1、DEUTSCHE NORM10167Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Ber
2、lin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).March 2004Detection of Escherichia coli O157 in meat and meat productsDocument comprises 6 pages.ICS 07.100.30Nachweis von Escherichia coli O157 in Fleisch und FleischerzeugnissenIn keeping with current practice in standa
3、rds published by the International Organization for Standardization (ISO), a comma has been used throughout as the decimal marker.ContentsPageForeword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Scope. . . . . . . . . . . . . . . . .
4、 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Normative references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 P
5、rinciple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Reagents, culture media and antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6、. . . . . . . . . . . 37 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Test report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Annex A Flowchart showing the detection of Escher
7、ichia coli O157. . . . . . . . 5Annex B Sequence of steps for inoculating HC agar following immunomagnetic separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Forew
8、ordThis standard has been prepared by Technical Committee Mikrobiologische Lebensmitteluntersuchung einschlielich Schnellverfahren of the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee). This standard is to be used in conjuncti
9、on with DIN EN ISO 16654, November 2001 edition.AmendmentsThis standard differs from the December 1998 edition as follows:a) Taking into account the specications of DIN EN ISO 16654, the modied tryptic soy broth for the enrichment is no longer specied.b) The standard has been editorially revised and
10、 references have been updated.Previous editionsDIN 10167: 1998-07, 1998-12.English price group 9 www.din.de www.beuth.de07.05 9637683!,bov“This standard, together with DIN EN ISO 16654, November 2001 edition, supersedes December 1998 edition.Page 2 DIN 10167:2004-031 ScopeThis standard species a met
11、hod of detecting Escherichia coli O157 (E. coli, for short) in meat and meat products using brilliant-green bile lactose broth (BRILA) as an additional enrichment medium and haemor-rhagic colitis agar (HC agar) as a second selective culture medium for subculturing. The specications given in DIN EN I
12、SO 16654 apply when using modied tryptone soya broth with novobiocin (mTSB + N) for enrichment and cexim tellurite sorbitol MacConkey agar (CT-SMAC) as the culture medium for subculturing.NOTE: This method may also be used to detect sorbitol-positive strains of Escherichia coli O157.2 Normative refe
13、rencesThis standard incorporates, by dated or undated reference, provisions from other publications. These norma-tive references are cited at the appropriate places in the text, and the titles of the publications are listed be-low. For dated references, subsequent amendments to or revisions of any o
14、f these publications apply to this standard only when incorporated in it by amendment or revision. For undated references, the latest edition of the publication referred to applies.DIN EN ISO 6887-1 Microbiology of food and animal feeding stuffs Preparation of test samples, initial sus-pension and d
15、ecimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions (ISO 6887-1:1999)DIN EN ISO 6887-2 Microbiology of food and animal feeding stuffs Preparation of test samples, initial sus-pension and decimal dilutions for micr
16、obiological examination Part 2: Specic rules for the preparation of meat and meat products (ISO 6887-2:2003)DIN EN ISO 16654 Microbiology of food and animal feeding stuffs Horizontal method for the detection of Escherichia coli O157 (ISO 16654:2001)3 ConceptEscherichia coli O157Microorganisms which
17、form typical colonies on the surface of a selected culture medium, produce indole and agglutinate with antiserum against the O157 antigen.4 PrincipleSamples are enriched in brilliant-green bile lactose broth and then separated by means of immunomagnetic particles coated with antibodies to E. coli O1
18、57. Samples are then subcultured on haemorrhagic colitis agar (see Annex B).Sorbitol-negative and MUG-negative colonies and sorbitol-positive and MUG-positive colonies are aggluti-nated with O157-antiserum. After elimination of agglutination, agglutinating strains are sent to a reference laboratory
19、for further differentiation or analysis for pathogenicity factors 1).5 Reagents, culture media and antisera5.1 GeneralTo obtain reproducible results, clearly identied culture media components and analytical grade reagents or dry culture media shall be used. The manufacturers instructions shall be st
20、rictly followed. The water used shall be either glass-distilled or demineralized and at least of equivalent purity. It shall contain no components likely to affect microbial growth.NOTE: Ready-to-use reagents or culture media are commercially available and may be used provided they are of a composit
21、ion similar to that of the culture media described here and are of equivalent efcacy.5.2 Enrichment medium5.2.1 Brilliant-green bile lactose broth (BRILA)5.2.1.1 Compositiona) Enzymatic digest of casein 10 gb) Lactose 10 gc) Ox bile, bacteriology-grade 20 gd) Brilliant green 0,013 3 ge) Water 1 000
22、ml1) Information on addresses of reference laboratories is obtainable from Normenausschuss Lebensmittel und landwirtschaftliche Produkte of DIN, Burggrafenstrasse 6, 10787 Berlin, Germany.Page 3 DIN 10167:2004-035.2.1.2 PreparationDissolve the components in the specied quantity of water and steriliz
23、e the solution at (121 1) C for 15 min-utes. Adjust the pH value so that after sterilization it is 7,2 0,1 at 25 C.5.3 Subculture medium5.3.1 Haemorrhagic colitis agar (HC agar)5.3.1.1 Compositiona) Enzymatic digest of casein 20 gb) Bile salts No. 3 1,12 gc) Sodium chloride 5 gd) Sorbitol 20 ge) 4-m
24、ethlyumbelliferyl-b-D-glucuronide 0,05 gf) Bromocresole purple 0,015 gg) Agar 12 g to 18 g *)h) Water 1 000 ml5.3.1.2 PreparationDissolve the components in the specied quantity of water and sterilize the solution at (121 1) C for 15 min-utes. Adjust the pH value so that after sterilization it is 7,2
25、 0,1 at 25 C.Cool the agar to between 45 C and 50 C, transfer 15 ml to 20 ml aliquots to Petri dishes with a diameter of 90 mm and allow to solidify.If the poured agar plates are not required immediately after preparation, they may be kept in the dark at 4 C to 8 C for a maximum of two weeks.5.3.1.3
26、 Nutrient agarSee subclause 5.4 of DIN EN ISO 16654.5.3.1.4 Tryptone/tryptophane mediumSee subclause 5.5 of DIN EN ISO 16654.5.4 Reagents and antisera5.4.1 Kovcs indole reagentSee subclause 5.6 of DIN EN ISO 16654.5.4.2 Anti-Escherichia coli O157 immunomagnetic particles 2)See subclause 5.7 of DIN E
27、N ISO 16654.5.4.3 0,01 mol/l modied phosphate buffer solution, of pH 7,2.5.4.4 Antisera 2)See subclause 5.10 of DIN EN ISO 16654.6 ApparatusIn addition to the equipment specied in subclause 6 of DIN EN ISO 16654, the following are required:6.1 UV lamp, operating at a wavelength of 366 nm.6.2 Cotton
28、buds, having a head of rmly wrapped cotton-viscose wool.6.3 Microscope slides, made of glass, measuring 75 mm 25 mm.6.4 Shaking incubator, capable of being controlled at (37 1) C, operating at a speed of 100 min1.*) Depending on the gel strength.2) Information on sources of supply is obtainable from
29、 Normenausschuss Lebensmittel und landwirtschaftliche Produkte of DIN, Burggrafenstrasse 6, 10787 Berlin, Germany.Page 4 DIN 10167:2004-037 Procedure7.1 SamplingSee clause 7 of DIN EN ISO 16654.7.2 Sample preparationThe sample shall be prepared as specied in DIN EN ISO 6887-1 and DIN EN ISO 6887-2.7
30、.3 Enrichment and isolationSee owchart in Annex A.7.3.1 EnrichmentPreheat the brilliant-green bile lactose broth to 37 C. Then add, under sterile conditions, 25 g of the pretreated sample to 225 ml of the broth in a 500 ml conical ask. Incubate the ask in the shaking incubator at 37 C for six hours,
31、 and subsequently for a further twelve to eighteen hours.NOTE: Current ndings show that an enrichment time of only six hours is needed prior to immunomagnetic separation, since growth of the accompanying microora is signicantly inhibited after six hours, and the rst analytical results are available
32、as soon as the following day. If a positive result is obtained after enrich-ment for only six hours, analysis after incubation for twelve to eighteen hours is not necessary.7.3.2 Immunomagnetic separationSee subclause 9.3 of DIN EN ISO 16654.7.3.3 Isolation of E. coli O157 on HC agarInoculate half o
33、f the surface of two predried HC agar plates with 50 l in each case of dissolved and resus-pended magnetic bead-bacterial complex using a cotton bud, as illustrated in Annex B. Then carry out the double streak procedure on each plate as described in Annex B using inoculation loops, to enable growth
34、of well-isolated colonies over the agar. Incubate the HC agar plates at 41 C for 16 h to 18 h.NOTE 1: The inoculation technique described, in combination with the 24-hour enrichment, will frequently fail to achieve sufcient separation of individual colonies, particularly on HC agar. Comparative stud
35、ies of dif-ferent inoculation techniques have conrmed that growth of individual colonies with sufcient separation can be achieved using a modied technique, as described in Annex B. In the rst step of this technique, inoculate only one third, not half, of the plate with a cotton bud. Utilize all of t
36、he remaining agar surface for the two following loop streak steps as described in Annex B.7.4 DetectionAfter the incubation, examine the agar plates for the presence of typical E. coli O157 colonies.On HC agar, typical colonies have a translucent blue appearance, corresponding to the colour of the a
37、gar medium (sorbitol-negative). They have a diameter greater than 1 mm and are not uorescent (MUG negative) when examined under UV light of long wavelength (366 nm). Sorbitol-positive O157 colonies have a yellow ap-pearance and are uorescent (MUG positive) when examined under UV light of the same wa
38、velength.7.5 ConrmationNOTE: Commercially available miniaturized biochemical kits that permit the identication of E.coli and latex agglutination kits for E. coli O157 may be used, provided appropriate tests with known positive and nega-tive strains are carried out to conrm performance. Alternatively
39、, immunochromatographical (lateral ow) assays for E. coli O157 may be used.7.5.1 Selection of coloniesTo conrm the result, take ve typical sorbitol-negative and sorbitol-positive colonies from each plate. If there are fewer than ve typical colonies on one plate, use all the colonies for conrmation.
40、Streak the selected colo-nies on the surface of predried agar plates (as in subclause 5.3.1.3), to enable growth of well-isolated colonies. Incubate the inoculated agar plates at 37 C for 18 h to 24 h. Use only pure cultures from the nutrient agar plates for the biochemical conrmation and the serolo
41、gical identication.7.5.2 Biochemical conrmation: Indole formationSee subclause 9.5.2 of DIN EN ISO 16654.NOTE: Other methods of detecting indole may be used if they have proved suitable.7.5.3 Serological identicationSee subclause 9.5.3 of DIN EN ISO 16654.Page 5 DIN 10167:2004-037.5.4 Further differ
42、entiationFor further differentiation (detection of the H antigen) and to analyze samples for pathogenicity factors (in particular verotoxins, adhesion factors, enterohaemolysin plasmid), the isolates should be sent to a reference laboratory (see Annex A).8 Test reportSee clause 12 of DIN EN ISO 1665
43、4.Annex AFlowchart showing the detection of Escherichia coli O15725 g sample material+225 ml BRILA, heated to 37 CHomogenization and incubation at 37 C for 6 h and a further 12 h to 18 hImmunomagnetic separationInoculation of the HC agar with 50 l of washed and resuspend-ed magnetic bead-bacterial c
44、omplex to obtain isolated coloniesIncubation at 41 C for 16 h to 18 hTaking of ve sorbitol-negative and ve sorbitol-positive colonies and incubation at 37 C for 18 h to 24 hConrmation of pure colonies by indole formation or by commer-cially available biochemical identication kits and by serological
45、identication with antiserum to E. coli O157Further analyses in a reference laboratoryPage 6 DIN 10167:2004-03Annex BSequence of steps for inoculating HC agar following immunomagnetic separationKey:1 Distribute the magnetic bead-bacterial complex (50 l) over half of the agar surface using a sterile c
46、otton bud2 Using an inoculation loop, make 30 to 40 streaks extending into the third quadrant3 Using an inoculation loop, make 20 to 30 streaks extending into the fourth quadrantNOTE: Following the 12 h to 18 h period of further enrichment, it is recommended that only one third of the surface of the
47、 nutrient medium be inoculated for subsequent subculturing, in order to obtain well-isolated colonies.Figure B.1: Sequence of steps for inoculating HC agar following immunomagnetic separationBibliographySzabo, R. A., Todd, E. C. D. and Jean, A., Method to isolate Escherichia coli O157:H7 from food. J. Food Pro-tect. 1986: 49, 768772.Zadik, P. M., Chapman, P. A. and Siddons, C. A., Use of tellurite for the selection of verocytotoxigenic Es-cherichia coli O157. J. Med. Microbiol. 1993: 39, 155158.
