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1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS 5763-13:1998 ICS 07.10
2、0.30; 65.120; 67.040 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Methods for Microbiological examination of food and animal feeding stuffs Part 13: Enumeration of Escherichia coli: colony-count technique at 44 C using membranes Licensed Copy: sheffieldun sheffieldun, na, F
3、ri Dec 01 13:53:19 GMT+00:00 2006, Uncontrolled Copy, (c) BSI This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 June 1998 BSI 1998 First published No
4、vember 1989 First revision June 1998 The following BSI references relate to the work on this standard: Committee reference AW/9 Draft for comment 96/500899 DC ISBN 0 580 29547 8 BS 5763-13:1998 Amendments issued since publication Amd. No.DateText affected Committees responsible for this British Stan
5、dard The preparation of this British Standard was entrusted to Technical Committee AW/9, Microbiology, upon which the following bodies were represented: Association of Clinical Pathologists Association of Public Analysts BLWA Ltd (the Association of the Laboratory Supply Industry) Campden and Chorle
6、ywood Food Research Association Dairy Industry Federation Department of Agriculture Northern Ireland Department of Health (Health Aspects of the Environment and Food Division) Food and Drink Federation Institute of Brewing Institute of Food Research Laboratory of the Government Chemist Leatherhead F
7、ood Research Association Meat and Livestock Commission Ministry of Agriculture, Fisheries and Food Natural Resources Institute Public Health Laboratory Service Society for Applied Bacteriology United Dairy Farmers University of Bristol Licensed Copy: sheffieldun sheffieldun, na, Fri Dec 01 13:53:19
8、GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS 5763-13:1998 BSI 1998i Contents Page Committees responsibleInside front cover Forewordii 1Scope1 2Normative references1 3Terms and definitions1 4Principle1 5Diluent, culture media and reagents1 6Apparatus2 7Sampling3 8Preparation of test sample3 9Procedu
9、re3 10Calculation and expression of results4 11Test report4 Bibliography4 Table 1 Composition of mineral-modified glutamate agar2 Table 2 Composition of tryptone bile agar2 Table 3 Composition of Vracko and Sherris indole detection reagent2 Licensed Copy: sheffieldun sheffieldun, na, Fri Dec 01 13:5
10、3:19 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii BSI 1998 BS 5763-13:1998 Foreword This part of BS 5763 was prepared by Technical Committee AW/9, Microbiology. It supersedes BS 5763-13:1989, which is withdrawn. This British Standard is based on ISO 6391:1997, Meat and meat products Enumeration of
11、Escherichia coli Colony-count technique at 44 C using membranes, published by the International Organization for Standardization (ISO). The technical content of this part of BS 5763 agrees with ISO 6391, with the following exceptions: the title and Scope have been modified to reflect the applicabili
12、ty of the method to food and animal feeding stuffs, not solely to meat and meat products; references in the text to international standards for sampling of meat and meat products have been deleted, and clauses 7 to 10 have been modified accordingly. It is recommended that BS 5763-0 Methods for micro
13、biological examination of food and animal feeding stuffs General laboratory practices be read in conjunction with this part of BS 5763. Although, for well-accepted statistical reasons, the limit for the lowest number of colonies counted per plate of selective medium is set at 15, for practical purpo
14、ses it is often desirable to establish an estimated count of lower numbers of Escherichia coli. The confidence limits of such determinations are to be found in BS 5763-0. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are respons
15、ible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, pages 1 to 4, an inside back cover and a back cover. Licensed Copy: sheffie
16、ldun sheffieldun, na, Fri Dec 01 13:53:19 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BSI 19981 BS 5763-13:1998 1 Scope This part of BS 5763 describes a method for the enumeration of viable Escherichia coli present in products intended for human consumption or feeding of animals. The method will dete
17、ct Escherichia coli (biotype 1) and lactose non-fermenting or anaerogenic variants 1, 2. NOTE 1 This method may not be appropriate for the examination of products having a high content of aerobic spore-forming bacteria. NOTE 2 Some pathogenic strains of Escherichia coli are unable to grow at 44 C, a
18、nd will not be detected by this method. 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this part of this British Standard. For dated references, subsequent amendments to, or revisions of, any of these public
19、ations do not apply. For undated references, the latest edition of the publication referred to applies. BS 5763-0, Microbiological examination of food and animal feeding stuffs General laboratory practices. BS 5763-6, Microbiological examination of food and animal feeding stuffs Preparation of dilut
20、ions. 3 Terms and definitions For the purposes of this British Standard, the following term and definition applies. 3.1 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 B
21、ritish Standard 4 Principle 4.1 Resuscitation Inoculation of a specified quantity of the initial suspension onto cellulose acetate membranes overlaid on mineral-modified glutamate agar, and incubation at 37 C for 4 h. NOTEThis procedure enables Escherichia coli damaged by storage under frozen, dried
22、 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, if present in the test sample, which would otherwise interfere with indole production during the subsequent isolation stage. 4.2 Iso
23、lation Transfer of membranes from the resuscitation stage on mineral-modified glutamate agar to tryptone bile agar and incubation at 44 C for 18 h to 20 h. 4.3 Detection Demonstration of the presence of Escherichia coli on the membranes by the production of indole by each colony. 4.4 Calculation Cal
24、culation of the number of Escherichia coli per millilitre or per gram of sample from the number of indole-positive colonies obtained on membranes at dilution levels chosen to give a significant result. 5 Diluent, culture media and reagents 5.1 General Current laboratory practice shall be in accordan
25、ce with BS 5763-0. 5.2 Diluent The dilutions shall be prepared in accordance with BS 5763-6. 5.3 Resuscitation media: mineral-modified glutamate agar 5.3.1 Composition The composition of the mineral-modified glutamate agar shall be in accordance with Table 1. 5.3.2 Preparation Dissolve the ammonium
26、chloride in the water. Add the other components and heat to boiling. Adjust the pH so that after sterilization it is 6.7 at 25 C. Transfer 100 ml volumes of the medium to suitable containers (6.6) and sterilize for 10 min in the autoclave (6.2) set at 121 C. 5.3.3 Preparation of agar plates Pour 12
27、ml to 15 ml of the medium, cooled to 47 C in the water bath (6.11), into sterile Petri dishes (6.7) and allow to solidify. NOTE 1 The plates may be stored at between 0 C and +5 C for up to one week. Immediately before use, dry the agar plates carefully e.g. in the drying cabinet or oven (6.5) set at
28、 50 C, preferably with the lids off and agar surface downwards, until any droplets have disappeared from the surface of the medium. Do not dry them any further. NOTE 2 The agar should be dry enough so that 1 ml of inoculum is totally absorbed into the membrane/agar within 15 min. Licensed Copy: shef
29、fieldun sheffieldun, na, Fri Dec 01 13:53:19 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 2 BSI 1998 BS 5763-13:1998 Table 1 Composition of mineral-modified glutamate agar Sodium glutamate6.35 g Lactose10.0 g Sodium formate0.25 g L(2) (cystine)0.02 g L(2) (aspartic acid)0.024 g L(+) (arginine)0.02 g T
30、hiamine0.001 g Nicotinic acid0.001 g Pantothenic acid0.001 g Magnesium sulfate (MgSO4.7H2O)0.100 g Ammonium iron(III) citrate at least 15 % Fe (m/m) 0.010 g Calcium chloride (CaCl2.2H2O)0.010 g Dipotassium hydrogen phosphate0.90 g Ammonium chloride2.5 g Agar1 000 ml Water12 g to 18 ga a Depending on
31、 the gel strength of the agar. 5.4 Selective medium: tryptone bile agar 3 5.4.1 Composition The composition of the tryptone bile agar shall be in accordance with Table 2. 5.4.2 Preparation Dissolve the components in the water and heat to boiling. Adjust the pH so that after sterilization it is 7.2 a
32、t 25 C. Transfer 100 ml volumes of the medium to suitable containers, and sterilize for 15 min in the autoclave (6.2) set at 121 C. Table 2 Composition of tryptone bile agar Tryptone (peptic digest of casein)20.0 ga Bile salts (refined)1.5 gb Agar12 g to 18 gc Water1 000 ml a Commercial brands that
33、favour indole formation should be used. b Oxoid Bile salts No. 3 is an example of a suitable product available commercially. (This information is given for the convenience of users of this British Standard and does not constitute an endorsement by BSI of this product.) c Depending on the gel strengt
34、h of the agar. 5.4.3 Preparation of agar plates Pour 12 ml to 15 ml of the medium, cooled to 47 C in the water bath (6.11), into sterile Petri dishes (6.7) and allow to solidify. NOTEThese plates may be stored at between 0 C and +5 C for up to 4 days. Immediately before use, dry the agar plates care
35、fully e.g. in the cabinet or oven (6.5) set at 50 C, preferably with the lids off and agar surface downwards, until any droplets have disappeared from the surface of the medium. Do not dry them any further. 5.5 Vracko and Sherris indole detection reagent 5.5.1 Composition The composition of the Vrac
36、ko and Sherris indole detection reagent shall be in accordance with Table 3. Table 3 Composition of Vracko and Sherris indole detection reagent p-Dimethylaminobenzaldehyde5 g Hydrochloric acid (HCl 1 mol/l)100 ml 5.5.2 Preparation Dissolve the reagent in the hydrochloric acid and store at room tempe
37、rature for no longer than 1 month. 6 Apparatus 6.1 Usual microbiological laboratory apparatus. 6.2 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), conforming to BS 5763-0. 6.3 Incubator, capable of being maintained at 37 C 1 C. 6.4 Incubator, capable of being maintained at 4
38、4 C 0.5 C. 6.5 Drying cabinet or oven, ventilated by convection, capable of being maintained at 50 C 1C. 6.6 Test tubes, of dimensions 18 mm 3 180 mm, or flasks or bottles, ranging in capacity from 125 ml to 300 ml, for sterilization and storage of culture media. Licensed Copy: sheffieldun sheffield
39、un, na, Fri Dec 01 13:53:19 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BSI 19983 BS 5763-13:1998 6.7 Petri dishes, made of glass or plastics, of 90 mm to 100 mm diameter. 6.8 Cellulose acetate membranes, of 0.45 mm to 1.2 mm pore size and of 85 mm diameter. 6.9 Pipettes, sterile, calibrated for bact
40、eriological use, of 1 ml nominal capacity, graduated in divisions of 0.1 ml, and with an outflow opening of 2 mm to 3 mm in diameter. 6.10 Spreaders, sterile, made of plastics or glass, e.g. hockey sticks made from a glass rod of approximately 3.5 mm diameter, and 20 cm length, bent at right angles
41、about 3 cm from one end and with the cut ends made smooth by heating. 6.11 Water bath or similar apparatus, capable of being maintained at 47 C 2 C. 6.12 Longwave (365 nm) ultraviolet lamp, fitted with a suitable filter to remove UV radiation below 310 nm. 6.13 pH meter, accurate to 0.1 pH units at
42、25 C. 6.14 Sterile forceps. 7 Sampling Sampling is not part of the method specified in this British Standard. Sampling shall be carried out in accordance with the British Standard appropriate for the product concerned. NOTEIf there is no specific British Standard, it is recommended that the parties
43、concerned come to an agreement on this subject. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. 8 Preparation of test sample Prepare the sample in accordance with the British Standard appropriate for
44、the product concerned. NOTEIf there is no specific British Standard, it is recommended that the parties concerned come to an agreement on this subject. 9 Procedure 9.1 Test portion, initial suspension and dilutions See BS 5763-6 and the British Standard appropriate for the product concerned. 9.2 Res
45、uscitation 9.2.1 Using sterile forceps, aseptically place a cellulose acetate membrane (6.8) onto the dried surface of each of two dishes of the mineral-modified glutamate agar (5.3.1) taking care to avoid trapping air bubbles beneath the membranes. Gently flatten the membranes with a sterile spread
46、er (6.10). Using a sterile pipette (6.9), add 1 ml of the initial suspension to the centre of each membrane. Using a sterile spreader (6.10), spread the inoculum evenly over the whole membrane surface, avoiding any spillage from the membrane. 9.2.2 Using another sterile pipette (6.9), inoculate 1 ml
47、 of the further diluted initial suspension onto further membranes, as in 9.2.1. The time elapsing between the preparation of the test sample (clause 8) and the inoculation on to the membrane shall not exceed 45 min, unless stipulated otherwise in the relevant British Standard. 9.2.3 Leave the inocul
48、ated dishes in a horizontal position at room temperature for approximately 15 min until the inocula have soaked into the membranes, then incubate them at 37 C in the incubator (6.3) for 4 h 3 0,5 h with the membrane/agar surface uppermost. 9.3 Transfer to selective medium 9.3.1 Using sterile blunt-e
49、nded forceps, transfer the membranes, inoculated side uppermost, from the mineral-modified glutamate agar (5.3.1) to the tryptone bile agar (5.4). The moist membrane will adhere to the agar surface; avoid trapping air bubbles under the membrane. Do not use a spreader as this would disperse any micro-colonies present, giving a false high count. 9.3.2 Incubate the dishes at 44 C in the incubator (6.4) for 18 h to 24 h with the membrane/agar surface uppermost. Do not stack them more than three high. 9.4 Dete
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