DD-ISO-TS-16649-3-2005.pdf
DRAFT FOR DEVELOPMENT DD ISO/TS 16649-3:2005 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of “ “-glucuronidase- positive Escherichia coli Part 3: Most probable number technique using 5-bromo-4-chloro- 3-indolyl-“ “ - D-glucuronide ICS 07.100.30 ? Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI DD ISO/TS 16649-3:2005 This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 13 December 2005 © BSI 13 December 2005 ISBN 0 580 46814 3 National foreword This Draft for Development reproduces verbatim ISO/TS 16649-3:2005 This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on this provisional basis, so that information and experience of its practical application may be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can be reported to the international organization responsible for the Technical Specification. A review of this publication will be initiated not later than 3 years after its publication by the international organization so that a decision can be taken on its status at the end of its 3-year life. Notification of the start of the review period will be made in an announcement in the appropriate issue of Update Standards. According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into an international standard, to extend the life of the Technical Specification for another 3 years or to withdraw it. Comments should be sent in writing to the Secretary of BSI Technical Committee AW/9, Microbiology, at British Standards House, 389 Chiswick High Road, London W4 4AL, giving the document reference and clause number and proposing, where possible, an appropriate revision of the text. A list of organizations 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 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. Summary of pages This document comprises a front cover, an inside front cover, the ISO/TS title page, pages ii to iv, pages 1 to 8, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Reference number ISO/TS 16649-3:2005(E) TECHNICAL SPECIFICATION ISO/TS 16649-3 First edition 2005-09-15 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli Part 3: Most probable number technique using 5-bromo-4-chloro-3-indolyl- -D- glucuronide Microbiologie des aliments Méthode horizontale pour le dénombrement des Escherichia coli -glucuronidase positive Partie 3: Technique du nombre le plus probable utilisant le bromo-5-chloro-4-indolyl-3 -D-glucuronate DD ISO/TS 16649-3:2005 Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iii Foreword 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 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 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. 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 urgent 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 more 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/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 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 16649-3 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. ISO 16649 consists of the following parts, under the general title Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli: Part 1: Colony-count technique at 44 °C using membranes and 5-bromo-4-chloro-3-indolyl- -D- glucuronide Part 2: Colony-count technique at 44 °C using 5-bromo-4-chloro-3-indolyl-D-glucuronide Part 3: Most probable number technique using 5-bromo-4-chloro-3-indolyl-D-glucuronide (Technical Specification) DD ISO/TS 16649-3:2005 Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iv Introduction This Technical Specification is intended to provide general guidance for the examination of products not dealt with by existing International Standards and to be taken into account by organizations preparing microbiological methods of test for application to foods or to animal feeding stuffs. Because of the large variety of products within this field of application, these guidelines may not be appropriate in every detail for certain products, and for some other products it may be necessary to use different methods. Nevertheless, it is hoped that in all cases every attempt will be made to apply the provided guidelines as far as possible and that deviations from them will only be made if absolutely necessary for technical reasons. When this Technical Specification is next reviewed, account will be taken of all information then available regarding the extent to which the guidelines have been followed and the reasons for deviations from them in the case of particular products. The harmonization of test methods cannot be immediate, and for certain groups of products International Standards and/or national standards may already exist that do not comply with these guidelines. In cases where International Standards already exist for the product to be tested, they should be followed, but 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 these guidelines will be those necessary for well-established technical reasons. DD ISO/TS 16649-3:2005 Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 1 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli Part 3: Most probable number technique using 5-bromo-4-chloro-3- indolyl- -D-glucuronide 1 Scope This Technical Specification specifies a horizontal method for the enumeration of -glucuronidase-positive Escherichia coli, by means of the liquid-medium culture technique and calculation of the most probable number (MPN) after incubation at 37 °C, then at 44 °C. This Technical Specification 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. The method is suitable for the enumeration of cells of Escherichia coli that may have been subjected to stress arising from dehydration, freezing, exposure to a saline (such as marine) environment or damage by disinfectants such as chlorine-containing products. A limitation of the applicability of this Technical Specification is imposed by the susceptibility of the method to a large degree of variability. The method is to be applied and the results interpreted in the light of the information given in Clause 11. WARNING Strains of Escherichia coli that do not grow at 44 °C and, in particular, those that are -glucuronidase negative, such as Escherichia coli O157 and some other strains of pathogenic E. coli, will not be detected by the method described in this Technical Specification. 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 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 6887-2, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 2: Specific rules for the preparation of meat and meat products DD ISO/TS 16649-3:2005 Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 2 ISO 6887-3, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 3: Specific rules for the preparation of fish and fishery products ISO 6887-4, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 4: Specific rules for the preparation of products other than milk and milk products, meat and meat products, and fish and fishery products ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations ISO 8261, Milk and milk products General guidance for the preparation 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 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 apply. 3.1 -glucuronidase-positive Escherichia coli bacteria which, at 44 °C, form typical blue or blue-green colonies on tryptone bile glucuronide medium under the conditions specified in this Technical Specification 3.2 enumeration of -glucuronidase-positive Escherichia coli determination of the most probable number of -glucuronidase-positive Escherichia coli per millilitre or per gram of sample, when the test is carried out in accordance with this Technical Specification 4 Principle 4.1 Three tubes1) of double-strength liquid selective enrichment medium are inoculated with a specified quantity of the test sample if the initial product is liquid, or with a specified quantity of the initial suspension in the case of other products. 4.2 Three tubes1) of single-strength liquid enrichment medium are inoculated with a specified quantity of test sample if the initial product is liquid, or with a specified quantity of the initial suspension in the case of other products. Then, under the same conditions, the single-strength liquid enrichment medium is inoculated with decimal dilutions of the test sample or of the initial suspension. 4.3 The tubes of double- and single-strength medium are inoculated at 37 °C for 24 h. The tubes are examined for acid production, indicating lactose fermentation. 4.4 Each tube of selective enrichment medium showing acid production is subcultured to tryptone bile glucuronide agar. 1) Five tubes can be used, see 9.2.1. DD ISO/TS 16649-3:2005 Licensed Copy: London South Bank University, London South Bank University, Sun Dec 10 13:26:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 3 4.5 The tryptone bile glucuronide agar is incubated at 44 °C for 20 h to 24 h. The tryptone bile glucuronide agar is examined for the presence of blue or blue-green colonies, indicating the presence of -glucuronidase- positive Escherichia coli. 4.6 The most probable number of -glucuronidase-positive Escherichia coli (see ISO 7218) are determined according to the number of tubes of selective enrichment medium whose subcultures have produced blue or blue-green colonies on tryptone bile glucuronide agar. 5 Dilution fluids and culture media For current laboratory practice, see ISO 7218. 5.1 Dilution fluids See the appropriate part of ISO 6887 or ISO 8261. 5.2 Culture media 5.2.1 Mineral-modified glutamate medium (selective enrichment medium) 5.2.1.1 Composition a) b) Double-strength medium Single-strength medium
