SIST EN 13783:2002

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.OXRURVFHQWQHJDDLebensmittel - Nachweis der Bestrahlung von Lebensmitteln mit Epifluoreszenz-Filtertechnik/aerober mesophiler Keimzahl (DEFT/APC) - ScreeningverfahrenProduits alimentaires - Détection d'aliments ionisés en utilisant la technique d'épifluorescence apres filtration et dénombrement de la flore aérobie sur milieu gélosé (DEFT/APC) - Méthode par criblageFoodstuffs - Detection of irradiated food using Direct Epifluorescent Filter Technique/Aerobic Plate Count (DEFT/APC) - Screening method67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 13783:2001SIST EN 13783:2002en01-junij-2002SIST EN 13783:2002SLOVENSKI
STANDARD



SIST EN 13783:2002



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 13783November 2001ICS 07.100.30; 67.050English versionFoodstuffs - Detection of irradiated food using DirectEpifluorescent Filter Technique/Aerobic Plate Count(DEFT/APC) - Screening methodProduits alimentaires - Détection d'aliments ionisés enutilisant la technique d'épifluorescence après filtration etdénombrement de la flore aérobie sur milieu gélosé(DEFT/APC) - Méthode par criblageLebensmittel - Nachweis der Bestrahlung vonLebensmitteln mit Epifluoreszenz-Filtertechnik/aerobermesophiler Keimzahl (DEFT/APC) - ScreeningverfahrenThis European Standard was approved by CEN on 29 September 2001.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the 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 translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards 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 STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2001 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 13783:2001 ESIST EN 13783:2002



EN 13783:2001 (E)2ContentspageForeword.21Scope.32Normative references.33Principle.34Reagents.35Apparatus.66Sampling technique.77Procedure.88Evaluation.99Limitations.1010Validation.1011Test report.11Annex A (informative)
Further information on the applicability.12Annex B (informative)
Practical example for calculation of the microscope factor.13Annex C (informative)
Flow diagram of the procedure.14Bibliography.15ForewordThis European Standard has been prepared by Technical Committee CEN /TC 275 "Food Analysis - HorizontalMethods", the secretariat of which is held by DIN.This European Standard shall be given the status of a national standard, either by publication of an identical text orby endorsement, at the latest by May 2002, and conflicting national standards shall be withdrawn at the latest byMay 2002.This European Standard was elaborated on the basis of a protocol developed following a concerted actionsupported by the Commission of European Union (XII C.) (BCR), and a screening investigation carried out by theDanish National Food Agency and the local Environment and food Agency, MLK FYN, DK Odense. Experts andlaboratories from E.U. and EFTA countries, contributed jointly to the development of the concerted action protocol.WARNING: The use of this standard may involve hazardous materials, operations and equipment. This standarddoes not purport to address all the safety problems associated with its use. It is the responsibility of the user of thisstandard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.The annexes A, B and C are informative.This standard includes a Bibliography.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden,Switzerland and the United Kingdom.SIST EN 13783:2002



EN 13783:2001 (E)31 ScopeThis European Standard specifies a microbiological screening method for the detection of irradiation treatment ofherbs 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 astandardised method (e.g. EN 1788, prEN 13751) to specifically prove an irradiation treatment of the suspectedfood.The method has been successfully tested in interlaboratory tests with herbs and spices [1] to [5].2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate places in the text, and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).ISO 4833, Microbiology – General guidance for the enumeration of microorganisms - Colony count technique at30 °C.ISO 7218, Microbiology of food and animal feeding stuffs – General rules for microbiological examinations.3 PrincipleThe method is based on the comparison of the APC with the count obtained using DEFT. The APC gives thenumber of viable microorganisms in the sample after a possible irradiation and the DEFT count indicates the totalnumber of microorganisms, including non-viable cells, present in the sample. The difference between the DEFTcount and the APC count in spices treated with doses of 5 kGy to 10 kGy is generally about or above 3 to 4 logunits. Similar differences between DEFT and APC counts can be induced by other treatments of the foods leadingto death 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 themicroorganisms on the filter. The microorganisms are stained with a fluorochrome, acridine orange (AO), resultingin an orange and orange-yellow fluorescence under illumination with blue light at 450 nm to 490 nm. Thesemicroorganisms 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 counted. In parallel,the APC is determined from a second portion of the same test sample [6] to [10].4 Reagents4.1 GeneralDuring the analysis use only reagents of recognized analytical grade. All the reagents used in the DEFT and APCdeterminations should be sterilized by membrane filtration through 0,2 µm pore size membrane filters or byautoclaving.4.2 Peptone saline diluent4.2.1 CompositionSodium chloride8,5 gPeptone1,0 gDistilled or demineralized water1000 mlSIST EN 13783:2002



EN 13783:2001 (E)44.2.2 PreparationDissolve the components in the water. Adjust the pH, if necessary, so that after sterilization the final pH is 7,2 ± 0,2at 20 °C to 25 °C. Sterilize in the autoclave (5.13) at 121 °C ± 1°C for 15 min. The diluent may be stored in a glassbottle at 4 °C to 6°C for not more than two weeks.4.3 Buffer, pH 3,04.3.1 Citric acid solution, substance concentration c(C6H8O7 · H2O) = 0,1 mol/l4.3.1.1 CompositionCitric acid monohydrate21 gDistilled or demineralized water1000 ml4.3.1.2 PreparationDissolve 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(NaOH) = 0,1 mol/l4.3.2.1 CompositionSodium hydroxide 4,0 g orSodium hydroxide solution, 1 mol/l100 mlDistilled or demineralized water900 ml4.3.2.2 PreparationDissolve the sodium hydroxide or dilute the sodium hydroxide solution in the water. The solution may be stored in aglass bottle at 4 °C to 6 °C for not more than three months.4.3.3 Complete buffer, pH 3,04.3.3.1
CompositionCitric acid solution (4.3.1)100 mlSodium hydroxide solution (4.3.2)54 ml4.3.3.2 PreparationMix the citric acid solution and the sodium hydroxide solution. Adjust pH to 3,0 ± 0,2 with citric acid solution orsodium hydroxide solution. Sterilize the buffer through a membrane filter of pore size 0,2 µm (5.3) before use. Thesolution may be stored in a glass bottle at 4 °C to 6 °C for not more than three weeks.SIST EN 13783:2002



EN 13783:2001 (E)54.4 Acridine orange solution4.4.1 Buffer solution, pH 6,64.4.1.1 CompositionCitric acid solution (4.3.1)35,5 mlSodium hydroxide solution (4.3.2)100 ml4.4.1.2 PreparationMix the citric acid solution and the sodium hydroxide solution. Adjust pH to 6,6 ± 0,2 with citric acid solution orsodium hydroxide solution. Sterilize the buffer through a membrane filter of pore size 0,2 µm (5.3) before use. Thebuffer solution may be stored in a glass bottle at 4 °C to 6 °C for not more than three weeks.4.4.2 Complete acridine orange solution4.4.2.1 CompositionAcridine orange0,025 gBuffer, pH 6,6 (4.4.1)100 ml4.4.2.2
PreparationDissolve the acridine orange in the buffer solution (4.4.1). Sterilize the acridine orange solution through a 0,2 µmpore size membrane filter (5.3) before use. The solution may be stored in a brown glass bottle at 4 °C to 6 °C fornot 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 as a mutagenic substance, disposable gloves and face masks should be used whenweighing the stain.4.5 2-Propanol4.6 Triton® X-1001), 1 % cleaning solution4.6.1 CompositionTriton® X – 10010 mlDistilled or demineralized water1000 ml4.6.2 PreparationMix Triton X-100 with warm (80°C) water. Sterilize the solution through a 0,2 µm 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.
1) Triton® X-100 is an example of a suitable product available commercially. This information is only given for the convenienceof users of this International Standard and does not constitute an endorsement by CEN of this product.SIST EN 13783:2002



EN 13783:2001 (E)64.7 Tryptone-Yeast Extract-Glucose-Agar (Plate count agar)4.7.1 CompositionTryptone5,0 gYeast Extract2,5 gDextrose (Glucose)1,0 gAgar (according to the gel strength of the agar used) 12 g to 18 gDistilled or demineralized water1000 ml4.7.2 PreparationDissolve the components or dehydrated complete medium in the water while heating until boiling. Adjust the pH, ifnecessary, so that after sterilization the final pH is 7,2 ± 0,2 at 20 °C to 25 °C. Sterilize in the autoclave (5.13) at121 °C ± 1 °C for 15 min.5 ApparatusUsual laboratory apparatus in accordance with ISO 7218 and, in particular, the following:5.1 Apparatus for membrane filtration of sample suspensionsFiltration equipment made of stainless steel or glass should be used. The bottom filter should be of sintered glassor stainless steel intended for filters with a diameter of 25 mm (5.5). The filter tower volume should be at least10 ml. The filtration equipment is placed vertically on a suction flask or a manifold connected to a water pump orvacuum pump with a pressure regulator. The vacuum during filtration should usually be approximately 70 kPa.NOTEThe use of a water pump is not very suitable if the water pressure cannot be regulated.5.2 Filter funneland suitable suction flasks made of glass for sterile filtration of reagents and diluent.5.3 Membrane filters,cellulose ester or similar, pore size 0,2 µm, 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 µm, for prefiltration of samples.5.5 Membrane filters,white polycarbonate, diameter 25 mm, pore size 0,6 µm for membrane filtration of sample test 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 totalmagnification of 1250 x is achieved.)5.9 Microscope slide,e. g. 76 mm x 26 mm.SIST EN 13783:2002



EN 13783:2001 (E)75.10 Coverslip,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 graduations of 0,01 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 Whirlmixer,for mixing sample suspension and diluent.5.16 Water bath,for maintaining the culture media at the
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