1、 Reference numbers ISO 13559:2002(E) IDF 153:2002(E) ISO and IDF 2002INTERNATIONAL STANDARD ISO 13559 IDF 153 First edition 2002-11-01 Butter, fermented milks and fresh cheese Enumeration of contaminating microorganisms Colony-count technique at 30 C Beurre, laits ferments et fromage frais Dnombreme
2、nt des micro- organismes contaminants Technique par comptage des colonies 30 C ISO 13559:2002(E) IDF 153:2002(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces wh
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8、ghts 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 committees. Each member body interested in a subj
9、ect 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 Commission (IE
10、C) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committ
11、ees 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 International Standard may be the subject of patent rights. ISO
12、 shall not be held responsible for identifying any or all such patent rights. International Standard ISO 13559 IDF 153 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC Int
13、ernational. It is being published jointly by ISO and IDF and separately by AOAC International. ISO 13559:2002(E) IDF 153:2002(E) iv ISO and IDF 2002 All rights reservedForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every m
14、ember country. Every National Committee 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 Sta
15、ndards 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 National Committees casting a vote. International Standard ISO 13559 IDF 153 was prepared by Technical C
16、ommittee ISO/TC 34, Food products, 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. All work was carried out by the Joint ISO/IDF/AOAC
17、Action Team, Non-Pathogen Contaminants, under the aegis of its project leader, Mr D. van den Berg (NL). INTERNATIONAL STANDARD ISO 13559:2002(E) IDF 153:2002(E) ISO and IDF 2002 All rights reserved 1Butter, fermented milks and fresh cheese Enumeration of contaminating microorganisms Colony-count tec
18、hnique at 30 C 1 Scope This International Standard specifies a method for the enumeration of contaminating microorganisms by means of the colony-count technique at 30 C. The method is applicable to butter, fermented milks and fresh cheese. 2 Normative references The following normative documents con
19、tain provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to
20、investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 6887-1, Mic
21、robiology 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 7218, Microbiology of food and animal feeding stuffs General
22、 rules for microbiological examinations ISO 8261 IDF 122:2001, Milk and milk products General guidance for 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 preparati
23、on and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory 3 Term and definition For the purposes of this International Standard, the following term and definition applies. 3.1 contaminating microorganisms non-lactic acid
24、 bacteria, yeasts and moulds forming countable colonies under the conditions specified in this International Standard NOTE 1 Product-specific lactic acid bacteria will not be detected by this method. NOTE 2 In certain types of fermented milks, non-lactic acid bacteria, yeasts or moulds can be part o
25、f the microflora contributing to the desired characteristics of the product. In such cases, care should be taken when applying the method described in this International Standard. 4 Principle 4.1 Poured plates are prepared using a specified culture medium, followed by surface plating of a specified
26、quantity of an initial suspension of the product. Under the same conditions, poured plates are prepared, followed by surface plating of a specified quantity of decimal dilutions of an initial suspension. ISO 13559:2002(E) IDF 153:2002(E) 2 ISO and IDF 2002 All rights reserved4.2 The plates are aerob
27、ically incubated at 30 C 1 C for 72 h. 4.3 The number of microorganisms per gram of test sample is calculated from the number of colonies obtained on plates chosen at dilution levels so as to give a significant result. 5 Diluents, culture media and reagents 5.1 General For current laboratory practic
28、e, see ISO 7218. 5.2 Basic materials See ISO 8261 IDF 122:2001, subclause 5.1. 5.3 Diluents for general use See ISO 8261 IDF 122:2001, subclause 5.2. 5.4 Diluents for special purposes See ISO 8261 IDF 122:2001, subclause 5.3. 5.5 Distribution, sterilization and storage of diluents See ISO 8261 IDF 1
29、22:2001, subclause 5.4. 5.6 Culture medium 5.6.1 General All components of the culture medium shall be free from carbohydrates. The absence of carbohydrates in the culture medium used here is essential for the method described. The quality of the carbohydrate-free medium used shall be assured in acc
30、ordance with ISO/TS 11133-1. 5.6.2 Composition Peptone from casein 7,5 g Peptone from gelatin 7,5 g Sodium chloride (NaCl) 5,0 g Agar 1)10 g to 15 g Water 1 000 ml 5.6.3 Preparation 5.6.3.1 Preparation from commercial dehydrated complete medium Follow the manufacturers instructions. Adjust the pH, i
31、f necessary, so that after sterilization it is 7,5 0,1 at 25 C. For fermented milks, adjust the pH, if necessary, so that after sterilization it is 8,0 0,1 at 25 C. 1) Depending on the gel strength of the agar. ISO 13559:2002(E) IDF 153:2002(E) ISO and IDF 2002 All rights reserved 35.6.3.2 Preparati
32、on from dehydrated basic components Dissolve and disperse by heating in the water, in the following order, the peptone from casein, the peptone from gelatin and the sodium chloride. Add the agar and heat to boiling while stirring frequently until the agar is completely dissolved, or steam for about
33、30 min. Filter through filter paper, if necessary. Adjust the pH so that, after sterilization, it is 7,5 0,1 at 25 C. For fermented milks, adjust the pH, if necessary, so that after sterilization it is 8,0 0,1 at 25 C. 5.6.3.3 Distribution, sterilization and storage Dispense the medium into test tub
34、es (6.8), in quantities of 12 ml to 15 ml per tube, or into flasks or bottles (6.9), in quantities of 100 ml to 150 ml. Sterilize in an autoclave set at 121 C 1 C for 15 min. If the medium is to be used immediately, cool it in the water bath (6.5) to between 44 C and 47 C. If not used immediately, s
35、tore it in the dark at between 1 C and 5 C for no longer than 3 months. In order to avoid any delay when pouring the medium and before commencing the microbiological examination, completely melt the medium in a boiling water bath, then cool it in another water bath set at between 44 C and 47 C befor
36、e use. 6 Apparatus Disposable apparatus is an acceptable alternative to reusable glassware if it has suitable specifications. Reusable glassware should be capable of undergoing repeated sterilization and should be chemically inert. Sterilize all apparatus that will come into contact with the test sa
37、mple and the diluents or culture medium in accordance with ISO 8261. Usual microbiological equipment (see ISO 7218 and ISO 8261) and, in particular, the following. 6.1 Incubator, capable of operating at 30 C 1 C. 6.2 Oven or incubator, ventilated by convection, capable of operating at 50 C 1 C, or a
38、 laminar air-flow cabinet. 6.3 Petri dishes, made of glass or plastic, of diameter 90 mm to 100 mm, or, when an inoculum of more than 0,1 ml is used, dishes of diameter 140 mm. 6.4 Graduated pipettes, of nominal capacity of 1 ml 0,02 ml or 10 ml 0,2 ml. 6.5 Water baths, capable of operating at betwe
39、en 44 C and 47 C, and capable of boiling. 6.6 Colony-counting equipment, consisting of an illuminated base with a 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, accurate to 0,1 pH unit at 25 C, with re
40、adability to 0,01 units. 6.8 Test tubes, with plugs or caps, of capacity of approximately 20 ml. 6.9 Bottles or flasks, with plugs or caps, of nominal capacity 150 ml to 250 ml. Bottles or flasks with non-toxic metal screw-caps may be used. 6.10 Sterile spreaders, with diameter of approximately 3,5
41、mm and length of 20 cm, bent at right angles about 3 cm from one end. The cut ends should be made smooth by heating. ISO 13559:2002(E) IDF 153:2002(E) 4 ISO and IDF 2002 All rights reserved7 Sampling It is important that the laboratory receive a sample which is truly representative and has not been
42、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. 8 Procedure 8.1 Preparation of test portion and initial suspension 8.1.1 General For general requirements see ISO 6887-1, an
43、d for specific requirements see ISO 8261. Take normal aseptic precautions. The operations described in 8.1 and 8.2 shall not be carried out in direct sunlight. 8.1.2 Butter See ISO 8261:2001, subclause 8.2.6. 8.1.3 Fresh cheese See ISO 8261:2001, subclause 8.2.4. 8.1.4 Fermented milks See ISO 8261:2
44、001, subclause 8.2.9. 8.2 Further decimal dilutions See ISO 8261. 8.3 Preparation of plates Pour 12 ml to 15 ml of the prepared medium (5.6) into Petri dishes (6.3) and allow to solidify. Dry the plates, preferably with the lids off and the agar surface facing downwards, in an oven or incubator (6.2
45、) set at 50 C for 30 min. See also ISO 7218. As an option, Petri dishes with a diameter of 140 mm may be used (6.3) when an inoculum of 0,1 ml is used. Instead of drying in an oven or incubator, the plates may also be dried in a laminar air-flow cabinet (6.2) for 30 min. 8.4 Inoculation and incubati
46、on 8.4.1 Transfer to each of two prepared plates (8.3), by means of a sterile pipette (6.4), 0,1 ml of the initial suspension of the product. 8.4.2 Repeat this operation using further decimal dilutions. 8.4.3 Carefully spread the inoculum as quickly as possible over the surface of the plate, taking
47、care not to touch the sides of the dish, using a sterile spreader (6.10). Use one sterile spreader for each plate. Leave the plates, with the lids on, for approximately 15 min on the bench to allow absorption of the inoculum into the plates. ISO 13559:2002(E) IDF 153:2002(E) ISO and IDF 2002 All rig
48、hts reserved 58.4.4 Invert the prepared dishes and place them in the incubator (6.1) set at 30 C for 72 h 2 h. Do not stack the dishes more than six high. Stacks of plates shall be separated from one another and from the walls and top of the incubator (see ISO 7218). 8.5 Counting of colonies Using t
49、he colony-counting equipment (6.6), count the colonies after incubation (8.4.4). Count the colonies characteristic for contaminating microorganisms in each dish containing not more than 150 colonies. Do not count pin-point colonies as these are not typical for contaminants. Information as to the source of contamination may be obtained by examination of the colonies present. It can be valuable, therefore, to record the types of colonies present (e.g. pure or mixe
