1、 Reference numberISO/TS 21872-1:2007(E)ISO 2007TECHNICAL SPECIFICATION ISO/TS21872-1First edition2007-04-15Microbiology of food and animal feeding stuffs Horizontal method for the detection of potentially enteropathogenic Vibrio spp. Part 1: Detection of Vibrio parahaemolyticus and Vibrio cholerae M
2、icrobiologie des aliments Mthode horizontale pour la recherche des Vibrio spp. potentiellement entropathognes Partie 1: Recherche de Vibrio parahaemolyticus et Vibrio cholerae ISO/TS 21872-1:2007(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing poli
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7、va 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2007 All rights reservedISO/TS 21872-1:2007(E) ISO 2007 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope 1 2 Normative references 1 3 Terms and definition
8、s .2 4 Principle2 4.1 General2 4.2 First enrichment in a liquid selective medium2 4.3 Second enrichment in a liquid selective medium 2 4.4 Isolation and identification .2 4.5 Confirmation.2 5 Culture media and reagents .3 5.1 Enrichment medium: Alkaline saline peptone water (ASPW) .3 5.2 Solid selec
9、tive isolation media.3 5.3 Saline nutrient agar (SNA) 3 5.4 Reagent for detection of oxidase.3 5.5 Saline triple sugar iron (TSI) agar 3 5.6 Saline medium for detection of ornithine decarboxylase (ODC) 3 5.7 Saline medium for detection of lysine decarboxylase (LDC)3 5.8 Saline medium for detection o
10、f arginine dihydrolase (ADH) 3 5.9 Reagent for detection of -galactosidase .3 5.10 Saline medium for detection of indole.4 5.11 Saline peptone waters.4 5.12 Sodium chloride solution4 6 Apparatus and glassware .4 7 Sampling.4 8 Preparation of test sample4 9 Procedure (see Annex A) .4 9.1 Test portion
11、 and initial suspension .4 9.2 First selective enrichment 5 9.3 Second selective enrichment .5 9.4 Isolation and identification .5 9.5 Confirmation.6 10 Expression of results 10 11 Test report 10 Annex A (normative) Diagram of procedure.11 Annex B (normative) Composition and preparation of the cultu
12、re media and reagents.12 Bibliography 19 ISO/TS 21872-1:2007(E) iv ISO 2007 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 carri
13、ed 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 committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. I
14、SO collaborates 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 Internati
15、onal Standards. 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. In other circumstances, particularly when there is an urg
16、ent market requirement for such documents, a technical committee may decide to publish other types of normative document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by m
17、ore than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO
18、/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed
19、 into an International Standard or be withdrawn. 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/TS 21872-1 was prepared by Technical Committee I
20、SO/TC 34, Food products, Subcommittee SC 9, Microbiology. ISO/TS 21872 consists of the following parts, under the general title Microbiology of food and animal feeding stuffs Horizontal method for the detection of potentially enteropathogenic Vibrio spp.: Part 1: Detection of Vibrio parahaemolyticus
21、 and Vibrio cholerae Part 2: Detection of species other than Vibrio parahaemolyticus and Vibrio cholerae ISO/TS 21872-1:2007(E) ISO 2007 All rights reserved vIntroduction Because of the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain
22、 products. In this case, different methods that are specific to these products may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt will be made to apply this horizontal method as far as possible. When this Technical Specification is next reviewed, account
23、 will be taken of all information then available regarding the extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of particular products. The harmonization of test methods cannot be immediate and, for certain groups of products, Inter
24、national Standards and/or national standards may already exist that do not comply with this horizontal method. It is hoped that when such standards are reviewed they will be changed to comply with this Technical Specification so that eventually the only remaining departures from this horizontal meth
25、od will be those necessary for well-established technical reasons. TECHNICAL SPECIFICATION ISO/TS 21872-1:2007(E) ISO 2007 All rights reserved 1Microbiology of food and animal feeding stuffs Horizontal method for the detection of potentially enteropathogenic Vibrio spp. Part 1: Detection of Vibrio p
26、arahaemolyticus and Vibrio cholerae WARNING In order to safeguard the health of laboratory personnel, it is essential that tests for detection of Vibrio spp., and the particularly toxigenic Vibrio cholerae, be conducted only in laboratories equipped for this purpose and under the supervision of an e
27、xperienced microbiologist, and that great care be exercised in the disposal of contaminated material. 1 Scope This part of ISO/TS 21872 specifies a horizontal method for the detection of the two main pathogenic Vibrio species causing intestinal illness in humans: V. parahaemolyticus and V. cholerae.
28、 It is applicable to products intended for human consumption and the feeding of animals, and environmental samples in the area of food production and food handling. NOTE Reasons for not applying this method are discussed in the Introduction. 2 Normative references The following referenced documents
29、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 6887 (all parts), Microbiology of food and animal feeding stuffs Preparation of
30、test samples, initial suspension and decimal dilutions for microbiological examination ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations ISO 8261, Milk and milk products General guidance for the preparation of test samples, in
31、itial suspensions and decimal dilutions for microbiological examination ISO/TS 21872-1:2007(E) 2 ISO 2007 All rights reserved3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 potentially enteropathogenic Vibrio parahaemolyticus and Vibrio chole
32、rae microorganisms which form typical colonies on solid selective media and which possess the described biochemical characteristics when the test is performed in accordance with this part of ISO/TS 21872 3.2 detection of potentially enteropathogenic Vibrio parahaemolyticus and Vibrio cholerae determ
33、ination of the presence or absence of Vibrio parahaemolyticus and Vibrio cholerae, in a specified quantity of product, when the test is performed in accordance with this part of ISO/TS 21872 4 Principle 4.1 General The detection of Vibrio parahaemolyticus and Vibrio cholerae requires four successive
34、 phases (see also Annex A). NOTE Vibrio parahaemolyticus and Vibrio cholerae can be present in small numbers and are often accompanied by a much larger number of other microorganisms belonging to the Vibrionaceae family or to other families. Consequently, two successive selective enrichments are nec
35、essary in order to detect the target organisms. 4.2 First enrichment in a liquid selective medium The enrichment medium (alkaline saline peptone water, ASPW) (5.1) is inoculated with the test portion at ambient temperature. It is incubated at 37 C for 6 h 1 h for deep frozen products, or at 41,5 C f
36、or 6 h 1 h for fresh products. 4.3 Second enrichment in a liquid selective medium The enrichment medium (ASPW) is then inoculated with the culture obtained in 4.2. It is incubated at 41,5 C for 18 h 1 h. 4.4 Isolation and identification The following two solid selective media are inoculated with the
37、 cultures obtained in 4.2 and in 4.3: thiosulfate citrate bile and sucrose agar (TCBS); another appropriate solid selective medium (left to the choice of the laboratory), complementary to the TCBS medium, allowing the detection of Vibrio parahaemolyticus and Vibrio cholerae. The TCBS is incubated at
38、 37 C, then examined after 24 h 3 h. The second selective medium is incubated according to the manufacturers recommendations. 4.5 Confirmation The presumptive colonies of Vibrio parahaemolyticus and Vibrio cholerae isolated in 4.4 are subcultured, then confirmed by means of appropriate biochemical t
39、ests. ISO/TS 21872-1:2007(E) ISO 2007 All rights reserved 35 Culture media and reagents For general laboratory practice, see ISO 7218. NOTE On account of the large number of culture media and reagents, for clarity of the text, their composition and preparation are given in Annex B. 5.1 Enrichment me
40、dium: Alkaline saline peptone water (ASPW) See B.1. 5.2 Solid selective isolation media 5.2.1 First medium: Thiosulfate, citrate, bile and sucrose (TCBS) agar See B.2. 5.2.2 Second medium The selection of the second medium is left to the choice of the test laboratory. Preparation of the medium shoul
41、d be strictly according to the manufacturers instructions. EXAMPLES Soya peptone triphenyl tetrazolium chloride agar (TSAT) and sodium dodecyl sulfate polymyxin sucrose (SDSPS) agar (mCPC, CPC, CC agars are not recommended for isolation of V. parahaemolyticus). 5.3 Saline nutrient agar (SNA) See B.3
42、. 5.4 Reagent for detection of oxidase See B.4. 5.5 Saline triple sugar iron (TSI) agar See B.5. 5.6 Saline medium for detection of ornithine decarboxylase (ODC) See B.6. 5.7 Saline medium for detection of lysine decarboxylase (LDC) See B.7. 5.8 Saline medium for detection of arginine dihydrolase (A
43、DH) See B.8. 5.9 Reagent for detection of -galactosidase See B.9. ISO/TS 21872-1:2007(E) 4 ISO 2007 All rights reserved5.10 Saline medium for detection of indole See B.10. 5.11 Saline peptone waters See B.11. 5.12 Sodium chloride solution See B.12. 6 Apparatus and glassware NOTE Disposable equipment
44、 is acceptable in the same way as reusable glassware, if the specifications are similar. Usual microbiology laboratory equipment (see ISO 7218) and, in particular, the following. 6.1 Incubator, adjustable to 37 C 1 C. 6.2 Incubator or water bath, adjustable to 41,5 C 1 C. 6.3 Water bath, adjustable
45、from 44 C to 47 C. 6.4 Water bath, adjustable to 37 C 1 C. It is recommended to use water baths (6.2, 6.3 and 6.4) containing an antibacterial agent. 7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sa
46、mpling is not part of the method specified in this part of ISO/TS 21872. See the International Standard specific to the relevant product. If a specific International Standard does not exist, it is recommended that the relevant parties reach agreement on this subject. 8 Preparation of test sample Pre
47、pare the test sample in accordance with the relevant part of ISO 6887, and/or ISO 8261, and an International Standard concerning the product to be examined. If a specific International Standard does not exist, it is recommended that the relevant parties reach agreement on this subject. 9 Procedure (
48、see Annex A) 9.1 Test portion and initial suspension For the preparation of the initial suspension, use the first enrichment medium (ASPW) specified in 5.1. Take a test portion (x g or x ml), according to the sensitivity required, and homogenize it in 9x ml (or 9x g) of enrichment medium. ISO/TS 218
49、72-1:2007(E) ISO 2007 All rights reserved 5In the case of large quantities, the ASPW should be warmed to 37 C before inoculation with the test portion. If the dilution and the incubation cannot be carried out on the same day, store the initial suspension until the next day at a temperature of 5 C 3 C. In order to reduce the amount of examination work, where more than one 25 g test portion stemming from the same batch of food is to be examined, and where proof is available in
