1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58presumptive Escherichia coli Part 2: Colony-count technique at 44 C using membranesICS 07.100.30; 6
2、7.100.01Milk and milk products Enumeration of BRITISH STANDARDBS ISO 11866-2:2005BS ISO 11866-2:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 23 January 2006 BSI 23 January 2006ISBN 0 580 47493 3Cross-referencesThe British Standards whi
3、ch 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
4、 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.Summary of pagesThis document comprises a front cover, an inside front cover, the ISO title
5、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 publicationAmd. No. Date CommentsA list of organizations represented on this committee can be obtained on request to its secreta
6、ry. present to the responsible 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.National forewordThis British Standard reproduces verbat
7、im ISO 11866-2:2005 and implements it as the UK national standard. It supersedes BS ISO 11866-3:1997 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: aid enquirers to understand the text;Ref
8、erence numbersISO 11866-2:2005(E)IDF 170-2:2005(E)INTERNATIONAL STANDARD ISO11866-2IDF170-2Second edition2005-12-01Milk and milk products Enumeration of presumptive Escherichia coli Part 2: Colony-count technique at 44 C using membranes Lait et produits laitiers Dnombrement dEscherichia coli prsums
9、Partie 2: Technique par comptage des colonies obtenues sur membranes 44 C BS ISO 11866-2:2005ii iiiForeword 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 c
10、arried 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 wor
11、k. 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 Inter
12、national 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. Attention is drawn to the possibility that some of th
13、e 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 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
14、Federation (IDF). It is being published jointly 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 Hori
15、zontal method for the detection and enumeration 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
16、-methylumbelliferyl-D-glucuronide (MUG) Part 2: Colony-count technique at 44 C using membranes BS ISO 11866-2:2005iv 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 rig
17、ht 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 adopted by the Action Teams and Standing Committees are circulated
18、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 document may be the subject of patent rights. IDF shall not be hel
19、d 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 is being published jointly by IDF and ISO. All work was carried o
20、ut 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 the former part 1 has been replaced by ISO 7251:2005. BS ISO 11866-2
21、:20051Milk and milk products Enumeration of presumptive Escherichia coli Part 2: Colony-count technique at 44 C using membranes 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 app
22、licable to milk, liquid milk products, dried milk, dried sweet whey, dried buttermilk, lactose, acid casein, lactic casein and 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
23、specified in this part of ISO 11866IDF 170 is the preferred method for samples in which comparatively large numbers of presumptive 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 refere
24、nces 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 7218, Microbiology of food and animal fe
25、eding stuffs General requirements and guidance for microbiological examinations ISO 8261IDF 122, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination BS ISO 11866-2:20052 3 Terms and definitions For the
26、 purposes of this 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 Princip
27、le 4.1 Resuscitation 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 unde
28、r frozen, dried or 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 st
29、age. 4.2 Isolation 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 ind
30、ole by each colony. 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 f
31、luid, culture media 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, un
32、der conditions which do not produce any change in their composition. 5.2 Dilution fluid See ISO 8261IDF 122. BS ISO 11866-2:200535.3 Culture media and reagent 5.3.1 Resuscitation medium: Mineral-modified glutamate agar 5.3.1.1 Composition Sodium glutamate Lactose Sodium formate L()Cystine L()Asparti
33、c acid L(+)Arginine Thiamine Nicotinic acid Pantothenic acid Magnesium sulfate heptahydrate (MgSO47H2O) Ammonium iron(III) citrate aCalcium chloride dihydrate (CaCl22H2O) Dipotassium hydrogen phosphate (K2HPO4) Ammonium chloride Agar Water 6,35 g10,0 g0,25 g0,02 g0,02 g0,024 g0,001 g0,001 g0,001 g0,
34、100 g0,010 g0,010 g0,90 g2,5 g12 g to 18 g b1 000 mlaIron content of at least 15 % (mass fraction). bDepending on the gel strength of the agar. 5.3.1.2 Preparation Dissolve the ammonium chloride in the water. Add the other components and heat to boiling. Adjust the pH, if necessary, so that after st
35、erilization 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 Pour into sterile Petri dishes (6.12), 12 ml to 15 ml of the medium cooled to approximately 45 C, and allow it to
36、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 drying cabinet or the oven (6.3) set at 50 C for 30 min or until the droplets have disappeared from the surface
37、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 11866-2:20054 5.3.2 Selective medium: Tryptone-bile agar 5.3.2.1 Composition Tryptone Bile salts (refined) Agar Water 20,0 g1,5 g12 g to 18 g a1 000 mlaDepending
38、 on the gel strength of the agar. 5.3.2.2 Preparation Dissolve the components 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
39、.1) set at 121 C for 15 min. 5.3.2.3 Preparation of agar plates Pour 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 wit
40、h the lids removed and the agar surfaces facing downwards, in the oven (6.3) 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 gHydrochloric aci
41、d, c(HCl) = 1 mol/l 100 ml5.3.3.2 Preparation Dissolve the 4-dimethylaminobenzaldehyde 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 82
42、61IDF 122. Glassware shall be resistant to repeated sterilization. Usual microbiological 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 11866-2:200556.2 Incubators, capable of operating at 3
43、7 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 pore size and of 85 mm diameter. 6.6 L
44、ong-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 Pipettes, calibrated for bacteriolog
45、ical 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. 6.12 Petri dishes, made of glass or
46、 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
47、 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this part of ISO 11866IDF 170. A recommended sampling method is given in ISO 707IDF 50. 8 Preparation of
48、test sample Prepare the test sample according to the method given in ISO 8261IDF 122. 9 Procedure NOTE If it is required to check whether the repeatability requirement is met (see Clause 11) carry out two single determinations in accordance with 9.1 to 9.5. 9.1 Test portion, initial suspension and f
49、urther dilutions Prepare the test portion, initial suspension (primary dilution) and further dilutions according to the method given in ISO 8261IDF 122. BS ISO 11866-2:20056 9.2 Resuscitation 9.2.1 Using sterile forceps (6.7), aseptically place a cellulose acetate membrane (6.5) onto the dried surface of each of two plates of the glutamate agar (5.3.1.3), taking care to avoid trapping air bubbles beneath the membranes. Gently
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