SIST EN ISO 21567:2005

SLOVENSKI STANDARD
01-januar-2005
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti
Shigelle spp. (ISO 21567:2004)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection of
Shigella spp. (ISO 21567:2004)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den
Nachweis von Shigella spp. (ISO 21567:2004)
Microbiologie des aliments - Méthode horizontale pour la recherche de Shigella spp.
(ISO 21567:2004)
Ta slovenski standard je istoveten z: EN ISO 21567:2004
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 21567
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2004
ICS 07.100.30
English version
Microbiology of food and animal feeding stuffs - Horizontal
method for the detection of Shigella spp. (ISO 21567:2004)
Microbiologie des aliments - Méthode horizontale pour la
recherche de Shigella spp. (ISO 21567:2004)
This European Standard was approved by CEN on 20 October 2004.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
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© 2004 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21567:2004: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 21567:2004) has been prepared by Technical Committee ISO/TC 34
"Agricultural food products" in collaboration with Technical Committee CEN/TC 275 "Food
analysis - Horizontal methods", the secretariat of which is held by DIN.

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 2005, and conflicting national standards
shall be withdrawn at the latest by May 2005.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Cyprus,
Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice
The text of ISO 21567:2004 has been approved by CEN as EN ISO 21567:2004 without any
modifications.
INTERNATIONAL ISO
STANDARD 21567
First edition
2004-11-01
Microbiology of food and animal feeding
stuffs — Horizontal method for the
detection of Shigella spp.
Microbiologie des aliments — Méthode horizontale pour la recherche de
Shigella spp.
Reference number
ISO 21567:2004(E)
©
ISO 2004
ISO 21567:2004(E)
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ii © ISO 2004 – All rights reserved

ISO 21567:2004(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references . 1
3 Terms and definitions. 2
4 Principle . 2
4.1 General. 2
4.2 Enrichment in selective liquid medium . 2
4.3 Plating out and identification of colonies. 2
4.4 Biochemical and serological confirmation. 2
5 Culture media, reagents and antisera. 2
6 Apparatus and glassware. 3
7 Sampling . 3
8 Preparation of test sample. 3
9 Procedure (see diagram in Annex A) . 4
9.1 Test portion . 4
9.2 Enrichment. 4
9.3 Plating out and colony selection. 4
9.4 Confirmation of colonies. 4
9.5 Serological confirmation. 10
10 Expression of results. 11
11 Test report. 11
Annex A (normative) Diagram of test procedure . 12
Annex B (normative) Composition and preparation of culture media and reagents . 13
Annex C (normative) Description of Shigella colony morphology and colour on selective agars,
for both identification and quality control purposes . 26

ISO 21567:2004(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 21567 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
iv © ISO 2004 – All rights reserved

ISO 21567:2004(E)
Introduction
Because of the large variety of food and feed products, this horizontal method may not be appropriate in every
detail for certain products. In this case, different methods which are specific to these products may be used if
absolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this
horizontal method as far as possible.
When this International Standard is next reviewed, account will be taken of all information then available
regarding the extent to which this horizontal method has been followed and the reasons for deviations from
this method in the case of particular products.
The harmonization of test methods cannot be immediate and, for certain groups of products, International
Standards and/or national standards may already exist that do not comply with this horizontal method. It is
hoped that when such standards are reviewed they will be changed to comply with this International Standard
so that eventually the only remaining departures from this horizontal method will be those necessary for
well-established technical reasons.

INTERNATIONAL STANDARD ISO 21567:2004(E)

Microbiology of food and animal feeding stuffs — Horizontal
method for the detection of Shigella spp.
WARNING — In order to safeguard the health of laboratory personnel, it is essential that the whole of
this method is only carried out by skilled personnel using good laboratory practices and preferably
working in a containment facility. Relevant national Health and Safety Regulations relating to this
organism shall be adhered to. Care shall be taken in the disposal of all infectious materials.
1 Scope
This International Standard specifies a horizontal method for the detection of Shigella species.
Subject to the limitations discussed in the Introduction, this International Standard is applicable to
 products intended for human consumption and the feeding of animals, and
 environmental samples in the area of food production and food handling.
2 Normative references
The following referenced documents are indispensable for the application 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.
ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions
ISO 6887-2, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 2: Specific rules for the preparation of meat and
meat products
ISO 6887-3, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 3: Specific rules for the preparation of fish and
fishery products
ISO 6887-4, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 4: Specific rules for the preparation of products
other than milk and milk products, meat and meat products, and fish and fishery products
ISO 7218:1996, Microbiology of food and animal feeding stuffs — General rules for microbiological
examinations
ISO 8261, Milk and milk products — General guidance for the preparation of test samples, initial suspensions
and decimal dilutions for microbiological examination
ISO/TS 11133-1, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production
of culture media — Part 1: General guidelines on quality assurance of culture media in the laboratory
ISO/TS 11133-2, Microbiology of food and animal feeding stuffs — Guidelines on preparation and production
of culture media — Part 2: Practical guidelines on performance testing of culture media
ISO 21567:2004(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
Shigella
microorganisms which form colonies fitting the description of these species on the solid selective media used,
and which display the biochemical and serological characteristics described when tests are carried out in
accordance with this International Standard
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 International Standard
4 Principle
4.1 General
The detection of Shigella necessitates four successive stages (see Annex A).
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; and 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
For current laboratory practices, see ISO 7218, ISO/TS 11133-1 and ISO/TS 11133-2 for the preparation,
production and performance testing of culture media.
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 International Standard by autoclaving, storage and then re-heating for use may result in
loss of selectivity.
2 © ISO 2004 – All rights reserved

ISO 21567:2004(E)
6 Apparatus and glassware
Disposable equipment is an acceptable alternative to re-usable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
6.1 Apparatus for dry sterilization (oven) and wet sterilization (autoclave) of equipment.
See ISO 7218.
6.2 Drying cabinet or oven, ventilated by convection, and capable of operating at a set temperature
between 37 °C and 55 °C.
6.3 Incubators, capable of operating at (37 ± 1) °C and (41,5 ± 1) °C.
6.4 Modified atmosphere jars or anaerobic incubation cabinets, and related apparatus to achieve
anaerobic conditions, with a gas composition of < 1 % O and 9 % to 13 % CO . See ISO 7218.
2 2
6.5 Water baths, operating at a set temperature of (47 ± 3) °C, for the cooling of molten media prior to
plate pouring, and another set at (50 ± 1) °C (see B 6.3.2 and B 10.2.2).
6.6 Peristaltic homogenizer (stomacher) or rotary blender.
See ISO 7218.
6.7 Inoculation needles and loops, made of platinum/iridium or nickel/chrome, of diameter approximately
3 mm, or plastic disposable loops and needles of suitable specifications.
6.8 pH-meter, having an accuracy of calibration of ± 0,1 pH unit at 25 °C.
6.9 Flasks and bottles, with closures, of suitable capacities for use in the preparation of enrichment broths
and agars and their storage.
6.10 Measuring cylinders.
6.11 Tubes, 18 mm in diameter and 160 mm in length (plugged or with screw caps), or culture bottles, of
nominal capacity 30 ml and 10 ml, with non-toxic metallic caps with liners or plastic disposable caps.
6.12 Petri dishes, of diameter between 90 mm to 100 mm and diameter 140 mm.
6.13 Glass slides or plates, suitable for use in agglutination tests.
7 Sampling
It is important that the laboratory receive a sample that is truly representative and has not been damaged or
changed during transit or storage.
Sampling is not part of the method specified in this International Standard. See the specific International
Standard dealing with the product concerned. If there is no specific International Standard, it is recommended
that the parties concerned come to an agreement on this subject.
8 Preparation of test sample
Prepare the test sample in accordance with the appropriate part of ISO 6887 and/or ISO 8261.
Analysis of samples should begin as quickly as possible, as survival of Shigella is poor.
ISO 21567:2004(E)
9 Procedure (see diagram in Annex A)
9.1 Test portion
See the appropriate part of ISO 6887 and/or ISO 8261 dealing with the procedures for the different types of
products concerned.
9.2 Enrichment
In general add x g or x ml of test portion to 9x ml of Shigella broth containing 0,5 µg/ml of novobiocin (B.1.2) to
make a 1 in 10 dilution of the test sample. Homogenize the test portion in the broth using a peristaltic
homogenizer or rotary blender (6.6). Aseptically adjust the pH to 7,0 ± 0,2, if necessary.
Incubate (6.4) the Shigella broth under anaerobic conditions with caps and closures loose, or with equipment
giving an equivalent effect, so that gas exchange can readily occur without contamination at (41,5 ± 1) °C
(6.3) for 16 h to 20 h.
9.3 Plating out and colony selection
9.3.1 Using the cultures obtained in 9.2, gently mix the contents by hand and allow the larger particles to
settle.
Inoculate, by means of a loop (6.7), the surface of the following selective agars to obtain well-isolated
colonies: MacConkey agar (B.2.1) with low selectivity; XLD agar (B.2.2) with moderate selectivity; and
Hektoen enteric agar (B.2.3) with a greater selectivity.
9.3.2 Incubate (6.3) the plates at (37 ± 1) °C for between 20 h and 24 h.
9.3.3 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 agars used.
Shigella species can form a minority proportion of the total microbial flora when contaminating a food sample
or after enrichment. In these circumstances, the direct streaking of the enrichment broth onto one plate per
selective agar may fail to allow the detection of Shigella colonies. It may therefore be appropriate in some
circumstances (e.g. the investigation of foods implicated in illness) to consider the inoculation of either two
90 mm dishes or one large (140 mm) Petri dish (6.12) to increase the possibility of detection.
The colonies of some Enterobacteriaceae strains are very similar in appearance to those of Shigella. Any
typical or suspect colonies shall be confirmed (see 9.4) as Shigella species or not. Also in some
circumstances (e.g. foods implicated in food poisoning), it may be appropriate to investigate more than
five colonies from a plate to increase confidence in the absence of Shigella in the food sample tested.
Mark any typical or suspect colonies found on each plate.
If no typical colonies are seen and the growth of other microorganisms is weak (particularly on the more
selective agar), re-incubate the plates for a further 24 h. Examine them again for typical Shigella colonies.
Carry out the confirmation procedure described in 9.4.
9.4 Confirmation of colonies
9.4.1 General
Identification kits (currently commercially available) that have been shown by the user to be reliable for the
identification of the different species of Shigella may be used. Follow the manufacturer's instructions precisely.
4 © ISO 2004 – All rights reserved

ISO 21567:2004(E)
For confirmation, sub-culture from each dish of each selective medium (see 9.3) 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.
9.4.2 Purification of colonies
Streak the selected colonies onto the surface of nutrient agar plates (B.3) so as to gain well-isolated colonies.
Incubate (6.3) 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.
9.4.3 Biochemical confirmation
9.4.3.1 General
By means of an inoculation needle (6.7), inoculate the media specified in 9.4.3.2 to 9.4.3.9 respectively with
each of the cultures selected in 9.4.1 and record all the results.
9.4.3.2 Triple sugar iron agar (TSI slopes) (B.4)
Stab the butt and streak the agar slope.
Incubate (6.3) at (37 ± 1) °C for (24 ± 3) h.
Interpret the changes in the medium as follows:
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 1).
9.4.3.3 Semi-solid nutrient agar for motility tests (B.5)
Stab the semi-solid nutrient agar with a colony using an inoculation needle (6.7).
Incubate (6.3) tubes at (37 ± 1) °C for 18 h to 24 h.
ISO 21567:2004(E)
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.
9.4.3.4 Urea agar (B.6)
Streak the agar surface.
Incubate (6.3) 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.
9.4.3.5 L-Lysine decarboxylase medium (B.7)
Inoculate below the surface of the liquid broth.
Incubate (6.3) 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.
9.4.3.6 L-Ornithine decarboxylase medium (B.8)
Inoculate below the surface of the liquid broth.
Incubate (6.3) 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 1).
9.4.3.7 Detection of indole formation (B.9)
Inoculate a tube containing 5 ml of tryptone/tryptophan medium (B.9.1) with the pure culture.
Incubate (6.3) 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).
6 © ISO 2004 – All rights reserved

ISO 21567:2004(E)
9.4.3.8 Detection of ββββ-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 (6.3) 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.
Shigella sonnei is positive. S. dysenteriae and S. boydii give variable reactions and S. flexneri is negative.
(see Table 1).
9.4.3.9 Utilization of carbohydrates (B.11)
Inoculate each of the prepared carbohydrate broths with a small inoculum.
Incubate (6.3) 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 1 for the reactions of different Shigella species.
9.4.3.10 Interpretation of biochemical results
Strains within some Shigella species vary in their biochemical reactions (see Table 1), 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 de
...