnosti in števila kvasovk in plesni
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17707:2023
01-maj-2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti in števila kvasovk in plesni
Plant biostimulants - Determination of the yeast and mould content
Pflanzen-Biostimulanzien - Bestimmung des Gehalts an Hefen und Schimmelpilzen
Biostimulants des végétaux - Détermination de la teneur en levures et en moisissures
Ta slovenski standard je istoveten z: prEN 17707
ICS:
65.080 Gnojila Fertilizers
oSIST prEN 17707:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17707:2023
DRAFT
EUROPEAN STANDARD
prEN 17707
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2023
ICS 65.080 Will supersede CEN/TS 17707:2022
English Version
Plant biostimulants - Determination of the yeast and
mould content
Biostimulants des végétaux - Détermination de la Pflanzen-Biostimulanzien - Bestimmung des Gehalts an
teneur en levures et en moisissures Hefen und Schimmelpilzen
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 455.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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.
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 European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17707:2023 E
worldwide for CEN national Members.
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Contents Page
European foreword . 4
Introduction . 5
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Principles . 8
4.1 General . 8
4.2 Plate count method . 8
5 Diluent and culture media . 8
5.1 General . 8
5.2 Diluent . 8
5.3 Culture media . 9
6 Apparatus and glassware . 9
7 Handling of plant biostimulants and sampling . 9
8 Procedure . 9
8.1 General . 9
8.2 Test portion and initial suspension . 9
8.2.1 General . 9
8.2.2 Liquid - water based - formulations . 9
8.2.3 Liquid – oil based (emulsifiable concentrate - EC) formulations . 10
8.2.4 Solid - Wettable Powder (WP) formulations . 10
8.2.5 Solid - Water dispersible granules (WDG) formulations . 10
8.2.6 Solid – Pellets, granules, microgranules (slow release) formulations . 10
8.2.7 Solid - substrate . 10
8.3 Serial dilutions . 10
8.4 Plate-count methods . 10
8.4.1 Pour-plate method . 10
8.4.2 Surface spread method . 11
8.4.3 Incubation . 11
9 Counting of colonies . 11
10 Expression of results . 12
10.1 Method of calculation . 12
10.2 Interpretation . 13
11 Test report . 13
Annex A (informative) Diluent . 15
A.1 General . 15
A.2 Phosphate Buffered Saline (PBS). 15
A.2.1 Composition . 15
A.2.2 Preparation . 15
Annex B (normative) Culture media. 16
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B.1 General . 16
B.2 Sabouraud dextrose chloramphenicol agar medium (SDCA) . 16
B.2.1 Composition . 16
B.2.2 Preparation . 16
B.3 Potato dextrose agar (PDA) medium with antibiotics . 17
B.3.1 Composition . 17
B.3.2 Preparation . 17
B.4 Glucose -peptone (GP) agar medium with antibiotics . 17
B.4.1 Composition . 17
B.4.2 Preparation . 17
B.5 Malt extract agar (MEA) medium with antibiotics . 18
B.5.1 Composition . 18
B.5.2 Preparation . 18
Annex C (informative) Interlaboratory study . 19
C.1 Materials used in the interlaboratory study . 19
C.2 Results of the interlaboratory study . 20
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 . 22
Bibliography . 23
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European foreword
This document (prEN 17707:2023) 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 CEN Enquiry.
This document will supersede CEN/TS 17707:2022.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of EU
Directive(s) / Regulation(s).
For relationship with EU Directive(s) / Regulation(s), see informative Annex ZA, which is an
integral part of this document.
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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 SR M/564 and M/564 Amd1, also contributes to the
Communication on “Innovating for Sustainable Growth: A Bio economy for Europe”. The Working
Group 5 “Labelling and denominations”, was created to develop a work program as part of this
request. The technical committee CEN/TC 455 “Plant Biostimulants” was established to carry out
the work program that will prepare a series of standards. The interest in biostimulants has
increased significantly in Europe as a valuable tool to use in agriculture. Standardization was
identified as having an important role in order to promote the use of biostimulants. The work of
CEN/TC 455 seeks to improve the reliability of the supply chain, thereby improving the confidence
of farmers, industry, and consumers in biostimulants, and will promote and support
commercialisation of the European biostimulant industry.
Biostimulants used in agriculture can be applied in multiple ways: on soil, on plant, as seed
treatment, etc. A microbial plant biostimulant consists of a microorganism or a consortium of
microorganisms, as referred to in Component Material Category 7 of Annex II of the EU Fertilising
Products Regulation.
This document is applicable to all microbial biostimulants in agriculture.
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
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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 a horizontal method for the enumeration of yeasts and moulds present
in plant biostimulants intended for use in agriculture, by means of the colony count technique
after aerobic incubation at 25 °C ± 2,5 °C.
This document allows the enumeration of yeasts and moulds, in technical and formulated plant
biostimulant, both in liquid and solid state. The method is applicable to microbial plant
biostimulant except those composed of fungi or yeast to verify that the concentration of yeast and
moulds does not exceed the respective limits described in the EU Fertilisers Regulation [1].
If necessary, yeast and mould enumerated can be identified using suitable identification tests.
2 Normative references
There are no normative references in this document.
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
yeast
mesophilic aerobic microorganism which, using mycological agar medium under the conditions
described in this document, develops matt or shiny round colonies (3.3) on the surface of the
medium, usually having a regular outline and a more or less convex surface
3.2
mould
mesophilic aerobic filamentous microorganism which, on the surface of mycological agar medium
under the conditions described in this document, usually develops flat or fluffy spreading colonies
(3.3) often producing spores or conidia
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, 3.119 [4]]
3.4
product
portion of an identified plant biostimulant product received in the laboratory for testing
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3.5
sample
portion of the product (3.4) 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
3.7
sample dilution
dilution of the initial suspension (3.6)
4 Principles
4.1 General
This method aims enumeration of colonies on a selective agar medium.
4.2 Plate count method
The plate count consists in the following steps:
— preparation of poured plates, or spread plates using a specific culture medium. Depending on
the expected number of colonies, a specified quantity of the sample (if the product is liquid),
or of an initial suspension (in the case of other products), or decimal dilutions of the
sample/suspension are inoculated;
— aerobic incubation of the plates at 25 °C ± 2,5 °C for 3 days to 5 days;
— calculation of the number of colony-forming units (CFU) of yeasts and moulds per gram or
per millilitre of sample from the number of colonies obtained on plates chosen at dilution
levels producing countable colonies. Moulds and yeasts are counted separately, if necessary.
NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C, for 5 days to 7 days, using the culture
medium without antibiotic. If necessary, to distinguish yeast colonies from bacterial colonies, the identity
of any doubtful colonies is confirmed by examination with a binocular magnifier or microscope.
5 Diluent and culture media
5.1 General
The following diluents and culture media are suitable for enumeration of yeast and moulds. Other
diluents and culture media may be used if they have been demonstrated to be suitable for use.
Diluent and culture media may be prepared using the descriptions provided in this document or
from dehydrated culture media, according to the instructions from the manufacturer. The
instructions provided by the supplier should be followed.
NOTE Ready-to-use media can be used when their composition and/or growth yields are comparable
to those of the formulae given in the present document.
5.2 Diluent
See Annex A for the recipe of the diluent to be use in the preparation of the initial suspension and
further decimal dilutions.
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5.3 Culture media
See Annex B for the list and recipes of the possible media to be use in the inoculation by plating
technique of the initial suspension and the further decimal dilutions.
6 Apparatus and glassware
Use the laboratory equipment, apparatus and glassware typical of microbiological laboratory. See
1
prEN 17708:— for a detailed list.
7 Handling of plant biostimulants 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.
2
Sampling is not part of the method specified in this document: see prEN 17702-1 .
If necessary, the product to be tested may be equilibrated at room temperature before starting
the analysis.
8 Procedure
8.1 General
Use sterile material, equipment and aseptic techniques to prepare the sample, initial suspension
and dilutions. In the case of the preparation of the initial suspension, the time that elapse between
the end of the preparation and the moment the inoculum comes into contact with the culture
medium shall not exceed 45 min, unless specifically mentioned in the established protocols or
documents.
Appropriate negative controls (diluent-only) should be run concurrently with the sample serial
dilutions. This step can be performed by incubating an aliquot of the diluent (i.e. 9 ml) at the same
conditions of the test to verify the absence of turbidity to assess the sterility of the diluent. Or,
alternatively, can be spread 1 ml of the diluent over a surface of the same agar medium used in
the analysis. The plate is incubated at the same conditions of the test to verify the absence of
growth to assess the sterility of the diluent.
8.2 Test portion and initial suspension
8.2.1 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:
8.2.2 Liquid - water based - formulations
Dispense 25 ml of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex A)
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.
1
Under preparation.
2
Under preparation.
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8.2.3 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 A)
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.
8.2.4 Solid - Wettable Powder (WP) formulations
Dispense 25 g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex A)
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.
8.2.5 Solid - Water dispersible granules (WDG) formulations
Dispense 25 g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex A)
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.
8.2.6 Solid – Pellets, granules, microgranules (slow release) formulations
Dispense 25 g of' sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex A)
maintained at room temperature in a sterile bag and disperse them using a stomacher for
maximum 2 minutes and then repeat 3 times with 5 minutes interval where the bag is put in water
with ice.
8.2.7 Solid - substrate
Dispense 25 g of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex A)
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 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.
3
See prEN 17708:— for general rules in the preparation of the decimal dilutions.
8.4 Plate-count methods
8.4.1 Pour-plate method
In Petri dishes 85 mm to 100 mm in diameter add, using a sterile pipette, 1 ml of the initial
suspension and or sample dilutions and pour 15 ml to 20 ml of the melted Agar medium kept in a
water bath at no more than 48 °C.
For liquid products is possible to inoculate also 1ml of undiluted product.
Mix the sample with the medium carefully rotating or tilting the plates. Allow the mixture in the
Petri dishes to solidify on a horizontal surface at room temperature.
It is recommended to perform the counting using at least two Petri dishes.
3
Under preparation.
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8.4.2 Surface spread method
In Petri dishes 85 mm to 100 mm in diameter put 15 ml to 20 ml of melted agar medium kept in a
water bath at no more than 48 °C.
Allow plates to cool and solidify, for example in a microbiological cabinet or in an incubator.
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.
For liquid products is possible to inoculate 0,1 ml the undiluted product.
−1
To facilitate enumeration of low populations of yeasts and moulds, 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.
8.4.3 Incubation
Incubate the prepared plates (8.3.1 or 8.3.2) aerobically, lids uppermost, in an upright position in
the incubator at 25 °C ± 2,5 °C for 3 days to 5 days, or use the alternative condition (4.2). If
necessary, leave the agar plates to stand in diffuse daylight for 1 day to 2 days.
After incubation, the dishes shall, if possible, be examined immediately. Alternatively, they may
be stored, un less otherwise specified, for up to a maximum of 24 h in the refrigerator at
5 °C ± 3 °C.
NOTE 1 In certain cases, where there is a potential confusing particles from the product with counted
colonies, it can be useful to prepare duplicate dishes containing the same sample dilutions and agar medium
which are stored in the refrigerator for comparison with incubated dishes.
NOTE 2 An intermediate check can be performed where both yeast and mould are suspected or when
fast-growing moulds are present.
9 Counting of colonies
After incubation, count the colonies in the Petri dishes containing 15 colonies to 150 colonies, or
less than 15 colonies if are counted in the plates of the undiluted product or of the initial
suspension.
NOTE 1 Enumeration methods for yeasts and especially moulds are imprecise because they consist of a
mixture of mycelium and asexual and sexual spores. Numbers of colony-forming units depend on the degree
of fragmentation of mycelium and the proportion of spores able to grow on the plating medium.
NOTE 2 Nonlinearity of counts from dilution plating often occurs, i.e. 10-fold dilutions of samples often
do not result in 10-fold reductions in numbers of colonies recovered on plating media. This has been
attributed to fragmentation of mycelia and breaking of spore clumps during dilution in addition to
competitive inhibition when large numbers of colonies are present on plates.
CAUTION — The spores of moulds disperse in the air with great facility: handle the Petri
dishes with care to avoid development of satellite colonies which would give an
overestimation of population in the sample.
If necessary, carry out an examination with a binocular magnifier or with a microscope in order
to distinguish between cells of yeasts or moulds and bacteria from colonies.
Count the colonies of yeasts and the colonies of moulds separately, if necessary.
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For identification of yeast and moulds, select areas of fungal growth and remove for high
microscopic examination or inoculate on suitable isolation or identification media.
10 Expression of results
10.1 Method of calculation
Calculate the number, N, of microorganisms 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:
m
N= (1)
Vd⋅
c
N= (2)
Vd⋅
∑ c
N= (3)
V⋅+n1 0,1n2 ⋅ d
( )
where
m is the arithmetic mean of the counts obtained from the duplicates;
V is the volume of i
...
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