1、BRITISH STANDARD BS ISO 8870:2006 Milk and milk-based products Detection of thermonuclease produced by coagulase-positive staphylococci ICS 67.100.01 BS ISO 8870:2006 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 May 2006 BSI 2006 ISBN 0
2、 580 48366 5 National foreword This British Standard reproduces verbatim ISO 8870:2006 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: A list of organizations re
3、presented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by u
4、sing 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 correct application. Compliance with a British Standard does not of itself confer immunity
5、 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 informed; monitor related international and European developments and promulgate them in the
6、UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to v, a blank page, pages 1 to 7 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd.
7、 No. Date Comments Reference numbers ISO 8870:2006(E) IDF 83:2006(E)INTERNATIONAL STANDARD ISO 8870 IDF 83 First edition 2006-05-01 Milk and milk-based products Detection of thermonuclease produced by coagulase-positive staphylococci Lait et produits laitiers Recherche de la thermonuclase en provena
8、nce des staphylocoques coagulase positive BS ISO 8870:2006ii iiiContents Page Foreword iv Foreword. v 1 Scope1 2 Normative references1 3 Terms and definitions .1 4 Principle1 5 Culture media and reagents .1 6 Apparatus.4 7 Sampling.4 8 Procedure.5 9 Evaluation and interpretation.6 10 Test report6 Bi
9、bliography 7 BS ISO 8870:2006iv 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 inter
10、ested 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
11、 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 te
12、chnical 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. IS
13、O shall not be held responsible for identifying any or all such patent rights. ISO 8870|IDF 83 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. BS ISO 8
14、870:2006v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with
15、ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International 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 lea
16、st 50 % of IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 8870|IDF 83 was prepared by the International
17、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 work was carried out by the Joint ISO-IDF Action Team on Harmonization, of the Standing Committee on Microbiological methods of analys
18、is, under the aegis of its project leader, Mr H. Becker (DE). This edition of ISO 8870|IDF 83 cancels and replaces IDF 83A:1998, which has been editorially revised. BS ISO 8870:2006blank1 Milk and milk-based products Detection of thermonuclease produced by coagulase-positive staphylococci 1 Scope Th
19、is International Standard specifies a method for the detection of heat-stable DNase (thermonuclease) produced by coagulase-positive staphylococci in milk and milk-based products. The enzyme can be used as an indicator that staphylococcal growth has reached hazardous levels, and can reveal the potent
20、ial presence of staphylococcal enterotoxins. 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 am
21、endments) applies. ISO 8261|IDF 122, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 thermo
22、nuclease enzyme produced by coagulase-positive staphylococci, which hydrolyses deoxyribonucleic acid (DNA) under the conditions specified in this International Standard 4 Principle Milk, reconstituted milk powder or homogenized milk-based products are, after addition of skim milk powder, adjusted to
23、 pH 3,8 and centrifuged. The supernatant is precipitated with trichloroacetic acid. After centrifuging, the precipitate is adjusted to pH 8,5 and Tris buffer is added. The solution is then heated in a boiling water bath for 15 min and tested for thermonuclease activity in toluidine blue O-DNA agar.
24、The colour of the agar turns from blue to pink if thermonuclease cleaves the DNA molecule. This colour reaction is due to the metachromatic properties of toluidine blue O. 5 Culture media and reagents 5.1 Basic materials For current laboratory practice, see ISO 8261|IDF 122. BS ISO 8870:20062 Use on
25、ly reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. If the prepared media and reagents are not used immediately, store them, unless otherwise stated, in the dark at between 0 C and +5 C for no longer than 1 month
26、, under conditions which do not produce any change in their composition. 5.2 Toluidine blue O-DNA agar 5.2.1 Tris buffer 5.2.1.1 Composition Tris(hydroxymethyl)aminomethane (C 4 H 11 NO 3 ) 6,06 g Calcium chloride (CaCl 2 ) 0,11 g Water 1 000 ml 5.2.1.2 Preparation Dissolve the tris(hydroxymethyl)am
27、inomethane and the calcium chloride in 980 ml of water. Adjust the pH to 9,0 and add water to 1 000 ml. 5.2.2 Base medium 5.2.2.1 Composition Deoxyribonucleic acid (DNA) a0,3 g Sodium chloride (NaCl) 10,0 g Agar 10,0 g Tris buffer (5.2.1) 1 000 ml aThe DNA used shall have been proven to be suitable
28、for the detection of thermonuclease. 5.2.2.2 Preparation Boil the components in a water bath (6.2) until the DNA and the agar are completely dissolved (about 1,5 h). 5.2.3 Toluidine blue O solution 5.2.3.1 Composition Toluidine blue O (C 15 H 16 N 3 SCl) a0,31 g Water 10 ml aToluidine blue O (Colour
29、 Index: C.I. Basic Blue 17; C.I. No. 52040). 5.2.3.2 Preparation Dissolve the toluidine blue O in the water with minimal heating and filter it through gauze (6.12). BS ISO 8870:20063 5.2.4 Complete medium 5.2.4.1 Composition Base medium (5.2.2) 1 000 ml Toluidine blue O solution (5.2.3) 3 ml 5.2.4.2
30、 Preparation Cool the base medium to approximately 50 C. Add the filtered toluidine blue O solution and mix by swirling. The complete medium may be stored for several months in small portions in closed flasks or bottles in the dark at between 0 C and +5 C. 5.2.5 Preparation of agar plates Transfer t
31、o Petri dishes (6.8), 13 ml of the complete medium (5.2.4) or the stored medium preheated to approximately 50 C. Allow it to solidify. Prepared plates may be stored for about 2 months (bottom uppermost and protected from drying) in the dark at between 0 C and +5 C. 5.3 Hydrochloric acid, c(HCl) = 2
32、mol/l. 5.4 Sodium hydroxide, c(NaOH) = 2 mol/l. 5.5 Trichloroacetic acid, c(CCl 3 COOH) = 3 mol/l. Dissolve 49,02 g of trichloroacetic acid in 100 ml of water. 5.6 Skim milk powder Skim milk powder shall be free from thermonuclease. Test every new lot using the method described in this International
33、 Standard. 5.7 Brain heart infusion broth 5.7.1 Composition Enzymatic digest of animal tissues 10,0 g Dehydrated calf brain infusion 12,5 g Dehydrated beef heart infusion 5,0 g Glucose 2,0 g Sodium chloride 5,0 g Disodium hydrogen phosphate, anhydrous (Na 2 HPO 4 ) 2,5 g Water 1 000 ml BS ISO 8870:2
34、0064 5.7.2 Preparation Dissolve the components or the dehydrated complete medium in the water by heating, if necessary. Adjust the pH so that after sterilization it is 7,4 0,2 at 25 C. Transfer the culture medium in quantities of approximately 5 ml in tubes of appropriate capacity (6.11). Sterilize
35、the medium at 121 C for 15 min. 5.8 Test microorganism (positive control) Any thermonuclease positive strain of Staphylococcus aureus may be used as test microorganism. 6 Apparatus Use the apparatus required for the preparation of test samples as specified in ISO 8261|IDF 122, the usual microbiologi
36、cal laboratory apparatus and, in particular, the following. Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications. Re- usable glassware should be capable of undergoing repeated sterilization and should be chemically inert. 6.1 Incubator, capable of
37、 being maintained at 37 C 1 C. 6.2 Water baths, capable of operating at 50 C and of boiling (for heating and cooling of the medium and the solutions to suitable temperatures). 6.3 Centrifuge, capable of delivering a centrifugal force of more than 27 000 g. 6.4 Centrifuge, capable of delivering a cen
38、trifugal force of more than 2 800 g. 6.5 Hollow cylinder, made from metal, of diameter about 2 mm, for cutting wells into the toluidine blue O-DNA agar. 6.6 Micropipettes, of nominal capacity 0,01 ml. 6.7 Loops, of platinum-iridium or nickel-chromium, of diameter approximately 3 mm. 6.8 Petri dishes
39、, made of glass or plastic, of diameter between 90 mm and 100 mm. 6.9 Measuring cylinders, of nominal capacities 10 ml, 100 ml and 1 000 ml. 6.10 Bottles, with stoppers, for storing toluidine blue O-DNA agar. 6.11 Test tubes, of diameter 15 mm and length 100 mm. 6.12 Gauze (for example absorbent cot
40、ton gauze Ph. Eur. type 20) 1) . 7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. 1) Absorbent cotton gauze Ph. Eur. type 2 is an example of a suitable product available commercially. This information
41、is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of this product. BS ISO 8870:20065 Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707|IDF 50. Store te
42、st samples in such a way that deterioration and change in composition are prevented. 8 Procedure 8.1 Preparation of the test sample 8.1.1 Milk Adjust 100 ml of test sample to pH 3,8 with hydrochloric acid (5.3). 8.1.2 Milk-based products Suspend 20 g of test sample and 5 g of skim milk powder (5.6)
43、in 50 ml of water. Adjust the pH to 3,8 with hydrochloric acid (5.3). The addition of skim milk powder is not necessary for all milk-based products. It may be omitted, for instance, when skim milk powder, whole milk powder or caseins are examined. The addition is required in the case of whey powder,
44、 yoghurt and fruit yoghurt and fruit ice cream. For any other milk-based products, the addition of skim milk powder is recommended. For the examination of cheese, the following extraction procedure may be used as an alternative. Suspend 20 g of cheese in 100 ml of water (at 45 C) in a blender. After
45、 blending, adjust the pH to 4,5 using hydrochloric acid (6 mol/l). Transfer the suspension to a 750 ml flat-bottom flask. Add water up to a final mass of 12,5 times the cheese mass. Agitate the suspension overnight in a water bath set at 25 C for 16 h. Adjust the pH to 4,5 again. Centrifuge and filt
46、rate the suspension. Continue the procedure as described in 8.1.3 with 100 ml of filtrate. If skim milk powder is not added, suspend 20 g of test sample in 40 ml water. Adjust the pH to 3,8 with hydrochloric acid (5.3). 8.1.3 General procedure 8.1.3.1 Centrifuge (6.3) the test sample at between 27 0
47、00 g and 34 000 g at 5 C for 20 min. 8.1.3.2 Decant the supernatant. Add 0,05 ml of cold trichloroacetic acid (5.5) for each millilitre of sample and mix. 8.1.3.3 Centrifuge at between 27 000 g and 34 000 g at 5 C for 20 min. 8.1.3.4 Dissolve the sediment in 1 ml of Tris buffer (5.2.1). Adjust the p
48、H to 8,5 with sodium hydroxide (5.4). Dilute the solution to 2 ml with Tris buffer and mix. 8.1.3.5 Heat the solution in a boiling water bath (6.2) for 15 min. 8.2 Preparation of the test microorganism (positive control) 8.2.1 Inoculate the brain heart infusion broth (5.7) with the test microorganis
49、m (5.8). Incubate at 37 C for 24 h. Centrifuge (6.4) at between 2 800 g and 3 500 g for 15 min. Decant the supernatant. 8.2.2 Heat the supernatant in a boiling water bath (6.2) for 15 min. The heated supernatant may be stored at 5 C for about 4 weeks. BS ISO 8870:20066 If necessary, any Staphylococcus aureus strain (for example isolated from milk or milk-based products) may be tested for thermonuclease production in the same way as described for the test microo
