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1、BRITISH STANDARD BS ISO 11866-3:1997 Milk and milk products Enumeration of presumptive Escherichia coli Part 3: Colony-count technique at 44 C using membranes ICS 67.100.01 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-3:1997
2、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 May 1997 BSI 02-1999 ISBN 0 580 27530 2 National foreword This British Standard reproduces verbatim
3、 ISO 11866-3:1997 and implements it as the UK national standard. Together with BS ISO 11866-1:1997 and BS ISO 11866-2:1997 it supersedes BS 4285-3.8:1988 which is withdrawn.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 responsibility
4、to: aid enquirers 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 org
5、anizations represented 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 Stand
6、ards Correspondence 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
7、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 the ISO title page, page ii, pages 1 to 6, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have
8、had amendments incorporated. This will 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 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-3:1997 BSI 02-1999i C
9、ontents Page National forewordInside front cover Forewordii 1Scope1 2Normative references1 3Definition1 4Principle1 5Dilution fluid, culture media and reagent2 6Apparatus and glassware3 7Sampling4 8Preparation of test sample4 9Procedure4 10Calculation and expression of results5 11Repeatability5 12Te
10、st report6 Annex A (informative) BibliographyInside back cover Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii blank Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Licensed Copy:
11、 sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-3:1997 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 Internat
12、ional 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. International organizations, governmental and non-governmental, in liaison with I
13、SO, also take part in the work. ISO collaborates closely with the International Electrotechnical 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 In
14、ternational Standard requires approval by at least 75 % of the member bodies casting 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
15、 AOAC INTERNATIONAL. It will also be published by these organizations. ISO 11866 consists 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-methylu
16、mbelliferyl-b-D-glucuronide (MUG); Part 3: Colony-count technique at 44 C using membranes. The method specified in ISO 11866-3 is preferred for samples in which comparatively large numbers of presumptive Escherichia coli are suspected. Annex A of this part of ISO 11866 is for information only. Descr
17、iptors: Agricultural products, food products, dairy products, milk, tests, microbiological analysis, counting, coliform bacteria, bacteria count methods. Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-3:1997 BSI 02-19991 1 Scop
18、e This part of ISO 11866 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 and rennet casein;
19、 caseinate and dried acid whey; cheese and processed cheese; butter; frozen milk products (including edible ices); custard, desserts and cream. The method specified in this part of ISO 11866 is the preferred method for samples in which comparatively large numbers of presumptive Escherichia coli (mor
20、e than 100 per gram or 10 per millilitre) are suspected. CAUTION Some Escherichia coli pathogenic species do not grow at 44 C. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this part of ISO 11866. At the time of publ
21、ication, 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 standards indicated below. Members of IEC and ISO maintain registers o
22、f currently valid International Standards. ISO 6610:1992, Milk and milk products Enumeration of colony-forming units of micro-organisms Colony-count technique at 30 C. ISO 7218:1996, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. ISO 8261:1989, Milk an
23、d milk products Preparation of test samples and dilutions for 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 form indole-positive (pink) colonies on cellulose acetate memb
24、ranes overlaid on tryptone-bile agar, under the conditions specified in this part of ISO 11866 4 Principle Enumeration of presumptive Escherichia coli requires four successive stages. 4.1 Resuscitation Inoculation of a specified quantity of the test sample or initial suspension onto cellulose acetat
25、e membranes overlaid on mineral-modified glutamate agar, then incubation at 37 C for 4 h. NOTEThis procedure enables the presumptive Escherichia coli damaged by storage under frozen, dried or chill conditions, or damaged by heat or chemical processes, to be resuscitated. It also permits the diffusio
26、n 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 Isolation Transfer of membranes from the resuscitation stage on the mineral-modified glutamate agar to tryptone-bile a
27、gar. Incubation at 44 C for 18 h to 24 h. Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-3:1997 2 BSI 02-1999 4.3 Detection Demonstration of the presence of presumptive Escherichia coli on the membrane by the production of indo
28、le by each colony. 4.4 Calculation Calculation of the number of colony-forming units (CFU) of presumptive Escherichia coli per gram or per millilitre of sample from the number of indole-positive colonies obtained on membranes at dilution levels chosen so as to give a significant result. 5 Dilution f
29、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, u
30、nder conditions which do not produce any change in their composition. 5.2 Dilution fluid See ISO 8261. 5.3 Culture media and reagent 5.3.1 Resuscitation medium: Mineral-modified glutamate agar 5.3.1.1 Composition 5.3.1.2 Preparation Dissolve the ammonium chloride in the water. Add the other componen
31、ts 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 Pour into sterile Petri dishes (6.12), 12 ml
32、to 15 ml of the medium cooled to approximately 45 C, and allow 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 drying cabinet or the oven (6.3) set at 50 C
33、for 30 min or until the droplets have disappeared from the surface of the medium. NOTEThe agar should be dry enough not to allow excess moisture to appear within 15 min of spreading the inoculum (1 ml). Sodium glutamate Lactose Sodium formate L()Cystine L()Aspartic acid L(+)Arginine Thiamine Nicotin
34、ic acid Pantothenic acid Magnesium sulfate heptahydrate (MgSO47H2O) Ammonium iron(III) citratea Calcium chloride dihydrate (CaCl22H2O) Dipotassium hydrogen phosphate (K2HPO4) 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
35、g 2,5 g 12 g to 18 gb 1 000 ml a Iron content of at least 15 % (m/m). b Depending on the gel strength of the agar. Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-3:1997 BSI 02-19993 5.3.2 Selective medium: Tryptone-bile agar 5.
36、3.2.1 Composition 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 (6.1) set at 121 C
37、 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 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
38、 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 5.3.3.2 Preparation Dissolve the 4-dimethylaminobenzaldehyde in the
39、 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 8261. Glassware shall be resistant to repeated sterilization. Usual microbiological laboratory
40、 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. 6.2 Incubators, 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
41、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 Long-wave ultraviolet (UV) lamp, of wavelength between 360 nm and 366 nm, fitted with a suitable filter to remove UV rad
42、iations 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 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 dia
43、meter. 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 plastic, of approximately 90 mm or approximately 100 mm diameter. Tryptone Bile salts (refined) Agar Water 20,0 g 1,5
44、 g 12 g to 18 ga 1 000 ml a Depending on the gel strength of the agar. 4-Dimethylaminobenzaldehyde Hydrochloric acid, c(HCl) = 1 mol/l 5,0 g 100 ml Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 26 02:11:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 11866-3:1997 4 BSI 02-1999 6.13 Spread
45、ers, 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 It is important that the laboratory receive a sample which is truly
46、representative and has not been damaged or changed during transport or storage. Sampling is not 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
47、 Procedure NOTEIf 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 further dilutions Prepare the test portion, initial suspension (primary dilution) and fur
48、ther dilutions according to the method given in ISO 8261. 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 memb
49、ranes. Gently flatten the membranes with a sterile spreader (6.13). Using a sterile pipette (6.9), add 1 ml of the test sample or the initial suspension to the centre of each membrane. Using a sterile spreader (6.13), 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 similar volumes of the further diluted test sample or initial suspension onto other membranes, as specified in 9.2.