1、BRITISH STANDARD BS ISO 6611:2004 Milk and milk products Enumeration of colony-forming units of yeasts and/or moulds Colony-count technique at 25 C ICS 07.100.30; 67.100.01 Incorporating corrigendum July 2010National foreword This British Standard is the UK implementation of ISO 6611:2004. It supers
2、edes BS 4285-3.6:1986 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the
3、 necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immunity from legal obligations. BS ISO 6611:2004 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 1 Novem
4、ber 2004 BSI 2010 Amendments/corrigenda issued since publication Date Comments 31 July 2010 Supersession text added ISBN 978 0 580 71485 6 Reference numbers ISO 6611:2004(E) IDF 94:2004(E) OSI DI dnaF 4002INTERNATIONAL STANDARD ISO 6611 IDF 94 Second edition 2004-10-15 Milk and milk products Enumera
5、tion of colony-forming units of yeasts and/or moulds Colony-count technique at 25 C Lait et produits laitiers Dnombrement des units formant colonie de levures et/ou moisissures Comptage des colonies 25 C IS:1166 O4002(E) ID:49 F4002(E) DPlcsid Fremia ihTs PDF file may ctnoian emdebt dedyfepcaes. In
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11、o gr Plbuisdehi n Switlrez dnaii ISO dnaID 4002 FA ll rihgtsser edevrISO 6611:2004 (E) BS ISO 6611:2004IS:1166 O4002(E) ID:49 F4002(E) I SO dna ID 4002 F All irhgts seredevr iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (I
12、SO 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, gov
13、ernmental 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
14、 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 membe
15、r 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 6611IDF 94 was prepared by Technical Committee ISO/TC 34, Food products,
16、 Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. This edition of ISO 6611IDF 94 cancels and replaces ISO 6611:1992, of which it consti
17、tutes a minor revision. ISO 6611:2004 (E) BS ISO 6611:2004IS:1166 O4002(E) ID:49 F4002(E) iv I SO dna ID 4002 F All irhgts seredevrForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committ
18、ee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams an
19、d Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the National Committees casting a vote. ISO 6611IDF 94 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and
20、milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Group of Experts, Enumeration of yeasts and moulds in dairy pro
21、ducts (E34), under the aegis of its chairman, Mr J.J. Devoyod (FR). This edition of ISO 6611IDF 94 cancels and replaces IDF 94B:1990, of which it constitutes a minor revision. ISO 6611:2004 (E) BS ISO 6611:2004NITERNATNOIAL STANDARD IS:1166 O4002(E) ID:49 F4002(E)I SO dna ID 4002 F All irhgts serede
22、vr 1Milk and milk products Enumeration of colony-forming units of yeasts and/or moulds Colony-count technique at 25 C 1 Scope This International Standard specifies a method for the detection and enumeration of colony-forming units (CFU) of viable yeasts and/or moulds in milk and milk products by mea
23、ns of the colony-count technique at 25 C. The method is applicable to milk, liquid milk products, dried milk, dried sweet whey, dried buttermilk, lactose, cheese, acid casein, lactic casein, rennet casein, caseinate, acid whey powder, butter, frozen milk products (including edible ices), custard, de
24、sserts, fermented milk and cream. NOTE This method is not suitable for a large number of thermolabile yeasts (in fresh cheese). In such cases the agar-surface-plating method is preferred. 2 Normative references The following referenced documents are indispensable for the application of this document
25、. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for micro
26、biological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations ISO 8261IDF 122:2001, Milk and milk products General guidance for the preparation o
27、f test samples, initial suspensions and decimal dilutions for microbiological examination ISO 6611:2004 (E) BS ISO 6611:2004ID:49 F4002(E) 2 I SO dna ID 4002 F All irhgts seredevr3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 yeasts and moul
28、ds microorganisms which at 25 C form colonies in a selective medium under the conditions specified in this International Standard 4 Principle 4.1 Poured plates are prepared using a specified selective culture medium and a specified quantity of the test sample if the initial product is liquid, or of
29、an initial suspension in the case of other products. Other plates are prepared, under the same conditions, using decimal dilutions of the test sample or of the initial suspension. 4.2 The plates are aerobically incubated at 25 C for 5 days. 4.3 The number of colony-forming units (CFU) of yeasts and/
30、or moulds per gram or per millilitre of product is calculated from the number of colonies obtained on plates chosen at dilution levels so as to give a significant result. 5 Diluents and culture medium For general guidance, see ISO 7218. 5.1 Basic materials See ISO 8261IDF 122. 5.1.1 Diluents For dil
31、uents for general use and diluents for special purposes, see ISO 8261IDF 122. 5.1.2 Distribution, sterilization and storage of diluents See ISO 8261IDF 122. 5.2 Yeast extract/dextrose/oxytetracycline/agar medium 5.2.1 Basic medium 5.2.1.1 Components Yeast extract powder 5,0 g Dextrose (C 6 H 12 O 6
32、) 20,0 g Agar 10 g to 15 g a Water 900 ml aDepending on the gel strength of the agar. ISO 6611:2004 (E) BS ISO 6611:2004IS:1166 O4002(E) ID:49 F4002(E) I SO dna ID 4002 F All irhgts seredevr 35.2.1.2 Preparation Dissolve the components or dehydrated complete medium in the water, by heating if necess
33、ary. Adjust the pH, if necessary, so that after sterilization it is 6,6 at 25 C. Sterilize in an autoclave (6.1) at 121 C 1 C for 15 min. 5.2.2 Oxytetracycline hydrochloride solution 5.2.2.1 Components Oxytetracycline hydrochloride (C 22 H 30 O 11 HCl) 50 mg Water 50 ml 5.2.2.2 Preparation Dissolve
34、the oxytetracycline hydrochloride in the water. The solution shall be freshly prepared before use. Sterilize the solution by means of filtration. 5.2.3 Complete medium 5.2.3.1 Components Oxytetracycline hydrochloride solution 10 ml Basic medium 90 ml 5.2.3.2 Preparation Cool the sterilized basic med
35、ium (5.2.1) to 45 C. Just before use, bring the oxytetracycline hydrochloride solution (5.2.2) to 45 C and add 10 ml of this solution aseptically to 90 ml of the basic medium. 5.3 Yeast extract/dextrose/chloramphenicol/agar medium 5.3.1 Components Yeast extract powder 5,0 g Dextrose (C 6 H 12 O 6 )
36、20,0 g Chloramphenicol (C 11 H 12 Cl 2 N 2 O 5 ) 0,1 g aAgar 12 g to 15 g bWater 1 000 ml aIn order to obtain a final concentration of 100 g/ml of medium. bDepending on the gel strength of the agar. 5.3.2 Preparation Dissolve the components in the water by heating, if necessary. Adjust the pH, if ne
37、cessary, so that after sterilization it is 6,6 at 25 C. ISO 6611:2004 (E) BS ISO 6611:2004IS:1166 O4002(E) ID:49 F4002(E) 4 I SO dna ID 4002 F All irhgts seredevrDispense the agar medium into suitable containers (6.8). Sterilize in an autoclave (6.1) at 121 C 1 C for 15 min. 6 Apparatus and glasswar
38、e CAUTION Sterilize all apparatus that will come into contact with the test sample, the diluents, the dilutions or the culture medium in accordance with ISO 8261 IDF 122:2001, 6.1. Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications. Usual microb
39、iological laboratory equipment, the apparatus required for the preparation of test samples and dilutions as specified in ISO 8261IDF 122 and, in particular, the following. 6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). See ISO 7218. 6.2 Incubator, capable of operating a
40、t 25 C 1 C. 6.3 Petri dishes, of 90 mm to 100 mm diameter. 6.4 Graduated pipettes, plugged with cotton wool, calibrated to deliver 1 ml 0,02 ml, or 10 ml 0,2 ml or 11 ml 0,2 ml. 6.5 Water bath, capable of operating at 45 C 1 C. 6.6 Colony-counting equipment, consisting of an illuminated base with a
41、dark background, fitted with a magnifying lens to be used at a magnification of 1,5, and a mechanical or electronic digital counter. 6.7 pH-meter, temperature-compensated, accurate to 0,1 pH units at 25 C. 6.8 Culture bottles or flasks. Bottles or flasks with non-toxic metal screwcaps may be used. 7
42、 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707. In cheeses that are matur
43、ed with a yeast or mould coat, it may be desirable to exclude the coat from the sample for analysis. In these instances, the coat may be removed using a sterile scalpel or knife before sampling is commenced. 8 Procedure 8.1 General In order to improve the precision of the method, the preparation of
44、dilutions should be carefully standardized. Factors that affect precision are as follows: type of blending equipment; blending time; ISO 6611:2004 (E) BS ISO 6611:2004IS:1166 O4002(E) ID:49 F4002(E) I SO dna ID 4002 F All irhgts seredevr 5 diluent; time allowed for large particles to settle; mixing
45、time allowed in the preparation of decimal dilutions. CAUTION Usual aseptic precautions shall be taken. The operations described in 8.2 and 8.3 shall not be carried out in sunlight. 8.2 Preparation of the test sample and primary dilution See ISO 8261IDF 122. 8.3 Further decimal dilutions See ISO 826
46、1IDF 122. 8.4 Duration of the procedure See ISO 6887-1. 8.5 Inoculation and incubation 8.5.1 Take two sterile Petri dishes (6.3). Transfer to each dish, by means of a sterile pipette (6.4), 1 ml of the test sample, if liquid, or 1 ml of the initial suspension in the case of other products. 8.5.2 Tak
47、e two further sterile Petri dishes. Transfer to each dish, by means of another sterile pipette, 1 ml of the 10 1dilution (liquid product) or 1 ml of the 10 2dilution (other products). 8.5.3 If necessary, repeat this operation using further decimal dilutions. 8.5.4 Pour about 15 ml of the medium cont
48、aining oxytetracycline hydrochloride (5.2) or the medium containing chloramphenicol (5.3), previously melted and maintained at 45 C in the water bath (6.5), into each Petri dish. 8.5.5 Carefully mix the inoculum with the medium by rotating the Petri dishes, and allow the mixture to solidify by leavi
49、ng the Petri dishes to stand on a cool horizontal surface. 8.5.6 The time taken between the preparation of the first dilution and the mixing of the inoculum with the medium shall not exceed 15 min. 8.5.7 Prepare a sufficient number of control plates to check the sterility. 8.5.8 After inverting the prepared dishes (8.5.5), place them (while keeping in an upright position) in the i
