1、BRITISH STANDARDBS ISO 4832:2006Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of coliforms Colony-count techniqueICS 07.100.30g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g
2、48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58Incorporating corrigendum June 2009BS ISO 4832:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 16 March 2006National forewordAmendments/corrigenda issued since publicationDa
3、te Comments 30 June 2009Addition to details in the National foreword BSI 2009 ISBN 978 0 580 66961 3This British Standard is the UK implementation of ISO 4832:2006. It supersedes BS 4285-3.7:1987 and BS 5763-2:1991 which are withdrawn.The UK participation in its preparation was entrusted to Technica
4、l Committee AW/9, Microbiology.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standa
5、rd cannot confer immunity from legal obligations.supersessionINTERNATIONALSTANDARDISO4832Third edition2006-02-15Reference numberISO 4832:2006(E)Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of coliforms Colony-count techniqueMicrobiologie des aliments Mthode ho
6、rizontale pour le dnombrement des coliformes Mthode par comptage des coloniesBS ISO 4832:2006ii iiiForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies(ISO member bodies). The work of preparing International Standards is normally car
7、ried out through ISOtechnical committees. Each member body interested in a subject for which a technical committee has beenestablished has the right to be represented on that committee. International organizations, governmental andnon-governmental, in liaison with ISO, also take part in the work. IS
8、O collaborates closely with the InternationalElectrotechnical Commission (IEC) on all matters of electrotechnical standardization.International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.The main task of technical committees is to prepare International
9、 Standards. Draft International Standardsadopted by the technical committees are circulated to the member bodies for voting. Publication as anInternational 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
10、of this document may be the subject of patentrights. ISO shall not be held responsible for identifying any or all such patent rights.ISO 4832 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology.This third edition of ISO 4832 cancels and replaces ISO 4832:199
11、1 and ISO 5541-1:1986. The main changesare follows: the alternative procedure of incubation at 35 C has been deleted (see 4.2); a confirmation test in brilliant green lactose bile broth has been introduced (see 5.4 and 9.4).Considering the nature of the changes to the previous edition of this Intern
12、ational Standard, it is considered thatthe validation of alternative methods based on ISO 4832:1991 is not affected by this revision.BS ISO 4832:2006ivIntroductionBecause of the large variety of food and feed products, this horizontal method may not be appropriate in everydetail for certain products
13、. In this case, different methods which are specific to these products may be used ifabsolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply thishorizontal method as far as possible.When this International Standard is next reviewed, account will be t
14、aken of all information then availableregarding the extent to which this horizontal method has been followed and the reasons for deviations from thismethod in the case of particular products.The harmonization of test methods cannot be immediate, and for certain groups of products InternationalStanda
15、rds and/or national standards may already exist that do not comply with this horizontal method. It ishoped that when such standards are reviewed, they will be changed to comply with this International Standardso that eventually the only remaining departures from this horizontal method will be those
16、necessary for well-established technical reasons.The technique described in this International Standard is more precise than that described in ISO 48311, butdoes not allow a microbiological examination to be carried out on such a large test portion. It is therefore thepreferred method when large num
17、bers of coliforms are present. Moreover, since the definition of “coliforms”adopted in the two documents is different, the microorganisms enumerated are not necessarily the same. Forany particular product, the method to be chosen will be specified in the International Standard dealing with thatprodu
18、ct.For the purposes of a practicable test method, the definition of “coliforms” given in Clause 3 and used as thebasis for the procedure is not necessarily identical to corresponding definitions given in other published texts.The method described in this International Standard will, on average, dete
19、ct only about of strains of themicroorganisms referred to in other publications as “(presumptive) coliforms” (e.g. certain strains of Citrobacter,Enterobacter, Klebsiella) (see Reference 2).90 %BS ISO 4832:20061Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of c
20、oliforms Colony-count technique1ScopeThis International Standard gives general guidelines for the enumeration of coliforms. It is applicable to products intended for human consumption and for the feeding of animals, and environmental samples in the area of food production and food handling,by means
21、of the technique of counting colonies after incubation on a solid medium at or at .NOTE The temperature is subject to agreement between the parties concerned. In the case of milk and milk products, thetemperature of incubation is .This technique is recommended when the number of colonies sought is e
22、xpected to be more than 100 permillilitre or per gram of the test sample.2 Normative referencesThe following referenced documents are indispensable for the application of this document. For datedreferences, only the edition cited applies. For undated references, the latest edition of the referenced
23、document(including any amendments) applies.ISO 6887 (all parts), Microbiology of food and animal feeding stuffs Preparation of test samples, initialsuspension and decimal dilutions for microbiological examinationISO 7218:1), Microbiology of food and animal feeding stuffs General requirements and gui
24、dance formicrobiological examinationsISO 8261, Milk and milk products General guidance for the preparation of test samples, initial suspensionsand decimal dilutions for microbiological examinationISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production
25、ofculture media Part 1: General guidelines on quality assurance for the preparation of culture media in thelaboratoryISO/TS 11133-2:2003, Microbiology of food and animal feeding stuffs Guidelines on preparation andproduction of culture media Part 2: Practical guidelines on performance testing of cul
26、ture media1) To be published.30C37C30CBS ISO 4832:200623 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1coliformsbacteria which, at the specified temperature (i.e. or , as agreed) form characteristic colonies incrystal violet neutral red bile lac
27、tose agar, and which in the confirmation test cause fermentation of lactose withthe production of gas under the test conditions specified in this International Standard4Principle4.1 Two poured plates are prepared using a solid selective culture medium, with a specified quantity of the testsample if
28、the initial product is liquid, or with a specified quantity of an initial suspension in the case of otherproducts.Other pairs of poured plates are prepared under the same conditions, using decimal dilutions of the test sampleor of the initial suspension.4.2 The plates are incubated at or (as agreed)
29、 for .4.3 The characteristic colonies are counted and, if required, a number of colonies are confirmed byfermentation of lactose.4.4 The number of coliforms per millilitre or per gram of sample is calculated from the number of characteristiccolonies obtained in the plates chosen (see ISO 7218).5 Cul
30、ture media and diluents5.1 GeneralSee ISO 7218, ISO/TS 11133-1 and ISO/TS 11133-2 for the preparation, production and performance testingof culture media.5.2 DiluentsSee ISO 6887 (relevant part), ISO 8261 or the specific International Standard dealing with the product underexamination.5.3 Solid sele
31、ctive medium: Crystal violet neutral red bile lactose (VRBL) agar5.3.1 CompositionEnzymatic digest of animal tissuesYeast extractLactose (C12H22O11H2O)Sodium chlorideBile saltsNeutral redCrystal violetAgarato WateraDepending of the gel strength of the agar.30C37C30C37C24h7g3g 10 g5g1,5 g0,03 g0,002
32、g12 g 18 g1 000 mlBS ISO 4832:200635.3.2 PreparationProceed as follows in order to conserve the selectivity power and specificity of the medium.Thoroughly mix the components or the dehydrated complete medium in the water and leave to stand for severalminutes. Adjust the pH so that, after boiling, it
33、 is at . Heat until boiling, stirring from time to time.Allow to boil for . Immediately cool the medium in the water bath (6.5) at to .To avoid overheating, do not heat the medium for too long nor reheat it. Consequently, do not sterilize it in theautoclave, and check the sterility of the medium at
34、the time of use (see 9.2.2).Use the medium within of its preparation.5.3.3 Performance testing for the quality assurance of the culture mediumFor the definitions of selectivity and productivity, refer to ISO/TS 11133-1. Performance testing relating to crystalviolet neutral red bile lactose (VRBL) ag
35、ar is given in ISO/TS 11133-2:2003, Table B.1.5.4 Confirmation medium: Brilliant green lactose bile broth5.4.1 Composition5.4.2 PreparationDissolve the components of the dehydrated complete medium in the water by heating gently if necessary in awater bath (6.5). If necessary, adjust the pH so that,
36、after sterilization, it is at .Dispense the medium, in quantities of , in test tubes (6.7) containing Durham tubes (6.8). Sterilize in anautoclave (6.1) at for . The Durham tubes shall not contain air bubbles after sterilization.6 Apparatus and glasswareUsual microbiological laboratory equipment (se
37、e ISO 7218) and, in particular, the following.6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).See ISO 7218.6.2 Incubator, capable of operating at or .6.3 Petri dishes, made of glass or plastic, of diameter to .6.4 Total-delivery pipettes, having nominal capacities of .6.5
38、 Water bath, or similar apparatus, capable of operating at to or at .6.6 Colony-counting equipment, consisting of an illuminated base and a mechanical or electronic digitalcounter.Enzymatic digest of caseinLactose (C12H22O11H2O)Dehydrated ox bileBrilliant greenWater7,4 0,2 25C2 min 44C47C4h10 g 10 g
39、20 g0,013 3 g1 000 ml7,2 0,2 25C10 ml121C 15 min30C 1C37C 1C90 mm 100 mm1ml44C47C 100CBS ISO 4832:200646.7 Test tubes, of dimensions approximately .6.8 Durham tubes, of dimensions appropriate for use with the test tubes (6.7).6.9 Bottles or flasks, for boiling and storage of culture media.6.10 pH-me
40、ter, accurate to pH unit at .6.11 Loop, of platinum-iridium or nickel-chromium, approximately in diameter, or disposable loops.7 SamplingSampling should have been carried out in accordance with the specific International Standard appropriate tothe product concerned. If there is no specific Internati
41、onal Standard, it is recommended that the partiesconcerned come to an agreement on this subject.8 Preparation of test samplePrepare the test sample in accordance with ISO 6887 (relevant part), ISO 8261 or the specific InternationalStandard appropriate to the product concerned. If there is no specifi
42、c International Standard, it is recommendedthat the parties concerned come to an agreement on this subject.9 Procedure9.1 Test portion, initial suspension and dilutionsPrepare the test portion, initial suspension (primary dilution) and further dilutions in accordance with ISO 6887(relevant part), IS
43、O 8261 or the specific International Standard appropriate to the product concerned.9.2 Inoculation and incubation9.2.1 Prepare two dishes for the liquid product and/or from each dilution chosen. Transfer, with a sterile pipette(6.4), of liquid product or the appropriate dilutions to the centre of ea
44、ch dish. Use another sterile pipette toinoculate each dilution into the dishes.9.2.2 Pour about of the VRBL medium (5.3), at to , into each Petri dish. The time elapsingbetween the end of the preparation of the initial suspension (or of the dilution if the product is liquid) andthe moment when the m
45、edium is poured into the dishes shall not exceed .Carefully mix the inoculum with the medium and allow the mixture to solidify, with the Petri dishes standing on acool horizontal surface.Also prepare a control plate with of the medium for checking its sterility.9.2.3 After complete solidification, p
46、our about of the VRBL medium (5.3), at to , onto thesurface of the inoculated medium. Allow to solidify as described above.9.2.4 Invert the prepared dishes and incubate them in the incubator (6.2) set at or (as agreed) for.16 mm 160 mm0,1 25C3mm1ml15 ml 44C47C10115 min15 ml4ml 44C47C30C37C24 h 2hBS
47、ISO 4832:200659.3 EnumerationAfter the specified period of incubation (see 9.2.4), select the Petri dishes with, if possible, 10 or more coloniesand fewer than 150 colonies. Count, using the colony-counting equipment (6.6), the purplish red colonies with adiameter of at least (sometimes surrounded b
48、y a reddish zone of precipitated bile). These areconsidered as typical colonies of coliforms and do not require further confirmation.For details of the colony-count technique, see ISO 7218.Also count and confirm atypical colonies (e.g. of smaller size), and all colonies derived from milk products th
49、atcontain sugars other than lactose, immediately after the incubation period according to 9.4. Conversion ofsugars other than lactose may result in colonies with an appearance that looks similar to the typical coliforms.NOTE The appearance of a reddish zone of precipitated bile around the colonies depends on the type of coliform and thequality of the medium.9.4 ConfirmationInoculate five colonies of each atypical type, if available
