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1、BRITISH STANDARD BS ISO 11866-2:2005 Milk and milk products Enumeration of presumptive Escherichia coli Part 2: Colony-count technique at 44 C using membranes ICS 07.100.30; 67.100.01 ? Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO
2、11866-2:2005 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 23 January 2006 BSI 23 January 2006 ISBN 0 580 47493 3 National foreword This British Standard reproduces verbatim ISO 11866-2:2005 and implements it as the UK national standard. It
3、 supersedes BS ISO 11866-3:1997 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-ref
4、erences 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
5、 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 British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the resp
6、onsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover,
7、 the ISO title page, pages ii to iv, pages 1 to 9 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: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 200
8、6, Uncontrolled Copy, (c) BSI Reference numbers ISO 11866-2:2005(E) IDF 170-2:2005(E) INTERNATIONAL STANDARD ISO 11866-2 IDF 170-2 Second edition 2005-12-01 Milk and milk products Enumeration of presumptive Escherichia coli Part 2: Colony-count technique at 44 C using membranes Lait et produits lait
9、iers Dnombrement dEscherichia coli prsums Partie 2: Technique par comptage des colonies obtenues sur membranes 44 C BS ISO 11866-2:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26
10、 03:15:17 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 committee
11、s. 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 Inter
12、national 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 St
13、andards 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. Attention is drawn to the possibility that some of the elements of this document may be the sub
14、ject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 11866-2IDF 170-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jo
15、intly by ISO and IDF. This edition of ISO 11866-2IDF 170-2 cancels and replaces ISO 11866-3:1997, of which it constitutes a minor revision. ISO 11866-1:1997 has been cancelled and replaced by ISO 7251:2005, Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumer
16、ation of presumptive Escherichia coli Most probable number technique. ISO 11866IDF 170 consists of the following parts, under the general title Milk and milk products Enumeration of presumptive Escherichia coli: Part 1: Most probable number technique using 4-methylumbelliferyl-D-glucuronide (MUG) Pa
17、rt 2: Colony-count technique at 44 C using membranes BS ISO 11866-2:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iv Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Commi
18、ttee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards a
19、dopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this
20、document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 11866-2IDF 170-2 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It
21、 is being published jointly by IDF and ISO. All work was carried out by the Joint ISO/IDF/AOAC Group of Experts on Pathogenic contaminants (E102), under the aegis of its chairman, Mrs R. Lodi (IT). This edition of ISO 11866-2IDF 170-2 cancels and replaces the former part 3 of IDF 170A:1999, while th
22、e former part 1 has been replaced by ISO 7251:2005. BS ISO 11866-2:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 1 Milk and milk products Enumeration of presumptive Escherichia coli Part 2: Colony-count technique at 44 C using membran
23、es 1 Scope This part of ISO 11866IDF 170 specifies a method for the enumeration of presumptive Escherichia coli by means of a colony-count technique at 44 C. The method is applicable to milk, liquid milk products, dried milk, dried sweet whey, dried buttermilk, lactose, acid casein, lactic casein an
24、d rennet casein, caseinate and dried acid whey, cheese and processed cheese, butter, frozen milk products (including edible ices), and custard, desserts and cream. The method specified in this part of ISO 11866IDF 170 is the preferred method for samples in which comparatively large numbers of presum
25、ptive Escherichia coli (more than 100 per gram or 10 per millilitre) are suspected. CAUTION Some pathogenic strains of Escherichia coli do not grow at 44 C. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only th
26、e edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations ISO 8261IDF 122, Milk and milk products Gener
27、al guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination BS ISO 11866-2:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 2 3 Terms and definitions For the purposes of this
28、 document, the following terms and definitions apply. 3.1 presumptive Escherichia coli bacteria which at 44 C form indole-positive (pink) colonies on cellulose acetate membranes overlaid on tryptone-bile agar, under the conditions specified in this part of ISO 11866IDF 170 4 Principle 4.1 Resuscitat
29、ion A specified quantity of the test sample or initial suspension is inoculated onto cellulose acetate membranes overlaid on mineral-modified glutamate agar, then they are incubated at 37 C for 4 h. NOTE This procedure enables the presumptive Escherichia coli damaged by storage under frozen, dried o
30、r chill conditions, or damaged by heat or chemical processes, to be resuscitated. It also permits the diffusion of high concentrations of any fermentable carbohydrate present in the test sample which would otherwise interfere with indole production during the subsequent isolation stage. 4.2 Isolatio
31、n The membranes from the resuscitation stage on the mineral-modified glutamate agar are transferred to tryptone-bile agar. They are incubated at 44 C for 18 h to 24 h. 4.3 Detection The presence of presumptive Escherichia coli on the membrane is demonstrated by the production of indole by each colon
32、y. 4.4 Calculation The number of colony-forming units (CFU) of presumptive Escherichia coli per gram or per millilitre of sample is calculated from the number of indole-positive colonies obtained on membranes at dilution levels chosen so as to give a significant result. 5 Dilution fluid, culture med
33、ia and reagent 5.1 General For current laboratory practice, see ISO 7218 and ISO 8261. If the prepared culture media and reagents are not used immediately, they shall, unless otherwise stated, be stored in the dark at a temperature between 0 C and +5 C for no longer than 1 month, under conditions wh
34、ich do not produce any change in their composition. 5.2 Dilution fluid See ISO 8261IDF 122. BS ISO 11866-2:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 3 5.3 Culture media and reagent 5.3.1 Resuscitation medium: Mineral-modified glut
35、amate agar 5.3.1.1 Composition Sodium glutamate Lactose Sodium formate L()Cystine L()Aspartic acid L(+)Arginine Thiamine Nicotinic acid Pantothenic acid Magnesium sulfate heptahydrate (MgSO47H2O) Ammonium iron(III) citrate a Calcium chloride dihydrate (CaCl22H2O) Dipotassium hydrogen phosphate (K2HP
36、O4) Ammonium chloride Agar Water 6,35 g 10,0 g 0,25 g 0,02 g 0,02 g 0,024 g 0,001 g 0,001 g 0,001 g 0,100 g 0,010 g 0,010 g 0,90 g 2,5 g 12 g to 18 g b 1 000 ml a Iron content of at least 15 % (mass fraction). b Depending on the gel strength of the agar. 5.3.1.2 Preparation Dissolve the ammonium chl
37、oride in the water. Add the other components and heat to boiling. Adjust the pH, if necessary, so that after sterilization it is 6,7 at 25 C. Transfer 100 ml volumes of the medium to suitable containers. Sterilize in the autoclave (6.1) set at 115 C for 10 min. 5.3.1.3 Preparation of agar plates Pou
38、r into sterile Petri dishes (6.12), 12 ml to 15 ml of the medium cooled to approximately 45 C, and allow it to solidify. The plates may be stored at 0 C to +5 C for up to 4 days. Immediately before use, dry the plates, preferably with the lids removed and the agar surfaces facing downwards, in the d
39、rying cabinet or the oven (6.3) set at 50 C for 30 min or until the droplets have disappeared from the surface of the medium. The agar should be dry enough not to allow excess moisture to appear within 15 min of spreading the inoculum (1 ml). BS ISO 11866-2:2005 Licensed Copy: sheffieldun sheffieldu
40、n, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 4 5.3.2 Selective medium: Tryptone-bile agar 5.3.2.1 Composition Tryptone Bile salts (refined) Agar Water 20,0 g 1,5 g 12 g to 18 g a 1 000 ml a Depending on the gel strength of the agar. 5.3.2.2 Preparation Dissolve the component
41、s in the water and heat to boiling. Adjust the pH, if necessary, so that after sterilization it is 7,2 at 25 C. Transfer aliquots of up to 500 ml of the medium to suitable containers. Sterilize the medium in the autoclave (6.1) set at 121 C for 15 min. 5.3.2.3 Preparation of agar plates Pour into st
42、erile Petri dishes (6.12), 12 ml to 15 ml of the medium cooled to approximately 45 C, and allow it to solidify. The plates may be stored at 0 C to +5 C for up to 4 days. Immediately before use, dry the plates, preferably with the lids removed and the agar surfaces facing downwards, in the oven (6.3)
43、 set at 50 C for 30 min or until the droplets have disappeared from the surface of the medium. 5.3.3 Indole detection reagent (Vracko and Sherris reagent) 5.3.3.1 Composition 4-Dimethylaminobenzaldehyde 5,0 g Hydrochloric acid, c(HCl) = 1 mol/l 100 ml 5.3.3.2 Preparation Dissolve the 4-dimethylamino
44、benzaldehyde in the hydrochloric acid by heating, if necessary. The reagent may be stored in the dark at 0 C to +5 C for a maximum period of 3 months. 6 Apparatus and glassware For general requirements, see ISO 7218 and ISO 8261IDF 122. Glassware shall be resistant to repeated sterilization. Usual m
45、icrobiological laboratory apparatus and, in particular, the following. 6.1 Autoclave, capable of operating at 115 C 1 C and at 121 C 1 C. For details, see ISO 7218. BS ISO 11866-2:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 03:15:17 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 5 6.2 In
46、cubators, capable of operating at 37 C 1 C and at 44 C 0,5 C. 6.3 Drying cabinet or oven, ventilated by convection, capable of operating at 50 C 1 C. 6.4 Refrigerator (for storage of prepared media and reagent), capable of operating at 0 C to 5 C. 6.5 Cellulose acetate membranes, 0,45 m to 1,2 m por
47、e size and of 85 mm diameter. 6.6 Long-wave ultraviolet (UV) lamp, of wavelength between 360 nm and 366 nm, fitted with a suitable filter to remove UV radiation below 310 nm. 6.7 Blunt-ended forceps, sterile, of approximately 12 cm length. 6.8 pH-meter, accurate to within 0,1 pH units at 25 C. 6.9 P
48、ipettes, calibrated for bacteriological use, with 1 ml nominal capacity, graduated in divisions of 0,1 ml and with an outflow opening of 2 mm to 3 mm diameter. 6.10 Measuring cylinders, for preparation of the media and reagent. 6.11 Bottles or flasks, for sterilization and storage of culture media.
49、6.12 Petri dishes, made of glass or plastic, of approximately 90 mm or approximately 100 mm diameter. 6.13 Spreaders, made of glass or plastic, for example hockey sticks made from a glass rod of approximately 3,5 mm diameter and 20 cm length, bent at right angles about 3 cm from one end and with the cut ends made smooth by heating. 7 Sampling A representative sample should have been sent to the lab