oSIST prEN 17715:2023
(Main)Plant biostimulants - Detection of Shigella spp.
Plant biostimulants - Detection of Shigella spp.
This European Standard establishes the methodology for the determination of Shigella spp. in plant biostimulant products in accordance to the future Regulation of EU fertilising products.
Pflanzen-Biostimulanzien - Nachweis von Shigella spp.
Dieses Dokument liefert ein Verfahren zum Nachweis, dass das Pathogen Shigella spp. in mikrobiellen Pflanzen-Biostimulanzien nicht in einer Konzentration vorhanden ist, die die jeweiligen Grenzwerte überschreitet, die in der EU Düngeprodukteverordnung festgelegt sind.
Das Nachweisverfahren für Shigella-Pathogene ist nicht trennscharf, und eine Quantifizierung wird selten durchgeführt. Der Nachweis erfolgt in der Regel mit einem Anreicherungsmedium und anschließender Subkultivierung auf verschiedenen Selektivmedien.
Biostimulants des végétaux - Détection de Shigella spp.
Le présent document fournit une méthode permettant de vérifier que le pathogène Shigella spp n’est pas présent dans les biostimulants microbiens des végétaux à une concentration dépassant les limites correspondantes indiquées dans le Règlement UE sur les fertilisants.
La méthode de détection des pathogènes Shigella n’est pas sensible et la quantification est rarement réalisée. La détection est généralement réalisée en utilisant un milieu d’enrichissement, puis en procédant à un repiquage sur une variété de milieux sélectifs.
Rastlinski biostimulanti - Ugotavljanje prisotnosti Shigella spp.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17715:2023
01-maj-2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti Shigella spp.
Plant biostimulants - Detection of Shigella spp.
Pflanzen-Biostimulanzien - Nachweis von Shigella spp.
Biostimulants des végétaux - Détection de Shigella spp.
Ta slovenski standard je istoveten z: prEN 17715
ICS:
65.080 Gnojila Fertilizers
oSIST prEN 17715:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17715:2023
DRAFT
EUROPEAN STANDARD
prEN 17715
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2023
ICS 65.080 Will supersede CEN/TS 17715:2022
English Version
Plant biostimulants - Detection of Shigella spp.
Biostimulants des végétaux - Détection de Shigella spp. Pflanzen-Biostimulanzien - Nachweis von Shigella spp.
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 NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
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 17715:2023 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 General . 7
4.2 Enrichment in selective liquid medium . 7
4.3 Plating out and identification of colonies. 7
4.4 Biochemical and serological confirmation . 7
5 Culture media, reagents and antisera . 7
6 Apparatus and glassware . 8
7 Sampling . 8
8 Procedure. 8
8.1 General . 8
8.2 Test portion . 8
8.3 Enrichment . 8
8.3.1 General . 8
8.3.2 Liquid formulations. 8
8.3.3 Solid formulations . 8
8.4 Plating out and colony selection . 9
8.5 Confirmation of colonies . 9
8.5.1 General . 9
8.5.2 Purification of colonies . 9
8.5.3 Biochemical confirmation . 9
8.5.4 Additional biochemical differentiation . 13
8.6 Serological confirmation . 14
8.6.1 Antigenic differentiation . 14
8.6.2 Agglutination tests . 15
8.6.3 Definitive confirmation . 15
9 Expression of results . 15
10 Test report . 15
Annex A (normative) Diagram of test procedure . 16
Annex B (normative) Composition and preparation of culture media and reagents . 17
B.1 General . 17
B.2 Selective enrichment broth . 17
B.3 Selective differential agars . 18
B.4 Nutrient agar . 20
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B.5 Triple iron sugar (TSI) agar . 20
B.6 Semi-solid nutrient agar . 21
B.7 Urea agar (Christensen’s medium) . 21
B.8 L-Lysine decarboxylase medium . 22
B.9 L-Ornithine decarboxylase medium . 22
B.10 Reagents for indole reaction . 23
B.11 β-Galactosidase reagent . 23
B.12 Bromocresol purple broth . 24
B.13 Saline solution . 25
B.14 Sodium acetate agar . 25
B.15 Christensen's citrate agar . 25
B.16 Mucate broth . 26
B.17 Antisera of Shigella species . 27
Annex C (normative) Description of Shigella colony morphology and colour on selective
agars, for both identification and quality control purposes . 28
Annex D (informative) Interlaboratory study . 29
D.1 Materials used in the interlaboratory study . 29
D.2 Results of the interlaboratory study . 30
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 . 31
Bibliography . 32
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European foreword
This document (prEN 17715:2023) 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 17715:2022.
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”. Working Group 5 “Labelling and denominations”, was
created to develop a work program as part of this request.
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 commercialization 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 Fertilising Products Regulation.
This document is applicable to all microbial biostimulants in agriculture.
The table 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
— 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
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 provides a method for verifying that the pathogen Shigella spp. is not present in microbial
plant biostimulants in a concentration that exceeds the respective limits outlined in the EU Regulation on
Fertilising Products.
The detection method for Shigella pathogens is not sensitive and quantification is rarely performed.
Detection is usually performed using an enrichment medium followed by subculturing onto a variety of
selective media.
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.
1
EN 17708:— , Plant biostimulants — Preparation of sample for microbial analysis
2
EN 17724:— , Plant biostimulants — Terminology
3 Terms and definitions
2
For the purposes of this document, the terms and definitions given in EN 17724:— and the following
apply.
ISO and IEC maintain terminology 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
Shigella spp.
microorganisms which form typical colonies on solid selective media described and which display the
morphological, physiological and biochemical characteristics described when the analysis is carried out
in accordance with this document
3.2
detection of Shigella spp.
determination of the presence or absence of these microorganism in a particular mass of product, when
tests are carried out in accordance with this document
3.3
interlaboratory study
study performed by multiple laboratories testing identical samples at the same time, the results of which
are used to estimate alternative-method performance parameters
Note 1 to entry: The aim of an interlaboratory study is to determine the variability of the results obtained in
different laboratories using identical samples.
[SOURCE: ISO 16140-1:2016, 2.33]
1
Under preparation
2
Under preparation
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3.4
sensitivity
number of samples found positive divided by the number of samples tested at a given level of
contamination.
Note 1 to entry: The results are thus dependent on the level of contamination of the sample.
3.5
specificity
number of samples found negative divided by the number of blank samples tested
3.6
predictive positive value
PPV
ratio of positive samples identified as positive to all those with positive test results (including samples
that were incorrectly identified as positive)
3.7
predictive negative value
NPV
ratio of negative samples identified as negative to all those with negative test results (including samples
that were incorrectly identified as negative)
4 Principle
4.1 General
Detection of Shigella spp. will be conducted in accordance with the sections specified in
EN ISO 21567:2004 and with the following four successive stages (see Annex A, Figure A.1).
4.2 Enrichment in selective liquid medium
A test portion is inoculated into Shigella broth containing 0,5 µg/ml of novobiocin, then incubated
anaerobically at (41,5 ± 1) °C for 16 h to 20 h.
4.3 Plating out and identification of colonies
From the enrichment culture obtained, three selective differential media are inoculated: MacConkey agar
with low selectivity; XLD agar with moderate selectivity; Hektoen enteric agar with the greatest
selectivity. All are incubated at 37 °C for 20 h to 24 h.
4.4 Biochemical and serological confirmation
Typical and suspect colonies are selected from each of the three selective agars. The colonies are purified
on nutrient agar, then biochemical and serological characterizations are carried out using the tests
described.
5 Culture media, reagents and antisera
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or
demineralized water or water of equivalent purity.
See Annex B for descriptions of all media, reagents and antisera.
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Commercially available dehydrated media should give more consistent results than media prepared from
their component parts in the laboratory. Follow the manufacturer’s instructions exactly, as small changes
in preparation can significantly change the performance of selective media. Excessive heating of the
selective agars used in this document by autoclaving, storage and then re-heating for use may result in
loss of selectivity.
6 Apparatus and glassware
Disposable equipment is an acceptable alternative to re-usable glassware if it has suitable specifications.
1
Usual microbiological laboratory equipment according to EN 17708:— shall be used.
7 Sampling
Sampling is not part of the method specified in this document (see the specific European Standard dealing
3
with the product concerned EN 17702-1:— ).
It is important that the laboratory receives a sample which is representative and has not been damaged
or changed during transport or storage.
8 Procedure
8.1 General
The presence or absence of Shigella pathogens in at least 25 g or 25 mL of the product under test will be
evaluated.
8.2 Test portion
1
See the appropriate part of EN 17708:— .
8.3 Enrichment
8.3.1 General
A representative sample of the product to be will be prepared according to following procedure, which
takes into consideration the different formulations of biostimulants based products.
8.3.2 Liquid formulations
Dispense 25 g of sample in 225 ml of sterile Shigella broth containing 0,5 µg/ml of novobiocin (B.2.2).
Soon after take the entire suspension and proceed then with the incubation under anaerobic conditions
at (41,5 ± 1) °C for 16 h to 20 h.
8.3.3 Solid formulations
Dispense 25 g of sample in 225 ml of sterile Shigella broth containing 0,5 µg/ml of novobiocin (B.2.2).
Soon after, take the entire suspension and process them in a stomacher for 2 min at highest speed.
Incubate the Shigella broth under anaerobic conditions at (41,5 ± 1) °C for 16 h to 20 h.
3
Under preparation
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8.4 Plating out and colony selection
8.4.1 Using the culture obtained in 8.3, gently mix the contents by hand and allow the larger particles to
settle.
Inoculate, by means of a loop, the surface of the following selective agars to obtain well-isolated colonies:
MacConkey agar (B.3.1) with low selectivity; XLD agar (B.3.2) with moderate selectivity; and Hektoen
enteric agar (B.3.3) with a greater selectivity.
8.4.2 Incubate the plates at (37 ± 1) °C for between 20 h and 24 h.
The appearance of different Shigella species can vary on the same selective agar. See Annex C for a
description of Shigella colonies on the different selective agar used.
Mark any typical or suspect colonies found on each plate.
If no typical colonies are seen and the growth of the other microorganisms is weak, re-incubate the plates
for a further 24 h. Examine them again for the typical Shigella colonies.
Carry out the confirmation procedure described in 8.5.
8.5 Confirmation of colonies
8.5.1 General
Identification kits (currently commercially available) that have been proven by the user to be reliable for
the identification of the different species of Shigella may be used. Follow the manufacturer’s instruction
precisely.
For confirmation, sub-culture from each dish of each selective medium (see 8.4) five marked typical or
suspect colonies.
If on one dish there are fewer than five typical or suspect colonies, take all the marked colonies for
confirmation.
Use pure cultures for biochemical and serological confirmation.
8.5.2 Purification of colonies
Streak the selected colonies onto the surface of nutrient agar plates (B.4) so as to gain well-isolated
colonies. Incubate the plates at (37 ± 1) °C for 18 h to 24 h.
If the cultures on nutrient agar are mixed, sub-culture the suspect colony onto a further plate of nutrient
agar and incubate at (37 ± 1) °C for 18 h to 24 h to obtain the pure culture.
Shigella sonnei can give two colony types on the same agar plate: a smooth round domed colony
(phase 1), and a flat irregular colony with a mat surface (phase 2).
NOTE It is possible to first test the most characteristic colony from each selective agar plate. If positive, it is
not necessary to test other colonies. If negative, progress through the other selected colonies until either all are
negative or a positive is found.
8.5.3 Biochemical confirmation
8.5.3.1 General
By means of an inoculation needle, inoculate the media specified in 8.5.3.2 to 8.5.3.9 respectively with
each of the cultures selected in 8.5.1 and record all the results.
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8.5.3.2 Triple sugar iron agar (TSI slopes) (B.5)
Stab the butt and streak the agar slope.
Incubate at (37 ± 1) °C for (24 ± 3) h.
Interpret the changes in the medium as shown in Table 2.
Table 2 — Interpretation of triple sugar iron agar test
Area of slope Appearance Indication
Butt Yellow Glucose fermented: positive
Red or unchanged Glucose not fermented: negative
Black
Formation of hydrogen sulfide: positive
Bubbles or cracks
Gas formation
Slant surface Yellow Lactose and/or sucrose utilized: positive
Red or unchanged Lactose and sucrose not utilized: negative
Typical Shigella cultures show a yellow butt (acid formation) and no gas bubbles, there is no change in
the colour of the slant (no utilization of lactose or sucrose) and no hydrogen sulfide production
(see Table 3).
8.5.3.3 Semi-solid nutrient agar for motility tests (B.6)
Stab the semi-solid nutrient agar with a colony using an inoculation needle.
Incubate tubes at (37 ± 1) °C for 18 h to 24 h.
Examine the line of inoculation for spreading growth. Non-motile microorganisms will give a discrete
line; motile strains will give diffuse growth away from the inoculum line.
All Shigella species are non-motile.
8.5.3.4 Urea agar (B.7)
Streak the agar surface.
Incubate at (37 ± 1) °C for (24 ± 3) h and examine at intervals.
If urea is hydrolysed, a rose-pink to deep cerise colour develops from the release of ammonia by the
decomposition of the urea with a change in the colour of the pH indicator. There is no change in colour of
the agar with a negative reaction.
Shigella species do not hydrolyse urea.
8.5.3.5 L-Lysine decarboxylase medium (B.8)
Inoculate below the surface of the liquid broth. Incubate at (37 ± 1) °C for (24 ± 3) h.
Turbidity and a purple colour after incubation indicate a positive reaction; yellow indicates a negative
result.
Shigella species do not decarboxylate lysine.
NOTE The use of a paraffin overlay in the tubes can help to ensure anaerobic conditions.
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8.5.3.6 L-Ornithine decarboxylase medium (B.9)
Inoculate below the surface of the liquid broth. Incubate at (37 ± 1) °C for (24 ± 3) h.
If a purple colour develops, the test is positive; a yellow colour means a negative result.
Shigella sonnei decarboxylates ornithine, but other Shigella species do not (see Table 3).
8.5.3.7 Detection of indole formation (B.10)
Inoculate a tube containing 5 ml of tryptone/tryptophan medium (B.10.1) with the pure culture.
Incubate at (37 ± 1) °C for (24 ± 3) h.
After incubation, add 1 ml of Kovac’s reagent (B.10.2).
The formation of a red ring within 10 min indicates indole formation, and a yellow/brown colour
indicates a negative reaction.
Shigella sonnei is negative whilst other strains give variable reactions (see Table 1).
8.5.3.8 Detection of β-galactosidase (B.11)
Suspend a loopful of the purified culture from the nutrient agar into 0,25 ml of saline solution (B.12) in a
screw cap bottle or test tube.
Add one drop of toluene and shake to mix well.
Put the tube in an incubator set at 37 °C and leave for several minutes. Add 0,25 ml of the complete
reagent and mix.
Replace in the incubator set at 37 °C and leave for (24 ± 3) h, examining at intervals.
A yellow colour indicates the formation of β-galactosidase, which can occur in as little as 20 min.
Sigella sonnei is positive. S. dysenteriae and S. boydii give variable reactions and S. flexneri is negative. (see
Table 1).
8.5.3.9 Utilization of carbohydrates (Bromocresol purple broth) (B.12)
Inoculate each of the prepared carbohydrate broths with a small inoculum.
Incubate at (37 ± 1) °C for (24 ± 3) h.
A positive reaction when carbohydrate is utilized gives a change in the pH indicator from purple to yellow.
See Table 3 for the reactions of different Shigella species.
8.5.3.10 Interpretation of biochemical results
Strains within some Shigella species vary in their biochemical reactions (see Table 3), therefore
interpretation based only on biochemical results is difficult and serotyping is essential to establish
identity.
Shigella are Gram-negative bacilli, 2 µm to 4 µm by 0,5 µm in size, but often show a tendency to shorter
cocco-bacillary forms and typically do not produce gas from glucose. They are non-motile, do not produce
hydrogen sulfide or decarboxylate lysine, and are lactose negative at 24 h. The other tests described
above give variable reactions or differing reactions according to the species.
Within the genus Shigella, mannitol discriminates Shigella dysenteriae (negative) from other species and
L-ornithine decarboxylase differentiates Shigella sonnei (positive) from other species.
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Table 3 — Biochemical differentiation and confirmation of Shigella species from Escherichia coli,
Hafnia and Providencia species
Test
H2S from TSI − − − − −
...
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