1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58presumptive Escherichia coli Part 1: Most probable number technique using 4-methylumbelliferyl- -D-
2、glucuronide (MUG) ICS 07.100.30; 67.100.01Milk and milk products Enumeration of BRITISH STANDARDBS ISO 11866-1:2005BS ISO 11866-1:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 24 January 2006 BSI 24 January 2006ISBN 0 580 47494 1Cross-r
3、eferencesThe 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 Standard
4、s 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.Summary of pagesThis document comprises a front cover, an
5、inside front cover, the ISO title page, pages ii to iv, pages 1 to 11 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
6、 obtained on request to its secretary. 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
7、 British Standard reproduces verbatim ISO 11866-1:2005 and implements it as the UK national standard. It supersedes BS ISO 11866-2:1997 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: aid e
8、nquirers to understand the text;Reference numbersISO 11866-1:2005(E)IDF 170-1:2005(E)INTERNATIONAL STANDARD ISO11866-1IDF170-1Second edition2005-12-01Milk and milk products Enumeration of presumptive Escherichia coli Part 1: Most probable number technique using 4-methylumbelliferyl-D-glucuronide (MU
9、G) Lait et produits laitiers Dnombrement dEscherichia coli prsums Partie 1: Technique du nombre le plus probable avec utilisation de 4-mthylumbellifryl-D-glucuronide (MUG) BS ISO 11866-1:2005ii iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national
10、 standards 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. Internationa
11、l organizations, 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
12、given in the 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 lea
13、st 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 subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 11866-1IDF 170-1 was prepared by Technical Committee
14、 ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, in collaboration with the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. This edition of ISO 11866-1IDF 170-1 cancels and replaces ISO 11866-2:1997, of which it constitutes a minor revision. IS
15、O 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 enumeration of presumptive Escherichia coli Most probable number technique. ISO 11866IDF 170 consists of the following parts, under the general titl
16、e Milk and milk products Enumeration of presumptive Escherichia coli: Part 1: Most probable number technique using 4-methylumbelliferyl-D-glucuronide (MUG) Part 2: Colony-count technique at 44 C using membranes BS ISO 11866-1:2005iv Foreword IDF (the International Dairy Federation) is a worldwide fe
17、deration of the dairy sector with a National Committee 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
18、and milk products. Draft International Standards adopted 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
19、the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 11866-1IDF 170-1 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food produ
20、cts, Subcommittee SC 5, Milk and milk products. It 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 11866-1IDF 170-1 cancels and rep
21、laces the former part 2 of IDF 170A:1999, while the former part 1 has been replaced by ISO 7251:2005. BS ISO 11866-1:20051Milk and milk products Enumeration of presumptive Escherichia coli Part 1: Most probable number technique using 4-methylumbelliferyl- -D-glucuronide (MUG) 1 Scope This part of IS
22、O 11866IDF 170 specifies a combined method for the enumeration of presumptive Escherichia coli and of presumptive coliforms by means of a culture technique involving a liquid medium with MUG, and calculation of the number of presumptive Escherichia coli and/or coliforms per gram or per millilitre by
23、 the most probable number (MPN) technique after incubation at 30 C. It is a more rapid method than that described in ISO 7251 as the incubation time is reduced (omission of several enrichment steps). The method is applicable to milk, liquid milk products, dried milk, dried sweet whey, dried buttermi
24、lk, 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 specified in this part of ISO 11866IDF 170 is preferred for samples in which compar
25、atively low numbers of presumptive Escherichia coli and/or other presumptive coliforms (less than 100 per gram or 10 per millilitre) are suspected. CAUTION The applicability of this part of ISO 11866IDF 170 is limited by the susceptibility of the method to a large degree of variability. The method s
26、hould, therefore, be applied and the results interpreted in the light of the information given in Clause 12. NOTE The methods described in ISO 4831 apply for the enumeration of coliforms for reference purposes. BS ISO 11866-1:20052 2 Normative references The following referenced documents are indisp
27、ensable 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 feeding stuffs General requirements and guidance for
28、 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 3 Terms and definitions For the purposes of this document, the following terms and definitions apply.
29、3.1 presumptive Escherichia coli bacteria which at 30 C cleave 4-methylumbelliferyl-D-glucuronide (MUG), with the production of fluorescence, and which produce indole from tryptophan, under the conditions specified in this part of ISO 11866IDF 170 3.2 coliforms bacteria which at 30 C cause fermentat
30、ion of lactose with the production of gas under the conditions specified in this part of ISO 11866IDF 170 4 Principle 4.1 Three tubes of double-strength liquid selective enrichment medium are inoculated with a specified quantity of test sample if the initial product is liquid, or with a specified qu
31、antity of the initial suspension in the case of other products. 4.2 Three tubes of single-strength liquid selective enrichment medium are inoculated 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
32、products. Then, under the same conditions, the single-strength medium is inoculated with specified quantities of decimal dilutions of the test sample or of the initial suspension. 4.3 The tubes of double- and single-strength medium are incubated at 30 C for 24 h to 48 h. 4.4 Those tubes showing fluo
33、rescence and formation of indole are identified as being positive for presumptive Escherichia coli. 4.5 Those tubes showing gas formation are identified as being positive for presumptive coliforms. 4.6 The MPN index is determined from the numbers of positive tubes (4.4) of selected dilutions by mean
34、s of an MPN table (Annex A) and the most probable number (MPN) of presumptive Escherichia coli per gram or per millilitre of the original sample is calculated. 4.7 The MPN index is determined from the numbers of positive tubes (4.5) of selected dilutions by means of an MPN table (Annex A) and the mo
35、st probable number (MPN) of coliforms per gram or per millilitre of the original sample is calculated. BS ISO 11866-1:200535 Dilution fluid, culture media and reagents 5.1 General For current laboratory practice, see ISO 7218 and ISO 8261IDF 122. If the prepared culture media and reagents are not us
36、ed 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 which do not produce any change in their composition. 5.2 Dilution fluid See ISO 8261IDF 122. 5.3 Culture media 5.3.1 Modified lauryl sulfate
37、tryptose broth (selective enrichment medium) 5.3.1.1 Composition a) Double-strength medium b) Single-strength medium Tryptose Lactose Dipotassium hydrogen phosphate (K2HPO4) Potassium dihydrogen phosphate (KH2PO4) Sodium chloride Sodium lauryl sulfate CH3(CH2)11OSO3Na 4-Methylumbelliferyl-D-glucuron
38、ide (MUG) Tryptophan Water 40,0 g 10,0 g 5,5 g 5,5 g 10,0 g 0,2 g 0,2 g 2,0 g 1 000 ml 20,0 g 5,0 g 2,75 g 2,75 g 5,0 g 0,1 g 0,1 g 1,0 g 1 000 ml 5.3.1.2 Preparation Dissolve the components or the dehydrated complete medium in the water, by heating if necessary. Adjust the pH, if necessary, so that
39、 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-strength medium, and to test tubes of dimensions 20 mm 200 mm (6.2) containing inverted Durham tubes (6.3) in the c
40、ase 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. BS ISO 11866-1:20054 5.4 Indole reagent (Kovacs reagent) 5.4.1 Composition 4-Dimethylaminobenzaldehyde 5,0 g2-Methylbutan-1-ol or
41、pentan-1-ol 75,0 mlHydrochloric acid (20= 1,18 g/ml to 1,19 g/ml 25,0 ml5.4.2 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). Cool and add the hydrochloric acid. Protect from light and store at approxima
42、tely 4 C. The colour of the reagent shall be light yellow to light brown. 5.5 Sodium hydroxide solution, c(NaOH) 0,5 mol/l. 5.5.1 Composition Sodium hydroxide 2 gWater 100 ml5.5.2 Preparation Dissolve the sodium hydroxide in the water. 6 Apparatus and glassware For general requirements, see ISO 7218
43、 and ISO 8261IDF 122. Glassware shall be resistant to repeated sterilization. Usual microbiological laboratory apparatus and, in particular, the following. 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
44、200 mm, or flasks or bottles of suitable capacity. Test tubes should be checked for absence of autofluorescence before being used. 6.3 Durham tubes, of a size suitable for use in the test tubes (6.2). 6.4 Incubator, capable of maintaining a temperature of 30 C 1 C at all points within it. 6.5 Water
45、bath, capable of operating at between 50 C and 55 C. BS ISO 11866-1:200556.6 Long-wave ultraviolet (UV) lamp, of wavelength between 360 nm and 366 nm, preferably in a UV cabinet or in a dark room, or covered by a box or a carton which provides dark conditions. NOTE Short-wave UV (germicidal) lamps a
46、re unsatisfactory. 6.7 pH-meter, accurate to within 0,1 pH units at 25 C. 6.8 Total-delivery pipettes, with nominal capacities of 1 ml and 10 ml. 6.9 Vortex mixer 7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport o
47、r 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 test sample Prepare the test sample according to the method given in ISO 8261IDF 122. 9 Procedure 9.1 Test portion, initial suspension a
48、nd further dilutions Prepare the test portion, initial suspension (primary dilution) and further decimal dilutions according to the method given in ISO 8261IDF 122. Prepare a sufficient number of dilutions to ensure that all tubes for the final dilution will yield a negative result. 9.2 Inoculation
49、of selective enrichment medium 9.2.1 Take three tubes of double-strength enrichment medium 5.3.1.1 a). Using a sterile pipette (6.8), transfer to each of these tubes 10 ml of the test sample if liquid, or 10 ml of the initial suspension (primary dilution) in the case of other products. 9.2.2 Then take three tubes of single-strength enrichment medium 5.3.1.1 b). Using a fresh sterile pipette (6.8), transfer to each of these tub
