1、BRITISH STANDARD Foodstuffs - Detection of irradiated food using Direct Epifluorescent Filter Technique/Aerobic Plate Count Screening method (DEFT/APC) - The European Standard EN 13783:2001 has the status of a British Standard ICs 67.050 BS EN 13783:2002 present to the responsible European committee
2、 any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. - A list of organizations represented on this committee can be obtained on request to its secretary. Cross-referen
3、ces The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalog
4、ue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. This British Standard. havinp. - been
5、 prepared under the direction of the Consumer Products and Services Sector Summary of pages This document comprises a front cover, an inside front cover, the EN title page, Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 24 January 2
6、002 pages 2 to 15 and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. O BSI 24 January 2002 ISBN O 580 38936 7 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 13783 November 2001 ICs 07.100.30; 67.050 English version Foodstuffs - Dete
7、ction of irradiated food using Direct Epifluorescent Filter Technique/Aerobic Plate Count (DEFT/APC) - Screening method Produits alimentaires - Dtection daliments ioniss en utilisant la technique dpifluorescence aprs filtration et dnombrement de la flore arobie sur milieu glos (DEFT/APC) - Mthode pa
8、r criblage Lebensmittel - Nachweis der Bestrahlung von Lebensmitteln mit Epifluoreszenz-Filtertechnik/aerober mesophiler Keimzahl (DEFT/APC) - Screeningverfahren This European Standard was approved by CEN on 29 September 2001 CEN members are bound to comply with the CENKENELEC Internal Regulations w
9、hich stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Management Centre or to any CEN member. This European Sta
10、ndard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official versions. CEN members are the national stand
11、ards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION EUROPISCHES KOMITEE FR NORMUNG COMIT EUROPEN DE NORMAL
12、ISATION Management Centre: rue de Stassart, 36 B-1050 Brussels O 2001 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 13783:2001 E EN 13783:2001 (E) Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagen
13、ts . 3 5 Apparatus . 6 6 Sampling technique . 7 7 Procedure . 8 8 Eva1 uat io n .9 9 Limitations 10 1 O Validation 1 O 11 Test report II Annex A (informative) Further information on the applicability . 12 Annex B (informative) Practical example for calculation of the microscope factor . 13 Annex C (
14、informative) Flow diagram of the procedure 14 Bibliography 15 Foreword This European Standard has been prepared by 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 standard, e
15、ither by publication of an identical text or by endorsement, at the latest by May 2002, and conflicting national standards shall be withdrawn at the latest by May 2002. This European Standard was elaborated on the basis of a protocol developed following a concerted action supported by the Commission
16、 of European Union (XII C.) (BCR), and a screening investigation carried out by the Danish National Food Agency and the local Environment and food Agency, MLK FYN, DK Odense. Experts and laboratories from E.U. and EFTA countries, contributed jointly to the development of the concerted action protoco
17、l. WARNING: The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and de
18、termine the applicability of regulatory limitations prior to use. The annexes A, B and C are informative. This standard includes a Bibliography. According to the CENKENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Stan
19、dard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. 2 EN 13783:2001 (E) 1 Scope This European Standard specifies a microbiological screening method fo
20、r the detection of irradiation treatment of herbs and spices, using the combined direct epifluorescent filter technique (DEFT) and aerobic plate count (APC). The DEFT/APC technique is not radiation specific, therefore, it is recommended to confirm positive results using a standardised method (e.g. E
21、N 1788, prEN 13751) to specifically prove an irradiation treatment of the suspected food. The method has been successfully tested in interlaboratory tests with herbs and spices I to 5. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other pub
22、lications. These normative references are cited at the appropriate places in the text, and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revi
23、sion. For undated references the latest edition of the publication referred to applies (including amendments). IS0 4833, Microbiology - General guidance for the enumeration of microorganisms - Colony count technique at 30 OC. IS0 721 8, Microbiology of food and animal feeding stuffs - General rules
24、for microbiological examinations. 3 Principle The method is based on the comparison of the APC with the count obtained using DEFT. The APC gives the number of viable microorganisms in the sample after a possible irradiation and the DEFT count indicates the total number of microorganisms, including n
25、on-viable cells, present in the sample. The difference between the DEFT count and the APC count in spices treated with doses of 5 kGy to 10 kGy is generally about or above 3 to 4 log units. Similar differences between DEFT and APC counts can be induced by other treatments of the foods leading to dea
26、th of microorganisms, e. g. heat, thus positive results shall be confirmed. A known volume of sample is filtered through a membrane filter at reduced pressure in order to concentrate the microorganisms on the filter. The microorganisms are stained with a fluorochrome, acridine orange (AO), resulting
27、 in an orange and orange-yellow fluorescence under illumination with blue light at 450 nm to 490 nm. These microorganisms are counted using an epifluorescence microscope to give the DEFT count. However, microorganisms which were non-viable before irradiation show green fluorescence and are not count
28、ed. In parallel, the APC is determined from a second portion of the same test sample 6 to IO. 4 Reagents 4.1 General During the analysis use only reagents of recognized analytical grade. All the reagents used in the DEFT and APC determinations should be sterilized by membrane filtration through 0,2
29、pm pore size membrane filters or by autoclaving. 4.2 Peptone saline diluent 4.2.1 Composition Sodium chloride 83 g Peptone 1,o g Distilled or demineralized water 1000 ml 3 EN 13783:2001 (E) 4.2.2 Preparation Dissolve the components in the water. Adjust the pH, if necessary, so that after sterilizati
30、on the final pH is 7,2 f 0,2 at 20 “C to 25 “C. Sterilize in the autoclave (5.13) at 121 “C f 1C for 15 min. The diluent may be stored in a glass bottle at 4 “C to 6C for not more than two weeks. 4.3 Buffer, pH 3,O 4.3.1 Citric acid solution, substance concentration c(CgHgO7 . H20) = 0,l mol/l 4.3.1
31、 .I Composition Citric acid monohydrate 21 g Distilled or demineralized water 1000 ml 4.3.1.2 Preparation Dissolve the citric acid monohydrate in the water. The solution may be stored in a sterilized glass bottle at 4 “C to 6 “C for not more than three months. 4.3.2 Sodium hydroxide solution, c(Na0H
32、) = 0,l mol/l 4.3.2.1 Composition Sodium hydroxide solution, 1 mol/l 100 ml 4.3.2.2 Preparation Dissolve the sodium hydroxide or dilute the sodium hydroxide solution in the water. The solution may be stored in a glass bottle at 4 “C to 6 “C for not more than three months. 4.3.3 Complete buffer, pH 3
33、,O 4.3.3.1 Composition Citric acid solution (4.3.1) Sodium hydroxide solution (4.3.2) 100 ml 54 ml 4.3.3.2 Preparation Mix the citric acid solution and the sodium hydroxide solution. Adjust pH to 3,O f 0,2 with citric acid solution or sodium hydroxide solution. Sterilize the buffer through a membran
34、e filter of pore size 0,2 pm (5.3) before use. The solution may be stored in a glass bottle at 4 “C to 6 “C for not more than three weeks. 4 EN 13783:2001 (E) 4.4 Acridine orange solution 4.4.1 Buffer solution, pH 6,6 4.4.1 .I Composition Citric acid solution (4.3.1) Sodium hydroxide solution (4.3.2
35、) 353 ml 100 ml 4.4.1.2 Preparation Mix the citric acid solution and the sodium hydroxide solution. Adjust pH to 6,6 f 0,2 with citric acid solution or sodium hydroxide solution. Sterilize the buffer through a membrane filter of pore size 0,2 pm (5.3) before use. The buffer solution may be stored in
36、 a glass bottle at 4 “C to 6 “C for not more than three weeks. 4.4.2 Complete acridine orange solution 4.4.2.1 Composition Acridine orange 0,025 g Buffer, pH 6,6 (4.4.1) 100 ml 4.4.2.2 Preparation Dissolve the acridine orange in the buffer solution (4.4.1). Sterilize the acridine orange solution thr
37、ough a 0,2 pm pore size membrane filter (5.3) before use. The solution may be stored in a brown glass bottle at 4 “C to 6 “C for not more than one week. NOTE 1 Concentrated acridine orange solution is commercially available and is recommended for safety reasons. NOTE 2 As acridine orange is regarded
38、 as a mutagenic substance, disposable gloves and face masks should be used when weighing the stain. 4.5 2-Propanol 4.6 Triton X-IOOI), 1 % cleaning solution 4.6.1 Composition Triton X - 1 O0 Distilled or demineralized water 10 ml 1000 ml 4.6.2 Preparation Mix Triton X-100 with warm (80C) water. Ster
39、ilize the solution through a 0,2 pm pore size membrane filter (5.3). The solution may be stored in a glass bottle at 4 “C to 6 “C for not more than three weeks. Triton X-100 is an example of a suitable product available commercially. This information is only given for the convenience of users of thi
40、s International Standard and does not constitute an endorsement by CEN of this product. 5 EN 13783:2001 (E) 4.7 Tryptone-Yeast Extract-Glucose-Agar (Plate count agar) 4.7.1 Composition Tryptone 5,O g Yeast Extract 23 g Dextrose (Glucose) 1,o g Agar (according to the gel strength of the agar used) Di
41、stilled or demineralized water 12 g to 18 g 1000 ml 4.7.2 Preparation Dissolve the components or dehydrated complete medium in the water while heating until boiling. Adjust the pH, if necessary, so that after sterilization the final pH is 7,2 f 0,2 at 20 “C to 25 “C. Sterilize in the autoclave (5.13
42、) at 121 “C f 1 “C for 15 min. 5 Apparatus Usual laboratory apparatus in accordance with IS0 7218 and, in particular, the following: 5.1 Apparatus for membrane filtration of sample suspensions Filtration equipment made of stainless steel or glass should be used. The bottom filter should be of sinter
43、ed glass or stainless steel intended for filters with a diameter of 25 mm (5.5). The filter tower volume should be at least 10 ml. The filtration equipment is placed vertically on a suction flask or a manifold connected to a water pump or vacuum pump with a pressure regulator. The vacuum during filt
44、ration should usually be approximately 70 kPa. NOTE The use of a water pump is not very suitable if the water pressure cannot be regulated. 5.2 Filter funnel and suitable suction flasks made of glass for sterile filtration of reagents and diluent. 5.3 Membrane filters, cellulose ester or similar, po
45、re size 0,2 pm, e.g. diameter 30 mm and/or 47 mm, for sterile filtration of reagents. 5.4 Membrane filters, polypropylene, diameter 25 mm, pore size 10 pm, for prefiltration of samples. 5.5 Membrane filters, white polycarbonate, diameter 25 mm, pore size 0,6 pm for membrane filtration of sample test
46、 solution. 5.6 Sterile fast filter paper, for filtration of spice samples. 5.7 Epifluorescence microscope, with suitable light and filter combination (450 nm to 490 nm ) 5.8 Optics, 100 x immersion objective, ocular with magnification of 10 x. (Using a tube magnification of 1,25 a total magnificatio
47、n of 1250 x is achieved.) 5.9 Microscope slide, e. g. 76 mm x 26 mm. 6 5.10 Coverslip, EN 13783:2001 (E) e. g. 25 mm x 50 mm, with a thickness corresponding to the requirements of the objective. 5.11 Immersion oil, non-fluorescing. Refractive index 1,515 to 1,518. 5.12 Stage micrometer, with graduat
48、ions of 0,Ol mm, for measuring the diameter of the microscope field of view. 5.13 Autoclave, for sterilization of diluent and culture medium. 5.14 Oven, for dry heat sterilization of glassware. 5.15 Whirl mixer, for mixing sample suspension and diluent. 5.16 Water bath, for maintaining the culture m
49、edia at the appropriate temperature. 5.17 Incubator, capable of maintaining a temperature of 30 OC f 1 OC. 5.18 Glass bottles, with screw caps, for storing of reagents (DEFT). 5.19 Test tubes, for dilution series of sample test solution (APC). 5.20 Pipettes, 1 ml, 2 ml, 5 ml and 20 ml. 5.21 Petri dishes, 0 90 mm (APC). 5.22 Colony counter (APC) 5.23 Forceps 5.24 Optional apparatus/equipment for semi-automatic or automatic DEFT counting consisting of image analyser, video camera for the microscope, TV monitor, keyboard and printer, microscope autofocus (automatic)
