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1、BRITISH STANDARD BS ISO 11866-1:1997 Milk and milk products Enumeration of presumptive Escherichia coli Part 1: Most probable number technique ICS 67.100.01 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-1:1997 This British Sta
2、ndard, 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 May 1997 BSI 02-1999 ISBN 0 580 27528 0 National foreword This British Standard reproduces verbatim ISO 11866-1:199
3、7 and implements it as the UK national standard. Together with BS ISO 11866-2:1997 and BS ISO 11866-3:1997 it supersedes BS 4285-3.8:1988 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibity to: aid enquirers
4、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 and European developments and promulgate them in the UK. A list of organizations represe
5、nted on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondenc
6、e Index”, or using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself
7、 confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the ISO title page, page ii, pages 1 to 8 and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will
8、be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No.DateComments Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-1:1997 BSI 02-1999i Contents Page National forewordInside fr
9、ont cover Forewordii 1Scope1 2Normative references1 3Definition1 4Principle1 5Dilution fluid, culture media and reagent2 6Apparatus and glassware3 7Sampling4 8Preparation of test sample4 9Procedure4 10Selection of dilutions5 11Determination, calculation and expression of results6 12Precision6 13Test
10、 report6 Annex A (normative) Determination of most probable number7 Annex B (informative) Bibliography8 Table 1 Examples of the selection of positive results for calculating MPN values6 Table A.1 MPN indexes and confidence limits7 Table A.2 Explanation of categories for results8 Licensed Copy: sheff
11、ieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii blank Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy
12、, (c) BSI BS ISO 11866-1:1997 ii BSI 02-1999 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 memb
13、er 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 Ele
14、ctrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 castin
15、g a vote. This part of ISO 11866 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 5, Milk and milk products, in collaboration with the International Dairy Federation (IDF) and AOAC INTERNATIONAL, and will also be published by these organizations. ISO 11866 c
16、onsists of the following parts, under the general title Milk and milk products Enumeration of presumptive Escherichia coli: Part 1: Most probable number technique; Part 2: Most probable number technique using 4-methylumbelliferyl-D-glucuronide (MUG); Part 3: Colony-count technique at 44 C using memb
17、ranes. The method specified in ISO 11866-1 is preferred for samples in which comparatively low numbers of Escherichia coli are suspected. Annex A forms an integral part of this part of ISO 11866. Annex B is for information only. Descriptors: Agricultural products, food products, dairy products, milk
18、, tests, microbiological analysis, counting, coliform bacteria, bacteria count methods. Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-1:1997 BSI 02-19991 1 Scope This part of ISO 11866 specifies a method for the enumeration of
19、 presumptive Escherichia coli by means of the culture technique involving a liquid medium, and calculation of the number of presumptive Escherichia coli per gram or per millilitre by the most probable number (MPN) technique after incubation at 37 C then incubation at 44 C. The method is applicable t
20、o 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); custard, desserts and cream. The method specified in
21、this part of ISO 11866 is preferred for samples in which comparatively low numbers of presumptive Escherichia coli (less than 100 per gram or 10 per millilitre) are suspected. CAUTION Some Escherichia coli pathogenic species do not grow at 44 C. The applicability of this part of ISO 11866 is limited
22、 by the susceptibility of the method to a large degree of variability. The method should, therefore, be used and the results interpreted in the light of the information given in clause 12. 2 Normative references The following standards contain provisions which, through reference in this text, consti
23、tute provisions of this part of ISO 11866. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this part of ISO 11866 are encouraged to investigate the possibility of applying the most recent editions of the standar
24、ds indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 7218:1996, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. ISO 8261:1989, Milk and milk products Preparation of test samples and dilutions for
25、microbiological examination. 3 Definition For the purposes of this part of ISO 11866, the following definition applies. 3.1 presumptive Escherichia coli bacteria which at 44 C cause fermentation of lactose with the production of gas, and which at 44 C produce indole from tryptophan, when the test is
26、 carried out in accordance with the method specified in this part of ISO 11866 4 Principle 4.1 Inoculation of three tubes of double-strength liquid selective enrichment medium 5.3.1.1 a) with a specified quantity of test sample if the initial product is liquid, or with a specified quantity of the in
27、itial suspension in the case of other products. 4.2 Inoculation of three tubes of single-strength liquid selective enrichment medium 5.3.1.1 b) 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 produ
28、cts. Then, under the same conditions, inoculation of single-strength medium 5.3.1 b) with decimal dilutions of the test sample or of the initial suspension. 4.3 Incubation of the tubes of double- and single-strength medium at 37 C for 24 h to 48 h. Examination of the tubes for gas formation. License
29、d Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-1:1997 2 BSI 02-1999 4.4 Inoculation, from the tubes of double- and single-strength medium which have given rise to gas formation, of a new series of tubes containing the second selective
30、 medium (5.3.2). 4.5 Incubation at 44 C for 24 h to 48 h and examination of the new series of tubes (4.4) for gas formation. 4.6 Inoculation, from the tubes of liquid selective medium (4.5) which have given rise to gas formation, of a new series of tubes containing tryptone water (5.4). 4.7 Incubati
31、on at 44 C for 24 h to 48 h and examination of this new series of tubes (4.6) for indole production. 4.8 Identification of those tubes inoculated originally in 4.1 and/or 4.2, which show in step 4.5 production of gas from the second selective medium at 44 C and in step 4.7 formation of indole from t
32、ryptone water at 44 C, as being positive for presumptive Escherichia coli. 4.9 Determination of the MPN index from the numbers of positive tubes (4.8) of selected dilutions by means of an MPN table (Annex A) and calculation of the most probable number (MPN) of presumptive Escherichia coli per gram o
33、r per millilitre of the original sample. 5 Dilution fluid, 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 temperatur
34、e between 0 C and + 5 C for no longer than 1 month, under conditions which do not produce any change in their composition. 5.2 Dilution fluid See ISO 8261. 5.3 Culture media 5.3.1 Lauryl sulfate tryptose broth (selective enrichment medium) 5.3.1.1 Composition 5.3.1.2 Preparation Dissolve the compone
35、nts or the dehydrated complete medium in the water, by heating if necessary. Adjust the pH, if necessary, so that after sterilization it is 6,8 at 25 C. Transfer the media in quantities of 10 ml to tubes of dimensions 16 mm 160 mm (6.2) containing inverted Durham tubes (6.3) in the case of single-st
36、rength medium, and to test tubes of dimensions 20 mm 200 mm (6.2) containing inverted Durham tubes (6.3) in the case of the double- strength medium. Sterilize for 15 min in the autoclave (6.1) set at 121 C. The inverted Durham tubes shall not contain air bubbles after sterilization. 5.3.2 EC broth (
37、second selective medium) a) Double-strength medium b) Single-strength medium Tryptone Lactose Dipotassium hydrogen phosphate (K2HPO4) Potassium dihydrogen phosphate (KH2PO4) Sodium chloride Sodium lauryl sulfate CH3(CH2)11OSO3Na Water 40,0 g 10,0 g 5,5 g 5,5 g 10,0 g 0,2 g 1 000 ml 20,0 g 5,0 g 2,75
38、 g 2,75 g 5,0 g 0,1 g 1 000 ml Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-1:1997 BSI 02-19993 5.3.2.1 Components 5.3.2.2 Preparation Dissolve the components or the dehydrated complete medium in the water, by heating if nece
39、ssary. Adjust the pH, if necessary, so that after sterilization it is 6,8 at 25 C. Transfer the medium in quantities of 10 ml to test tubes of dimensions 16 mm 160 mm (6.2) containing inverted Durham tubes (6.3). Sterilize for 15 min in the autoclave (6.1) set at 121 C. The inverted Durham tubes sha
40、ll not contain air bubbles after sterilization. 5.4 Tryptone water 5.4.1 Components 5.4.2 Preparation Dissolve the components in the water, by heating if necessary. Adjust the pH, if necessary, so that after sterilization it is 7,3 at 20 C. Dispense the medium in quantities of 5 ml to 10 ml into tub
41、es of dimensions 16 mm 160 mm (6.2). Sterilize for 15 min in the autoclave (6.1) set at 121 C. 5.5 Indole reagent (Kovacs reagent) 5.5.1 Composition 5.5.2 Preparation Dissolve the 4-dimethylaminobenzaldehyde in the alcohol by heating gently to between 50 C and 55 C by means of the water bath (6.5).
42、Cool and add the hydrochloric acid. Protect from light and store at approximately 4 C. The colour of the reagent shall be light yellow to light brown. 6 Apparatus and glassware For general requirements, see ISO 7218 and ISO 8261. Glassware shall be resistant to repeated sterilization. Usual microbio
43、logical laboratory apparatus and, in particular, the following. Tryptose or trypticase Lactose Bile salts (No. 3)a Dipotassium hydrogen phosphate (K2HPO4) Potassium dihydrogen phosphate (KH2PO4) Sodium chloride Water 20,0 g 5,0 g 1,5 g 4,0 g 1,5 g 5,0 g 1 000 ml a See reference 3. Tryptone Sodium ch
44、loride Water 10,0 g 5,0 g 1 000 ml 4-Dimethylaminobenzaldehyde 2-Methylbutan-1-ol or pentan-1-ol Hydrochloric acid (20 1,18 g/ml to 1,19 g/ml) 5,0 g 75,0 ml 25,0 ml Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:12:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-1:1997 4 BSI 02
45、-1999 6.1 Autoclave, capable of operating at 121 C 1 C. For details, see ISO 7218. 6.2 Test tubes, of dimensions approximately 16 mm 160 mm and 20 mm 200 mm, or flasks or bottles of suitable capacity. 6.3 Durham tubes, of size suitable for use in the test tubes (6.2). 6.4 Water bath, capable of oper
46、ating at 44 C 0,5 C. 6.5 Water bath, capable of operating at between 50 C and 55 C. 6.6 Incubator, capable of maintaining a temperature of 37 C 1 C at all points within it. 6.7 Loops, made of platinum/iridium or nickel/chromium or plastic, approximately 3 mm in diameter, or sterile disposable bags.
47、6.8 pH-meter, accurate to within 0,1 pH units at 25 C. 6.9 Total-delivery pipettes, with nominal capacities of 1 ml and 10 ml. 7 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is no
48、t part of the method specified in this part of ISO 11866. A recommended sampling method is given in ISO 707. 8 Preparation of test sample Prepare the test sample according to the method given in ISO 8261. 9 Procedure 9.1 Test portion, initial suspension and dilutions Prepare the test portion, initia
49、l suspension (primary dilution) and further decimal dilutions according to the method given in ISO 8261. Prepare a sufficient number of dilutions to ensure that all the tubes for the final dilution will yield a negative result. 9.2 Selective enrichment medium 9.2.1 Inoculation 9.2.1.1 Take three tubes of double-strength enrichment medium 5.3.1.1 a). Using a sterile pipette (6.9), transfer to each of these tubes 10 ml of the test sample if liquid, or 10 ml of the initia
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