【AS澳大利亚标准】AS 5013.24.2 Food microbiology Method 24.2 Microbiology of food and animal fee

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1、AS 5013.24.22009iAS 5013.24.22009Australian StandardFood microbiologyMethod 24.2: Microbiology of food and animal feeding stuffsHorizontal method for the detection and enumeration of Listeria monocytogenesEnumeration method(ISO 11290-2:1998, MOD)PREFACEThis Standard was prepared by the Standards Aus

2、tralia 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 microbiology, Metho

3、d 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 and animal feedi

4、ng 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 are listed in Ap

5、pendix ZZ.The objective of this revision is to establish a horizontal method for the enumeration ofListeria 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 International Standard number ap

6、pears 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 replaced by references t

7、o equivalentAustralian Standards as follows:www.standards.org.auiiiReference to International Standard Australian StandardISOAS6887Microbiology of food and animal feeding stuffsPreparation of test samples, initial suspension and decimal dilutions for microbiological examination6887-1 Part 1: General

8、 rules for thepreparation of the initial suspension and of decimal dilutions7218Microbiology of food and animal feeding stuffsGeneral rules for microbiological examination11290Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenes

9、11290-1 Part 1: Detection method5013Food microbiology5013.11.1Method 11.1: Microbiology of food animal feeding stuffPreparation of test samples initial suspension and decimal dilutions for microbiological examinationGeneral rules for the preparation of the initial suspension and decimal dilutions501

10、3.14Method 14: Microbiology of food and animal feeding stuffsGeneral rules for microbiological examinations5013.24.1Method 24.1: Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenesDetection method (ISO 11290-1:1996, MOD)The lab

11、oratory 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 Standard is downloaded from Standard SharingThe terms normative and informative have bee

12、n used 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.INTRODUCTIONBecause of the large variety of food and feed pro

13、ducts, 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 should be made to apply this horizontal me

14、thod 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 the reasons for deviations from this method in the case of particular products.The harmonization of

15、 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 to comply with this part of ISO 11290 so th

16、at eventually the only remaining departures from this horizontal method will be those necessary for well-established technical reasons.vThe Standard is downloaded from Standard SharingNOTESwww.standards.org.au Standards AustraliaWARNING In order to safeguard the health of laboratory personnel, it is

17、 strongly recommended that tests for detecting Listeria monocytogenes 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 lab

18、oratory staff are made aware of the particular risk to the developing foetus presented by infection of the mother through exposure to Listeria mono- cytogenes. National legislation may involve more specific demands.1ScopeThis part of ISO 11290 specifies a horizontal method for the enumeration of Lis

19、teria monocytogenes.NOTE The method also allows enumeration 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 consump

20、tion or animal foodstuffs.In general (see note in 9.2.1), 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.2Normative referencesThe following standards contain provisions w

21、hich, through reference to the text, constitute provisions 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 possibilit

22、y of applying the most recent editions of standards indicated 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 microbiolo

23、gical examination Part 1: General rules for the preparation 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 metho

24、d for the detection and enumeration of Listeria monocytogenes Part 1: Detection method.1) To be published. (Revision of ISO 6887:1993)27The Standard is downloaded from Standard Sharing3DefinitionsFor the purposes of this part of ISO 11290, the following definitions apply.3.1Listeria monocytogenesmic

25、roorganisms 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 112903.2enumeration of Listeria monocytogenesdetermination

26、 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 112904PrincipleWithin the limits of this part of ISO 11290, the enumeration of Listeria monocytogenes requires six su

27、ccessive 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 specified quantity of the test sam

28、ple 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.4.5 Confirmation of presumpti

29、ve 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.5Culture media and reagentsFor current laboratory practice, see ISO 7218.NOTE Because of the lar

30、ge number of culture media and reagents, it has been considered preferable, for clarity of the text, to describe them in annex B.6Apparatus and glasswareUsual microbiological equipment (see ISO 7218) and, in particular, the following.6.1 Apparatus for dry sterilization (oven) or wet sterilization (a

31、utoclave)See ISO 7218.www.standards.org.au Standards Australia6.2 Drying cabinet or incubator, capable of being maintained 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 (op

32、tional);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 platinum/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,

33、enabling measurements to be made which are accurate to 0,1 pH unit.6.8 Test tubes or flasks, of appropriate capacity, 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

34、,1 ml and 0,5 ml divisions.6.10 Petri dishes, of diameter 90 mm and 140 mm.6.11 Jars, suitable for microaerobic 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 ill

35、umination test (optional) See annex B.6.14 Microscope, preferably with phase-contrast.7SamplingSampling 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 con

36、cerned come to an agreement on this subject.It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage (see ISO 7218).The Standard is downloaded from Standard Sharing8Preparation of test samplePrepare the test sa

37、mple in accordance 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.9ProcedureWARNING Whenever a choice is given between 35 C or 37 C for th

38、e incubation temperature, this temperature shall be agreed between the parties concerned and recorded in the test report.9.1 Test portion, initial suspension and dilutionsSee ISO 6887-1 and any specific International Standard appropriate to the product concerned.Use as diluent for preparing the init

39、ial suspension 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

40、 on the 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

41、min at 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 incubation9.2.1 Transfer, by means of a sterile pipette (6.9), 0,1 ml of the initial suspension (9.1) to e

42、ach of two dishes ofPALCAM 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 by

43、 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 duplicate

44、s 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 about15 min at ambient te

45、mperature 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. IncubatePALCAM agar dishes either microaerobically in a jar (6.11) containing the gas mixture (6.12) or aerobically.9.3 Enumeration of characte

46、ristic colonies9.3.1 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

47、for a given sample, by beginning with the higher dilution.9.3.2 For dishes incubated microaerobically, after incubation leave the PALCAM agar dishes (9.3.1) to air for 1 h to allow the agar to regain its pink to purple colour.9.3.3 After 24 h the characteristic colonies of Listeria spp. grow as smal

48、l or very small greyish green or olive green colonies, sometimes with black centres, but always with black halos. After 48 h Listeria spp. appear in the form of green colonies, about 1,5 mm to 2 mm in diameter, with a central depression and surrounded by a black halo.9.3.4 Count all the colonies pre

49、sumed to be Listeria spp. (9.3.3) on each dish containing less than 150 characteristic or non-characteristic colonies.9.4 Confirmation of Listeria spp.9.4.1 Selection of colonies for confirmation9.4.1.1 After the period of incubation (9.3.1), keep the dishes containing less than 150 presumptive List

50、eria spp. colonies, at all dilutions and, if possible, at two successive dilutions.Select five of the presumptive colonies on each plate retained. If there are fewer than five presumptive colonies on a dish, select for confirmation all presumptive colonies.9.4.1.2 Streak the selected colonies onto t

51、he surface of predried plates of tryptone soya yeast extract agar(TSYEA) (B.4) in a manner which will allow well-separated colonies to develop.Place the plates in the incubator 6.3 c) set at 35 C or 37 C for 18 h to 24 h or until growth is satisfactory.Typical colonies are 1 mm to 2 mm in diameter,

52、convex, colourless and opaque with an entire edge. If the colonies are not well separated, pick a typical Listeria spp. colony onto another TSYEA plate. Carry out the following tests from colonies of a pure culture on the TSYEA.NOTE The Henry illumination test may be conducted if necessary (see anne

53、x C and note in B.4.2). The colonies then appear bluish with a granular surface.9.4.2 Catalase reactionTake a colony separated in 9.4.1.2 and suspend it in a drop of hydrogen peroxide solution (B.10) on a slide. The immediate formation of gas bubbles indicates a positive reaction (see ISO 7218).9.4.

54、3 Gram stainingPerform the Gram stain on a colony separated in 9.4.1.2 (see ISO 7218). Listeria spp. are revealed asGram-positive slim, short rods (of approximately 0,4 m to 0,5 m diameter, and 1 m to 2 m length).9.4.4 Motility test (if necessary)3)Take a colony separated in 9.4.1.2 and suspend it i

55、n a tube containing TSYEB (B.5).Incubate in the incubator 6.3 b) set at 25 C for 8 h to 24 h until a cloudy medium is observed.Deposit a drop of the above culture using a loop (6.5) onto a clean glass microscope slide. Place a coverslip on top and examine it with the microscope (6.14). Listeria spp.

56、 appear as slim, short rods with tumbling motility.Cultures grown above 25 C may fail to exhibit this motion. Always compare them to a known culture. Cocci, large rods, or rods with rapid swimming motility are not Listeria spp.3) This examination is not necessary in all cases if the analysis is carr

57、ied out by a microbiologist who regularly works on the detection of L. monocytogenes.The Standard is downloaded from Standard SharingAs an alternative test for motility, using an inoculating needle (6.5), stab the motility agar (B.8) with a typical colony on TSYEA (9.4.1.2). Incubate it for 48 h in

58、the incubator 6.3 b) set at 25 C.Examine for growth around the stab. Listeria spp. are motile, giving a typical umbrella-like growth pattern immediately below the surface of the agar. If growth is not sufficient, incubate for up to an additional 5 days and examine the culture during this time.9.5 Co

59、nfirmation of L. monocytogenes9.5.1 Haemolysis test9.5.1.1 If the morphological and physiological characteristics and catalase reaction are indicative of Listeria spp., determine the haemolytic reaction on sheep blood agar dishes (B.6).Before use, thoroughly dry the blood agar surface, then mark the

60、 agar into squares. For each culture, take a colony separated in 9.4.1.2 and stab one labelled square using a wire (6.5). Also stab positive (L. monocytogenes) and negative (L. innocua) control cultures.After incubation at 35 C or 37 C for 24 h 2 h, examine the test strains and controls. L. monocyto

61、genes show narrow, clear, light zones of -haemolysis4); L. innocua show no clear zone around the stab. L. seeligeri show a weak zone of -haemolysis. L. ivanovii usually show wide, clearly delineated zones of -haemolysis. Examine the plates by transparency to compare test cultures with controls.9.5.1

62、.2 The haemolytic reaction may also be carried out by using the CAMP test (9.5.3), or by using red cells in suspension as follows. Disperse the colony in 150 l of TSYEB (B.5); incubate at 35 C or 37 C for 2 h. Add 150 l of sheep blood red cell suspension (B.12). Incubate at 35 C or 37 C for between 15 min and 60 min, then refrigerate at +3 C 2 C