SIST EN ISO 21871:2006

SLOVENSKI STANDARD
01-april-2006
Mikrobiologija živil in krme – Horizontalna metoda za ugotavljanje nizkega števila
domnevno Bacillus cereus – MPN tehnika pri nizkem številu in metoda odkrivanja
(ISO 21871:2006)
Microbiology of food and animal feeding stuffs - Horizontal method for the determination
of low numbers of presumptive Bacillus cereus - Most probable number technique and
detection method (ISO 21871:2006)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur
Bestimmung niedriger Zahlen von präsumtivem Bacillus cereus - Verfahren der
wahrscheinlichsten Keimzahl (MPN) und Nachweisverfahren (ISO 21871:2006)
Microbiologie des aliments - Méthode horizontale pour le dénombrement de Bacillus
cereus présumés en petit nombre - Technique du nombre le plus probable et méthode
de recherche (ISO 21871:2006)
Ta slovenski standard je istoveten z: EN ISO 21871:2006
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 21871
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2006
ICS 07.100.30
English Version
Microbiology of food and animal feeding stuffs - Horizontal
method for the determination of low numbers of presumptive
Bacillus cereus - Most probable number technique and detection
method (ISO 21871:2006)
Microbiologie des aliments - Méthode horizontale pour le Mikrobiologie von Lebensmitteln und Futtermitteln -
dénombrement de Bacillus cereus présumés en petit Horizontales Verfahren zur Bestimmung niedriger Zahlen
nombre - Technique du nombre le plus probable et von präsumtivem Bacillus cereus - Verfahren der
méthode de recherche (ISO 21871:2006) wahrscheinlichsten Keimzahl (MPN) und
Nachweisverfahren (ISO 21871:2006)
This European Standard was approved by CEN on 28 November 2005.
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, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
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© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21871:2006: E
worldwide for CEN national Members.

Foreword
This document (EN ISO 21871:2006) 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 July 2006, and conflicting national standards
shall be withdrawn at the latest by July 2006.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice
The text of ISO 21871:2006 has been approved by CEN as EN ISO 21871:2006 without any
modifications.
INTERNATIONAL ISO
STANDARD 21871
First edition
2006-01-15
Microbiology of food and animal feeding
stuffs — Horizontal method for the
determination of low numbers of
presumptive Bacillus cereus —
Most probable number technique and
detection method
Microbiologie des aliments — Méthode horizontale pour le
dénombrement de Bacillus cereus présumés en petit nombre —
Technique du nombre le plus probable et méthode de recherche

Reference number
ISO 21871:2006(E)
©
ISO 2006
ISO 21871:2006(E)
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ii © ISO 2006 – All rights reserved

ISO 21871:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 2
4.1 Enumeration method. 2
4.2 Detection method. 2
5 Diluent, culture media and reagents. 2
5.1 General. 2
5.2 Diluent. 2
5.3 Liquid selective enrichment medium: Tryptone soya polymyxin broth (TSPB) . 3
5.4 Solid selective medium: Polymyxin pyruvate egg yolk mannitol bromothymol blue agar
(PEMBA). 4
5.5 Solid selective medium: Mannitol egg yolk polymyxin agar (MYP) . 6
5.6 Staining solutions for microscopic identification . 7
5.7 Sheep blood agar. 8
6 Apparatus and glassware . 8
7 Sampling. 9
8 Preparation of test sample. 9
9 Procedure . 9
9.1 Enumeration method. 9
9.2 Detection method. 11
10 Calculation and expression of results. 12
10.1 Enumeration method for the determination of the most probable number (MPN) . 12
10.2 Detection method. 12
11 Test report . 12
Annex A (normative) Diagram of enumeration procedure. 13
Bibliography . 14

ISO 21871:2006(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 21871 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
iv © ISO 2006 – All rights reserved

ISO 21871:2006(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 21871:2006(E)

Microbiology of food and animal feeding stuffs — Horizontal
method for the determination of low numbers of presumptive
Bacillus cereus — Most probable number technique and
detection method
1 Scope
This International Standard specifies a horizontal method for the detection or the enumeration of low numbers
of viable presumptive Bacillus cereus by means of the most probable number technique. 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 (all parts), Microbiology of food and animal feeding stuffs — Preparation of test samples, initial
suspension and decimal dilutions for microbiological examination
ISO 7218, 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 for the preparation 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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
presumptive Bacillus cereus
microorganism that forms typical or atypical colonies on the surface of selective culture media and which gives
positive confirmation reactions under the conditions specified in this International Standard
NOTE For the purpose of a practical test method, this definition of presumptive Bacillus cereus, used as a basis for
the procedure, does not exclusively describe strains of Bacillus cereus. In particular, the confirmatory test is inadequate to
distinguish between Bacillus cereus and other closely related but less commonly encountered Bacillus species such as
Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides and Bacillus pseudomycoides.
ISO 21871:2006(E)
4 Principle
4.1 Enumeration method
4.1.1 Inoculation of three tubes of double-strength liquid selective enrichment medium [5.3.1.1 a)] with a
specified quantity of the primary dilution (initial suspension).
4.1.2 Inoculation of three tubes of single-strength liquid selective enrichment medium [5.3.1.1 b)] with a
specified quantity of the primary dilution (initial suspension). Then, under the same conditions, inoculation of
single-strength liquid selective enrichment medium [5.3.1.1 b)] with specified quantities of decimal dilutions of
the primary dilution (initial suspension).
4.1.3 Incubation of the tubes of double and single-strength liquid selective enrichment medium (5.3) for 48 h
at 30 °C.
4.1.4 Inoculation of the solid selective medium (5.4 or 5.5) from the liquid selective enrichment medium
(5.3).
4.1.5 Incubation of the solid selective medium (5.4 or 5.5) for 18 h to 48 h at 37 °C (5.4) or 30 °C (5.5) and
examination of the plates to check for the presence of colonies which, from their characteristics, are
considered to be presumptive Bacillus cereus.
4.1.6 Confirmation of suspected colonies by means of haemolysis (9.1.5.3) or by microscopic examination
(9.1.5.4).
4.1.7 Calculation of the most probable number of presumptive Bacillus cereus per gram or per millilitre of
sample from selected dilutions by reference to most probable number tables.
4.2 Detection method
4.2.1 Inoculation of a liquid selective enrichment medium (5.3) with a specified quantity of the initial
suspension of the test sample.
4.2.2 Incubation of the tube for 48 h at 30 °C.
4.2.3 Inoculation of a solid selective medium (5.4 or 5.5) from the liquid selective enrichment medium (5.3).
4.2.4 Incubation of the solid selective medium (5.4 or 5.5) for 18 h to 48 h at 37 °C (5.4) or 30 °C (5.5) and
examination of the plates to check for the presence of colonies which, from their characteristics, are
considered to be presumptive Bacillus cereus.
4.2.5 Confirmation of suspected colonies by means of haemolysis (9.1.5.3) or by microscopic examination
(9.1.5.4).
4.2.6 The results are given as the “presence” or “absence” of presumptive Bacillus cereus in grams or
millilitres of product.
5 Diluent, culture media and reagents
5.1 General
For current laboratory practice, see ISO 7218, ISO/TS 11133-1 and ISO/TS 11133-2.
5.2 Diluent
See ISO 6887 (all parts), ISO 8261 and any specific standard dealing with the product to be examined.
2 © ISO 2006 – All rights reserved

ISO 21871:2006(E)
5.3 Liquid selective enrichment medium: Tryptone soya polymyxin broth (TSPB)
(see Reference [1])
5.3.1 Base medium
5.3.1.1 Composition
a) b)
Double-strength Single-strength
medium medium
Enzymatic digest of casein 34,0 g 17,0 g
Enzymatic digest of soya 6,0 g 3,0 g
Sodium chloride (NaCl) 10,0 g 5,0 g
Glucose 5,0 g 2,5 g
Dipotassium hydrogen phosphate (K HPO ) 5,0 g 2,5 g
2 4
Water 1 000 ml 1 000 ml
5.3.1.2 Preparation
Dissolve the ingredients or the complete base medium in the water by heating and shaking. Adjust the pH, if
necessary, so that after sterilization it is 7,3 ± 0,2 at 25 °C.
Dispense the media in quantities of 10 ml [double-strength medium (5.3.1.1 a)] and 9 ml [single-strength
medium (5.3.1.1 b)] into tubes of appropriate capacity [e.g. 16 mm × 160 mm (6.7)].
Sterilize in an autoclave (6.1) at 121 °C for 15 min.
5.3.2 Polymyxin B sulfate solution
5.3.2.1 Composition
Polymyxin B sulfate 500 000 units (equivalent to about 0,05 g)
Water 50 ml
5.3.2.2 Preparation
Dissolve the polymyxin B sulfate in the water. Sterilize by filtration.
5.3.3 Complete medium
Immediately before use, add 200 µl (double-strength medium) or 100 µl (single-strength medium) of the
polymyxin B sulfate solution (5.3.2) to each of the tubes containing base medium (5.3.1).
5.3.4 Performance testing for the quality assurance of the culture medium
For the definition of selectivity and productivity refer to ISO/TS 11133-2. Table 1 introduces
...