1、BRITISH STANDARDBS EN ISO 21871:2006Microbiology of food and animal feeding stuffs Horizontal method for the determination of low numbers of presumptive Bacillus cereus Most probable number technique and detection methodThe European Standard EN ISO 21871:2006 has the status of a British StandardICS
2、07.100.30g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 21871:2006This British Standard was published under the authority of the Standard
3、s Policy and Strategy Committee on 1 February 2006 BSI 1 February 2006ISBN 0 580 47208 6National forewordThis British Standard is the official English language version of EN ISO 21871:2006. It is identical with ISO 21871:2006.The UK participation in its preparation was entrusted to Technical Committ
4、ee AW/9, Microbiology, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary.Cross-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI
5、Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its
6、 correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests in
7、formed; monitor related international and European developments and promulgate them in the UK.Summary of pagesThis document comprises a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, the ISO title page, pages ii to v, a blank page, pages 1 to 14, an inside back
8、cover and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 21871January 2006ICS 07.100.30English VersionMicrobiology of food and
9、animal feeding stuffs - Horizontalmethod for the determination of low numbers of presumptiveBacillus cereus - Most probable number technique and detectionmethod (ISO 21871:2006)Microbiologie des aliments - Mthode horizontale pour lednombrement de Bacillus cereus prsums en petitnombre - Technique du
10、nombre le plus probable etmthode de recherche (ISO 21871:2006)Mikrobiologie von Lebensmitteln und Futtermitteln -Horizontales Verfahren zur Bestimmung niedriger Zahlenvon prsumtivem Bacillus cereus - Verfahren derwahrscheinlichsten Keimzahl (MPN) undNachweisverfahren (ISO 21871:2006)This European St
11、andard 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 EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concernin
12、g such nationalstandards 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 translationunder the responsibility of a CEN member into its own languag
13、e and notified to the Central Secretariat has the same status as the officialversions.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,
14、Netherlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2006 CEN All rights of exploitatio
15、n in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 21871:2006: EForeword 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 - Hori
16、zontal 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 200
17、6. 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,
18、 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. EN ISO 21871:2006Reference numberIS
19、O 21871:2006(E)INTERNATIONAL STANDARD ISO21871First edition2006-01-15Microbiology 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 Mthode horizontale
20、pour le dnombrement de Bacillus cereus prsums en petit nombre Technique du nombre le plus probable et mthode de recherche EN ISO 21871:2006ii iiiContents 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 Dete
21、ction 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 e
22、gg 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 Enu
23、meration 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 EN ISO 21871:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide fede
24、ration 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 commi
25、ttee. 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 accordan
26、ce 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
27、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 Co
28、mmittee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. EN ISO 21871:2006vIntroduction 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 p
29、roducts, 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
30、 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
31、 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. EN I
32、SO 21871:2006blank1Microbiology 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 enume
33、ration 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 N
34、ormative 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), Microbi
35、ology 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
36、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 c
37、ulture 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
38、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 tes
39、t 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 Bac
40、illus species such as Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides and Bacillus pseudomycoides. EN ISO 21871:20062 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
41、 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
42、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 m
43、edium (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 p
44、resumptive 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 mos
45、t 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 li
46、quid 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.
47、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
48、 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. EN ISO 21871:200635.3 Liquid selective enrichment medium: Tryptone soya polymyxin broth (TSPB) (see Reference 1) 5.
49、3.1 Base medium 5.3.1.1 Composition a) Double-strength medium b) Single-strength 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 (K2HPO4) 5,0 g 2,5 g 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
