1、 Reference numbers ISO 6730:2005(E) IDF 101:2005(E) ISO and IDF 2005INTERNATIONAL STANDARD ISO 6730 IDF 101 Second edition 2005-09-15 Milk Enumeration of colony- forming units of psychrotrophic microorganisms Colony-count technique at 6,5 C Lait Dnombrement des units formant colonie de micro-organis
2、mes psychrotrophes Technique par comptage des colonies 6,5 C ISO 6730:2005(E) IDF 101:2005(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 which are embedded ar
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5、mittees. In the unlikely event that a problem relating to it is found, please inform the ISO Central Secretariat at the address given below. ISO and IDF 2005 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electr
6、onic or mechanical, including photocopying and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41
7、 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2005 All rights reservedISO 6730:2005(E) IDF 101:2005(E) ISO and IDF 2005 All rights reserved iii
8、Contents Page Foreword iv 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 1 5 Diluents and culture medium 2 5.1 General. 2 5.2 Basic materials 2 5.3 Diluents for general use. 2 5.4 Distribution, sterilization and storage 2 5.5 Culture medium. 2 6 Apparatus and glasswar
9、e 3 7 Sampling 4 8 Procedure 4 8.1 General. 4 8.2 Preparation of the test sample and primary dilution 4 8.3 Further decimal dilutions. 4 8.4 Duration of the procedure 4 8.5 Inoculation and incubation 4 8.6 Interpretation. 5 9 Expression of results . 5 10 Repeatability 6 11 Test report . 7 Bibliograp
10、hy . 8 ISO 6730:2005(E) IDF 101:2005(E) iv ISO and IDF 2005 All rights reservedForeword 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 I
11、SO 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, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates
12、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 Directives, Part 2. The main task of technical committees is to prepare International Standards.
13、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 member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this d
14、ocument may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 6730 IDF 101 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
15、being published jointly by ISO and IDF. This edition of ISO 6730 IDF 101 cancels and replaces ISO 6730:1992, of which it constitutes a minor revision. ISO 6730:2005(E) IDF 101:2005(E) ISO and IDF 2005 All rights reserved v Foreword IDF (the International Dairy Federation) is a worldwide federation o
16、f 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 ISO in the development of standard methods of analysis and sampling for milk and milk p
17、roducts. 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 least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possib
18、ility 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 6730 IDF 101 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommit
19、tee 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/AOAC Group of Experts on Non-pathogenic contaminants with classical techniques (E 22), under the aegis of its chairman, Mr H. Asperger (AT). This edition cancels and replaces
20、 IDF 101A:1991. INTERNATIONAL STANDARD ISO 6730:2005(E) IDF 101:2005(E) ISO and IDF 2005 All rights reserved 1 Milk Enumeration of colony-forming units of psychrotrophic microorganisms Colony-count technique at 6,5 C 1 Scope This International Standard specifies a method for the enumeration of colon
21、y-forming units (CFU) of psychrotrophic microorganisms in raw and heat-treated milk by means of the colony-count technique at 6,5 C. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies.
22、 For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6887-1:1999, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for t
23、he preparation of the initial suspension and decimal dilutions ISO 7218:1996, Microbiology of food and animal feeding stuffs General rules for microbiological examinations ISO 8261:2001, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilu
24、tions for microbiological examination 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 psychrotrophic microorganisms bacteria, yeasts and moulds forming countable colonies under the conditions specified in this International Standard 4 Princip
25、le 4.1 Poured plates are prepared using a specified culture medium and a specified quantity of the test sample. Other plates are prepared under the same conditions, using decimal dilutions of the test sample. 4.2 The plates are aerobically incubated at 6,5 C for 10 days. 4.3 The number of colony-for
26、ming units (CFU) of microorganisms per millilitre of sample is calculated from the number of colonies obtained on plates chosen at dilution levels so as to give a significant result. ISO 6730:2005(E) IDF 101:2005(E) 2 ISO and IDF 2005 All rights reserved5 Diluents and culture medium 5.1 General For
27、general guidance, see ISO 7218. 5.2 Basic materials See ISO 8261. 5.3 Diluents for general use See ISO 8261. 5.4 Distribution, sterilization and storage See ISO 8261. 5.5 Culture medium 5.5.1 Components Tryptone 5,0 g Yeast extract 2,5 g Glucose monohydrate (C 6 H 12 O 6 H 2 O) 1,0 g Skimmed milk po
28、wder a1,0 g Agar 10 g to 15 g bWater 1 000 ml aThe skimmed milk powder shall be free from inhibitory substances. This should be proved by comparative tests using skimmed milk powder known to be free from such substances. bDepending on the gel strength of the agar. 5.5.2 Preparation 5.5.2.1 Preparati
29、on from commercial dehydrated complete medium Follow the manufacturers instructions but, in all cases, add the skimmed milk powder, even if the manufacturer considers such an addition unnecessary. Adjust the pH, if necessary, so that after sterilization it is 7,0 at 25 C. 5.5.2.2 Preparation from de
30、hydrated basic components Dissolve and disperse in the water, in the following order: the yeast extract, tryptone, glucose and, finally, the skimmed milk powder. NOTE Heating the water will assist in this procedure. Add the agar and heat to boiling, stirring frequently until the agar is completely d
31、issolved, or steam for about 30 min. If the solution is not clear, filter it through filter paper. ISO 6730:2005(E) IDF 101:2005(E) ISO and IDF 2005 All rights reserved 3 Adjust the pH, if necessary, so that after sterilization it is 7,0 at 25 C. 5.5.2.3 Distribution, sterilization and storage Dispe
32、nse the medium into test tubes (6.9) 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 (6.1) at 121 C 1 C for 15 min. If the medium is to be used immediately, cool it to 45 C in the water bath (6.5). If not, before
33、beginning the microbiological examination, in order to avoid any delay when pouring the medium, completely melt the medium in a hot water bath (6.6) then cool it to 45 C in the water bath (6.5). (See also 8.5.4.) Store the medium in the dark at a temperature between 0 C and +5 C for no longer than 3
34、 months after preparation. In order to check the temperature of the agar, it is recommended to place a thermometer into a portion of 15 g/l agar solution in a separate container identical to that used for the medium. This temperature control solution should be exposed to the same heating and cooling
35、 operations as the medium itself. 6 Apparatus and glassware CAUTION Sterilize all apparatus that will come into contact with the test sample, the diluent, the dilutions or the culture medium in accordance with ISO 8261. Disposable apparatus is an acceptable alternative to re-usable glassware if it h
36、as suitable specifications. Usual microbiological laboratory equipment, the apparatus required for the preparation of test samples and dilutions as specified in ISO 8261 and, in particular, the following. 6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). See ISO 7218. 6.2
37、Incubator, capable of operating at 6,5 C 0,5 C. 6.3 Petri dishes, made of glass or plastic, 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 Water b
38、ath, capable of operating at more than 100 C. 6.7 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. 6.8 Temperature-compensated pH-meter, accurate to
39、within 0,1 pH units at 25 C. 6.9 Test tubes, of approximately 20 ml capacity (or flasks or bottles of suitable capacity), and flasks or bottles of 150 ml to 250 ml capacity, for sterilization and storage of the culture medium. Bottles or flasks with non-toxic metal screw caps may be used. ISO 6730:2
40、005(E) IDF 101:2005(E) 4 ISO and IDF 2005 All rights reserved7 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
41、sampling method is given in ISO 707 IDF 50. 8 Procedure 8.1 General In order to improve the precision of the method, the preparation of dilutions should be carefully standardized. Factors that affect precision are type of blending equipments, blending time, diluent, time allowed for large particles
42、to settle, and mixing 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 8261:2001, 8.2. 8.3 Further deci
43、mal dilutions See ISO 6887-1 and ISO 8261:2001, 8.3. Other dilution series may be used (e.g. a primary dilution of 10 ml of test sample in 90 ml of diluent, or 11 ml of test sample in 99 ml of diluent). The accuracy and precision of the method are greater when the larger quantities of sample and dil
44、uent are used. 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. 8.5.2 Take two further sterile Petri dishes. Transfer to each dish, by means of
45、another sterile pipette, 1 ml of the 10 1dilution of the test sample. 8.5.3 If necessary, repeat this operation using further decimal dilutions. 8.5.4 Check that the temperature of the culture medium (5.5) does not exceed 46 C. ISO 6730:2005(E) IDF 101:2005(E) ISO and IDF 2005 All rights reserved 5
46、If the culture medium is at a temperature greater than 46 C, it may damage or kill the psychrotrophic microflora of the sample. If any damage is expected, spread plating plus a low incubation temperature should be used. Pour 12 ml to 15 ml of the culture medium (5.5) into each Petri dish. If 15 ml i
47、s insufficient to obtain a homogeneous distribution of the organisms, a volume of 20 ml should be used. 8.5.5 Carefully mix the inoculum with the medium by rotating the Petri dishes, and allow the mixture to solidify by leaving the Petri dishes to stand on a cool horizontal surface. 8.5.6 The time t
48、aken 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 Invert the prepared dishes and place them in the incubator (6.2) set at 6,5 C for 10 days. To p
49、revent spreading, some precautions should be taken, such as the addition of an overlayer of culture medium after solidifying, or the addition of a drop of glycerol on filter paper in the lid of the dish. 8.5.9 Do not stack the dishes more than six high. Separate the stacks of dishes from one another and from the walls and top of the incubator. 8.6 Interpretation 8.6.1 Count the colonies on each plate (see 9.1), using the colony-counting equipment (6.7). Examine
