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1、 i AS 5013.32009 ISO 4831:2006 www.standards.org.au Australian Standard Food microbiology Method 3: Microbiology of food and animal feeding stuffsHorizontal method for the detection and enumeration of coliforms Most probable number technique PREFACE This Standard was prepared by the Standards Austra
2、lia Committee FT-024, Food Products and Constituted Subcommittee FT-024-01, Food Microbiology, to supersede AS 5013.3 2004, Food microbiology, Method 3: MicrobiologyGeneral guidance for the enumeration of coliformsMost probable number technique. This Standard is identical with and reproduced from IS
3、O 4831:2006, Microbiology of food and animal feeding stuffsHorizontal method for the detection and enumeration of coliformsMost probable number technique. The objective of this Standard is to provide general guidance for the detection and the enumeration of coliforms present in products intended for
4、 human consumption or feeding of animals, by means of calculation of the most probable number (MPN) after incubation at 30C or 37C in a liquid medium, this temperature forming the subject of agreement between parties concerned. Clauses 4 (Principle), 9 (Procedure) and 10 (Expression of results) of A
5、S 5013.32004 have been technically revised. The main changes are as follows: (a) The alternative procedure of incubation at 35C has been deleted. (b) Detection and enumeration of coliforms are covered (Clauses 4 and 9). (c) Description of the MPN and the CCT have been omitted (Clause 10) and referen
6、ce is given to AS 5013.14 (ISO 7218). As this Standard is reproduced from an international standard, the following applies: (i) Its number appears on the cover and title page while the international standard number appears only on the cover. (ii) In the source text this International Standard should
7、 read this Australian Standard. (iii) A full point substitutes for a comma when referring to a decimal marker. (iv) Substitute mL for ml wherever it appears. References to International Standards should be replaced by references to Australian Standards, as follows: AS 5013.32009 ii Reference to Inte
8、rnational Standard Australia Standard ISO AS 6887 6887-1 Microbiology of food and animal feeding stuffsPreparation of test samples, initial suspension and decimal dilutions for microbiological examinations Part 1: General rules for the preparation of the initial suspension and decimal dilutions 5013
9、 5013.11.1 Food microbiology Method 11.1: Microbiology of food and animal feeding stuffs Preparation of test samples initial suspension and decimal dilutions for microbiological examinationGeneral rules for the preparation of the initial suspension and decimal dilutions 6887-2 Part 2: Specific rules
10、 for the preparation of meat and meat products 5013.11.2 Method 11.2: Microbiology of food and animal feeding stuffs Preparation of test samples initial suspension and decimal dilutions for microbiological examination Specific rules for the preparation of meat and meat products 6887-3 Part 3: Specif
11、ic rules for the preparation of fish and fishery products 5013.11.3 Method 11.3: Microbiology of food and animal feeding stuffs Preparation of test samples initial suspension and decimal dilutions for microbiological examination Specific rules for the preparation of fish and fishery products 6887-4
12、Part 4: Specific rules for the preparation of products other than milk and milk products, meat and meat products and fish and fishery products 5013.11.4 Method 11.4: Microbiology of food and animal feeding stuffs Preparation of test sample initial suspension and decimal dilutions for microbiological
13、 examination Specific rules for the preparation of products other than milk and milk products, meat and meat products and fish and fishery products 7218 Microbiology of food and animal feeding stuffsGeneral requirements and guidance for microbiological examinations 5013.14 Method 14: Microbiology of
14、 food and animal feeding stuffsGeneral rules for microbiological examinations 8261 Milk and milk productsGeneral guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination 5013.17 Method 17: Milk and milk products General guidance for the
15、preparation of test samples, initial suspensions and decimal dilutions for microbiological examination Only international references that have been adopted as Australian Standards have been listed. The laboratory should have a clearly defined quality control system to ensure that the apparatus, cult
16、ure media, reagents and technique are suitable for the test. The use of positive controls is part of this system. ii ii The Standard is downloaded from Standard Sharing iii The method given in this Standard is for use for all foods for the enumeration of coliforms. The term normative has been used
17、in this Standard to define the application of the annex to which it applies. A normative annex is an integral part of a Standard. iii iii iv INTRODUCTION ISO 4831:2006(E) iv ISO 2006 All rights reserved Introduction Because of the large variety of food and feed products, this horizontal method may n
18、ot 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 should be made to apply this horizontal method as far as possible. When this
19、 International Standard 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 particular products. The harmonization of test methods cannot be imm
20、ediate 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 to comply with this International Standard so that eventually the onl
21、y remaining departures from this horizontal method will be those necessary for well-established technical reasons. The technique described in this International Standard is less precise than that described in ISO 4832 1, but allows a microbiological examination to be carried out on a larger test por
22、tion, thus permitting a lower number of coliforms per gram or per millilitre of product to be detected. Moreover, since the definition of “coliforms” adopted in the two documents is different, the microorganisms enumerated are not necessarily the same. For any particular product, the method to be ch
23、osen will be specified in the International Standard dealing with that product. For the purposes of a practicable test method, the definition of “coliforms” given in Clause 3 and used as the basis for the procedure is not necessarily identical to corresponding definitions given in other published te
24、xts. A proportion of strains of the microorganisms described in other published texts as “coliforms” (including Escherichia coli) fail to produce enough gas to be detectable by use of a Durham tube (i.e. “anaerogenic strains”). Therefore the method described in this International Standard will not d
25、etect all strains of the microorganisms referred to in other publications as “(presumptive) coliforms” (e.g. certain strains of Citrobacter, Enterobacter, Klebsiella) (see Reference 2). iv iv The Standard is downloaded from Standard Sharing ISO 2006 All rights reserved 1 Microbiology of food and an
26、imal feeding stuffs Horizontal method for the detection and enumeration of coliforms Most probable number technique 1 Scope This International Standard gives general guidelines for the detection and the enumeration of coliforms. It is applicable to products intended for human consumption and for the
27、 feeding of animals, and environmental samples in the area of food production and food handling. Enumeration is carried out by calculation of the most probable number (MPN) after incubation in a liquid medium at 30 C or 37 C. NOTE The temperature is subject to agreement between the parties concerned
28、. In the case of milk and milk products, the temperature of incubation is 30 C. This enumeration method is applicable when the number sought is expected to be in the range 1 to 100 per millilitre or per gram of test sample. A limitation on the applicability of this International Standard is imposed
29、by the susceptibility of the method to a large degree of variability. The information given in Clause 11 provides guidance on the applicability of the method and on the interpretation of the results. 2 Normative references The following referenced documents are indispensable for the application of t
30、his document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6887 (all parts), Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decim
31、al dilutions for microbiological examination ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations ISO 8261, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions fo
32、r microbiological examination ISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory ISO/TS 11133-2:2003, Microbiology of food and
33、animal feeding stuffs Guidelines on preparation and production of culture media Part 2: Practical guidelines on performance testing of culture media 1 1 www.standards.org.au Standards Australiawww.standards.org.au Standards Australia ISO 4831:2006(E) 2 ISO 2006 All rights reserved 3 Terms and defini
34、tions For the purposes of this document, the following terms and definitions apply. 3.1 coliforms bacteria which, at the specified temperature (i.e. 30 C or 37 C, as agreed) cause fermentation of lactose with the production of gas under the test conditions specified in this International Standard 3.
35、2 detection of coliforms determination of the presence or absence of these bacteria, in a particular quantity of product, when tests are carried out in accordance with the method specified in this International Standard 3.3 enumeration of coliforms most probable number of coliforms found per millili
36、tre or per gram of the test sample, when the test is carried out in accordance with the method specified in this International Standard 4 Principle 4.1 Detection of coliforms 4.1.1 A tube of selective enrichment broth is inoculated with the test portion and incubated at 30 C or 37 C (as agreed) for
37、24 h or 48 h. 4.1.2 A tube of confirmation medium is inoculated from the tube obtained in 4.1.1 when opacity and/or gas formation has been noted, and incubated at 30 C or 37 C (as agreed) for 24 h or 48 h. 4.1.3 The presence of coliforms is confirmed in the case that opacity and gas formation have b
38、een noted after examination of the tube obtained in 4.1.2. 4.2 Enumeration by the MPN technique 4.2.1 Three tubes of double-strength liquid selective enrichment medium are inoculated with a specified quantity of the test sample if the initial product is liquid, or with a specified quantity of an ini
39、tial suspension in the case of other products. 4.2.2 Three tubes of single-strength liquid selective enrichment medium are inoculated with a specified quantity of the test sample if the initial product is liquid, or with a specified quantity of an initial suspension in the case of other products. Th
40、en, under the same conditions, further tubes of single-strength medium are inoculated with decimal dilutions of the test sample or of the initial suspension. 4.2.3 The tubes containing double-strength selective enrichment medium are incubated at 30 C or 37 C (as agreed) for 24 h, and the tubes conta
41、ining single-strength medium are incubated for 24 h or 48 h, after which period these tubes are examined for gas formation or opacity preventing the detection of gas formation. 4.2.4 A series of tubes of the confirmation medium are inoculated with the cultures from the tubes of double-strength selec
42、tive enrichment medium, and with the cultures from the tubes of single-strength selective enrichment medium in which gas formation or opacity preventing the detection of gas formation has been noted. 4.2.5 The tubes from 4.2.4 are incubated at 30 C or 37 C (as agreed) for 24 h or 48 h and the tubes
43、are examined for gas formation. 4.2.6 The most probable number of coliforms per millilitre or per gram of sample (i.e. the MPN) is calculated from the number of tubes in the new series (4.2.5) showing gas formation. A table for determination of most probable numbers is used. 2 2 www.standards.org.au
44、 Standards Australiawww.standards.org.au Standards Australia The Standard is downloaded from Standard Sharing ISO 4831:2006(E) ISO 2006 All rights reserved 3 5 Culture media and diluents 5.1 General See ISO 7218, ISO/TS 11133-1 and ISO/TS 11133-2 for the preparation, production and performance test
45、ing of culture media. 5.2 Diluents See ISO 6887 (relevant part), ISO 8261 or the specific International Standard dealing with the product under examination. 5.3 Selective enrichment medium: Lauryl sulfate tryptose broth 5.3.1 Composition a) Double-strength mediumb) Single-strength medium Enzymatic d
46、igest of milk and animal proteins40 g 20 g Lactose (C12H22O11H2O) 10 g 5 g Dipotassium hydrogen phosphate (K2HPO4) 5,5 g 2,75 g Potassium dihydrogen phosphate (KH2PO4) 5,5 g 2,75 g Sodium chloride 10 g 5 g Sodium lauryl sulfate 0,2 g 0,1 g Water 1 000 ml 1 000 ml 5.3.2 Preparation Dissolve the diffe
47、rent components 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 0,2 at 25 C. Dispense the media in quantities of 10 ml into tubes of dimensions of approximately 16 mm 160 mm (6.4) containing Durham tubes (6.5
48、) in the case of single-strength medium, and into test tubes of dimensions of approximately 20 mm 200 mm (6.4) not containing Durham tubes (6.5) in the case of the double-strength medium. Sterilize in an autoclave set at 121 C for 15 min. The Durham tubes shall not contain air bubbles after steriliz
49、ation. 5.3.3 Performance testing for the quality assurance of the culture medium For the definitions of selectivity and productivity, refer to ISO/TS 11133-1. Performance testing relating to lauryl sulfate tryptose broth is given in ISO/TS 11133-2:2003, Table B.1. 3 3 www.standards.org.au Standards Australiawww.standards.org.au Standards Australia ISO 4831:2006(E) 4 ISO 2006 All rights reserved 5.4 Confirmation medium: Brilliant green l