FprCEN/TS 17715
(Main)Plant biostimulants - Detection of Shigella spp
Plant biostimulants - Detection of Shigella spp
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 Fertilizing 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.
Biostimulanzien für die pflanzliche Anwendung - Nachweis von Shigella spp
Dieses Dokument liefert ein Verfahren, mit dem nachgewiesen werden kann, dass das Pathogen Shigella spp. in mikrobiellen Biostimulanzien für die pflanzliche Anwendung 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.
[Not translated]
General Information
Standards Content (sample)
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17715:2021
01-november-2021
[Not translated]
Plant biostimulants - Detection of Shigella spp
Biostimulanzien für die pflanzliche Anwendung - Nachweis von Shigella spp
Ta slovenski standard je istoveten z: FprCEN/TS 17715
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17715:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17715
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Detection of Shigella spp
Biostimulanzien für die pflanzliche Anwendung -
Nachweis von Shigella spp
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 17715:2021 E
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Contents Page
European foreword ........................................................................................................................................... 3
Introduction ........................................................................................................................................................ 4
1 Scope ........................................................................................................................................................ 5
2 Normative references ........................................................................................................................ 5
3 Terms and definitions ....................................................................................................................... 5
4 Principle ................................................................................................................................................. 5
4.1 General .................................................................................................................................................... 5
4.2 Enrichment in selective liquid medium ...................................................................................... 6
4.3 Plating out and identification of colonies ................................................................................... 6
4.4 Biochemical and serological confirmation ................................................................................ 6
5 Culture media, reagents and antisera .......................................................................................... 6
6 Apparatus and glassware ................................................................................................................. 6
7 Sampling ................................................................................................................................................. 6
8 Procedure ............................................................................................................................................... 6
8.1 General .................................................................................................................................................... 6
8.2 Test portion ........................................................................................................................................... 6
8.3 Enrichment ............................................................................................................................................ 7
8.3.1 General .................................................................................................................................................... 7
8.3.2 Liquid formulations ............................................................................................................................ 7
8.3.3 Solid formulations ............................................................................................................................... 7
8.4 Plating out and colony selection .................................................................................................... 7
8.5 Confirmation of colonies................................................................................................................... 7
8.5.1 General .................................................................................................................................................... 7
8.5.2 Purification of colonies ..................................................................................................................... 7
8.5.3 Biochemical confirmation ................................................................................................................ 8
8.5.4 Additional biochemical differentiation .................................................................................... 12
8.6 Serological confirmation ............................................................................................................... 13
8.6.1 Antigenic differentiation ............................................................................................................... 13
8.6.2 Agglutination tests ........................................................................................................................... 14
8.6.3 Definitive confirmation .................................................................................................................. 14
9 Expression of results ....................................................................................................................... 14
10 Test report .......................................................................................................................................... 14
Annex A (normative) Diagram of test procedure ............................................................................... 15
Annex B (normative) Composition and preparation of culture media and reagents ........... 16
Annex C (normative) Description of Shigella colony morphology and colour on selective
agars, for both identification and quality control purposes ............................................. 27
Bibliography ..................................................................................................................................................... 28
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European foreword
This document (FprCEN/TS 17715:2021) 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
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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 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 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 Fertilizing 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
— 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.---------------------- Page: 6 ----------------------
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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 Fertilizing 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.
FprCEN/TS 17708, Plant biostimulants – Preparation of sample for microbial analysis
FprCEN/TS 17724, Plant Biostimulants – TerminologyEN ISO 21567:2004, Microbiology of food and animal feeding stuffs – Horizontal method for the detection
of Shigella spp (ISO 21567:2004)EN ISO 11133:2014, Microbiology of food, animal feed and water - Preparation, production, storage and
performance testing of culture media3 Terms and definitions
For the purposes of this document, the terms and definitions given in FprCEN/TS 17724 and the
following 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
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 document3.2
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 document4 Principle
4.1 General
Detection of Shigella spp. will be conducted in accordance with the refer sections of EN ISO 21567:2004
and with the following four successive stages (see Annex A, Figure A.1).As amended by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020
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4.2 Enrichment in selective liquid medium
A test portionis 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.
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.
Usual microbiological laboratory equipment according to FprCEN/TS 17708 shall be used.
7 SamplingSampling is not part of the method specified in this document (see the specific European Standard
dealing with the product concerned).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
See the appropriate part of FprCEN/TS 17708.
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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 formulationsDispense 25 g of sample in 225 mL of sterile Shigella broth containing 0,5 µg/ml of novobiocin (B.2.1).
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.2.1).
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.
8.4 Plating out and colony selection8.4.1 Using the culture obtained in 8.2, 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 8.3) 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.3) 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
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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 8.4.3.2 to 8.4.3.9 respectively with
each of the cultures selected in 8.4.1 and record all the results.8.5.3.2 Triple sugar iron agar (TSI slopes) (B.4)
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.5)
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.6)
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.
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8.5.3.5 L-Lysine decarboxylase medium (B.7)
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.
8.5.3.6 L-Ornithine decarboxylase medium (B.8)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.9)Inoculate a tube containing 5 ml of tryptone/tryptophan medium (B.9.1) with the pure culture.
Incubate at (37 ± 1) °C for (24 ± 3) h.After incubation, add 1 ml of Kovac’s reagent (B.9.2).
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.10)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 (B.11)
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.
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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 speciesTest Escherichia Hafnia Providencia Shigella Shigella Shigella Shigella
coli sonnei flexneri dysenteriae boydii
species
H S from TSI − − − − − − −
Gas from glucose (TSI) + V + − − e e
Motility + V V − − − −
Urease − − V − − − −
L-Lysine decarboxylase V + − − − − i
L-Ornithine V + − + − − −
decarboxylase
Indole formation + − + − d d d
V (61 %) V (44 %) V (29 %)
β-Galactosidase + V - + (95 %) - f f
V (50 %) V (11 %)
Acid from:
Dulcitol V V − − g g g
V (9,4 %) V (4,5 %) V (6,7 %)
Glucose + + − + (100 %) + (100 %) + (100 %) + (100 %)
Lactose V V − c a − a
Mannitol + + V + (99 %) b − + (98 %)
+ (94 %)
Melibiose V V V − V V V
Raffinose V V − c V (53 %) − −
− (2,5 %)
Salicin V V − − − − −
Sorbitol + − − − V (31 %) V (29 %) V (42 %)
Sucrose V − V c − − −
− (1,5 %)
Xylose + + − − − h V (57 %)
V (4,0 %)
V: strains variable within or between serovars of a species and, where given, (x %) indicates percentage of positive
strains .Some strains of S. flexneri serovar 2a and S. boydii 9 produce acid.
Some strains of S. flexneri serovars 4 and 6b do not produce acid.
Shigella sonnei produces acid after several days incubation.
Some serotypes of Shigella dysenteriae and S. flexneri serovar 6 and S. boydii are negative.
Strains of S. flexneri and S. boydii serovars 13 and 14 produce acid and gas.Strains of S. dysenteriae serovar 1 and S. boydii serovar 13 are always positive.
Strains of S. dysenteriae serovar 5 and S. flexneri serovar 6 are positive.Strains of S. dysenteriae serovars 8 and 10 are positive and 4 and 6 are variable.
Only strains of S. boydii serovar 13 are positive.From Ewing W.H. and Lindberg A.A. Serology of the Shigella. In: Methods in Microbiology (Ed. Bergan T.), Vol. 14,
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8.5.4 Additional biochemical differentiation
8.5.4.1 General
It is recommended to carry out additional biochemical differentiation tests for a better identification of
the strains: some strains of Escherichia coli and Shigella species are similar.8.5.4.2 Sodium acetate
Streak the slope of the sodium acetate medium (B.13) with the pure culture (8.4.1). Use a straight wire
to minimize the amount of culture medium transferred with the inoculum, or use an inoculation needle.
Incubate under aerobic conditions for 2 days at (37 ± 1) °C.Examine the green medium for growth: a positive result is found when the medium turns blue. Look for
the growth, a blue colour indicates a positive reaction.If no growth occurs, incubate the culture for 2 additional days at (37 ± 1) °C. Examine the medium again.
Shigella species do not grow or grow very poorly. Strains of E. coli give blue colonies with the
surrounding medium blue/green.8.5.4.3 Christensen's citrate
Inoculate the slant surface of the Christensen’s citrate (B.14) using an inoculation needle with a pure
culture (8.4.1). Minimize as far as possible the quantity of medium transferred with the inoculum.
Incubate aerobically for 2 days at (37 ± 1) °C.Examine to detect a cream/pink growth. In this case, the medium changes to red.
If no growth occurs, incubate the culture for a further 2 days and examine again.
Shigella species do not grow.8.5.4.4 Sodium mucate
Inoculate the test broth (B.15.1) and the control broth (B.15.2) with the pure culture (8.4.1). Incubate
aerobically for 2 days at (37 ± 1) °C.Examine the medium to detect growth and colour development. A blue colour indicates a negative
reaction and a yellow/straw colour indicates a positive reaction.If no growth occurs in the test broth, incubate the culture for a further 2 days at (37 ± 1) °C. Examine
the medium again.Shigella sonnei shows variable reactions but other Shigella species are negative.
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Table 4 — Additional biochemical tests to differentiate some strains of Shigella spp. and
Escherichia coliSpecies Biochemical reactions (growth) for a determined period after
incubation
Sodium acetate Christensen's citrate Sodium mucate
+ % at 2 + % after 2 + % at 2 + % after 2 + % at 2 + % after 2
days days days days days days
S. dysenteriae − (0) − (0) − (0) − (0) − (0) − (0)
S. flexneri − (0) − (0) − (0) − (0) − (0) − (0)
S. boydii − (0) − (0) − (0) − (0) − (0) − (0)
S. sonnei − (0) − (0) − (0) − (0) V (6,4) V (36,7)
E. coli V (83,8) + (93,5) V (>15,3) V (>34,2) + (91,6) + (93,0)
+ > 90 % strains positive.
> 90 % strains negative.
V Variable results with between 10 % to 89 % of strains positive.
% Percentage of positive strains after determined incubation.
a th
From Bacteriological Analytical Manual, 8 Edition (Revised 1997), FDA, USA.
8.6 Serological confirmation
8.6.1 Antigenic differentiation
Shigella species are non-motile and therefore do not hav
...
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