1、INTERNATIONAL STANDARD IS0 6785 IDF 93 Second edition 200t -05-1 5 Milk and milk products - Detection of Salmonella spp. Lait et produits laitiers - Recherche de Salmonella spp. Reference numbers Fe= 2$y IS0 6785:2001 (E) IDF 93:2001 (E) 0 IS0 and IDF 2001 - -, - - I - =w= *. i IS0 6785:2001 (E) IDF
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7、IS0 and IDF 2001 -All rights reserved IS0 6785:2001(E) IDF 93:2001(E) Contents Page 1 Scope . 2 Normative reference . 3 Terms and definitions 4 Principle 4.1 General 4.2 Pre-enrichment in non-selective liquid medium . 4.3 Enrichment in selective liquid media . 4.4 Streaking out and recognition 4.5 C
8、onfirmation 5 Culture media, reagents and sera 6 Apparatus and glassware . 7 Sampling . 8 Preparation of test sample 9 Procedure . i 9.1 9.2 9.3 9.4 9.5 10 11 12 13 Safety precautions . Test portion and pre-enrichment Enrichment Streaking out and recognition Confirmation Control cultures Expression
9、of results Safety precautions Test report An nexes A Diagram of procedure . B Specification for brilliant green C Standard method for streaking agar plates . Bibliography . O IS0 and IDF 2001 -Ail rights reserved 1 1 1 1 1 1 1 2 2 2 12 13 13 13 13 13 14 14 15 18 18 18 19 20 21 22 23 iii IS0 6785:200
10、1(E) IDF 93:2001 (E) Foreword I IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 technical committees. Each member body interested in
11、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. IS0 collaborates closely with the International Eiectrotechnical Commissi
12、on (IEC) on all matters of electrotechnical standardization. I I International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as
13、an international Standard requires approval by at least 75 O/O of 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. IS0 shall not be held responsible for identifying any or all such patent
14、 rights. international Standard IS0 6785 I iDF 93 was prepared by Technical Committee ISOTTC 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 IS0 and IDF and separate
15、ly by AOAC International. This second edition cancels and replaces the first edition (IS0 67851 985), which has been technically revised. Annexes A and B form a normative part of this International Standard. Annex C is for information only. iv O IS0 and IDF 2001 -All rights reserved IS0 6785:2001 (E
16、) IDF 93:2001(E) Foreword 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 collaborate
17、s with IS0 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 International Standa
18、rd requires approval by at least 50% of National Committees casting a vote. International Standard IS0 6785 I IDF 93 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 Inter
19、national. It is being published jointly by IS0 and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team on Harmonization, of the Standing Committee on Microbial methods of analysis, under the aegis of its project leader, Mr. H. Becker (DE). This fo
20、urth edition cancels and replaces the third edition (IDF 93:1995). O IS0 and IDF 2001 - Ali rights reserved V INTERNATIONAL STANDARD IS0 6785:2001 (E) IDF 93:2001(E) Milk and milk products - Detection of Salmonella spp. 1 Scope This International Standard specifies a method for the detection of Salm
21、onella spp. in milk and milk products. 2 Normative reference The following normative document contains 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
22、 not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the normative document indicated below. For undated references, the latest edition of the normative document referred to applies. Membe
23、rs of IS0 and IEC maintain registers of currently valid International Standards. IS0 8261 I IDF 122, Milk and milk products - General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination. 3 Terms and definitions For the purposes of t
24、his International Standard, the following terms and definitions apply. 3.1 Salmonella microorganisms which form typical colonies on solid selective media and which display the biochemical and serological characteristics described when tests are carried out in accordance with this International Stand
25、ard 3.2 detection of Salmonella detection of the presence or absence of these microorganisms, in a particular mass or volume, when tests are carried out in accordance with this International Standard 4 Principle 4.1 General The detection of Salmonella necessitates four successive stages (see annex A
26、). 4.2 Pre-enrichment in non-selective liquid medium Inoculation of the pre-enrichment medium with the test portion, and incubation at 37 OC for 16 h to 20 h. 4.3 Enrichment in selective liquid media Inoculation of Rappaport-Vassiliadis modified magnesium chloride/malachite green medium and of selen
27、itekystine medium with the culture obtained in 4.2. 0 IS0 and IDF 2001 -All rights resewed 1 IS0 6785:2001(E) IDF 93:2001(E) Incubation of the Rappaport-Vassiliadis modified magnesium chloride/malachite green medium in the water bath or incubator (6.4) set at 413 OC for 24 h and then a further 24 h.
28、 Incubation of the selenite/cystine medium in the incubator (6.3) set at 37 OC for 24 h and then a further 24 h. 4.4 Streaking out and recognition From the cultures obtained (4.3), inoculation of two selective solid media (brilliant greedphenol red agar and any other suitable solid selective medium)
29、. NOTE Suitable media allow the recovery of lactose-fermenting Salmonella strains. Incubation of the brilliant green/phenol red agar in the incubator (6.3) set at 37 OC and examination after 20 h to 24 h and, if necessary, again after 40 h to 48 h to check the presence of colonies which, from their
30、characteristics, are considered to be presumptive Salmonella. Incubation of the second selective solid medium at the appropriate temperature and examination after the appropriate time to check the presence of colonies which, from their characteristics, are considered to be presumptive Salmonella. 4.
31、5 Confirmation Subculturing of colonies of presumptive Salmonella (4.4) and confirmation by means of appropriate biochemical and serological tests. 5 Culture media, reagents and sera In order to improve the reproducibility of the results, it is recommended that, for the preparation of culture media,
32、 dehydrated basic components or dehydrated complete media are used. In that case, follow the manufacturers instructions rigorously. Use only reagents of recognized analytical grade, unless otherwise specified. The pH values given refer to a temperature of 25OC. Adjustments, if necessary, are made by
33、 adding either hydrochloric acid c (HCI) = 1 mol/l or sodium hydroxide solution c (NaOH) = 1 mol/l. If not used immediately, store the prepared culture media and reagents under conditions that do not produce any change in their composition, in the dark at a temperature between O OC and + 5 OC, for n
34、o longer than 1 month, unless otherwise stated. 5.1 Water Use distilled or demineralized water or water of equivalent purity. The water shall be free from substances that might inhibit the growth of microorganisms under the test conditions specified in this International Standard. 2 O IS0 and IDF 20
35、01 -All rights reserved IS0 6785:2001 (E) IDF 93:2001 (E) - Enzymatic digest of soya 50 g Sodium chloride (NaCI) 890 9 Potassium dihydrogen phosphate (KH2P04) 1,4g Dipotassium hydrogen phosphate (K2HP04) 092 9 Water 1000ml 5.2 Culture media 5.2.1 Pre-enrichment medium: Buffered peptone water 5.2.1.1
36、 Composition Peptone 103 g Sodium chloride (NaCI) 5,o 9 (Na2HP04-l 2H20) 990 9 Potassium dihydrogen phosphate (KH2P04) 1,5g Disodium hydrogen phosphate dodecahydrate Water 1 O00 ml 5.2.1.2 Preparation Dissolve the components in the water by heating. Adjust the pH so that after sterilization it is 7,
37、O f 0,l. Transfer the medium in quantities of 225 ml into flasks (6.9) of capacity 500 ml (or multiples of 225 ml into flasks of suitable capacity). Sterilize in the autoclave (6.1) set at 121 OC for 15 min. Cool to room temperature. 5.2.2 First selective enrichment medium: Rappaport-Vassiliadis mod
38、ified magnesium chloride/malachite green medium (RVS broth) 5.2.2.1 Solution A 5.2.2.1.1 Composition 5.2.2.1.2 Preparation Dissolve the components in the water by heating to about 70 OC. Prepare solution A on the day of preparation of the complete RVS medium. 5.2.2.2 Solution B 5.2.2.2.1 Composition
39、 Magnesium chloride hexahydrate (MgCI2.6H20) 400,O g Water 1000ml 5.2.2.2.2 Preparation Dissolve the magnesium chloride in the water. As this salt is very hygroscopic, it is advisable to dissolve the entire contents of MgC12.6H20 from a newly opened container. For instance, 250 g of MgC12.6H20 is ad
40、ded to 625 ml of water, giving a solution of total volume of 795 ml and a concentration of about 0,3 g/ml of MgC12.6H20. Solution B can be stored in an airtight brown glass bottle at room temperature for at least 2 years. O IS0 and IDF 2001 - All rights reserved 3 IS0 685:2001 (E) IDF 93:2001(E) Sol
41、ution A (5.2.2.1) 1 O00 ml Solution B (5.2.2.2) 100 ml Solution C (5.2.2.3) 10 ml - 5.2.2.3 Solution C 5.2.2.3.1 Composition Malachite green oxalate 0,4 g 100 ml Water 5.2.2.3.2 Preparation Dissolve the malachite green oxalate in the water. Solution C can be stored in a brown glass bottle at room te
42、mperature for at least 8 months. 5.2.2.4 Complete medium 5.2.2.4.1 Composition 5.2.2.4.2 Preparation Add to 1 O00 ml of solution A, 100 ml of solution B and 10 ml of solution C. Adjust the pH, if necessary, so that after sterilization it is 5,2 f 0,l. Dispense 10 ml quantities of the thus-obtained s
43、olution into test tubes (6.9) or into sterile flasks (6.8) of suitable capacity to obtain the portions necessary for the test. Sterilize in the autoclave (6.1) set at 115 OC for 15 min. Store the prepared medium in the refrigerator at 3 OC zk 2 OC. 5.2.3 Second selective enrichment medium: Selenite/
44、cystine medium WARNING - Extreme care should be taken with the laboratory use of selenite solutions because of their potentially toxic effect. Do not pipette by mouth under any circumstances. 5.2.3.1 Base 5.2.3.1.1 Composition Tryptone 5,O g Lactose 4,o 9 Disodium hydrogen phosphate dodecahydrate (N
45、a2HP04.1 2H20) 10,o 9 Sodium hydrogen selenite 40 g Water 1 O00 ml 5.2.3.1.2 Preparation Dissolve the first three basic components in the water by boiling for 5 min. After cooling, add the sodium hydrogen selenite. Adjust the pH, if necessary, to 7,O 4 0,l. Do not sterilize. 4 O IS0 and IDF 2001 -Al
46、l rights reserved IS0 6785:2001 (E) IDF 93:2001(E) 7 Meat extract powder 5,O g Peptone 10,o 9 Yeast extract powder 3,O g Disodium hydrogen phosphate (Na2HP04) 1,og Sodium dihydrogen phosphate (NaH2P04) 0,6 g Agar 12g to 18ga Water 900 ml a Depending on the gel strength of the agar. 5.2.3.2 L-Cystine
47、 solution 5.2.3.2.1 Composition L-Cystine 091 9 Sodium hydroxide solution, c (NaOH) = 1 mol/l 15 ml Sterile water approx. 85 ml 5.2.3.2.2 Preparation Add the components to a sterile 100 ml one-mark volumetric flask. Dilute to the mark with sterile water. Do not sterilize. 5.2.3.3 Complete medium 5.2
48、.3.3.1 Composition Base (5.2.3.1) L-Cystine solution (5.2.3.2) 1 O00 ml 10 ml 5.2.3.3.2 Preparation Add the L-cystine solution aseptically to the base. Adjust the pH, if necessary, to 7,O f 0,l. Dispense the medium aseptically into sterile flasks of suitable capacity to obtain the portions necessary
49、 for the test. The medium may be used until a red precipitate occurs. 5.2.4 First selective solid medium: Brilliant greedphenol red agar (Edel and Kampelmacher) 5.2.4.1 Base 5.2.4.1.1 Composition 5.2.4.1.2 Preparation Dissolve the components or the dehydrated complete base in the water by heating, if necessary. Adjust the pH, if necessary, so that after sterilization it is 7,O f 0,l. Transfer the base to tubes (6.9) or flasks (6.8) of appropriate capacity. Sterilize in the autoclave (6.1) set at 121 OC for 15 min. O IS0 and IDF 2001 -All rights reserved 5 IS0 67852001 (E) IDF
