EN 17715:2024
(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.
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.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.
Pflanzen-Biostimulanzien - Nachweis von Shigella spp.
Dieses Dokument liefert ein Verfahren zum Nachweis, dass das Pathogen Shigella spp. in mikrobiellen Pflanzen-Biostimulanzien nicht vorhanden ist.
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.
Dieses Dokument ist für die Mischungen von Düngeprodukten anwendbar, in denen eine Mischung aus mindestens zwei der folgenden Kategorien von Komponenten-EU Düngeprodukten besteht: Düngemittel, Kalkdünger, Bodenverbesserungsmittel, Kultursubstrate, Pflanzen-Biostimulanzien und wenn die folgende Kategorie Pflanzen-Biostimulanzien prozentual den höchsten Massen- oder Volumenanteil in der Mischung bzw., bei flüssiger Form, den höchsten Trockenmasseanteil aufweist. Wenn Pflanzen-Biostimulanzien nicht den höchsten prozentualen Anteil in der Mischung ausmachen, ist die für den höchsten prozentualen Anteil geltende Europäische Norm anwendbar. Falls eine Düngemittelmischung aus Komponenten gleicher Menge besteht oder falls die für die Mischung verwendeten Komponenten-EU Düngeprodukte identische Formulierungen haben, entscheidet der Anwender, welche Norm angewendet wird.
Biostimulants des végétaux - Recherche 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.
La méthode de recherche des pathogènes Shigella n’est pas sensible et la quantification est rarement réalisée. La recherche est généralement réalisée en utilisant un milieu d’enrichissement, puis en procédant à un repiquage sur une variété de milieux sélectifs.
Le présent document est applicable aux mélanges de fertilisants constitués d’au moins deux composants appartenant aux catégories de fertilisants UE suivantes : engrais, amendements minéraux basiques, amendements du sol, supports de culture, biostimulants des végétaux et, dans lesquels la catégorie Biostimulants des végétaux représente le pourcentage le plus élevé du mélange en masse ou en volume, ou dans le cas d’un liquide, en masse sèche. Si les biostimulants des végétaux ne représentent pas le pourcentage le plus élevé du mélange, la Norme européenne relative au composant représentant le pourcentage le plus élevé du mélange s’applique. Dans le cas où un mélange de fertilisants contient des composants en quantité égale ou dans le cas où les composants des fertilisants UE utilisés pour le mélange ont des formulations identiques, l’utilisateur décide quelle norme appliquer.
Rastlinski biostimulanti - Ugotavljanje prisotnosti Shigella spp.
Ta dokument določa metodo, s katero se preverja neprisotnost patogena Shigella spp. v mikrobnih rastlinskih biostimulantih.
Metoda ugotavljanja prisotnosti patogenov Shigella ni občutljiva in kvantifikacija se redko izvaja. Prisotnost patogenov se običajno ugotavlja z uporabo obogatitvenega gojišča, ki mu sledi subkultura na različnih
selektivnih gojiščih.
Ta dokument se uporablja za mešanice sredstev za gnojenje, ki vsebujejo najmanj dve od naslednjih kategorij sredstev za gnojenje EU: gnojila, sredstva za apnenje, izboljševalci tal, rastni substrati in rastlinski biostimulanti, pri čemer rastlinski biostimulanti predstavljajo največji masni ali prostorninski odstotek mešanice ali, v primeru tekoče oblike, odstotek suhe mase. Če rastlinski biostimulanti ne predstavljajo največjega odstotka v mešanici, se uporablja evropski standard za največji odstotek mešanice. Če vsebuje mešanica sredstev za gnojenje sestavine v enakih količinah ali če imajo posamezna sredstva za gnojenje EU, uporabljena za mešanico, enake formulacije1, standard za uporabo izbere uporabnik.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2025
Nadomešča:
SIST-TS CEN/TS 17715:2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti Shigella spp.
Plant biostimulants - Detection of Shigella spp.
Pflanzen-Biostimulanzien - Nachweis von Shigella spp.
Biostimulants des végétaux - Recherche de Shigella spp.
Ta slovenski standard je istoveten z: EN 17715:2024
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17715
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2024
EUROPÄISCHE NORM
ICS 65.080 Supersedes CEN/TS 17715:2022
English Version
Plant biostimulants - Detection of Shigella spp.
Biostimulants des végétaux - Recherche de Pflanzen-Biostimulanzien - Nachweis von
Shigella spp. Shigella spp.
This European Standard was approved by CEN on 26 August 2024.
This European Standard was corrected and reissued by the CEN-CENELEC Management Centre on 11 December 2024.
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. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists 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.
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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17715:2024 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Culture media, reagents and antisera . 7
6 Apparatus and glassware . 7
7 Sampling . 7
8 Procedure. 7
9 Expression of results . 14
10 Test report . 14
Annex A (normative) Diagram of test procedure . 16
Annex B (normative) Composition and preparation of culture media and reagents . 17
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
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. 32
Bibliography . 33
European foreword
This document (EN 17715:2024) has been prepared by Technical Committee CEN/TC 455 “Plant
Biostimulants”, the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2025, and conflicting national standards shall be
withdrawn at the latest by May 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes CEN/TS 17715:2022.
— the introduction has been updated and Table 1 has been removed;
— Clause 2, Normative references, has been updated;
— Annex B and Annex C have been revised;
— Annex D on the interlaboratory study has been added;
— Annex ZA has been added.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
For the relationship with EU Legislation, see informative Annex ZA, which is an integral part of this
document.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: 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 the United
Kingdom.
Introduction
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 [1] laying down rules on the making available on the market
of EU fertilising products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as SR M/564 and relevant amendments, also contributes to the
Communication on “Innovating for Sustainable Growth: A Bio economy for Europe”. The interest in plant
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.
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.
1 Scope
This document provides a method for verifying that the pathogen Shigella spp. is not present in microbial
plant biostimulants.
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.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of
the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers,
Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest
percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant
Biostimulants is not the highest percentage in the blend, the European Standard for the highest
percentage of the blend applies. In case a blend of fertilizing products is composed of components in
equal quantity or in case the component EU fertilising products used for the blend have identical
formulations , the user decides which standard to apply.
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.
EN 17708:2024, Plant biostimulants — Preparation of sample for microbial analysis
EN ISO 21567:2004, Microbiology of food and animal feeding stuffs — Horizontal method for the detection
of Shigella spp. (ISO 21567:2004)
EN 17702-1:2024, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
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 terms and definitions 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
An example of such a blend is a product with 2 claimed functions consisting of a non-microbial plant biostimulant
and an organic fertilizer composed of 1 kg/kg of plant biostimulant from
seaweed.
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020.
3.2
detection of Shigella spp.
determination of the presence or absence of these microorganisms 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 [2]]
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
positive predictive 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
negative predictive 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. shall be conducted in accordance with the sections specified in
EN ISO 21567:2004 and with the following four successive stages (according to Annex A, Figure A.1).
4.2 Enrichment in selective liquid medium
A test portion shall be inoculated with Shigella broth (B.2.1) containing 0,5 µg/ml of novobiocin, then
incubated anaerobically at 41,5 °C ± 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 shall be inoculated: MacConkey
agar with low selectivity; XLD agar with moderate selectivity; Hektoen enteric agar with the greatest
selectivity. All shall be incubated at 37 °C ± 1°C for 20 h to 24 h.
4.4 Biochemical and serological confirmation
Typical and suspect colonies shall be selected from each of the three selective agars. The colonies shall
be purified on nutrient agar, then biochemical and serological characterizations shall be 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.
All media, reagents and antisera that shall be used are specified in Annex B.
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 can 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 EN 17708:2024 shall be used.
7 Sampling
Sampling is not part of the method specified in this document (the specific European Standard dealing
with the product concerned EN 17702-1:2024 shall be used).
The laboratory shall receive 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 shall be
evaluated.
8.2 Test portion
The appropriate part of EN 17708:2024 shall be used.
8.3 Enrichment
The appropriate part of EN 17708:2024 shall be used.
The incubation shall be under anaerobic conditions at 41,5 °C ± 1 °C for 16 h to 20 h.
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 °C ± 1 °C for 20 h to 24 h.
The appearance of different Shigella species can vary on the same selective agar. The morphology and
colour of Shigella colonies on the different selective agar used shall be evaluated according to Annex C.
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 instructions
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) or of the same medium (B.3) so
as to gain well-isolated colonies. Incubate the plates at 37 °C ± 1 °C for 18 h to 24 h.
If the cultures are not pure, sub-culture the suspect colony onto a further plate and incubate at
37 °C ± 1 °C for 18 h to 24 h to obtain a 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).
The most characteristic colony from each selective agar plate may be tested first. If positive, other
colonies shall not be tested. 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/or 8.5.2 and record all the results.
If commercially available, consumables (e.g. miniaturized strips) allow to biochemically identify the
strains of Shigella spp., they may be used instead of the tests from 8.5.3.3 to 8.5.3.9.
8.5.3.2 Triple sugar iron agar (TSI slopes) (B.5)
Stab the butt and streak the agar slope.
Incubate at 37 °C ± 1 °C for 18 h to 24 h.
Interpret the changes in the medium as shown in Table 1.
Table 1 — 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 spp. 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 2).
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 the tubes at 37 °C ± 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.
NOTE All Shigella species are non-motile.
8.5.3.4 Urea agar (B.7)
Streak the agar surface.
Incubate at 37 °C ± 1 °C for 18 to 24 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.
NOTE 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 °C ± 1 °C for 18 to 24 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.9)
Inoculate below the surface of the liquid broth. Incubate at 37 °C ± 1 °C for 18 to 24 h.
If a purple colour develops, the test is positive; a yellow colour means a negative result.
NOTE Shigella sonnei decarboxylates ornithine, but other Shigella species do not (see Table 2).
8.5.3.7 Detection of indole formation (B.10)
Inoculate a tube containing at least 3 ml of tryptone/tryptophan medium (B.10.1) with the pure culture.
Incubate at 37 °C ± 1 °C for 18 to 24 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.
NOTE Shigella sonnei is negative whilst other strains give variable reactions (see Table 2).
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.13) 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 ± 1°C and leave for several minutes. Add 0,25 ml of the complete
reagent and mix.
Replace in the incubator set at 37 °C ± 1°C and leave for 18 to 24 h, examining at intervals.
A yellow colour indicates the formation of β-galactosidase, which can occur in as little as 20 min.
NOTE Sigella sonnei is positive. S. dysenteriae and S. boydii give variable reactions and S. flexneri is negative.
(see Table 2).
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 °C ± 1 °C for 18 h to 24 h.
A positive reaction when carbohydrate is utilized gives a change in the pH indicator from purple to yellow.
See Table 2 for the reactions of the different Shigella species.
8.5.3.10 Interpretation of biochemical results
Strains within some Shigella species vary in their biochemical reactions (see Table 2), therefore
interpretation based only on biochemical results is difficult and serotyping is essential to establish
identity.
Shigella spp. 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.
Table 2 — Biochemical differentiation and confirmation of Shigella species from Escherichia coli,
Hafnia and Providencia species
Test
H2S from TSI − − − − − − −
e e
Gas from glucose (TSI) + V + − −
Motility + V V − − − −
Urease − − V − − − −
i
L-Lysine decarboxylase V + − − − −
L-Ornithine decarboxylase V + − + − − −
d d d
Indole formation + − + −
V (61 %) V (44 %) V (29 %)
f f
β-Galactosidase
+ V - + (95 %) - V (50 %) V (11 %)
Acid from:
g g g
Dulcitol V V − −
V (9,4 %) V (4,5 %) V (6,7 %)
Glucose + + − + (100 %) + (100 %) + (100 %) + (100 %)
c a a
Lactose V V − −
b
Mannitol + + V + (99 %) − + (98 %)
+ (94 %)
Melibiose V V V − V V V
c
Raffinose
V V − V (53 %) − −
− (2,5 %)
Salicin V V − − − − −
Sorbitol + − − − V (31 %) V (29 %) V (42 %)
c
Sucrose
V − V − − −
− (1,5 %)
h
Xylose + + − − − V (57 %)
V (4,0 %)
V: strains variable within or between serovars of a species and, where given, (x %) indicates percentage of positive
strains .
a
Some strains of S. flexneri serovar 2a and S. boydii 9 produce acid.
b
Some strains of S. flexneri serovars 4 and 6b do not produce acid.
c
S. sonnei produces acid after several days’ incubation.
d
Some serotypes of S. dysenteriae and S. flexneri serovar 6 and S. boydii are negative.
e
Strains of S. flexneri and S. boydii serovars 13 and 14 produce acid and gas.
f
Strains of S. dysenteriae serovar 1 and S. boydii serovar 13 are always positive.
g
Strains of S. dysenteriae serovar 5 and S. flexneri serovar 6 are positive.
h
Strains of S. dysenteriae serovars 8 and 10 are positive and 4 and 6 are variable.
i
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,
Academic Press, 1984 [3].
Escherichia
coli
Hafnia
species
Providencia
Shigella
sonnei
Shigella
flexneri
Shigella
dysenteriae
Shigella
boydii
8.5.4 Additional biochemical differentiation
8.5.4.1 General
In case of doubt, additional biochemical differentiation tests should be carried out 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.14) with the pure culture (8.5.2). 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 two days at 37 °C ± 1 °C.
Examine the green medium for growth: a positive result is found when the medium turns blue.
If no growth occurs, incubate the culture for two additional days at 37 °C ± 1 °C. Examine the medium
again.
NOTE 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.15) using an inoculation needle with a pure
culture (8.5.2). Minimize as far as possible the quantity of medium transferred with the inoculum.
Incubate aerobically for two days at 37 °C ± 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 two days and examine again.
NOTE Shigella species do not grow.
8.5.4.4 Sodium mucate
Inoculate the test broth (B.16.1) and the control broth (B.16.2) with the pure culture (8.5.2). Incubate
aerobically for two days at 37 °C ± 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 two days at 37 °C ± 1 °C. Examine
the medium again.
NOTE Shigella sonnei shows variable reactions but other Shigella species are negative.
For further details on reactions of Shigella species, see Table 3.
a
Table 3 — Additional biochemical tests to differentiate some strains of Shigella spp. and
Escherichia coli
Species Biochemical reactions (growth) for a determined period after
incubation
Sodium acetate Christensen's citrate Sodium mucate
+ % at + % after + % at + % after + % at + % after
two days two days two days two days two days two 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 with positive reaction.
- > 90 % strains with negative reaction.
V Variable results with between 10 % to 89 % of strains with positive reaction.
% Percentage of strains with positive reaction after determined incubation.
a
From Bacteriological Analytical Manual, 8th Edition (Revised 1997), FDA, USA [4].
8.6 Serological confirmation (optional)
8.6.1 Antigenic differentiation
Shigella species are non-motile and therefore do not have flagella antigens. Differentiation within and
between species depends upon the analysis of distinct somatic group “O” and specific “O” type antigens
(see Table 4).
Growth from a fresh culture on nutrient agar is required (see 8.5.2). Carry out agglutination tests on clean
glass slides or plates of glass (Clause 6) of the appropriate size.
Table 4 — Antigenic differentiation within the Shigella species
Antigenic
Shigella species Serovars (specific antigen designation)
group
S. dysenteriae A 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13
S. flexneri B 1a, 1b, 2a, 2b, 3a, 3b, 3c, 4a, 4b, 5a, 5b, 6, X, Y
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
S. boydii C
17, 18
S. sonnei D 1
The group antigens (A, B, C, D) can contain minor antigens that can cross react with other group antigens;
...
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