1、BRITISH STANDARD BS ISO 16649-2:2001 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase- positive Escherichia coli Part 2: Colony-count technique at 44 C using 5-bromo-4-chloro-3-indolyl -D-glucuronide ICS 07.100.30 NO COPYING WITHOUT BSI PERMISSIO
2、N EXCEPT AS PERMITTED BY COPYRIGHT LAWBS ISO 16649-2:2001 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Policy and Strategy Committee on 23 October 2001 BSI 23 October 2001 IS
3、BN 0 580 38593 0 National foreword This British Standard reproduces verbatim ISO 16649-2:2001 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: A list of organizations repre
4、sented 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 Corresponde
5、nce Index”, or by 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 i
6、tself confer immunity from legal obligations. 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 an
7、d promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 8, an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendmen
8、ts issued since publication Amd. No. Date CommentsReference number ISO 16649-2:2001(E) INTERNATIONAL STANDARD ISO 16649-2 First edition 2001-04-15 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli Part 2: Colony-count tec
9、hnique at 44 C using 5-bromo-4-chloro-3-indolyl -D-glucuronide Microbiologie des aliments Mthode horizontale pour le dnombrement des Escherichia coli -glucuronidase positive Partie 2: Technique de comptage des colonies 44 C au moyen de 5-bromo-4-chloro-3-indolyl -D-glucuronateISO 16649-2:2001(E) ii
10、ISO 16649-2:2001(E)iii 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 member body interested in
11、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 Electrotechnical Commissi
12、on (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an I
13、nternational Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this part of ISO 16649 may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights
14、. International Standard ISO 16649-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. ISO 16649 consists of the following parts, under the general title Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of -glucuronidas
15、e-positive Escherichia coli: Part 1: Colony-count technique at 44 C using membranes and 5-bromo-4-chloro-3-indolyl -D-glucuronide Part 2: Colony-count technique at 44 C using 5-bromo-4-chloro-3-indolyl -D-glucuronide Part 3: Most probable number techniqueISO 16649-2:2001(E) iv Introduction Because o
16、f the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain products. In this case, different methods which are specific to these products may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt sho
17、uld be made to apply this horizontal method as far as possible. When this part of ISO 16649 is next reviewed, account will be taken of all information then available regarding the extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of
18、particular products. The harmonization of test methods cannot be immediate and, for certain groups of products, International Standards and/or national standards may already exist that do not comply with this horizontal method. It is hoped that when such standards are reviewed they will be changed t
19、o comply with this part of ISO 16649 so that eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons. This International Standard describes two horizontal methods (ISO 16649-1 and ISO 16649-2) for the enumeration of -glucuro
20、nidase-positive Escherichia coli. The user may choose either ISO 16649-1 or ISO 16649-2. Either part is for general application. However, ISO 16649-1 should be used for foodstuffs which may contain severely stressed cells.INTERNATIONAL STANDARD ISO 16649-2:2001(E)1 Microbiology of food and animal fe
21、eding stuffs Horizontal method for the enumeration of -glucuronidase-positive Escherichia coli Part 2: Colony-count technique at 44 C using 5-bromo-4-chloro-3-indolyl -D-glucuronide 1 Scope This part of ISO 16649 specifies a horizontal method for the enumeration of -glucuronidase-positive Escherichi
22、a coli in products intended for human consumption or for the feeding of animals. It uses a colony-count technique at 44 C on a solid medium containing a chromogenic ingredient for detection of the enzyme -glucuronidase. WARNING Strains of Escherichia coli which do not grow at 44 C and, in particular
23、, those that are -glucuronidase negative, such as Escherichia coli O157, will not be detected. 2 Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this part of ISO 16649. For dated references, subsequent amendmen
24、ts to, or revisions of, any of these publications do not apply. However, parties to agreements based on this part of ISO 16649 are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of
25、the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: G
26、eneral rules for the preparation of the initial suspension and decimal dilutions. ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. 3 Terms and definitions For the purposes of this part of ISO 16649, the following terms and definitions apply. 3.
27、1 -glucuronidase-positive Escherichia coli bacteria which at 44 C form typical blue colony on tryptone-bile-glucuronide medium (TBX) under the conditions specified in this part of ISO 16649 3.2 enumeration of -glucuronidase-positive Escherichia coli determination of the number of colony-forming unit
28、s (CFU) of -glucuronidase-positive Escherichia coli, per millilitre or per gram of sample, when test and calculations are carried out in accordance with this part of ISO 16649ISO 16649-2:2001(E) 2 4P r i n c i p l e 4.1 Duplicate plates of tryptone-bile-glucuronic medium (TBX) are inoculated with th
29、e specified quantity of the test sample or the initial suspension. Under the same conditions, using decimal dilutions of the test sample or of the initial suspension, two plates per dilution are inoculated. The dishes are incubated for 18 h to 24 h at 44 C 1 C then examined to detect the presence of
30、 colonies which, from their characteristics, are considered to be -glucuronidase-positive Escherichia coli. 4.2 The number of colony-forming units (CFU) of -glucuronidase-positive Escherichia coli per gram or per millilitre of sample is calculated (see clause 10). 5 Diluent and culture media For cur
31、rent laboratory practice, see ISO 7218. 5.1 Diluent See ISO 6887-1 or the specific International Standard dealing with the product to be examined. 5.2 Culture medium: Tryptone-bile-glucuronic medium (TBX) 5.2.1 Composition Enzymatic digest of casein 20,0 g Bile salts No. 3 1,5 g 5-Bromo-4-chloro-3-i
32、ndolyl -D-glucuronic acid (BCIG) 144 mol a Dimethyl sulfoxide (DMSO) b 3ml Agar 9gto1 8g c Water 1 000 ml a e.g. 0,075 g of cyclohexylammonium salt. b Dimethyl sulfoxide is harmful by inhalation and contact. The use of a fume cupboard when handling is advised. Because of this toxicity, a diluent rec
33、ommended by the manufacturer may be used. c Depending on the gel strength of the agar. 5.2.2 Preparation Dissolve the BCIG in the dimethyl sulfoxide or in the diluent recommended by the manufacturer. Dissolve all components in the water and heat to boiling. Adjust the pH, if necessary, so that after
34、 sterilization, it is 7,2 0,2 at 25 C. Sterilize the medium in the autoclave set at 121 C for 15 min. Immediately cool the medium in the water bath (6.3) at 44 Cto4 7C.ISO 16649-2:2001(E)3 6 Apparatus and glassware Usual microbiological equipment (see ISO 7218) and, in particular, the following. 6.1
35、 Apparatus for dry sterilization (oven) or wet sterilization (autoclave). 6.2 Incubators, capable of operating at 44 C 1 C. 6.3 Water-bath, capable of being maintained at 44Cto47C. 6.4 Test tubes, flasks or bottles of suitable capacity. 6.5 Pipettes or micropipettes, total delivery (blow out), havin
36、g wide openings and having a nominal capacity of 1 ml and 10 ml, graduated respectively in 0,1 ml and 0,5 ml divisions. 6.6 Petri dishes, of approximately 90 mm diameter. 6.7 pH-meter, capable of measuring to an accuracy of 0,1 pH unit. Its minimum measuring threshold shall be 0,01 pH unit. The pH-m
37、eter shall be equipped with either manual or automatic temperature equalization. 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 not part of the method specified in this part of
38、 ISO 16649. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject. 8 Preparation of test sample Prepare the test sample in accordance with the specific International Standard appropriate to the product concerned. If there is
39、 no specific International Standard available, 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 ISO 6887-1 and any specific International Standard appropriate to the product. 9.2 Inoculation and incu
40、bation 9.2.1 Using a sterile pipette or a micropipette (6.5), transfer to a sterile Petri dish (6.6) 1 ml of the test sample (if liquid), or 1 ml of the initial dilution (10 1 ) in the case of other products. Inoculate two plates per dilution. Repeat the procedure with the further decimal dilutions,
41、 if necessary, using a new sterile pipette for each dilution.ISO 16649-2:2001(E) 4 9.2.2 Pour into each Petri dish approximately 15 ml of the TBX medium (5.2), previously cooled at 44Ct o4 7 C in the water bath (6.3). Carefully mix the inoculum with the medium and allow the mixture to solidify, with
42、 the Petri dishes standing on a cool horizontal surface. The time which elapses between the distribution of the inoculum in a dish and pouring of the medium shall not exceed 15 min. 9.2.3 Invert the inoculated dishes (9.2.2) so that the bottom is uppermost and place them in an incubator (6.2) se ta
43、t44C for 18 h to 24 h. The total incubation time shall not be longer than 24 h. WARNING If the presence of stressed cells is suspected, incubate for an initial period of 4 h at 37 C, and then raise the incubation temperature to 44 C for 18 h to 24 h. The incubation temperature shall not exceed 45 C.
44、 9.3 Counting the colony-forming units After the specified period of incubation (9.2.3) count the typical CFU of -glucuronidase-positive Escherichia coli in each dish containing less than 150 typical CFU and less than 300 total (typical and non-typical) CFU. If they form part of the retained dishes,
45、 the dishes containing 0 typical CFU should be taken into consideration in the different calculation methods defined in clause 10. 10 Expression of results 10.1 General The calculation in 10.2 takes into account those cases most frequently encountered when conducting tests in accordance with good la
46、boratory practice. Some special, fairly improbable, cases can arise (e.g. very different CFU numbers between the two dishes from the same dilution, or very different proportions from that of the dilution factor between the dishes from two successive dilutions). It is then necessary that the counting
47、 results be examined, interpreted and possibly rejected by a competent microbiologist. 10.2 Calculation For a result to be valid, in general it is considered that it is necessary to count the CFU on at least one dish containing as a minimum 15 blue CFU. Calculate N, the number of CFU of -glucuronida
48、se-positive Escherichia coli present in the test sample per millilitre or per gram, as the weighted mean from two successive dilutions using the following equation: 12 (0 , 1) a N Vn n d (1) where a is the sum of the CFU counted on all the dishes retained from two successive dilutions, at least one
49、of which contains a minimum 15 blue CFU; n 1 is the number of dishes retained at the first dilution; V is the volume of inoculum, in millilitres, applied to each dish;ISO 16649-2:2001(E)5 n 2 is the number of dishes retained at the second dilution; d is the dilution factor corresponding to the first dilution retained d = 1 in the case (liquid products) where the directly inoculated test sample is retained. Round off the results to t
