1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58antimicrobial residues Tube diffusion testICS 67.100.01Milk and milk products Determination of DRAF
2、T FOR DEVELOPMENTDD ISO/TS 26844:2006DD ISO/TS 26844:2006This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 29 December 2006 BSI 2006ISBN 0 580 49853 0an international Standard, to extend the life of the Technical Specification or to withdr
3、aw it. Comments should be sent to the Secretary of the responsible BSI Technical Committee at British Standards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products.A list of organi
4、zations represented on AW/5 can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Amendments issued since publicationAmd. No. Date Commentsthat UK experience can be report
5、ed to the international organization responsible for its conversion to an international standard. A review of this publication will be initiated not later than 3 years after its publication by the international organization so that a decision can be taken on its status. Notification of the start of
6、the review period will be made in an announcement in the appropriate issue of Update Standards.According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into National forewordThis Draft for Development was published
7、 by BSI. It is the UK implementation of ISO/TS 26844:2006. This publication is not to be regarded as a British Standard.It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on this provisional basis, so that information and exper
8、ience of its practical application can be obtained.Comments arising from the use of this Draft for Development are requested so Reference numbersISO/TS 26844:2006(E)IDF/RM 215:2006(E)TECHNICAL SPECIFICATION ISO/TS26844IDF/RM215First edition2006-11-15Milk and milk products Determination of antimicrob
9、ial residues Tube diffusion test Lait et produits laitiers Dtermination de rsidus antimicrobiens Test de dissmination en tube DD ISO/TS 26844:2006ii iiiContents Page Foreword iv Foreword. v 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 1 5 Test organism, culture medi
10、a, standard solutions and control samples . 2 6 Apparatus and glassware 6 7 Sampling 7 8 Preparation of test sample. 7 9 Procedure 7 9.1 Control samples 7 9.2 Test tube preparation . 7 9.3 Incubation 8 9.4 Interpretation. 8 10 Confirmation (optional) 8 10.1 General. 8 10.2 Presumptive confirmation o
11、f beta-lactams8 10.3 Presumptive confirmation of sulfonamides. 9 10.4 Confirmation of other inhibitors 9 11 Expression of results . 9 12 Precision 9 13 Test report . 9 Annex A (informative) Data from collaborative studies . 10 Annex B (informative) Preparation of test-organism suspension. 11 Bibliog
12、raphy . 13 DD ISO/TS 26844:2006iv 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 int
13、erested 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 Electrotechnic
14、al 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
15、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. In other circumstances, particularly when there is an urgent market requirement for such documents, a technical commit
16、tee may decide to publish other types of normative document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting
17、a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether
18、it will be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn. Attention is
19、 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/TS 26844IDFRM 215 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and m
20、ilk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. DD ISO/TS 26844:2006vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee h
21、as the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. The main task of technical committees is to prepare International Standards. Draft Inte
22、rnational Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the IDF National Committees casting a vote. In other circumstances, particularly when there is
23、an urgent market requirement for such documents, a Standing Committee may decide to publish an other type of normative document which is called by IDF: Reviewed method. Such a method represents an agreement between the members of a Standing Committee and is accepted for publication if it is approved
24、 by at least 50 % of the committee members casting a vote. A Reviewed method is equal to an ISO/PAS or ISO/TS and will, therefore, also be published jointly under ISO conditions. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF
25、 shall not be held responsible for identifying any or all such patent rights. ISO/TS 26844IDF/RM 215 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All
26、 work was carried out by the Joint ISO-IDF Action Team on Veterinary residues, of the Standing Committee on Analytical methods for additives and contaminants, under the aegis of its project leaders, Mr H. Stegeman (NL) and Mr J. Kerkhof (NL). DD ISO/TS 26844:2006blank1Milk and milk products Determin
27、ation of antimicrobial residues Tube diffusion test 1 Scope This Technical Specification (Reviewed Method) specifies a microbiological inhibitor test for the detection of a broad variety of antimicrobials in milk and milk products. The method is applicable to raw milk, heat-treated milk and reconsti
28、tuted dried milk. 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 4833
29、, Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of micro-organisms Colony-count technique at 30 C ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations ISO 13969IDF 183, Milk and milk product
30、s Guidelines for a standardized description of microbial inhibitor tests ISO 18330IDF 188, Milk and milk products Guidelines for a standardized description of immunoassays or receptor assays for the detection of antimicrobial residues 3 Terms and definitions For the purposes of this document, the fo
31、llowing terms and definitions apply. 3.1 antimicrobial substances substances that show an inhibition in the procedure specified in this document 3.2 limits of detection concentration level at which a defined percentage of positive samples is detected EXAMPLE 95 %. 4 Principle A milk sample is added
32、to two test tubes with agar media containing Geobacillus stearothermophilus ATCC 10149 (identical to NIZO strain C953). The test tubes differ from each other in pH, in added supplements and synergistic antibiotics. Incubation resulting in normal growth of the organism causes the pH DD ISO/TS 26844:2
33、0062 indicator in the agar to change colour from purple to yellow. When substances that are inhibitory to the growth of microorganisms are present in the milk, the colour of the pH indicator will remain purple. Test tube A (pH 7,0; chloramphenicol) shows an improved sensitivity for tetracycline resi
34、dues, and test tube B (pH 8,0; trimethoprim) for beta-lactams, macrolides, aminoglycosides, sulfonamides and trimethoprim residues. 5 Test organism, culture media, standard solutions and control samples Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or de
35、ionized water or water of equivalent purity. 5.1 Test organism Use a suspension of Geobacillus stearothermophilus ATCC 10149 (identical to NIZO strain C953)1)adjusted to a viable count of approximately 5 000 000 colony-forming units/ml (see Annex B for preparation). Check the quality of each new bat
36、ch of the test-organism suspension by determining the sensitivity for the standard solutions mentioned in Table 1. Table 1 Standard solutions for testing the sensitivity of the test-organism suspension Standard solution Content g/kg milk Benzylpenicillin (Penicillin-G) 2 Sulfadiazine 150 Neomycin 30
37、 Erythromycin 10 Oxytetracycline 100 Perform the check with standard solutions and control milk in 5-fold, according to the procedure described in Clause 9. Determine the sensitivity of the test-organism suspension for benzylpenicillin and oxytetracycline with tube A (5.2.5) and the sensitivity for
38、sulfadiazine, neomycin and erythromycin with tube B (5.2.6). A positive result should be obtained in all test tubes. 5.2 Culture media In order to improve the reproducibility of the method, it is recommended to use dehydrated basic components or dehydrated complete media for the preparation of cultu
39、re media. Follow the manufacturers instructions. 5.2.1 Basic medium 5.2.1.1 Components Casein trypton 5,0 g Yeast extract 2,5 g Glucose, anhydrous 1,0 g Agar 10 g to 15 g Water 1 000 ml NOTE The basic dehydrated medium is commercially available as Plate Count Agar. 1) Suspension of Geobacillus stear
40、othermophilus ATCC 10149 or NIZO strain C953 is an example of a product available commercially. This information is given for the convenience of users of this Technical Specification and does not constitute an endorsement by ISO or IDF of this product. DD ISO/TS 26844:200635.2.1.2 Preparation Dissol
41、ve the components in the water by heating. Adjust the pH so that after sterilization it is 7,0 0,2. Autoclave the medium at 121 C 1 C for 15 min. The thus-prepared basic medium may be kept for a maximum of 3 months if stored in the dark at 0 C to +5 C. 5.2.2 Bromocresolpurple solution 5.2.2.1 Compon
42、ents Bromocresolpurple 250 mg Ethanol, 96% 5 ml Water 100 ml 5.2.2.2 Preparation Dissolve the bromocresolpurple in the ethanol. Dilute with water to 100 ml. The bromocresolpurple solution may be kept for a maximum of 6 months if stored in the dark at 0 C to +5 C. 5.2.3 Chloramphenicol (CAP) solution
43、 5.2.3.1 Components Chloramphenicol 20,0 mg Methanol 5 ml Water 100 ml 5.2.3.2 Preparation Dissolve the chloramphenicol in the methanol. Dilute with water to 100 ml. The chloramphenicol solution may be kept for a maximum of 1 month if stored in the dark at 0 C to +5 C. 5.2.4 Trimethoprim (TMP) solut
44、ion 5.2.4.1 Components Trimethoprim 25,0 mg Ethanol, 96 % 5 ml Water 1 000 ml 5.2.4.2 Preparation Dissolve the trimethoprim in the ethanol. Dilute with water to 1 000 ml. The TMP solution may be kept for a maximum of 1 month if stored in the dark at 0 C to +5 C. DD ISO/TS 26844:20064 5.2.5 Test tube
45、s A (pH 7) Melt the basic medium (5.2.1). Cool the medium in a water bath (6.3) to 63 C 1 C. Add 1,5 ml of CAP solution (5.2.3) and 2 ml of bromocresolpurple solution (5.2.2) to 100 ml of the preheated basic medium, while keeping the medium in the water bath set at 63 C. Mix the medium well. Keeping
46、 the medium in the water bath set at 63 C, adjust the pH (see 6.5) to 7,0 0,1 at that temperature by using 1 mol/l NaOH or 1 mol/l HCl. Subsequently, add per 100 ml of medium such an amount (approx. 2 ml) of test-organism suspension (5.1) that in the absence of antimicrobial substances the colour ch
47、ange does appear after incubation in the water bath at 63 C for 4 h 15 min 30 min. Mix and dispense the test medium in portions of 1 ml in tubes (6.4) and leave the medium to solidify. The test tubes A may be stored at 0 C to +5 C for a maximum of 3 days, provided that the tubes are covered to avoid
48、 evaporation (e.g. with parafilm). 5.2.6 Test tubes B (pH 8) Melt the basic medium (5.2.1). Cool the medium in a water bath (6.3) to 63 C 1 C. Add 0,6 ml of TMP solution (5.2.4) and 2 ml of bromocresolpurple solution (5.2.2) to 100 ml of the preheated basic medium, while keeping the medium in the wa
49、ter bath set at 63 C. Mix the medium well. Keeping the medium in the water bath set at 63 C, adjust the pH (see 6.5) to 8,00 0,02 at that temperature by using 1 mol/l NaOH or 1 mol/l HCl. Take care that while adjusting, the pH of the medium does not exceed a value of 8,05. Subsequently, add an amount (approx. 2 ml) of test-organism suspension (5.1) per 100 ml of medium so that in the absence of antimicrobial substances the colour change appears after the incubation in the water
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