1、 Collection of SANS standards in electronic format (PDF) 1. Copyright This standard is available to staff members of companies that have subscribed to the complete collection of SANS standards in accordance with a formal copyright agreement. This document may reside on a CENTRAL FILE SERVER or INTRA
2、NET SYSTEM only. Unless specific permission has been granted, this document MAY NOT be sent or given to staff members from other companies or organizations. Doing so would constitute a VIOLATION of SABS copyright rules. 2. Indemnity The South African Bureau of Standards accepts no liability for any
3、damage whatsoever than may result from the use of this material or the information contain therein, irrespective of the cause and quantum thereof. ICS 07.100.30 ISBN 0-626-13948-1 SANS 16654:2003 Edition 1 ISO 16654:2001 Edition 1 SOUTH AFRICAN NATIONAL STANDARD Microbiology of food and animal feedi
4、ng stuffs Horizontal method for the detection of Escherichia coli O157 This national standard is the identical implementation of ISO 16654:2001 and is adopted with the permission of the International Organization for Standardization. Published by Standards South Africa 1 dr lategan road groenkloof p
5、rivate bag x191 pretoria 0001 tel: 012 428 7911 fax: 012 344 1568 international code + 27 12 www.stansa.co.za Standards South Africa 2003 SANS 16654:2003 Edition 1 ISO 16654:2001 Edition 1 Table of changes Change No. Date Scope National foreword This South African standard was approved by National C
6、ommittee STANSA SC 5140.26, Microbiological evaluation of foods, feeds and beverages, in accordance with procedures of Standards South Africa, in compliance with annex 3 of the WTO/TBT agreement. Reference numberISO 16654:2001(E)ISO 2001INTERNATIONALSTANDARDISO16654First edition2001-05-01Microbiolog
7、y of food and animal feedingstuffs Horizontal method for thedetection of Escherichia coli O157Microbiologie des aliments Mthode horizontale pour la recherche desEscherichia coli O157ISO 16654:2001(E) ISO 2001 All rights reserved iiiContents PageForeword.ivIntroduction.v1 Scope 12 Normative reference
8、s 13 Term and definition .14 Principle25 Culture media, reagents and antisera .26 Apparatus and glassware .77 Sampling.88 Preparation of test sample89 Procedure (see annex A)89.1 Test portion and initial suspension .89.2 Enrichment .89.3 Immunomagnetic separation (IMS)89.4 Plating out onto selective
9、 agars and identification of E. coli O157 colonies99.5 Confirmation.109.6 Further characterization1110 Quality assurance1110.1 Test strains for quality assurance purposes1110.2 Culture method 1111 Expression of results 1112 Test report 11Annex A (normative) Diagram of procedure 12Bibliography13ISO 1
10、6654:2001(E)iv ISO 2001 All rights reservedForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISOmember bodies). The work of preparing International Standards is normally carried out through ISO technicalcommittees. Each member bo
11、dy interested in a subject for which a technical committee has been established hasthe right to be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotec
12、hnicalCommission (IEC) on all matters of electrotechnical standardization.International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.Publicat
13、ion as an International 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 of this International Standard may be the subject ofpatent rights. ISO shall not be held responsible for identifying any or all such
14、 patent rights.International Standard ISO 16654 was prepared by Technical Committee ISO/TC 34, Food products,Subcommittee SC 9, Microbiology.Annex A forms a normative part of this International Standard.ISO 16654:2001(E) ISO 2001 All rights reserved vIntroductionBecause of the large variety of food
15、and feed products, this horizontal method may not be appropriate in everydetail for certain products. In this case, different methods specific to these products may be used if absolutelynecessary for justified technical reasons. Nevertheless, every attempt should be made to apply this horizontalmeth
16、od as far as possible.When this International Standard is next reviewed, account will be taken of all information then available regardingthe extent to which this horizontal method has been followed and the reasons for deviations from this method in thecase of particular products.The harmonization o
17、f test methods cannot be immediate, and for certain groups of products InternationalStandards and/or national standards may already exist that do not comply with this horizontal method. It is hopedthat when such standards are reviewed they will be changed to comply with this International Standard s
18、o thateventually the only remaining departures from this horizontal method will be those necessary for well-establishedtechnical reasons.INTERNATIONAL STANDARD ISO 16654:2001(E) ISO 2001 All rights reserved 1Microbiology of food and animal feeding stuffs Horizontalmethod for the detection of Escheri
19、chia coli O157WARNING Escherichia coli O157 can cause severe life-threatening illness and has a low infective dose.Laboratory-acquired infections have been reported.In order to safeguard the health of laboratory personnel, it is essential that the whole of this method becarried out only by skilled p
20、ersonnel using good laboratory practices and preferably working in acontainment facility. Relevant national Health and Safety Regulations relating to this organism must beadhered to.Care must be taken in the disposal of all infectious materials.1 ScopeThis International Standard specifies a horizont
21、al method for the detection of Escherichia coli serogroup O157.Subject to the limitations discussed in the introduction, this International Standard is applicable to productsintended for human consumption or for animal feeding stuffs.2 Normative referencesThe following normative documents contain pr
22、ovisions which, through reference in this text, constitute provisions ofthis International Standard. For dated references, subsequent amendments to, or revisions of, any of thesepublications do not apply. However, parties to agreements based on this International Standard are encouraged toinvestigat
23、e the possibility of applying the most recent editions of the normative documents indicated below. Forundated references, the latest edition of the normative document referred to applies. Members of ISO and IECmaintain registers of currently valid International Standards.ISO 6887-1, Microbiology of
24、food and animal feeding stuffs Preparation of test samples, initial suspension anddecimal dilutions for microbiological examination Part 1: General rules for the preparation of the initialsuspension and decimal dilutions.ISO 7218, Microbiology of food and animal feeding stuffs General rules for micr
25、obiological examinations.3 Term and definitionFor the purposes of this International Standard, the following term and definition applies.3.1Escherichia coli O157E. coli O157microorganisms which form typical colonies on the surface of the plating-out medium used in this InternationalStandard, and whi
26、ch produce indole and agglutinate specifically with antiserum against the O157 antigenNOTE 1 Sorbitol-positive E. coli O157 strains are not detected on CT-SMAC (5.2) media.NOTE 2 Some indole-negative mutations have been found.ISO 16654:2001(E)2 ISO 2001 All rights reserved4PrincipleThe detection of
27、Escherichia coli O157 necessitates four successive stages (see annex A).a) Enrichment of the test portion homogenized in modified tryptone soya broth containing novobiocin (mTSB + N)with incubation at 41,5 C Gb1 1 C for 6 h and subsequently for a further 12 h to 18h.b) Separation and concentration o
28、f microorganisms by means of immunomagnetic particles coated withantibodies to E. coli O157.c) Isolation by subculture of the immunomagnetic particles with adhering bacteria onto cefixime tellurite sorbitolMacConkey agar (CT-SMAC) and the users choice of a second selective isolation agar.d) Confirma
29、tion of sorbitol-negative colonies from CT-SMAC and colonies typical of E. coli O157 on the secondisolation agar, by indole production and agglutination with E. coli O157 antiserum.NOTE Further characterization, by for example pathogenic markers, of the positive isolates can be obtained by forwardin
30、gthem to an appropriate reference laboratory.5 Culture media, reagents and antiseraFor current laboratory practices, see ISO 7218.5.1 Enrichment medium: Modified tryptone soya broth with novobiocin (mTSB + N)See reference 1.5.1.1 Modified tryptone soya broth (mTSB)5.1.1.1 CompositionEnzymatic digest
31、 of casein 17,0 gEnzymatic digest of soya 3,0 gD(+)-glucose 2,5 gBile salts No. 3 1,5 gSodium chloride 5,0 gDipotassium hydrogen phosphate (K2HPO4) 4,0 gWater 1000 ml5.1.1.2 PreparationDissolve the components or the dehydrated complete medium in the water, by heating if necessary. Adjust the pH,usin
32、g the pH-meter (6.6), if necessary, so that after sterilization it is 7,4 Gb1 0,2 at 25 C.Dispense the medium in appropriate amounts in flasks or bottles (6.7).Sterilize for 15 min in the autoclave (6.1) set at 121 C.ISO 16654:2001(E) ISO 2001 All rights reserved 35.1.2 Novobiocin solution5.1.2.1 Co
33、mpositionNovobiocin 0,45 gWater 100 ml5.1.2.2 PreparationDissolve the novobiocin in the water and sterilize by membrane filtration.Prepare on the day of use.5.1.2.3 Preparation of the complete mediumImmediately before use, add 1 ml or 4 ml of novobiocin solution (5.1.2) to either 225 ml or 900 ml of
34、 cooled mTSB(5.1.1).The final concentration of novobiocin is 20 mg per litre of mTSB.5.2 First selective isolation medium: Cefixime tellurite sorbitol MacConkey agar (CT-SMAC)See reference 2.5.2.1 Base medium5.2.1.1 CompositionEnzymatic digest of casein 17,0 gEnzymatic digest of animal tissues 3,0 g
35、Sorbitol 10,0 gBile salts No. 3 1,5 gSodium chloride 5,0 gNeutral Red 0,03 gCrystal Violet 0,001 gAgar9gto18gaWater 1000 mlaDepending on the gel strength of the agar.5.2.1.2 PreparationDissolve the basic components or the complete dehydrated base in the water by boiling. Adjust the pH (6.6), ifneces
36、sary, so that after sterilization it is 7,1 Gb1 0,2 at 25 C.Sterilize for 15 min in the autoclave (6.1) set at 121 C.5.2.2 Potassium tellurite solution5.2.2.1 CompositionPotassium tellurite for bacteriological use 0,25 gWater 100 mlISO 16654:2001(E)4 ISO 2001 All rights reserved5.2.2.2 PreparationDi
37、ssolve the potassium tellurite in the water and sterilize by membrane filtration.This solution may be stored at room temperature for up to 1 month, but discard it if a white precipitate forms.5.2.3 Cefixime solution5.2.3.1 CompositionCefixime 5,0 mgWater 100,0 ml5.2.3.2 PreparationDissolve the cefix
38、ime in the water and sterilize by membrane filtration.NOTE Cefixime may need to be dissolved in ethanol.This solution may be stored at 3 C Gb1 2 C for 1 week.5.2.4 Complete medium5.2.4.1 CompositionBase medium (5.2.1) 1 000 mlPotassium tellurite solution (5.2.2) 1,0 mlCefixime solution (5.2.3) 1,0 m
39、l5.2.4.2 PreparationEither cool the freshly sterilized base medium (5.2.1) to between 44 Gb0C and 47 Gb0C (6.5), or melt it by steaming thepreviously sterilized and solidified base medium, then cool to between 44 Gb0C and 47 Gb0C.Add 1 ml of the tellurite solution and 1 ml of the cefixime solution t
40、o 1000 ml of the base medium. Mix and pourabout 15 ml amounts into sterile Petri dishes (6.15). Allow to solidify.The final concentration of tellurite is 2,5 mg/l and cefixime 0,05 mg/l.Immediately before use, dry the agar plates, preferably with the lids removed and with the agar surfaces facingdow
41、nwards, in an oven set at a temperature between 25 C and 50 C (6.2), until the droplets have disappearedfrom the surface of the medium. Do not dry them any further. The agar plates may also be dried in a laminar-flowsafety cabinet for 30 min with half-open lids, or overnight with the lids in place.I
42、f prepared in advance, the undried plates may be stored in the dark in plastic bags or other moisture-retentivecontainers, in a refrigerator at 3 C Gb1 2 C for up to 2 weeks.5.3 Second selective isolation mediumUse any other solid selective medium, at the choice of the laboratory, complementary to C
43、T-SMAC agar andespecially appropriate for the isolation of Escherichia coli O157.Immediately before use, dry the agar plates, preferably with the lids removed and with the agar surfaces facingdownwards, in an oven set at a temperature between 25 C and 50 C (6.2), until the droplets have disappearedI
44、SO 16654:2001(E) ISO 2001 All rights reserved 5from the surface of the medium. Do not dry them any further. The agar plates may also be dried in a laminar-flowsafety cabinet for 30 min with half-open lids, or overnight with the lids in place.If prepared in advance, the undried plates may be stored i
45、n the dark in plastic bags or other moisture-retentivecontainers, in a refrigerator at 3 C Gb1 2 C for a time that causes no change to its performance.5.4 Nutrient agar5.4.1 CompositionMeat extract 3,0 gPeptone 5,0 gAgar9gto18gaWater 1 000 mlaDepending on the gel strength of the agar.5.4.2 Preparati
46、onDissolve the components or the dehydrated complete medium in the water, by heating if necessary. Adjust the pH,if necessary, so that after sterilization it is 7,0 Gb1 0,2 at 25 C.Transfer the medium into flasks or bottles (6.7) of appropriate capacity.Sterilize for 15 min in the autoclave (6.1) se
47、t at 121 C.5.4.3 Preparation of nutrient agar platesTransfer about 15 ml of the molten, cooled medium (5.4.2) at between 44 C and 47 C (6.5) to Petri dishes andallow to solidify.Immediately before use, dry the agar plates, preferably with the lids removed and with the agar surfaces facingdownwards,
48、in an oven set at a temperature between 25 C and 50 C (6.2), until the droplets have disappearedfrom the surface of the medium. Do not dry them any further. The agar plates may also be dried in a laminar-flowsafety cabinet for 30 min with half-open lids, or overnight with the lids in place.If prepar
49、ed in advance, the undried plates may be stored in the dark, in plastic bags or other moisture-retentivecontainers, in a refrigerator at 3 C Gb1 2 C for up to 2 weeks.5.5 Tryptone/tryptophan medium5.5.1 CompositionTryptone 10,0 gSodium chloride 5,0 gDL-Tryptophan 1,0 gWater 1 000 ml5.5.2 PreparationDissolve the components in the water by boiling if necessary. Adjust the pH (6.6) so that after sterilization it is7,5 Gb1 0,2 at 25 C.ISO 16654:2001(E)6 ISO 2001 All rights reservedDispense in 5 ml amounts into test t
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