1、 Reference numbers ISO 8553:2004(E) IDF 131:2004(E) ISO and IDF 2004INTERNATIONAL STANDARD ISO 8553 IDF 131 First edition 2004-05-01 Milk Enumeration of microorganisms Plate-loop technique at 30 C Lait Dnombrement des micro-organismes Mthode de lanse sur botes de Petri 30 C ISO 8553:2004(E) IDF 131:
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7、copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2004 All rights reservedISO 8553:2004(E) IDF 131:2004(E) ISO and IDF 2004 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a worldwide federa
8、tion 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 subject for which a technical committee has been established has the right to be represented on that committ
9、ee. International organizations, governmental 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
10、 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 technical committees are circulated to the member bodies for voting. Publication as an International Standard requires ap
11、proval 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. ISO shall not be held responsible for identifying any or all such patent rights. ISO 8553|IDF 131 was prepared by Technic
12、al 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 being published jointly by ISO and IDF and separately by AOAC International. ISO 8553:2004(E) IDF 131:2004(E) iv ISO and IDF
13、 2004 All rights reservedForeword 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 col
14、laborates 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 and Standing Committees are circulated to the National Committees for voting. Publication as an Internationa
15、l Standard requires approval by at least 50 % of IDF National Committees casting a vote. ISO 8553|IDF 131 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. I
16、t 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 harmonization, of the Standing Committee on Microbiological methods of analysis, under the aegis of its project leader, Dr J. Floor (ZA)
17、. INTERNATIONAL STANDARD ISO 8553:2004(E) IDF 131:2004(E) ISO and IDF 2004 All rights reserved 1Milk Enumeration of microorganisms Plate-loop technique at 30 C 1 Scope This International Standard specifies a method for the enumeration of microorganisms in raw milk by using the plate-loop technique a
18、t 30 C. 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 4833, Microbio
19、logy of food and animal feeding stuffs Horizontal method for the enumeration of microorganisms Colony count technique at 30 C 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 prep
20、aration 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 m
21、edia in 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 defi
22、nitions apply. 3.1 microorganisms bacteria, yeasts and moulds forming countable colonies under the conditions specified in this International Standard 4 Principle 4.1 A poured plate is prepared using a specified culture medium and a specified quantity of the test sample taken by means of a calibrate
23、d wire loop. 4.2 The plate is aerobically incubated at 30 C for 72 h. 4.3 The number of microorganisms per millilitre of test sample is calculated from the number of colonies obtained on the plate (see Clause 10). ISO 8553:2004(E) IDF 131:2004(E) 2 ISO and IDF 2004 All rights reserved5 Diluents, cul
24、ture medium and reagent 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 digestion 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 1
25、8 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 medium Follow the manufacturers instructions but, in all cases, add the skimmed milk powder even if the
26、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 disperse in the water, in the following order, the yeast extract, the enzymatic digestion of casein,
27、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. Adjust the pH, if necessary, so that after sterilization it is 7,0 0,2 at 25 C. 5.3.2.3 Distribution, st
28、erilization and storage Dispense the medium into test tubes (6.8), in quantities of 12 ml to 15 ml per tube, or into flasks or bottles (6.9) of capacity not greater than 500 ml. Sterilize for 15 min in an autoclave (6.11) set at 121 C. If the medium is to be used immediately, cool it in a water bath
29、 (6.6) to between 44 C and 47 C before use. If not, store it in the dark at 3 C 2 C for no longer than three months under conditions which do not allow any change in its composition and properties. Before commencing the microbiological examination and in order to avoid any delay when pouring the med
30、ium, completely melt the stored medium, then cool it in a water bath (6.6) to between 44 C and 47 C before use (see 9.2.4). With regard to a temperature check of the medium and other requirements, see ISO 7218. ISO 8553:2004(E) IDF 131:2004(E) ISO and IDF 2004 All rights reserved 35.3.3 Performance
31、testing for the quality assurance of the culture medium Test the performance of the medium according to ISO/TS 11133-2. 5.4 Disinfecting solution Use a sodium hypochlorite solution containing 50 mg/l to 100 mg/l of active chlorine or a 1:1 mixture of ethanol (96 %) and acetone. Prepare a fresh disin
32、fecting solution daily. 6 Apparatus and glassware Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following. 6.1 Glassware Disposable glassware is an acceptable alternative to re-usable glassware if it has suitable specifications. Re-usable glassware shall be capabl
33、e of undergoing repeated sterilization and shall be chemically inert. 6.2 Loop inoculation equipment 6.2.1 Components The total equipment assembly cannot be purchased as a unit, but may be made up from the components listed in 6.2.1.1 to 6.2.1.3. Automatic equipment may also be used. 6.2.1.1 Platinu
34、m inoculating loop, calibrated to hold 0,001 ml, soldered to a Luer-lock hypodermic needle. Make a 30 bend about 3 mm to 4 mm from the loop, with the loop opening towards the hub. NOTE Suitable platinum inoculating loops are available from Gerber Instruments K. Schneider jerking the loop out rapidly
35、 causes more than 0,001 ml to adhere. It is necessary to use wide-mouth sample bottles and good illumination to facilitate the process. 9.2.2 Raise the cover of a sterile Petri dish (6.10). Insert the loop and depress the plunger causing 1 ml sterile diluent to flow across the charged loop, thus was
36、hing a measured amount of sample into the dish. Do not depress the plunger so rapidly that diluent fails to follow the shank and flow across the loop. NOTE Normally, the residue remaining on the loop after discharging the sample is not significant. However, imperfections in the welding of the loop o
37、r in the smoothness of the metal surface as in the case of old, damaged loops can lead to incomplete rinsing. Replace badly damaged loops. As a precaution, sterilize the loop after every set of 20 test samples by submerging it in a disinfecting solution (5.4). Do not sterilize the loop in a flame as
38、 this will significantly reduce its life. Discharge at least five 1 ml portions of diluent to waste before proceeding. 9.2.3 Immediately after every set of test samples and before flushing or sterilizing the loop, discharge 1 ml of sterile buffer into a Petri dish. Label this dish “Rinsing control”.
39、 No more than two colonies should develop on this plate during incubation (9.2.6). Additionally, pour one sterile Petri dish with agar medium (5.3) only. Label this dish “Agar control”. 9.2.4 Add 10 ml to 12 ml of melted medium (see 5.3.2.3) at a temperature of between 44 C and 47 C to each inoculat
40、ed dish. The time elapsing between discharging the loop and pouring the media into the dishes shall not exceed 15 min. 9.2.5 Mix immediately after pouring by rotating the dishes sufficiently to obtain evenly dispersed colonies after incubation. Allow the mixture to solidify by leaving the Petri dishes to stand on a cool horizontal surface. 9.2.6 Invert the prepared dishes. Place them for 72 h 3 h in the incubator (6.4) set at 30 C. Do not stack the dishes more than six high. Separate stacks of dishes from one another and from the walls and top of the incubator (see ISO 7218).
