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1、 i AS 5013.24.22009 www.standards.org.au Australian Standard Food microbiology Method 24.2: Microbiology of food and animal feeding stuffsHorizontal method for the detection and enumeration of Listeria monocytogenesEnumeration method (ISO 11290-2:1998, MOD) PREFACE This Standard was prepared by the
2、Standards Australia Committee FT-024, Food Products, and Constituted Subcommittee FT-024-01, Food Microbiology, to supersede in part AS/NZS 1766.2.151998, Food microbiology, Method 2.15: Examination of specific organismsListeria monocytogenes in dairy products and AS/NZS 1766.2.16.11998, Food microb
3、iology, Method 2.16.1: Examination for specific organismsFood and animal feeding stuffsHorizontal method for the detection and enumeration of Listeria monocytogenesDetection method. This Standard is an adoption with national modifications and reproduced from ISO 11290- 2:1998, Microbiology of food a
4、nd animal feeding stuffsHorizontal method for the detection and enumeration of Listeria monocytogenes, Part 2: Enumeration method, including Amendment 1: 2004 which is added at the end of the ISO text. ISO 11290-2:1998 was confirmed on 20 June 2005. Additional requirements for Australian conditions
5、are listed in Appendix ZZ. The objective of this revision is to establish a horizontal method for the enumeration of Listeria monocytogenes. As this Standard is reproduced from an International Standard, the following applies: (a) Its number appears on the cover and title page while the Internationa
6、l Standard number appears only on the cover. (b) In the source text this part of ISO 11290 should read this Australian Standard. (c) A full point substitutes for a comma when referring to a decimal marker. (d) Substitute mL for ml whenever it appears. References to International Standards should be
7、replaced by references to equivalent Australian Standards as follows: AS 5013.24.22009 ii Reference to International Standard Australian Standard ISO AS 6887 Microbiology of food and animal feeding stuffsPreparation of test samples, initial suspension and decimal dilutions for microbiological examin
8、ation 5013 Food microbiology 6887-1 Part 1: General rules for the preparation of the initial suspension and of decimal dilutions 5013.11.1 Method 11.1: Microbiology of food animal feeding stuffPreparation of test samples initial suspension and decimal dilutions for microbiological examinationGeneral
9、 rules for the preparation of the initial suspension and decimal dilutions 7218 Microbiology of food and animal feeding stuffsGeneral rules for microbiological examination 5013.14 Method 14: Microbiology of food and animal feeding stuffsGeneral rules for microbiological examinations 11290 Microbiolo
10、gy of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenes 5013.24.1 Method 24.1: Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenesDetection method (ISO 11290-1:1996, MO
11、D) 11290-1 Part 1: Detection method The laboratory should have a clearly defined quality control system to ensure that the apparatus, culture media, reagents and technique are suitable for the test. The use of positive controls is part of this system. The terms normative and informative have been us
12、ed in this Standard to define the application of the annex or appendix to which they apply. A normative annex or appendix is an integral part of a Standard, whereas an informative annex or appendix is only for information and guidance. ii ii The Standard is downloaded from Standard Sharing iii INTR
13、ODUCTION ISO 11290-2:1998(E) ISO iv Introduction Because of 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 j
14、ustified technical reasons. Nevertheless, every attempt should be made to apply this horizontal method as far as possible. When this part of ISO 11290 is next reviewed, account will be taken of all information then available regarding the extent to which this horizontal method has been followed and
15、the reasons for deviations from this method in the case of 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 th
16、at when such standards are reviewed they will be changed to comply with this part of ISO 11290 so that eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons. iii iii iv NOTES iv iv The Standard is downloaded from Standard
17、 Sharing 1 1 Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenes Part 2: Enumeration method WARNING In order to safeguard the health of laboratory personnel, it is strongly recommended that tests for detecting Listeria monocyto
18、genes are undertaken in properly equipped laboratories, under the control of a skilled microbiologist, and that great care is taken in the disposal of all contaminated materials. In particular, it is strongly recommended that female laboratory staff are made aware of the particular risk to the devel
19、oping foetus presented by infection of the mother through exposure to Listeria mono- cytogenes. National legislation may involve more specific demands. 1 Scope This part of ISO 11290 specifies a horizontal method for the enumeration of Listeria monocytogenes. NOTE The method also allows enumeration
20、of other Listeria species which may be used as indicators of the hygienic quality of food or feed products. Subject to the limitations discussed in the introduction, this part of ISO 11290 is applicable to products intended for human consumption or animal foodstuffs. In general (see note in 9.2.1),
21、the lower limit of enumeration of this method is 10 L. monocytogenes per millilitre of sample for liquid products, or 100 L. monocytogenes per gram of sample for other products. 2 Normative references The following standards contain provisions which, through reference to the text, constitute provisi
22、ons of this International Standard. At the time of publication, the editions indicated were valid. All Standards are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of standards ind
23、icated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 6887-1:1), Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the prepar
24、ation of the initial suspension and of decimal dilutions. ISO 7218:1996, Microbiology of food and animal feeding stuffs General rules for microbiological examination. ISO 11290-1:1996, Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria mono
25、cytogenes Part 1: Detection method. 1) To be published. (Revision of ISO 6887:1993) 1 1 www.standards.org.au Standards Australiawww.standards.org.au Standards Australia ISO 11290-2:1998(E) ISO 2 3 Definitions For the purposes of this part of ISO 11290, the following definitions apply. 3.1 Listeria m
26、onocytogenes microorganisms which form typical colonies on the solid selective medium described and which display the morphological, physiological and biochemical characteristics described when the analysis is carried out in accordance with this part of ISO 11290 3.2 enumeration of Listeria monocyto
27、genes determination of the number of colony-forming units (CFU) of Listeria monocytogenes (see 3.1), in a given quantity of product, when the analysis is carried out in accordance with this part of ISO 11290 4 Principle Within the limits of this part of ISO 11290, the enumeration of Listeria monocyt
28、ogenes requires six successive steps (see annex A for a flowchart). 4.1 Preparation of the initial suspension in one of the two diluents described, as necessary. 4.2 Resuscitation for 1 h at 20 C. 4.3 Surface plating, on the solid selective culture medium contained in two Petri dishes, of a specifie
29、d quantity of the test sample for liquid products or the initial suspension for other products. Preparation of other dishes, under the same conditions, using decimal dilutions of the test sample or initial suspension. 4.4 Incubation of the dishes at 35 C or 37 C and examination after 24 h and 48 h.
30、4.5 Confirmation of presumptive colonies of Listeria monocytogenes with the tests described. 4.6 From the number of confirmed colonies, calculation of the number of Listeria monocytogenes per gram or per millilitre of the test sample. 5 Culture media and reagents For current laboratory practice, see
31、 ISO 7218. NOTE Because of the large number of culture media and reagents, it has been considered preferable, for clarity of the text, to describe them in annex B. 6 Apparatus and glassware Usual microbiological equipment (see ISO 7218) and, in particular, the following. 6.1 Apparatus for dry steril
32、ization (oven) or wet sterilization (autoclave) See ISO 7218. .auCopyright ISO.au 2 2 www.standards.org.au Standards Australiawww.standards.org.au Standards Australia The Standard is downloaded from Standard Sharing ISO ISO 11290-2:1998(E) 3 6.2 Drying cabinet or incubator, capable of being maintai
33、ned between 25 C 1 C and 50 C 1 C. 6.3 Incubators, for maintaining the inoculated media, plates and tubes within the following temperature ranges: a)20 C 1 C (optional); b)25 C 1 C (optional); c)35 C 1 C or 37 C 1 C. 6.4 Water bath, capable of being maintained at 47 C 2 C. 6.5 Loops and wires of pla
34、tinum/iridium or nickel/chromium, or Pasteur pipettes or single-use loops. 6.6 Glass or plastic spreaders, sterile. 6.7 pH-meter, capable of being read to the nearest 0,01 pH unit at 25 C, enabling measurements to be made which are accurate to 0,1 pH unit. 6.8 Test tubes or flasks, of appropriate ca
35、pacity, for sterilization and storage of culture media and incubation of liquid media. 6.9 Total-delivery graduated pipettes, of nominal capacities 1 ml and 10 ml, graduated respectively in 0,1 ml and 0,5 ml divisions. 6.10 Petri dishes, of diameter 90 mm and 140 mm. 6.11 Jars, suitable for microaer
36、obic incubation (optional). 6.12 Gas mixture (optional), of specified composition for microaerobic incubation: 5 % to 12 % CO2, 5 % to 15 % O2, and N2 up to 100 %. 6.13 Equipment for the Henry illumination test (optional) See annex B. 6.14 Microscope, preferably with phase-contrast. 7 Sampling Sampl
37、ing is not part of the method specified in this part of ISO 11290. If there is no specific International Standard dealing with sampling of the product concerned, it is recommended that the parties concerned come to an agreement on this subject. It is important that the laboratory receive a sample wh
38、ich is truly representative and has not been damaged or changed during transport or storage (see ISO 7218). .auCopyright ISO.au 3 3 www.standards.org.au Standards Australiawww.standards.org.au Standards Australia ISO 11290-2:1998(E) ISO 4 8 Preparation of test sample Prepare the test sample in accor
39、dance with the specific International Standard appropriate to the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject. 9 Procedure WARNING Whenever a choice is given between 35 C or 37 C for the incubati
40、on temperature, this temperature shall be agreed between the parties concerned and recorded in the test report. 9.1 Test portion, initial suspension and dilutions See ISO 6887-1 and any specific International Standard appropriate to the product concerned. Use as diluent for preparing the initial sus
41、pension either buffered peptone water (B.1), or half-Fraser broth base medium (B.2). Half-Fraser broth base without the addition of selective agents may be used as a diluent for the food or feed sample when both the detection method (see ISO 11290-1) and this enumeration method are carried out on th
42、e same test sample. This procedure is to avoid the need to prepare two initial suspensions; the selective agents are added to the suspension once the test portion for enumeration has been used. Use of this procedure should be noted in the test report. Let the initial suspension stand for 1 h 5 min a
43、t 20 C 2 C by using, if necessary, the incubator 6.3 a), in order to resuscitate the stressed microorganisms. If a dilution range is used, prepare it after resuscitation. 9.2 Inoculation and incubation 9.2.1 Transfer, by means of a sterile pipette (6.9), 0,1 ml of the initial suspension (9.1) to eac
44、h of two dishes of PALCAM agar (B.3), dried beforehand if necessary in the incubator (6.2). Repeat the procedure using further decimal dilutions if necessary. NOTE When, for certain products, it is necessary to estimate low numbers of Listeria monocytogenes, the limit of enumeration can be lowered b
45、y a factor of 10 by examining 1,0 ml of the initial suspension. Distribute the 1 ml of inoculum either on the surface of the agar medium in a large Petri dish (140 mm) or over the surface of the agar medium in three small dishes (90 mm) using a sterile spreader (6.6). In both cases, prepare duplicat
46、es by using two large dishes or six small dishes. 9.2.2 Carefully spread the inoculum as quickly as possible over the surface of the agar plate without touching the sides of the dish with the spreader. Use a fresh sterile spreader for each plate.2) Leave the plates closed for about 15 min at ambient
47、 temperature for the inoculum to be absorbed into the agar. 9.2.3 Invert the dishes prepared in 9.2.2 and place them in an incubator 6.3 c) set at 35 C or 37 C. Incubate PALCAM agar dishes either microaerobically in a jar (6.11) containing the gas mixture (6.12) or aerobically. 9.3 Enumeration of ch
48、aracteristic colonies 9.3.1 After incubation for 24 h, and for an additional 18 h to 24 h if growth is slight or if no colonies are observed after 24 h of incubation, examine the dishes (9.2.3) for the presence of colonies presumed to be Listeria spp. (see 9.3.3). 2) It is possible to use the same spreader for a given sample, by beginning with the higher dilution. .auCopyright ISO.au 4 4 www.standards.org.au Standards Australiawww.standards.org.au Standards Australia The Standard is downloaded from Standard Sharing ISO ISO 11290-2:1998(E) 5 9.3.2 For dish