SIST EN 13624:2004
(Main)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal activity of chemical disinfectants for instruments used in the medical area - Test method and requirements (phase 2, step 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal activity of chemical disinfectants for instruments used in the medical area - Test method and requirements (phase 2, step 1)
This European Standard specifies a test method and the minimum requirements for fungicidal or yeasticidal activity of chemical disinfectant products that form a homogeneous, physically stable preparation when diluted with hard water or - in the case of ready-to-use products - with water. Products can only be tested at a concentration of 80 % or less as some dilution is always produced by adding the test organisms and interfering substance.
This European Standard applies to products that are used in the medical area for disinfecting instruments by immersion - even if they are not covered by the EEC/93/42 Directive on Medical Devices.
This European Standard applies to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example :
- in hospitals, in community medical facilities and in dental institutions ;
- in clinics of schools, of kindergartens and of nursing homes ;
and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test (see annex E).
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Prüfung der fungiziden Wirkung chemischer Desinfektionsmittel für Instrumente im humanmedizinischen Bereich - Prüfverfahren und Andforderungen (Phase 2, Stufe 1)
Diese Europäische Norm legt ein Prüfverfahren und Mindestanforderungen an die fungizide oder Hefen abtötende Wirkung von chemischen Desinfektionsmitteln fest, die bei Verdünnung mit Wasser standardisierter Härte als homogenes physikalisch stabiles Präparat vorliegen, bzw. bei gebrauchsfertigen Produkten bei der Verdünnung mit Wasser. Die Produkte können nur in einer Konzentration von 80 % oder darunter geprüft werden, da durch Zugabe der Prüfkeime und der Belastungssubstanz immer eine gewisse Verdünnung bewirkt wird.
Diese Europäische Norm gilt für Produkte, die zur Desinfektion von Instrumenten im humanmedizinischen Bereich durch Eintauchen verwendet werden - selbst wenn diese nicht in der Richtlinie 93/42/EWG über Medizinprodukte erfasst sind.
Diese Europäische Norm gilt für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist, z. B. bei der Patientenbetreuung in
3 Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
3 medizinischen Einrichtungen in Schulen, Kindergärten und Heimen;
und können auch am Arbeitsplatz oder im privaten Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung der Patienten dienen.
ANMERKUNG 1 Das beschriebene Verfahren dient zur Bestimmung der Wirksamkeit kommerziell erhältlicher Zubereitungen oder Wirkstoffe unter den praktischen Bedingungen, unter denen sie angewendet werden.
ANMERKUNG 2 Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1 (siehe Anhang E).
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide des désinfectants chimiques utilisés pour les instruments en médecine - Méthode d'essai et exigences (phase 2, étape 1)
La présente Norme Européenne spécifie une méthode d'essai et les exigences minimales relatives a l'activité fongicide et levuricide des désinfectants chimiques qui forment une préparation homogene, physiquement stable, lorsqu'ils sont dilués dans l'eau dure ou - dans le cas de produits prets a l'emploi - dans l'eau. Les produits ne peuvent etre soumis a l'essai qu'a une concentration inférieure ou égale a 80 %, car l'ajout des souches d'essai et de la substance interférente entraîne toujours une dilution.
La présente Norme Européenne s'applique aux produits utilisés dans le domaine médical pour désinfecter les instruments par immersion - meme s'ils ne sont pas couverts par la Directive CEE/93/42 sur les dispositifs médicaux.
La présente Norme Européenne s'applique dans les zones et les situations ou la désinfection est médicalement préconisée. De telles indications se rencontrent dans le cadre des soins apportés aux patients, par exemple :
3 dans les hôpitaux, centres de soins médicaux et cabinets dentaires ;
3 dans les infirmeries d'écoles, de jardins d'enfants et de maisons de retraite ;
et peuvent aussi se rencontrer sur les lieux de travail ou a domicile. Elles peuvent également concerner des services tels que des blanchisseries et des cuisines qui fournissent des produits directement aux patients.
NOTE 1 La méthode décrite vise a déterminer l'activité des formulations commerciales ou des substances actives dans leurs conditions d'utilisation.
NOTE 2 La méthode correspond a la phase 2/étape 1 (voir annexe E).
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za ocenjevanje fungicidnega delovanja kemičnih razkužil za instrumente, ki se uporabljajo v humani medicini - Preskusna metoda in zahteve (faza 2, stopnja 1)
General Information
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Prüfung der fungiziden Wirkung chemischer Desinfektionsmittel für Instrumente im humanmedizinischen Bereich - Prüfverfahren und Andforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide des désinfectants chimiques utilisés pour les instruments en médecine - Méthode d'essai et exigences (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal activity of chemical disinfectants for instruments used in the medical area - Test method and requirements (phase 2, step 1)11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 13624:2003SIST EN 13624:2004en,fr,de01-februar-2004SIST EN 13624:2004SLOVENSKI
STANDARD
SIST EN 13624:2004
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 13624December 2003ICS 11.080.20English versionChemical disinfectants and antiseptics - Quantitative suspensiontest for the evaluation of fungicidal activity of chemicaldisinfectants for instruments used in the medical area - Testmethod and requirements (phase 2, step 1)Antiseptiques et désinfectants chimiques - Essai quantitatifde suspension pour l'évaluation de l'activité fongicidedesdésinfectants chimiques utilisés pour les instruments enmédecine - Méthode d'essai et prescriptions (phase 2,étape 1)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Suspensionsversuch zur Prüfung derfungiziden Wirkung chemischer Desinfektionsmittel fürInstrumente im humanmedizinischen Bereich -Prüfverfahren und Andforderungen (Phase 2, Stufe 1)This European Standard was approved by CEN on 3 November 2003.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,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 13624:2003 ESIST EN 13624:2004
EN 13624:2003 (E)2ContentsPageForeword.3Introduction.41Scope.52Normative references.53Terms and definitions.54Requirements.65Test methods.75.1Principle.75.2Materials and reagents.75.3Apparatus and glassware.105.4Preparation of test organism suspensions and product test solutions.115.5Procedure for assessing the fungicidal activity of the product.145.6Experimental data and calculation.195.7Verification of methodology.235.8Expression of results and precision.245.9Interpretation of results - conclusion.255.10Test report.25Annex A (informative)
Referenced strains in national collections.27Annex B (informative)
Neutralizer and Rinsing liquids.28B.1Neutralizers.28B.2Rinsing liquids.29B.3Neutralizer added to the agar for counting :.29Annex C (informative)
Flow diagrams.30C.1Dilution neutralization method.30C.2Membrane filtration method.31Annex D (informative)
Example of a typical test report.34Annex E (informative)
Information on the application and interpretation of European standards onchemical disinfectants and antiseptics.36E.1Introduction.36E.2General guidelines for the application and interpretation of test methods in accordancewith European Standards for chemical disinfectants and antiseptics.36E.3Guide to the interpretation of tests for chemical disinfectants and antiseptics.37Annex ZA (informative)
Clauses of this European Standard addressing essential requirements orother provisions of EU Directives.38Bibliography.39SIST EN 13624:2004
EN 13624:2003 (E)3ForewordThis document (EN 13624:2003) has been prepared by Technical Committee CEN/TC 216 “Chemicaldisinfectants and antiseptics”, the secretariat of which is held by AFNOR.This European Standard shall be given the status of a national standard, either by publication of an identicaltext or by endorsement, at the latest by June 2004, and conflicting national standards shall be withdrawn atthe latest by June 2004.This document has been prepared under a mandate given to CEN by the European Commission and theEuropean Free Trade Association, and supports essential requirements of EU Medical Devices Directive93/42.For relationship with EU Directive, see informative annex ZA, which is an integral part of this document.A collaborative trial has been undertaken to provide a precision annex to this standard.Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different applications inthe medical field are in preparation.Annexes A, B, C, D, and E are informative.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, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands,Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and the United Kingdom.SIST EN 13624:2004
EN 13624:2003 (E)4IntroductionThis European Standard describes a suspension test for establishing whether a chemical disinfectant for useon instruments (surgical instruments, anaesthesia material, endoscopes etc.) has or does not have afungicidal activity in the area described in the scope.In this laboratory test chosen conditions include contact time, temperature, test organisms and interferingsubstances, i.e. conditions which may influence the action of chemical disinfectants in practical situations.The conditions are intended to cover general purposes and to allow reference between laboratories andproduct types. Each concentration of the chemical disinfectant found by this test corresponds to the chosenexperimental conditions. However, for some applications the instructions of use of a product may differ andtherefore additional test conditions need to be used.SIST EN 13624:2004
EN 13624:2003 (E)51 ScopeThis European Standard specifies a test method and the minimum requirements for fungicidal or yeasticidalactivity of chemical disinfectant products that form a homogeneous, physically stable preparation when dilutedwith hard water or - in the case of ready-to-use products - with water. Products can only be tested at aconcentration of 80 % or less as some dilution is always produced by adding the test organisms andinterfering substance.This European Standard applies to products that are used in the medical area for disinfecting instruments byimmersion - even if they are not covered by the EEC/93/42 Directive on Medical Devices.This European Standard applies to areas and situations where disinfection is medically indicated. Suchindications occur in patient care, for example :¾ in hospitals, in community medical facilities and in dental institutions ;¾ in clinics of schools, of kindergartens and of nursing homes ;and may occur in the workplace and in the home. It may also include services such as laundries and kitchenssupplying products directly for the patients.NOTE 1The method described is intended to determine the activity of commercial formulations or active substancesunder the conditions in which they are used.NOTE 2This method corresponds to a phase 2 step 1 test (see annex E).2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications.These normative references are cited at the appropriate places in the text and the publications are listedhereafter. For dated references, subsequent amendments to or revisions of any of these publications apply tothis European Standard only when incorporated in it by amendment or revision. For undated references thelatest edition of the publication referred to applies (including amendments).EN 12353, Chemical disinfectants and antiseptics – Preservation of microbial strains used for thedetermination of bactericidal and fungicidal activity.ISO 4793, Laboratory sintered (fritted) filters – Porosity grading, classification and designation.ISO 6710, Single-use containers for venous blood specimen collection.3 Terms and definitionsFor the purposes of this European Standard, the following terms and definitions apply.3.1productchemical agent or formulation used as chemical disinfectant or antisepticSIST EN 13624:2004
EN 13624:2003 (E)63.2fungicideproduct which kills fungi and their spores under defined conditionsAdjective : fungicidal3.3fungicidal activitycapability of a product to produce a reduction in the number of viable vegetative yeast cells and mould sporesof relevant test organisms under defined conditions3.4fungistatic activitycapability of a product to inhibit the growth of fungi under defined conditions3.5yeasticideproduct which kills yeasts under defined conditionsAdjective : yeasticidal3.6yeasticidal activitycapability of a product to produce a reduction in the number of viable yeast cells of relevant test organismsunder defined conditions3.7clean conditionsconditions representative of surfaces which have received a satisfactory cleaning programme and/or areknown to contain minimal levels of organic and/or inorganic substances3.8dirty conditionsconditions representative of surfaces which are known to or may contain, organic and/or inorganic substances4 RequirementsThe product, when diluted with hard water or - in the case of ready-to-use products - with water and tested inaccordance with clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution) or dirty conditions(3 g/l bovine albumin solution plus 3 ml/l washed sheep erythrocytes) according to its practical applicationsand under the obligatory test conditions (one or two selected test organisms, 20 °C, 60 min), shalldemonstrate at least a decimal log (lg) reduction in counts of 4. It is possible to test also the product asdelivered (highest test concentration = 80 %).The fungicidal activity shall be evaluated using the following two test organisms : Aspergillus niger andCandida albicans.The yeasticidal activity shall be evaluated using the following test organism : Candida albicans.Where indicated, additional specific fungicidal or yeasticidal activity shall be determined applying other contacttimes, temperatures and interfering substances (see 5.5.1.1) in order to take into account intended specificuse conditions.NOTEFor these additional conditions, the concentration defined as a result can be lower than the one obtainedunder the obligatory test conditions.SIST EN 13624:2004
EN 13624:2003 (E)75 Test methods5.1 Principle5.1.1A test suspension of fungi (yeast cells and mould spores) in a solution of an interfering substance isadded to a sample of the product as delivered and/or diluted with hard water (for ready to use products:water). The mixture is maintained at 20 °C ± 1 °C for 60 min ± 10 s (obligatory test conditions). At the end ofthis contact time, an aliquot is taken; the fungicidal and/or the fungistatic action in this portion is immediatelyneutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitableneutralizer cannot be found, membrane filtration is used. The numbers of surviving fungi in each sample aredetermined and the reduction is calculated.5.1.2The test is performed using the spores of Aspergillus niger and the vegetative cells of Candidaalbicans or only the vegetative cells of Candida albicans as test organisms (obligatory test conditions).5.1.3Additional and optional contact times and temperatures are specified. Additional interferingsubstances can be used.5.2 Materials and reagents5.2.1 Test organismsThe fungicidal activity shall be evaluated using the following two test organisms1) :¾ Candida albicansATCC 10231 ;¾ Aspergillus nigerATCC 16404.The yeasticidal activity shall be evaluated using only Candida albicans.NOTESee annex A for corresponding strain reference in some other culture collections.5.2.2 Culture media and reagents5.2.2.1 GeneralThe reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be freefrom substances that are toxic or inhibitory to the test organisms.NOTETo improve reproducibility, it is recommended that commercially available dehydrated material is used for thepreparation of culture media. The manufacturer's instructions relating to the preparation of these products should berigorously followed. For each culture medium and reagent a limitation for use should be fixed.5.2.2.2 WaterThe water shall be freshly glass distilled water and not demineralized water.Sterilize in the autoclave (see 5.3.1).NOTE 1Sterilization is not necessary if the water is used - e.g. for preparation of culture media - and subsequentlysterilized.
1)The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC).This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN ofthe product named.SIST EN 13624:2004
EN 13624:2003 (E)8NOTE 2If distilled water of adequate quality is not available, water for injections (see EP) can be used.5.2.2.3 Malt Extract Agar (MEA)Malt extract30,0 gSoja peptone, papaic digest or soybean meal3,0 gAgar15,0 gWater (see 5.2.2.2)to 1 000 mlSterilize in the autoclave (see 5.3.1). After sterilization the pH of the medium shall be equivalent to 5,6 ± 0,2when measured at 20 °C (see 5.3.2.4).NOTEIn special circumstances (problems with neutralization - see 5.5.1.2 and 5.5.1.3) it may be necessary to addneutralizer to MEA (see B.3).5.2.2.4 DiluentTryptone Sodium Chloride Solution :¾ tryptone, pancreatic digest of casein1,0 g ;¾ sodium chloride (NaCl)8,5 g ;¾ water (see 5.2.2.2)to 1 000 ml.Sterilize in the autoclave (see 5.3.1). After sterilization the pH of the diluent shall be equivalent to 7,0 ± 0,2when measured at 20 °C.5.2.2.5 NeutralizerThe neutralizer shall be validated for the product being tested in accordance with 5.5.1 and 5.5.2. Theneutralizer shall be sterile.NOTEInformation on neutralizers that have been found to be suitable for some categories of products is given in B.1.5.2.2.6 Rinsing liquid (for membrane filtration)The liquid shall be sterile, compatible with the filter membrane and capable of filtration through the filtermembrane under the test conditions described in 5.5.3.NOTEInformation on rinsing liquids that have been found to be suitable for some categories of products is given inB.2.5.2.2.7 Hard water for dilution of productsPrepare :¾ solution A : dissolve 19,84 g anhydrous magnesium chloride (MgCl2) or an equivalent of hydratedmagnesium chloride and 46,24 g anhydrous calcium chloride (CaCl2) or an equivalent of hydratedcalcium chloride in water (see 5.2.2.2) and dilute to 1 000 ml . Sterilize in the autoclave (see 5.3.1). Storethe solution at 2 °C to 8 °C for no longer than one month ;¾ solution B : dissolve 35,02 g sodium hydrogencarbonate (NaHC03) in water (see 5.2.2.2) and dilute to1 000 ml. Sterilize by membrane filtration (see 5.3.2.7). Store the solution at 2 °C to 8 °C for no longerthan one week ;SIST EN 13624:2004
EN 13624:2003 (E)9¾ hard water : for the preparation of 1 litre, place 600 ml to 700 ml water (see 5.2.2.2) in a 1000 mlvolumetric flask (see 5.3.2.12) and add 6,0 ml of solution A, then 8,0 ml of solution B. Mix and dilute to1 000 ml with water (see 5.2.2.2). The pH of the hard water shall be 7,0 ± 0,2. If necessary adjust the pHby using a solution of approximately 40 g/L (about 1 mol/L) of sodium hydroxide (NaOH) or approximately36,5 g/L (about 1 mol/L) of hydrochloric acid (HCl). The hard water shall be freshly prepared underaseptic conditions and used within 12 hours.NOTEWhen preparing the product test solutions (see 5.4.2) the addition of the product to this hard water produces adifferent final water hardness in each test tube. In any case the final hardness is lower than 300 mg/L of calcium carbonate(CaC03) in the test tube.5.2.2.8 Interfering substances5.2.2.8.1 GeneralThe interfering substance shall be chosen according to the conditions of use laid down for the product.The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.The ionic composition e.g. pH, calcium and/or magnesium hardness and chemical composition e.g. mineralsubstances, protein, carbohydrates, lipids, detergents shall be defined.NOTEIn the following the term “interfering substance” is used even if it contains more than one substance.5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent (see5.2.2.4).Sterilize by membrane filtration (see 5.3.2.7), keep in a refrigerator (at 2 °C to 8 °C) and use within 1 month.The final concentration of the bovine albumin in the test procedure (see 5.5) is 0,3 g/l.5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solutions – high concentration with sheeperythrocytes)Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent (see5.2.2.4).Sterilize by membrane filtration (see 5.3.2.7).Prepare at least 8,0 ml fresh sterile defibrinated sheep blood according to ISO 6710 or purchase such bloodfrom a commercial supplier (see 5.2.2.8.4). Centrifuge the erythrocytes at 800 g for 10 min. After discardingthe supernatant, resuspend erythrocytes in diluent (see 5.2.2.4). Repeat this procedure at least 3 times, untilthe supernatant is colourless. Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilizedbovine albumin solution (see above). To avoid later contamination this mixture should be split in portionsprobably needed per day and kept in separate containers for a maximum of 7 days in a refrigerator at 2 °C to8 °C.The final concentration of bovine albumin and sheep erythrocytes in the test procedure (see 5.5) shall be 3 g/land 3 ml/l respectively.5.2.2.8.4 Sterile defibrinated sheep bloodThe sterile defibrinated sheep blood can be acquired from a commercial supplier or prepared accordingto ISO 6710.SIST EN 13624:2004
EN 13624:2003 (E)105.3 Apparatus and glassware5.3.1 GeneralSterilize all glassware and parts of the apparatus that will come into contact with the culture media andreagents or the sample, except those which are supplied sterile, by one of the following methods :a) by moist heat, in the autoclave (see 5.3.2.1 a) by maintaining it at ()12130+ °C for a minimum holding timeof 15 min ;b) by dry heat, in the hot air oven (see 5.3.2.1 b) by maintaining it at 180 °C for a minimum holding time of30 min, at 170 °C for a minimum holding time of 1 h or at 160 °C for a minimum holding time of 2 h.5.3.2 Usual microbiological laboratory equipment2) and in particular, the following :5.3.2.1 Apparatus for sterilization :a) for moist heat sterilization, an autoclave capable of being maintained at ()12130+ °C for a minimum holdingtime of 15 min ;b) for dry heat sterilization, a hot air oven capable of being maintained at 180 °C for a minimum holding timeof 30 min, at 170 °C for a minimum holding time of 1 h or at 160 °C for a minimum holding time of 2 h.5.3.2.2 Water baths capable of being controlled at 20° C ± 1 °C, at 45 °C ± 1 °C (to maintain melted TSAin case of pour plate technique) and at additional test temperatures ± 1 °C (see 5.5.1).5.3.2.3 Incubator, capable of being controlled at 30 °C ± 1 °C.5.3.2.4 pH-meter, having an inaccuracy of calibration of not more than ± 0,1 pH units at 20 °C.The additional inaccuracy of the electrodes shall not be more than ± 0,1 pH units.NOTEFor measuring the pH of the agar-media (see 5.2.2.3) a puncture electrode or a flat membrane electrodeshould be used.5.3.2.5 Stopwatch5.3.2.6 Electromechanical agitator e.g.Vortex® mixer 3).5.3.2.7 Membrane filtration apparatus constructed of a material compatible with the product, with a filterholder of at least 50 ml volume, and suitable for use of filters with diameter 47 mm to 50 mm and 0,45 µmpore size (or 0,22 µm pore size for sterilization of hard water - see 5.2.2.7 and albumin – see 5.2.2.8).The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of themicro-organisms over the membrane and in order to prevent overlong filtration.The device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
2)Disposable equipment is an acceptable alternative to reusable glassware.3)Vortex® in an example of a suitable product available commercially. This information is given for the convenience ofusers of this standard and does not constitute an endorsement by CEN of this product.SIST EN 13624:2004
EN 13624:2003 (E)115.3.2.8 Containers : test tubes, culture bottles or flasks of suitable capacity.5.3.2.9 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml.Calibrated automatic pipettes may be used.5.3.2.10 Petri dishes of size 90 mm to 100 mm.5.3.2.11 Glass beads (Diameter : 3 mm to 4 mm).5.3.2.12 Volumetric flasks5.3.2.13 Orbital mechanical shaker5.3.2.14 Centrifuge (800 gN and 2000 gN).5.3.2.15 Roux bottles or similar flasks5.3.2.16 Fritted filter, porosity of 40 µm to 100 µm (see ISO 4793).5.4 Preparation of test organism suspensions and product test solutions5.4.1 Test organism suspensions (Test and validation suspension)5.4.1.1 GeneralTwo different test organism suspensions have to be prepared: the “test suspension” to perform the test andthe “validation suspension” to perform the controls and method validation.5.4.1.2 Stock culture of test organismsThe test organisms and their stock cultures shall be prepared and kept in accordance with the requirements ofEN 12353.5.4.1.3 Working culture of test organisms5.4.1.3.1 Candida albicans (yeast)In order to prepare the working culture of Candida albicans (see 5.2.1), subculture from the stock culture (see5.4.1.1) by streaking onto MEA slopes or plates (see 5.2.2.3) and incubate (see 5.3.2.3). After 42 h to 48 hprepare a second subculture from the first subculture in the same way and incubate for 42 h to 48 h. From thissecond subculture, a third subculture may be produced in the same way. The second and/or third subcultureis/are the working culture(s).If it is not possible to prepare the second subculture on a particular day, a 72 h subculture may be used forsubsequent subculturing, provided that the subculture has been kept in the incubator (see 5.3.2.3) during the72 h period.Never produce and use a fourth subculture.5.4.1.3.2 Aspergillus niger (mould)For Aspergillus niger use only the first subculture grown on MEA (see 5.2.2.3) in Roux bottles (see 5.3.2.15)and incubate for 7-9 days. No further subculturing is needed.SIST EN 13624:2004
EN 13624:2003 (E)125.4.1.4 Test suspension (“N”)5.4.1.4.1 Candida albicansa) Take 10 ml of diluent (see 5.2.2.4) and place in a 100 ml flask with 10 g of glass beads (see 5.3.2.11).Take the working culture (see 5.4.1.3.1) and transfer loopfuls of the cells into the diluent (see 5.2.2.4).Thecells should be suspended in the diluent by immersing the loop in the diluent and rubbing it against theside of the flask to dislodge the cells. Shake the flask for 3 min using a mechanical shaker (see 5.3.2.13).Aspirate the suspension from the glass beads and transfer to another tube.b) Adjust the number of cells in the suspension to 1,5 x 107 cfu/ml4) to 5,0 x 107 cfu/ml usingdiluent (see 5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension inthe water bath at q (see 5.5.1.1.a) and use within 2 h.NOTEThe use of spectrophotometer for adjusting the number of cells is highly recommended (about 620 nmwavelength – cuvette 10 mm path length). Each laboratory should therefore produce calibration data for each testorganism knowing that suitable values of optical density are found between 0,200 and 0,350. A colorimeter is a suitablealternative.c) For counting prepare 10-5and 10-6dilutions of the test suspension using diluent (see 5.2.2.4). Mix (see5.3.2.6).Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spreadplate technique.When using the pour plate technique, transfer about half of each 1 ml sample into separate Petri dishes(i.e. in duplicate = four plates) and add 15 ml to 20 ml melted MEA (see 5.2.2.3), cooled to 45 °C ± 1 °C.When using the spread plate technique spread each 1,0 ml sample – divided in portions of approximatelyequal size - on an appropriate number (at least two) of surface dried plates containing MEA (see 5.2.2.3).Incubation and counting (see 5.4.1.6).5.4.1.4.2 Aspergillus nigera) Take the working culture (see 5.4.1.3.2) and suspend the spores in 10 ml of sterile 0,05 % v/vpolysorbate 80 solution in water (see 5.2.2.2). Using a sterile glass spatula detach the conidiospores fromthe culture surface. The suspension is transferred into a conical flask and gently shaken by hand for oneminute together with glass beads (see 5.3.2.11). The suspension is filtered through a frittedfilter (see 5.3.2.16).b) Microscopic examination under x 400 magnification shall be carried out immediately after the preparationand just before the test, to show the absence of mycelia fragments and spore germination (check at leastten fields of view for absence of both).If germinated spores are present, the suspension shall be discarded.If mycelia are present, a washing process (centrifugation) shall be set up as follows: Transfer the filteredsuspension to centrifuge tubes. The filtered suspension is centrifuged (see 5.3.2.14) at 2000 g for 20 min.The conidiospores are washed at least twice by resuspension in di
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