1、BSI Standards PublicationM i c r o b i o l o g y o f t h e fo o dc h a i n H o r i z o n t a l m e t h o df o r t h e e n u m e r a t i o n o fm i c r o o r g a n i s m s Part 2: Colony count at 30C by thesurface plating techniqueBS EN ISO 4833-2:2013rt 2: C lony count at 30 d grees Cby the surface
2、plating technique Incorporating corrigendum February 2014BS EN ISO 4833-2:2013National forewordThis British Standard is the UK implementation of EN ISO 4833-2:2013. It is identical to ISO 4833-2:2013, incorporating corrigendum February 2014. Together with BS EN ISO 4833-1:2013 it supersedes BS EN IS
3、O 4833:2003, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on this committee can be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
4、of a contract. Users are responsible for its correct application. The British Standards Institution 2014. Published by BSI Standards Limited 2014ISBN 978 0 580 85906 9ICS 07.100.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published unde
5、r the authority of the Standards Policy and Strategy Committee on 31 October 2013.Amendments/corrigenda issued since publicationDate Text affected31 May 2014 Implementation of ISO corrigendum February 2014: clause 5.3.1 updatedBRITISH STANDARDEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO
6、4833-2 September 2013 ICS 07.100.30 English Version Microbiology of the food chain - Horizontal method for the enumeration of microorganisms - Part 2: Colony count at 30 degrees C by the surface plating technique (ISO 4833-2:2013)Microbiologie de la chane alimentaire - Mthode horizontale pour le dno
7、mbrement des micro-organismes -Partie 2: Comptage des colonies 30 degrs C par la technique densemencement en surface (ISO 4833-2:2013)Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren fr die Zhlung von Mikroorganismen - Teil 2: Koloniezhlung bei 30 C mittels Oberflchenverfah
8、ren (ISO 4833-2:2013) This European Standard was approved by CEN on 26 July 2013. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and
9、bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the re
10、sponsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav
11、Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE N
12、ORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 4833-2:2013: EBS EN ISO 4833-2:2013EN ISO 4833-2:2013 (E) 3 Forew
13、ord This document (EN ISO 4833-2:2013) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by DIN. This European Standard shall be given the status of a national
14、standard, either by publication of an identical text or by endorsement, at the latest by March 2014, and conflicting national standards shall be withdrawn at the latest by March 2014. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights
15、. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 4833:2003, together with EN ISO 4833-1:2013. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound
16、to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
17、Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 4833-2:2013 has been approved by CEN as EN ISO 4833-2:2013 without any modification. BS EN ISO 4833-2:2013ISO 4833-2:2013(E) ISO 2013 All rights reserved iiiContents Page1 Scope
18、 . 12 Normative references 13 Terms and definitions . 24 Principle 25 Culture media and diluents . 25.1 General . 25.2 Diluents . 25.3 Agar medium: plate count agar (PCA) 26 Apparatus . 37 Sampling 48 Preparation of test sample . 49 Procedure. 49.1 Test portion, initial suspension and dilutions 49.2
19、 Inoculation and incubation 49.3 Counting of colonies . 510 Expression of results 511 Test report . 5Annex A (normative) Surface colony count using a spiral plater 6Bibliography .12BS EN ISO 4833-2:2013INTERNATIONAL STANDARD ISO 4833-2:2013(E)Microbiology of the food chain Horizontal method for the
20、enumeration of microorganisms Part 2: Colony count at 30 C by the surface plating technique1 ScopeThis part of ISO 4833 specifies a horizontal method for enumeration of microorganisms that are able to grow and form colonies on the surface of a solid medium after aerobic incubation at 30 C. The metho
21、d is applicable to:a) products intended for human consumption or for animal feed;b) environmental samples in the area of food and feed production and food handling.This part of ISO 4833 is applicable to:1) products containing heat-sensitive organisms that are likely to form a significant proportion
22、of the total flora (e.g. psychrotrophic organisms in chilled and frozen foods, dried foods, other foods that may contain heat-sensitive organisms);2) products containing obligately aerobic bacteria that are likely to form a significant proportion of the total flora (e.g. Pseudomonas spp.);3) product
23、s that contain small particles that can prove difficult to distinguish from colonies in a pour plate;4) products whose intense colour prevents the recognition of colonies in a pour plate;5) products for which distinction between different types of colony is required as part of the assessment of food
24、 quality.In addition to the manual spread plating technique, this part of ISO 4833 also specifies the use of a spiral plater, a rapid method of performing surface colony counts (Annex A).The applicability of this part of ISO 4833 to the examination of certain fermented food and animal feeds is limit
25、ed and other media or incubation conditions can be more appropriate. However, this method can be applied to such products even though it is possible that the predominant microorganisms in these products are not detected effectively.For some matrices, the method described in this part of ISO 4833 can
26、 give different results to those obtained using the method described in ISO 4833-1.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For unda
27、ted 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 decimal dilutions for microbiological examinationISO 7218, Microbiology of food an
28、d animal feeding stuffs General requirements and guidance for microbiological examinations ISO 2013 All rights reserved 1BS EN ISO 4833-2:2013ISO 4833-2:2013(E)ISO 11133, Microbiology of food, animal feed and water Preparation, production, storage and performance testing of culture media3 Terms and
29、definitionsFor the purposes of this document, the following terms and definitions apply.3.1microorganismentity of microscopic size, encompassing bacteria, fungi, protozoa and virusesSOURCE: ISO/TS 11139:2006,32.26Note 1 to entry: For the purposes of this part of ISO 4833, microorganisms are bacteria
30、, yeasts and moulds that are able to produce colonies under the conditions specified in this part of ISO 4833.4 PrincipleA specified quantity of the test sample, or a specified quantity of an initial suspension in the case of other products, is surface plated on a solid agar culture medium contained
31、 in Petri dishes.Other plates are prepared under the same conditions using decimal dilutions of the test sample or of the initial suspension.The plates are incubated under aerobic conditions at 30 C for 72 h.The number of microorganisms per gram of sample or the number of microorganisms per millilit
32、re of sample is calculated from the number of colonies obtained on the plates containing fewer than 300 colonies.5 Culture media and diluents5.1 GeneralFollow ISO 11133 for the preparation, production and performance testing of culture media.5.2 DiluentsUse the diluent(s) specified in ISO 6887 for t
33、he product concerned or the specific International Standard dealing with the product under examination.5.3 Agar medium: plate count agar (PCA)5.3.1 Composition5,0 gYeast extract 2,5 gGlucose, anhydrous (C6H12O6) 1,0 gAgara9 g to 18 gWater 1 000 mlaDepending on the gel strength of the agar.When dairy
34、 products are examined, add skimmed milk powder at 1,0 g/l of the culture medium. The skimmed milk powder shall be free from inhibitory substances.2 ISO 2013 All rights reservedBS EN ISO 4833-2:2013Enzymatic digestion of caseinISO 4833-2:2013(E)5.3.2 PreparationDissolve the components or the dehydra
35、ted complete medium in the water, by heating if necessary. Mix thoroughly and leave to stand for several minutes.Adjust the pH (6.5), if necessary, so that after sterilization it is 7,0 0,2 at 25 C.Dispense the medium into flasks or bottles (6.9) of suitable capacity. Sterilize in an autoclave (6.1)
36、 at 121 C for 15 min.If the medium is to be used immediately, cool it in a water bath (6.4) maintained at 47 C to 50 C before use. If not, allow the medium to solidify in the flask or bottle. Before use, melt the medium completely in a boiling water bath, then cool it in the water bath (6.4) maintai
37、ned at 47 C to 50 C.5.3.3 Preparation of agar platesPour 15 ml to 20 ml of the medium into sterile Petri dishes (6.6) and allow to solidify.The plates may be stored at (5 3) C for up to 4 weeks.Immediately before use, the agar plates should be dried in accordance with ISO 11133.5.3.4 Performance tes
38、ting of the culture medium5.3.4.1 GeneralPlate count agar is a non-selective medium, used in this part of ISO 4833 as a pre-poured plate for surface inoculation. Productivity shall be tested according to ISO 11133.5.3.4.2 ProductivityIncubation (30 1) C for (72 3) h Control strains Escherichia coli
39、WDCM 00013 or WDCM 00012aWorld Data Centre for Microor-ganisms (WDCM)Bacillus subtilis subsp. spizizenii WDCM 00003aStaphylococcus aureus WDCM 00032 or WDCM 00034Reference medium Tryptone soya agarControl method QuantitativeCriterion Productivity ratio (PR) 0,7aThe strains to be used by the user lab
40、oratory (minimum). See Reference 15 for information on culture collection strain numbers and contact details.6 ApparatusDisposable apparatus is an acceptable alternative to re-usable glassware and plastic if it has suitable specifications.Usual microbiological laboratory equipment (see ISO 7218) and
41、 in particular the following.6.1 Oven for dry sterilization or autoclave for wet sterilization, used in accordance with ISO 7218.6.2 Drying cabinet or incubator, ventilated by convection, for drying plates, capable of being maintained between 37 C and 55 C, or laminar flow cabinet. ISO 2013 All righ
42、ts reserved 3BS EN ISO 4833-2:2013ISO 4833-2:2013(E)6.3 Incubator, capable of being maintained at (30 1) C.6.4 Water baths, capable of being maintained at 47 C to 50 C, and another capable of maintaining water at boiling point.6.5 pH-meter, accurate to within 0,1 pH unit at 25 C.6.6 Petri dishes, ma
43、de of glass or plastic, of diameter 90 mm to 100 mm, or140 mm.6.7 Total delivery graduated pipettes, sterile, of nominal capacities 0,1 ml and 1 ml, ISO 8351class A, or automatic pipettes, ISO 8655-2,2with use of sterile tips.6.8 Colony-counting equipment (optional), consisting of an illuminated bas
44、e and, optionally, a mechanical or electronic digital counter.6.9 Bottles or flasks, of appropriate capacity, for preparation, sterilization and, if necessary, storage of culture media.6.10 Spreaders, made of glass, plastic or steel, sterile, for spreading the inoculum on the surface of the culture
45、medium.7 SamplingSampling is not part of the method specified in this part of ISO 4833. See the specific International Standard dealing with the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject.It is
46、important the laboratory receive a truly representative sample which has not been damaged or changed during transport or storage.8 Preparation of test samplePrepare the test sample in accordance with the specific International Standard appropriate to the product concerned.9 Procedure9.1 Test portion
47、, initial suspension and dilutionsFollow the specifications of ISO 6887 or the specific International Standard appropriate to the product concerned.9.2 Inoculation and incubation9.2.1 Transfer, using a sterile pipette (6.7), 0,1 ml of the test sample, if the product is liquid, or of the initial susp
48、ension in the case of other products, to the centre of each of two agar plates (5.3). If plates from more than one dilution are prepared, this may be reduced to one agar plate (see ISO 7218).If, for certain products, it is desirable to count low numbers of organisms, the limits of detection can be r
49、aised by a factor of 10 by inoculating 1,0 ml of the test sample, if liquid, or 1.0 ml of the initial suspension for other products, either on the surface of one large agar plate (140 mm) or on the surface of three small agar plates (90 mm). In both cases, prepare duplicates by using two large plates or six small ones.9.2.2 Take one other agar plate (5.3). Use another sterile pipette (6.7) to dispense 0.1 ml of the 101dilution (liquid products) or 0,1 ml of the 102dilution (other products).9.2.3 If necess
