1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 13720:2010Meat and meat products Enumeration of presumptivePseudomonas spp. (ISO13720:2010)BS EN ISO 13720:2010 BRITISH STANDARDNational forewordThis British Standard i
2、s the UK implementation of EN ISO13720:2010. It supersedes BS 7857-1:1996 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee AW/9, Microbiology.A list of organizations represented on this committee can beobtained on request to its secretary.This publicatio
3、n does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 61033 2ICS 07.100.30; 67.120.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under t
4、he authority of theStandards Policy and Strategy Committee on 30 September 2010.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 13720 September 2010 ICS 07.100.30; 67.120.10 English Version Meat and meat products - Enumeration of presumpt
5、ive Pseudomonas spp. (ISO 13720:2010) Viande et produits base de viande - Dnombrement des Pseudomonas spp. prsomptifs (ISO 13720:2010) Fleisch und Fleischerzeugnisse - Zhlung von prsumtiven Pseudomonas spp. (ISO 13720:2010) This European Standard was approved by CEN on 20 February 2010. CEN members
6、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 t
7、o the CEN 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 Management Centre has the s
8、ame 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,
9、Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any m
10、eans reserved worldwide for CEN national Members. Ref. No. EN ISO 13720:2010: EBS EN ISO 13720:2010EN ISO 13720:2010 (E) 3 Foreword The text of ISO 13720:2010 has been prepared by Technical Committee ISO/TC 34 “Food products“ of the International Organization for Standardization (ISO) and has been t
11、aken over as EN ISO 13720:2010 by 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 March
12、 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent righ
13、ts. According to the CEN/CENELEC Internal Regulations, the national standards organizations 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, Ire
14、land, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 13720:2010 has been approved by CEN as a EN ISO 13720:2010 without any modification. BS EN ISO
15、13720:2010ISO 13720:2010(E) ISO 2010 All rights reserved iiiForeword 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 commit
16、tees. 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 In
17、ternational 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
18、 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
19、subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 13720 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. This second edition cancels and replaces the first edition (ISO 13720:1995), which has b
20、een technically revised. BS EN ISO 13720:2010BS EN ISO 13720:2010INTERNATIONAL STANDARD ISO 13720:2010(E) ISO 2010 All rights reserved 1Meat and meat products Enumeration of presumptive Pseudomonas spp. 1 Scope This International Standard specifies a method for the enumeration of presumptive Pseudom
21、onas spp. present in meat and meat products, including poultry. 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 documen
22、t (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,
23、 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 7218, Microbiology of food and animal feeding stuffs General requirements and
24、 guidance for microbiological examinations 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 f
25、ood 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 Pseudomonas spp. bacteria
26、 which at 25 C form colonies in cephalothin-sodium fusidate-cetrimide (CFC) agar and which show a positive oxidase reaction when tested according to the method described in this International Standard 4 Principle An initial suspension and decimal dilutions are prepared from the test sample. The soli
27、d selective medium, CFC agar, is inoculated with a specified quantity of the initial suspension of the product. BS EN ISO 13720:2010ISO 13720:2010(E) 2 ISO 2010 All rights reservedOther plates are prepared under the same conditions, using decimal dilutions of the initial suspension. The plates are i
28、ncubated at 25 C for 44 h 4 h. Colonies of presumptive Pseudomonas spp. are confirmed by the oxidase test (positive). The number of presumptive Pseudomonas spp. per millilitre, or per gram, of the test sample is calculated from the number of confirmed colonies per plate. 5 Diluent, culture medium an
29、d reagent 5.1 General For current laboratory practice, see ISO 7218; for preparation and testing of media, see ISO/TS 11133-1 and ISO/TS 11133-2. 5.2 Diluent See ISO 6887-1 and ISO 6887-2. 5.3 Cephalothin-sodium fusidate-cetrimide agar (see Reference 3) 5.3.1 Basic medium 5.3.1.1 Composition Enzymat
30、ic digest of gelatin 16,0 g Enzymatic digest of casein 10,0 g Potassium sulfate (K2SO4) 10,0 Magnesium chloride (MgCl2) 1,4 g Agara12,0 g to 18,0 gWater 1 000 ml aThe mass used depends on the gel strength of the agar. 5.3.1.2 Preparation Dissolve the basic components or the dehydrated basic medium i
31、n the water, by bringing to the boil. Adjust the pH (6.4), if necessary, so that after sterilization it is 7,2 0,2 at 25 C. Dispense the basic medium into flasks or bottles of suitable capacity (6.6). Sterilize for 15 min in the autoclave (6.1) at 121 C. 5.3.2 Inhibitor solutions Do not keep solutio
32、ns for more than 7 days at 5 C 3 C. 5.3.2.1 Cephalothin solution 5.3.2.1.1 Composition Cephalothin sodium salt 0,1 g Water 100 ml BS EN ISO 13720:2010ISO 13720:2010(E) ISO 2010 All rights reserved 35.3.2.1.2 Preparation Dissolve the cephalothin in the water and sterilize the solution by filtration.
33、5.3.2.2 Sodium fusidate solution 5.3.2.2.1 Composition Sodium fusidate 0,1 g Water 100 ml 5.3.2.2.2 Preparation Dissolve the sodium fusidate in the water and sterilize the solution by filtration. 5.3.2.3 Cetrimide solution 5.3.2.3.1 Composition Cetrimidea0,1 g Water 100 ml aMixture consisting chiefl
34、y of tetradecyltrimethylammonium bromide together with smaller amounts of dodecyltrimethylammonium bromide and cetrimonium (hexadecyltrimethylammonium) bromide. 5.3.2.3.2 Preparation Dissolve the cetrimide in the water and sterilize the solution by filtration. 5.3.3 Complete medium 5.3.3.1 Compositi
35、on Volume ml Final concentration g/ml Basic medium (5.3.1) 100 Cephalothin solution (5.3.2.1) 5 50 Sodium fusidate solution (5.3.2.2) 1 10 Cetrimide solution (5.3.2.3) 1 10 5.3.3.2 Preparation Add the inhibitor solutions to the basic medium, cooled in a water bath (6.3) to 47 C 2 C, then mix careful
36、ly. 5.3.4 Preparation of CFC agar plates Pour amounts of approximately 15 ml of the complete medium (5.3.3) into sterile Petri dishes (6.8) and allow to solidify. Immediately before use, the agar plates should be dried in accordance with ISO/TS 11133-1. If prepared in advance, the undried agar plate
37、s shall be kept for not more than 4 weeks at 5 C 3 C. BS EN ISO 13720:2010ISO 13720:2010(E) 4 ISO 2010 All rights reserved5.3.5 Performance testing For the definition of selectivity and productivity refer to ISO/TS 11133-1. The performance of CFC agar shall be tested according to the methods and cri
38、teria described in ISO/TS 11133-2. 5.3.5.1 Productivity Incubation: At 25 C 1 C for 44 h 4 h Strain: Pseudomonas fluorescens WDCM 001151)or Pseudomonas fragi WDCM 001161)Reference medium: Soybean-casein digest agar medium (TSA) Method of control: Quantitative Criteria: Productivity ratio PRW 0,5 5.3
39、.5.2 Selectivity Incubation: At 25 C 1 C for 44 h 4 h Strain: Escherichia coli WDCM 000131)Method of control: Quantitative Criteria: Total inhibition 5.4 Reagent for the detection of oxidase 5.4.1 Composition N,N,N,N-Tetramethyl-p-phenylenediamine dihydrochloride 1,0 g Water 100 ml 5.4.2 Preparation
40、 Dissolve the reagent in the water immediately before use. Commercially available discs or sticks may be used. In this case, follow the manufacturers recommendations. 6 Apparatus Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following. 6.1 Oven for dry sterilizati
41、on or autoclave for wet sterilization. 6.2 Incubator, capable of operating at 25 C 1 C. 1) Refer to the reference strain catalogue available (viewed 2010-07-19) on http:/www.wfcc.nig.ac.jp/WDCM_Reference_Strain_Catalogue for information on culture collection strain numbers and contact details BS EN
42、ISO 13720:2010ISO 13720:2010(E) ISO 2010 All rights reserved 56.3 Water bath, capable of operating at 47 C 2 C. 6.4 pH-meter, capable of measurements to an accuracy of 0,05 pH units. 6.5 Loops made of platinum-iridium alloy or equivalent sterile disposable loops. 6.6 Test tubes, bottles or flasks, o
43、f appropriate capacity. 6.7 Total-delivery pipettes, sterile, of nominal capacity 1 ml and graduated in 0,1 ml divisions, ISO 8351class A, or automatic pipettes, ISO 8655-22, with use of sterile tips. 6.8 Petri dishes, made of glass or plastic, of diameter 90 mm to 100 mm. 6.9 Spreaders, made of gla
44、ss or plastic, e.g. hockey sticks made from a glass rod of approximately 3,5 mm diameter and 200 mm length, bent at right angles about 30 mm from one end and with the cut ends annealed. 7 Sampling Sampling is not part of the method specified in this International Standard. If there is no specific In
45、ternational Standard dealing with sampling of the product concerned, it is recommended that the interested parties come to an agreement on this subject. It is important that the laboratory receive a sample which is truly representative and which has not been damaged or changed during transport or st
46、orage (see ISO 7218). 8 Preparation of test sample Prepare the test sample in accordance with ISO 6887-1 and ISO 6887-2 and/or the specific International Standard appropriate to the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come t
47、o an agreement on this subject. 9 Procedure 9.1 Test portion, initial suspension and dilutions Prepare the initial suspension and dilutions in accordance with ISO 6887-2. 9.2 Inoculation and incubation 9.2.1 In accordance with ISO 7218, one plate per dilution shall be used with at least two successi
48、ve dilutions. If only one dilution is performed, then two plates shall be used. 9.2.2 Take one CFC agar plate (5.3.4). Transfer, by means of a pipette (6.7), 0,1 ml of the initial suspension on to the plate. Take another CFC agar plate. Transfer, by means of another sterile pipette, 0,1 ml of the fi
49、rst decimal dilution of the initial suspension on to the plate. Repeat these operations with subsequent dilutions, using a clean sterile pipette for each decimal dilution. 9.2.3 Spread the liquid over the surface of the agar plate with a sterile spreader (6.9) until the surface is completely dry. BS EN ISO 13720:2010ISO 13720:2010(E) 6 ISO 2010 All rights reserved9.2.4 Incubate the dishes prepared in this way, with their lids downwards, in an incubator (6.2) at 25 C 1 C f
