1、BRITISH STANDARD BS ISO 8552:2004 Milk Estimation of psychrotrophic microorganisms Colony-count technique at 21 C (Rapid method) ICS 07.100.30 BS ISO 8552:2004 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 22 July 2004 BSI 22 July 2004 ISBN
2、 0 580 44114 8 National foreword This British Standard reproduces verbatim ISO 8552:2004 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
3、represented 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
4、 using 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 immuni
5、ty 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 the UK interests informed; monitor related international and European developments and promulgate them i
6、n the 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 6, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments i
7、ssued since publication Amd. No. Date Comments Reference numbers ISO 8552:2004(E) IDF 132:2004(E)INTERNATIONAL STANDARD ISO 8552 IDF 132 First edition 2004-05-01 Milk Estimation of psychrotrophic microorganisms Colony-count technique at 21 C (Rapid method) Lait Estimation des micro-organismes psychr
8、otrophes Technique par comptage des colonies 21 C (Mthode rapide) BSISO8552:2004IS:2558 O4002(E) ID:231 F002(4)E DPlcsid Fremia ihTs PDF file may ctnoian emdebt dedyfepcaes. In ccaocnadrw eith Aebods licensilop gnic,y this file mairp eb ynted iv roweb detu slahl ton ide ebtlnu deess teh typefaces wh
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14、 ID 4002 F All irhgts seredevr 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 int
15、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
16、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
17、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 document may be the subject of patent rights.
18、ISO shall not be held responsible for identifying any or all such patent rights. ISO 8552|IDF 132 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 International. It is bei
19、ng published jointly by ISO and IDF and separately by AOAC International. BSISO8552:2004IS:2558 O4002(E) ID:231 F002(4)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
20、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 Standards
21、 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 IDF National Committees casting a vote. ISO 8552|IDF 132 was prepared by Technical Committee ISO/TC 34, Food produ
22、cts, 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 Action Team on Microbiological
23、 harmonization, of the Standing Committee on Microbiological methods of analysis, under the aegis of its project leader, Dr J. Floor (ZA). This first edition of ISO 8552|IDF 132 cancels and replaces the first edition of IDF 132A:1991, which has been editorially and technically revised. BSISO8552:200
24、4IS:2558 O4002(E) ID:231 F002(4)E I SO dna ID 4002 F All irhgts seredevr vIntroduction The rapid method described in this International Standard is essentially an approximate method because the incubation conditions are such as to permit organisms other than psychrotrophs to be counted if they grow
25、sufficiently quickly at 21 C. Nevertheless, comparative trials in a number of laboratories have shown that the results obtained by the method described here (based on that of Reference 1) correlate well with those obtained by the method described in ISO 6730|IDF 101 (with incubation at 6,5 C for 10
26、days). The latter method (which will be replaced by the horizontal method ISO 17410) should be used if a more accurate enumeration of psychrotrophic microorganisms is required. BSISO8552:20044002:2558OSISBNITERNATNOIAL STANDARD IS:2558 O4002(E) ID:231 F002(4)EI SO dna ID 4002 F All irhgts seredevr 1
27、Milk Estimation of psychrotrophic microorganisms Colony-count technique at 21 C (Rapid method) 1 Scope This international Standard specifies a rapid method for estimating the number of psychrotrophic microorganisms by means of the colony-count technique at 21 C. The method is applicable to raw and p
28、asteurized 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 6730,
29、 Milk Enumeration of colony-forming units of psychrotrophic micro-organisms Colony count technique at 6,5 degrees C 1)ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations ISO 8261|IDF 122, Milk and milk products General guidance for the preparation
30、of test samples, initial suspensions and decimal dilutions for microbiological examination ISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in
31、the laboratory ISO/TS 11133-2, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 2: Practical guidelines on performance testing of culture media 3 Terms and definitions For the purposes of this document, the following terms and definitions
32、apply. 3.1 psychrotrophic microorganisms bacteria, yeasts and moulds forming countable colonies when incubated aerobically at 6,5 C for 10 days under the conditions specified in ISO 6730 1) Equivalent to IDF 101A:1991. BSISO8552:2004IS:2558 O4002(E) ID:231 F002(4)E 2 I SO dna ID 4002 F All irhgts se
33、redevr4 Principle 4.1 Poured plates are prepared using a specified culture medium and a specified quantity of an appropriate dilution of the test sample. 4.2 The plates are incubated aerobically at 21 C for 25 h. 4.3 The number of microorganisms per millilitre of sample is calculated from the number
34、 of colonies obtained on plates chosen at dilution levels so as to give a significant result. 5 Diluents and culture medium 5.1 General See ISO 7218 and ISO/TS 11133-1. 5.2 Diluents See ISO 8261|IDF 122. 5.3 Culture medium Plate count milk agar 5.3.1 Composition Yeast extract 2,5 g Enzymatic digesti
35、on of casein 5,0 g Skimmed milk powder a1,0 g Glucose, anhydrous (C 6 H 12 O 6 ) 1,0 g Agar 9 g to 18 g bWater 1 000 ml aThe skimmed milk powder shall be free from inhibitory substances. bDepending on the gel strength of the agar. 5.3.2 Preparation 5.3.2.1 Preparation from commercial dehydrated medi
36、um 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 0,2 at 25 C. 5.3.2.2 Preparation from dehydrated basic components Dissolve and d
37、isperse in the water, in the following order, the yeast extract, the enzymatic digestion of casein, the glucose and, finally, the skimmed milk powder. Heating the water will assist this procedure. Add the agar and heat to boiling while stirring frequently until the agar is completely dissolved. Adju
38、st the pH, if necessary, so that after sterilization it is 7,0 0,2 at 25 C. BSISO8552:2004IS:2558 O4002(E) ID:231 F002(4)E I SO dna ID 4002 F All irhgts seredevr 35.3.2.3 Distribution, sterilization and storage Dispense the prepared medium into test tubes (6.6), in quantities of 12 ml to 15 ml per t
39、ube, or into flasks or bottles (6.7) of capacity not greater than 500 ml. Sterilize for 15 min in an autoclave (6.10) set at 121 C. If the medium is to be used immediately, cool it in a water bath (6.4) to between 44 C and 47 C before use. If not, store the medium in the dark at 3 C 2 C for no longe
40、r than three months (see ISO 7218). Before commencing the microbiological examination and in order to avoid any delay when pouring the medium, completely melt the stored medium, then cool it in a water bath (6.4) at between 44 C and 47 C before use (see 8.2.4). With regard to the temperature check o
41、f the medium and other requirements, see ISO 7218. 5.3.3 Performance testing for the quality assurance of the culture medium Test the performance of the medium in accordance with ISO/TS 11133-2. 6 Apparatus and glassware Usual microbiological equipment (see ISO 7218) and, in particular, the followin
42、g. 6.1 Glassware Disposable glassware is an acceptable alternative to re-usable glassware if it has suitable specifications. Re-usable glassware shall be capable of undergoing repeated sterilization and shall be chemically inert. 6.2 Incubator, capable of operating at 21 C 1 C. 6.3 pH meter, having
43、an accuracy of calibration of 0,1 pH unit at 25 C. 6.4 Water bath, capable of operating at between 44 C and 47 C. 6.5 Colony-counting equipment, consisting, for example, of an illuminated base with a dark background, fitted with a magnifying lens to be used at a magnification of at least 2, and a me
44、chanical or electronic digital counter. 6.6 Test tubes, of approximate capacity 20 ml, with suitable stoppers. 6.7 Flasks or bottles, of appropriate capacity but not greater than 500 ml, with suitable stoppers. 6.8 Graduated pipettes, of nominal capacity 1 ml. 6.9 Petri dishes, made of glass or plas
45、tic, of diameter 90 mm to 100 mm. 6.10 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). See ISO 7218. 7 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is not
46、 part of the method specified in this International Standard. A recommended sampling method is given in ISO 707. BSISO8552:2004IS:2558 O4002(E) ID:231 F002(4)E 4 I SO dna ID 4002 F All irhgts seredevr8 Procedure 8.1 Preparation of primary dilution and further decimal dilutions Prepare the primary di
47、lution (10 1 ) and further decimal dilutions of the test sample in accordance with ISO 8261|IDF 122. Bacterial colonies obtained with this rapid method tend to be very small and are not readily detectable in a zero dilution plate because of opacity or cloudiness of the media. Therefore, dilute the t
48、est samples at least 10-fold (preferably 100-fold or more) in order to be able to also observe small pinpoint colonies. 8.2 Inoculation and incubation 8.2.1 Take two sterile Petri dishes (6.9). Transfer using a sterile pipette (6.8) 1 ml of the primary dilution (8.1) to each dish. 8.2.2 If necessary
49、, repeat the procedure with further decimal dilutions, using a new sterile pipette for each dilution. 8.2.3 If appropriate and possible, select only the critical dilution steps (at least two consecutive decimal dilutions) for the inoculation of the Petri dishes that will give colony counts of between 15 and 300 colonies per plate. 8.2.4 Pour about 12 ml to 15 ml of plate count medium (5.3.2.
