1、BRITISH STANDARD BS ISO 15213:2003 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of sulfite-reducing bacteria growing under anaerobic conditions ICS 07.100.30 BS ISO 15213:2003 This British Standard was published under the authority of the Standards Policy and
2、Strategy Committee on 16 May 2003 BSI 16 May 2003 ISBN 0 580 41892 8 National foreword This British Standard reproduces verbatim ISO 15213:2003 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has th
3、e responsibility to: 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 “Interna
4、tional 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. Compliance with a Brit
5、ish Standard does not of itself confer immunity 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 an
6、d European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, ISO title page, pages ii to iv, pages 1 to 6, an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was
7、 last issued. Amendments issued since publication Amd. No. Date CommentsINTERNATIONAL STANDARD ISO 15213 First edition 2003-05-01 Reference number ISO 15213:2003(E) Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of sulfite-reducing bacteria growing under anaerob
8、ic conditions Microbiologie des aliments Mthode horizontale pour le dnombrement des bactries sulfito-rductrices se dveloppant en conditions anarobiesBSISO15213:2003ii BSISO15213:2003 iii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards
9、 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 organiza
10、tions, 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 t
11、he 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
12、 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. ISO15213 was prepared by Technical Committee ISO/TC34, Food produc
13、ts, Subcommittee SC9, Microbiology. BSISO15213:2003iv Introduction Because of the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain products. In this case, different methods specific to these products may be used if absolutely necessar
14、y for justified technical reasons. Nevertheless, every attempt will be made to apply this horizontal method as far as possible. When this International Standard is next reviewed, account will be taken of all information then available regarding the extent to which this horizontal method has been fol
15、lowed 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 group of products International Standards and/or national standards may already exist that do not comply with this horizontal method. It is
16、 hoped that when such standards are reviewed they will be changed to comply with this International Standard so that eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons. BSISO15213:2003ANRETNIITOTS LANDNADRA ISO 12513002
17、:3)E( ISO 3002 r llAithgs reservde 1 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of sulfite-reducing bacteria growing under anaerobic conditions 1S c o p e This International Standard specifies a horizontal method for the enumeration of sulfite-reducing bacte
18、ria growing under anaerobic conditions. 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. 2 Normative references The following referenced documents are indispensable for the application
19、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 6887-1:1999, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decima
20、l dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 8261, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination ISO 7
21、218:1996, Microbiology of food and animal feeding stuffs General rules for microbiological examinations ISO 7218:1996/Amd.1:2001, Microbiology of food and animal feeding stuffs General rules for microbiological examinations Amendment 1 ISO/TS 11133-1, Microbiology of food and animal feeding stuffs G
22、uidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 sulfite-reducing bacteria growing
23、under anaerobic conditions bacteria forming countable typical colonies under the conditions specified in this International Standard 4 Principle 4.1 Two agar plates (or tubes) are prepared, using iron sulfite medium, and using a specified quantity of the test sample if the initial product is liquid,
24、 or using a specified quantity of an initial suspension in the case of other products. BSISO15213:20031ISO :31251(3002)E 2 ISO 3002 All rithgs reresvde Two other agar plates (or tubes) are prepared, under the same conditions, using decimal dilutions of the test sample or the initial suspension. 4.2
25、The plates (or tubes) are incubated under anaerobic conditions at for to (final reading after ), or possibly at if thermophilic bacteria are suspected. Typical black-coloured colonies are counted. The black colour of the colonies and the surrounding zone is due to the formation of iron(II) sulfide a
26、s a result of the reaction between sulfide ions and trivalent iron Fe(III) present in the medium. 4.3 The number of sulfite-reducing bacteria per millilitre or per gram of sample is calculated from the number of colonies obtained on the plates (or tubes). 5 Culture medium and diluent For current pra
27、ctices, see ISO 7218. 5.1 Plate count medium: Iron sulfite agar 5.1.1 Composition 5.1.2 Preparation Dissolve the ingredients in the water by heating. If necessary, adjust the pH so that after sterilization it is at . Pour portions of the medium into flasks. If the enumeration is performed by use of
28、tubes (6.5), pour or of medium into the tubes. Sterilize forin an autoclave set at . Just before use, de-aerate the medium. 5.2 Saline peptone diluent See ISO 6887-1:1999, 5.2.1. 6 Apparatus and glassware Usual microbiological equipment (see ISO 7218) and, in particular, the following. 6.1 Homogeniz
29、ation equipment, for samples of solid food (see ISO 7218). 6.2 Water bath, capable of being maintained at between and . Enzymatic digest of casein Pancreatic digest of soya Yeast extract Disodium disulfite (Na 2 S 2 O 5 ) Iron(III) ammonium citrate Agar to a Water a Depending on the gel strength of
30、the agar. 37 C 1 C 24 h 48 h 48 h 50 C 15 g 5g 5g 1g 1g 9 g 18 g 1 000 ml 7,6 0,2 25 C 250 ml 500 ml 20 ml 25 ml 15 min 121 C 44 C4 7 C BSISO15213:20032ISO 12513002:3)E( ISO 3002 r llAithgs reservde 3 6.3 Anaerobic jars, with equipment for generating an anaerobic atmosphere, and including a system t
31、o check the anaerobic conditions. 6.4 Incubator, capable of being maintained at and, if necessary, at . 6.5 Test tubes, of dimensions , and flasks or bottles of capacity . 7 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or change
32、d during transport or storage. Sampling is not part of the method specified in this International Standard. If there is no specific International Standard dealing with sampling of the product concerned, it is recommended that the parties concerned come to an agreement on this subject. 8 Preparation
33、of test sample Prepare the test samples in accordance with ISO 6887-1, ISO 8261 or the specific International Standard appropriate to the product concerned. If there is no specific International Standard available, it is recommended that the parties concerned come to an agreement on this subject. 9
34、Procedure 9.1 General A diagram of procedure is given in Annex A. 9.2 Test portion, initial suspension and dilutions See ISO 6887-1, ISO 8261 or any specific International Standard appropriate to the concerned product. Heat treatment of the initial suspension may be necessary to eliminate vegetative
35、 forms of spore-forming bacteria and/or non-spore-forming bacteria. Temperatures and heating times vary according to the actual need, from combinations producing a definite pasteurization effect at a moderate heat activation effect (e.g. for ), to boiling for several minutes. In this case, results c
36、ould be given as number of spores of sulfite- reducing bacteria growing under anaerobic conditions. 9.3 Inoculation Take two sterile Petri dishes. Using a sterile pipette, transfer to each dish of test sample if the product is liquid, or of the initial suspension in the case of other products. Take
37、two other sterile Petri dishes. Using a fresh sterile pipette, transfer to each dish of the first decimal dilution ( ) of the test sample if the product is liquid, or of the first decimal dilution of the initial suspension ( ) in the case of other products. Repeat the described procedure with the fu
38、rther dilutions, using a fresh sterile pipette for each dilution. Pour into each Petri dish approximately of iron sulfite agar (5.1) which has been cooled to to in the water bath (6.2). The time elapsing between inoculation of the Petri dishes and addition of the agar should not exceed . Carefully m
39、ix the inoculum with the medium by horizontal movements and allow the medium to solidify. After the medium has solidified, pour to of the same medium into the dish as an overlay. 37 C 1 C5 0 C 1 C 16 mm 160 mm 500 ml 75 C 20 min 1ml 1ml 1ml 10 1 1ml 10 2 15 min 44 C4 7 C 15 min 5ml 10ml BSISO15213:2
40、0033ISO :31251(3002)E 4 ISO 3002 All rithgs reresvde If tubes are used, inoculate a volume from each dilution into each of two tubes of medium kept at to . Mix gently without forming bubbles, and leave the medium to solidify in a cold water bath (6.2). After the medium has solidified, pour to of the
41、 same medium into each tube as an overlay. 9.4 Incubation After solidification, incubate the Petri dishes in anaerobic jars (6.3) at for to . If thermophilic bacteria are suspected, prepare a second set of Petri dishes (see 9.3). Incubate this set at . In the case of tubes, incubation in anaerobic j
42、ars is not necessary. 9.5 Counting of the colonies Read the results after and , depending on the degree of black colour and the growth rate of the microoganisms. Black colonies, possibly surrounded by a black zone, are counted as sulfite-reducing bacteria. NOTE 1 Diffuse, unspecific blackening of th
43、e medium may occur, especially when inoculation is performed in agar tubes instead of Petri dishes. The growth of anaerobic bacteria, which only produce hydrogen (not H 2 S), may also reduce the sulfite present and lead to a general blackening of the medium. Count colonies of sulfite-reducing bacter
44、ia in each dish containing less than 150 typical colonies and less than 300 total colonies. When the number of colonies is high, some tubes may be unreadable. In this case, only tubes where the colonies are clearly separate should be considered for counting. NOTE 2 This International Standard may be
45、 used to enumerate only Clostridium. After obtaining characteristic colonies, pick five of them from each dish, and confirm the genus Clostridium with confirmation tests (e.g. respiratory tests, spore- forming tests). 10 Expression of results and confidence limits See Amendment 1 to ISO 7218:1996. 1
46、1 Test report The test report shall specify: a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; c) the test method used, including the temperature of incubation, use of tubes, and any thermal treatment to destroy vegetative bacteria; d)
47、 all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the test results; e) the test results obtained. The test report shall also state if further tests are to be carried out by a reference labora
48、tory, or, if already carried out, what the results were. 1ml 44 C 47 C 2ml 3ml 37 C 1 C 24 h 48 h 50 C 1 C 24 h 48 h BSISO15213:20034ISO 12513002:3)E( ISO 3002 r llAithgs reservde 5 Annex A (normative) Diagram of procedure BSISO15213:20035ISO :31251(3002)E 6 ISO 3002 All rithgs reresvde Bibliography
49、 1 NMKL No. 95:1997, Sulfite-reducing Clostridia Determination in food 1) 1) This revised NMKL method has been elaborated by Reidar Skjelkvle. Oslo City Food Control Authority, Vestbyvn. 13, N-0976 Oslo, Norway. BSISO15213:20036BS ISO 15213:2003 BSI 389 Chiswick High Road London W4 4AL BSI British Standards Institution BSI is the independent national body responsible for preparing Br
