Chemical disinfectants - Quantitative suspension test for the evaluation of virucidal activity against bacteriophages of chemical disinfectants used in food and industrial areas - Test method and requirements (phase 2, step 1)

This European Standard specifies a test method (phase 2, step 1) and requirements for the minimum virucidal activity against bacteriophages of chemical disinfectants that form a homogeneous, physically stable preparation in hard water and that are used in food and industrial areas, excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues. This European Standard applies to least to the following: a) processing, distribution and retailing of:...

Chemische Desinfektionsmittel - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung gegenüber Bakteriophagen von chemischen Desinfektionsmitteln in den Bereichen Lebensmittel, und Industrie - Prüfverfahren und Anforderung (Phase 2, Stufe 1)

Diese Europäische Norm legt ein Prüfverfahren (Phase 2, Stufe 1) und Anforderungen an die viruzide Mindestwirkung von chemischen Desinfektionsmitteln gegenüber Bakteriophagen fest, die in hartem Wasser eine homogene, physikalisch stabile Lösung ergeben, und die im Lebensmittelbereich und in der Industrie benutzt werden, ausgenommen davon sind Bereiche und Situationen, in denen die Desinfektion medizinisch indiziert ist und Produkte, die auf lebenden Geweben benutzt werden.
Diese Europäische Norm gilt grundsätzlich für den folgenden Anwendungsbereich:
a)   Verarbeitung, Vertrieb und Verkauf von:
1)   Lebensmitteln tierischer Herkunft:
   Milch und Milchprodukte;
   Fleisch und Fleischprodukte;
   Fisch, Meerestiere und daraus hergestellte Zubereitungen;
   Eier und Eierprodukte;
   Tiernahrung;
   usw.
2)   Lebensmitteln pflanzlicher Herkunft:
   Getränke;
   Früchte, Gemüse und daraus hergestellte Zubereitungen (einschließlich Zucker, Destillationsprodukte usw.);
   Mehl, gemahlene und gebackene Produkte;
   Tiernahrung;
   usw.
b)   Andere industrielle Bereiche:
   Biotechnologie (Hefe, Proteine, Enzyme usw.).
Bei Anwendung dieser Europäischen Norm ist die Bestimmung der viruziden Wirkung gegenüber Bakteriophagen in dem unverdünnten Produkt nicht möglich, da immer eine bestimmte Verdünnung durch Zugabe der Impfkultur und der Belastungssubstanz auftritt.
Bei chemischen Desinfektionsmitteln, die ohne Verdünnung verwendet werden können, ist es nicht möglich zu bestimmen, ob diese Produkte bei einer Konzentration über 80 % eine viruzide Wirkung gegenüber Bakteriophagen besitzen.
ANMERKUNG   Das beschriebene Verfahren bestimmt die Wirksamkeit gewerblich hergestellter Produkte (Zubereitungen) oder anderer aktiver Substanzen auf Viren (Bakteriophagen) unter Anwendungsbedingungen.
Diese Europäische Norm gilt nur für Desinfektionsmittel, die die Validierungsprüfung bestehen (siehe Anhang A).

Désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité virucide, vis à vis des bactériophages, des désinfectants chimiques utilisés dans les domaines de l'agro-alimentaire et de l'industrie - Méthode d'essai et prescriptions (phase 2, étape 1)

La présente Norme européenne spécifie une méthode d'essai (phase 2, étape 1) et des prescriptions relatives à l'activité virucide minimale vis-à-vis des bactériophages des désinfectants chimiques qui forment une préparation homogène, physiquement stable dans l'eau dure et qui sont utilisés dans les domaines de l'agro-alimentaire et de l'industrie, à l'exception des secteurs et situations où la désinfection est médicalement prescrite et des produits utilisés sur des tissus vivants.
La présente Norme européenne s'applique au moins aux domaines suivants :
a)   fabrication, distribution et vente au détail de :
1)   produits alimentaires d'origine animale :
   lait et produits laitiers ;
   viande et produits dérivés de la viande ;
   poisson, fruits de mer et leurs produits dérivés ;
   �ufs et produits avicoles ;
   produits alimentaires pour animaux ;
   etc. ;
2)   produits alimentaires d'origine végétale :
   boissons ;
   fruits, légumes et leurs dérivés (y compris le sucre, la distillerie �) ;
   farine, minoterie et boulangerie ;
   produits alimentaires pour animaux ;
   etc. ;
b)   autre branche industrielle :
   biotechnologie (levure, protéines, enzymes,�.).
La présente Norme européenne ne permet pas de déterminer l'activité virucide vis-à-vis des bactériophages d'un produit non dilué puisqu'il y a toujours dilution lors de l'ajout de l'inoculum et de la substance interférente.
Dans le cas des désinfectants chimiques qui peuvent être utilisés sans dilution, il n'est pas possible de déterminer l'activité virucide de ces produits vis-à-vis des bactériophages à une concentration supérieure à 80 %.
NOTE   La méthode décrite est destinée à déterminer l'efficacité des formulations commerciales ou des substances actives sur les virus (bactériophages) dans les conditions dans lesquelles elles sont utilisées.
La présente Norme européenne s'applique uniquement aux désinfectants satisfaisant à l'essai de validation (voir annexe A).

Kemična razkužila – Kvantitativni suspenzijski preskus za ocenjevanje virucidnega delovanja kemičnih razkužil proti bakteriofagom v živilski in drugih industrijah – Preskusna metoda in zahteve (faza 2, stopnja 1)

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung gegenüber Bakteriophagen von chemischen Desinfektionsmitteln in den Bereichen Lebensmittel, und Industrie - Prüfverfahren und Anforderung (Phase 2, Stufe 1)Désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité virucide, vis a vis des bactériophages, des désinfectants chimiques utilisés dans les domaines de l'agro-alimentaire et de l'industrie - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants - Quantitative suspension test for the evaluation of virucidal activity against bacteriophages of chemical disinfectants used in food and industrial areas - Test method and requirements (phase 2, step 1)71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposesICS:Ta slovenski standard je istoveten z:EN 13610:2002SIST EN 13610:2003en01-november-2003SIST EN 13610:2003SLOVENSKI

STANDARD
SIST EN 13610:2003

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 13610December 2002ICS 11.080.20; 67.050; 71.100.35English versionChemical disinfectants - Quantitative suspension test for theevaluation of virucidal activity against bacteriophages ofchemical disinfectants used in food and industrial areas - Testmethod and requirements (phase 2, step 1)Désinfectants chimiques - Essai quantitatif de suspensionpour l'évaluation de l'activité virucide contre lesbactériophages des désinfectants chimiques utilisés dansle domaine de l'agro-alimentaire et dans l'industrie -Méthode d'essai et exigences (phase 2, étape 1)Chemische Desinfektionsmittel - QuantitativerSuspensionsversuch zur Bestimmung der viruzidenWirkung gegenüber Bakteriophagen von chemischenDesinfektionsmitteln in den Bereichen Lebensmittel, undIndustrie - Prüfverfahren und Anforderung (Phase 2, Stufe1)This European Standard was approved by CEN on 23 October 2002.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, Malta, 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© 2002 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 13610:2002 ESIST EN 13610:2003

EN 13610:2002 (E)2ContentspageForeword......................................................................................................................................................................3Introduction.................................................................................................................................................................41Scope..............................................................................................................................................................52Normative references....................................................................................................................................63Terms and definitions....................................................................................................................................64Requirements.................................................................................................................................................75Test methods..................................................................................................................................................75.1Principle..........................................................................................................................................................75.2Material and reagents....................................................................................................................................75.3Apparatus and glassware...........................................................................................................................125.4Preparation of host bacteria suspensions................................................................................................135.5Preparation of bacteriophage suspension................................................................................................145.6Product test solution...................................................................................................................................165.7Procedure.....................................................................................................................................................165.8Calculation and expression of results.......................................................................................................195.9Test report....................................................................................................................................................23Annex A (normative)

Test for validation of dilution-neutralization and molecular sieving methods...............25Annex B (informative)

Neutralizers..........................................................................................................................33Annex C (informative)

Example of a typical test report.........................................................................................35Annex D (informative)

Information on the application and interpretation of European standards onchemical disinfectants and antiseptics.....................................................................................................39Annex E (informative)

Example of plaques from lysates of phages P001 and P008..........................................41Bibliography..............................................................................................................................................................44SIST EN 13610:2003

EN 13610:2002 (E)3ForewordThis document (EN 13610:2002) has been prepared by Technical Committee CEN /TC 216, "Antiseptics andchemical disinfectants" 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 identical text orby endorsement, at the latest by June 2003, and conflicting national standards shall be withdrawn at the latest byJune 2003.In this European Standard the Annex A is normative and the Annexes 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, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain,Sweden, Switzerland and the United Kingdom.SIST EN 13610:2003

EN 13610:2002 (E)4IntroductionThis European Standard describes a suspension test method intended to establish whether a product proposed asa disinfectant in the fields described in clause 1 has or does not have virucidal activity against bacteriophages.NOTEVirulent bacteriophages (phages) lytic for (i.e. virulent for) starter cultures of Lactococcus lactis subsp. lactis usedfor the production of cheese and other fermented milk products are used as model viruses to test the virucidal activity of aproduct.The laboratory test closely simulates practical conditions of application. Chosen conditions (contact time,temperature, viruses [i.e. bacteriophages] in suspension, ...) reflect parameters which are found in practicalsituations including conditions which may influence the action of disinfectants.Each utilization concentration found from this test corresponds to defined experimental conditions.The conditions are intended to cover general purposes and to allow reference between laboratories and producttypes.However for some applications the recommendations of use may differ and therefore additional test conditionsneed to be used.SIST EN 13610:2003

EN 13610:2002 (E)51 ScopeThis European Standard specifies a test method (phase 2, step 1) and requirements for the minimum virucidalactivity against bacteriophages of chemical disinfectants that form a homogeneous, physically stable preparation inhard water and that are used in food and industrial areas, excluding areas and situations where disinfection ismedically indicated and excluding products used on living tissues.This European Standard applies at least to the following:a) processing, distribution and retailing of:1) food of animal origin:¾ milk and milk products;¾ meat and meat products;¾ fish, seafood, and their products;¾ eggs and egg products;¾ animal feeds;¾ etc.;2) food of vegetable origin:¾ beverages;¾ fruits, vegetables and their derivatives (including sugar, distillery ...);¾ flour, milling and baking;¾ animal feeds;¾ .…;b) other industrial areas:¾ biotechnology (yeast, proteins, enzymes, ...).Using this European Standard, it is not possible to determine the virucidal activity against bacteriophages ofundiluted product as some dilution is always produced by adding the inoculum and interfering substance.For chemical disinfectants that can be used without dilution it is not possible to determine whether these products,at a concentration above 80 % have a virucidal activity against bacteriophages.NOTEThe method described is intended to determine the activity of commercial formulations or active substances onviruses (bacteriophages) in the conditions in which they are used.This European Standard is applicable only to disinfectants, complying with the validation test (see annex A).SIST EN 13610:2003

EN 13610:2002 (E)62 Normative referencesNot applicable.3 Terms and definitionsFor the purposes of this European Standard, the following terms and definitions apply.3.1product (for chemical disinfection and/or antisepsis)chemical agent or formulation used as a chemical disinfectant or antiseptic[EN 1040:1997]3.2virucide against bacteriophagesproduct which inactivates the bacteriophagesNOTEThe adjective derived from "virucide" is "virucidal".3.3virucidal activity against bacteriophagescapability of a product to reduce the infectivity of intact bacteriophages belonging to reference bacteriophagestrains P001 and P008 for at least 4 lg under the conditions defined by this standard3.4infectivity of bacteriophagesthe ability of a bacteriophage to propagate in a suitable host bacterial cell resulting in the release of bacteriophageprogeny3.5inactivation of bacteriophagethe reduction of infectivity of a bacteriophage by a product specified as a chemical disinfectant3.6reference bacteriophage suspensionbacteriophage suspension of a defined virus strain maintained in reference centers and which should not bepassaged more than 10 times3.7stock bacteriophage suspensionbacteriophage suspension of a defined strain that has been multiplied on a large scale to obtain a bacteriophagesuspension revealing identical characteristics as the reference bacteriophage suspensionNOTEThis stock bacteriophage suspension is used to prepare a high-titer test bacteriophage suspension for thebacteriophage inactivation test.3.8high-titer bacteriophage suspensionthe high-titer bacteriophages suspension obtained from agar plates revealing confluent lysis in the bacterial lawnthat is used to prepare the bacteriophage test suspension in the virucidal testing of the disinfectant3.9bacteriophage test suspensionthe bacteriophage suspension of a defined titer that is used in the virucidal testing of the disinfectantSIST EN 13610:2003

EN 13610:2002 (E)74 RequirementsThe product diluted in hard water when tested in accordance with clause 5 shall demonstrate at least a 4 log10reduction of infectivity of bacteriophages when tested in the presence of a volume fraction of 1 % acidic whey(prepared from acidified low-fat milk) or optionally in the presence of a volume fraction of 1 % skim milk as theinterfering substance according to its practical applications and under the required test conditions (20 °C, 15 min, 2reference bacteriophage strains).The virucidal activity against bacteriophages shall be evaluated using the two virulent bacteriophages

Lactococcuslactis subsp. lactis bacteriophage P001 and Lactococcus lactis subsp. lactis bacteriophage P008.Both phages shall be propagated on the host strain Lactococcus lactis subsp. lactis F7/2.The determined virucidal concentration of the tested product is suggested as being suitable for practical situationsof use.Where appropriate, additional specific virucidal activity against bacteriophages shall be determined under otherconditions of time, temperature, additional strains and interfering substances in accordance with (see 5.7.1) inorder to take into account intended specific use conditions.NOTEFor these additional conditions, the concentration defined as a result can be lower than the one obtained under theinitial test conditions of 20 °C, 15 min, 2 selected bacteriophage reference strains.5 Test methods5.1 Principle5.1.1A test suspension of bacteriophages in a solution of interfering substances is added to a prepared sampleof the product under test diluted in hard water.The mixture is maintained at 20 °C ± 1 °C for 15 min ± 10 s (required obligatory test conditions).At the end of the contact time, aliquots are taken and the virucidal activity against bacteriophages in this portion isimmediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization with avalidated neutralizer. If a suitable neutralizer is not available for a specific product, removal of the product bymolecular sieving (i.e. gel filtration) shall be used.The number of surviving bacteriophage particles and the number of bacteriophage particles in the test suspensionare determined from appropriate dilution series with a factor of 10 prepared in medium in test tubes.5.1.2Additional and optional exposure times, temperatures and interfering substances are specified (see 5.7.1).NOTE 1The test described is based on an assessment (under specific conditions) which gives a reduction of at least99,99 % (4 lg) of the infectivity of the different phages after different contact times.NOTE 2For principal reasons, the result of an inactivation applied to a viral population is not necessarily equal to 100 %: i.e.,one cannot conclude that there is a 100 % inactivation when on conducting the experiment no infectious phage are found withina limit number of sampling.5.2 Material and reagents5.2.1 Test organismsThe virucidal activity against bacteriophages shall be evaluated using the two following bacteriophage strains :SIST EN 13610:2003

EN 13610:2002 (E)8¾ Lactococcus lactis subsp. lactis bacteriophage P001DSM 4262 1);¾ Lactococcus lactis subsp. lactis bacteriophage P008DSM 10567.Both phages shall be propagated on the following host strain:¾ Lactococcus lactis subsp. lactis F7/2DSM 4366.5.2.2 Culture media and reagents5.2.2.1 GeneralThe reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free fromsubstances that cause toxic or inactivating effects either to the bacteriophages or to the host bacteria used forcounting phage-derived plaques.NOTETo improve reproducibility, it is recommended that commercially available dehydrated material is used for thepreparation of culture media. The manufacturers instructions relating to the preparation of these products should be rigorouslyfollowed.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 1If the water is sterilized during the sterilization of the reagents, this is not necessary.NOTE 2If distilled water of adequate quality is not available, water for injectable preparation (European Pharmacopoeia) canbe used.5.2.2.3 M17-brothFor maintenance of bacterial host strain, propagation of bacteriophages and for formulation of phage diluent(see 5.2.2.6).Phytone peptone (from soya meal)5,00 gPolypeptone peptone (from casein & animal tissue)5,00 gBeef extract powder5,00 gYeast extract2,50 gD(+)-lactose5,00 gAscorbic acid0,50 gSodium-ß-glycerophosphate19,00 gMagnesium sulfate , 7 H2O0,25 gWater (see 5.2.2.2)1 000 mlSterilize in the autoclave (see 5.3.1). After sterilization the pH of the medium shall be equivalent to 7,0 ± 0,2 whenmeasured at 20 °C. When M17-broth is the diluent for neutralizer formulation (see 5.2.2.12 and Annex B), double

1)DSM 4262, DSM 10567 and DSM 4366 are the collection numbers of bacteriophage and bacterial strains supplied by theDSMZ (Deutsche Sammlung von Mikroorganismen und ZellKulturen). This information is given for the convenience of users ofthis standard and does not constitute an endorsement by CEN of the culture collection named. Corresponding strains suppliedby other culture collections may be used if they can be shown to lead to the same results.SIST EN 13610:2003

EN 13610:2002 (E)9concentrated M17-broth shall be used for preparation (i.e., all reagents shall be added in double concentration to1 000 ml water).5.2.2.4 M17-agar (bottom agar)Bottom agar for quantitative counting of lysis zones (plaques) obtained from single infective bacteriophage particlesin the bacterial lawn of the host bacteria.Add 15 g of agar to 1 000 ml of M17-broth (see 5.2.2.3). Dissolve the agar by boiling with constant stirring.Sterilize in the autoclave (see 5.3.1). After sterilization the pH of the medium shall be equivalent to 7,0 ± 0,2 whenmeasured at 20 °C. When the agar is cooled down to 47 °C ± 1 °C, add 10 ml of a sterile 1 mol/l CaCl2-stocksolution (see 5.2.2.8). Mix gently and pour 15 ml to 18 ml of agar into Petri dishes (see 5.3.2.10).5.2.2.5 Overlay agar (top agar, soft agar)For counting bacteriophages: Dissolve 6,5 g agar in 1 000 ml M17-broth (see 5.2.2.3) and heat until boiling withconstant stirring. Dispense the molten agar in test tubes (2,5 to 3 ml each).Sterilize in the autoclave (see 5.3.1).NOTEFor achieving clear phage-derived lysis zones (plaques) in the lawn of host bacterial cells only well-defined agarshould be used which is specified by the supplier for phage enumeration by the overlay technique (see 5.5.2 and 5.5.3).5.2.2.6 Phage diluent (on basis of ¼ strength Ringer’s solution)For preparing dilution series for titration of phage (counting of phage-derived lysis zones):¾ 1/4-strength Ringer’s solution:¾ sodium chloride2,250 g;¾ potassium chloride0,105 g;¾ calcium chloride, anhydrous0,06 g;¾ sodium hydrogen carbonate0,050 g;¾ water (see 5.2.2.2)to 1 000 ml.Add 10 ml M17-broth (see 5.2.2.3) to 90 ml of 1/4-strength Ringer’s solution.Sterilize in the autoclave (see 5.3.1). Before use, add 1 ml from an 1 mol/l CaCl2-stock solution (see 5.2.2.8) to100 ml of the dilution broth.NOTERinger’s solution can be prepared from ready-to-use tablets according to the supplier’s recommendations.5.2.2.7 SM-bufferFor resuspension and storage of intact phage particles:¾ Tris-HCl2,4 g;¾ NaCl5,8 g;SIST EN 13610:2003

EN 13610:2002 (E)10¾ MgSO4 , 7 H2O2,5 g;¾ water (see 5.2.2.2)to 1 000 ml.Adjust the pH of the buffer to 7,4 ± 0,1. Sterilize in the autoclave (see 5.3.1).5.2.2.8 CaCl2-stock solutions (1 mol/l and 0,05 mol/l)Dissolve either 110,99 or 5,55 g anhydrous CaCl2 in water (see 5.2.2.2) and dilute to 1 000 ml to obtain the 1 mol/lor the 0,05 mol/l stock solution, respectively. Sterilize in the autoclave (see 5.3.1).5.2.2.9 Lactic acid solution (a volume fraction of 10 %)For acidification of low-fat milk to prepare acidic whey.Dilute a volume fraction of 90 % stock solution of lactic acid with water (see 5.2.2.2) to obtain a volume fraction of10 % working solution. For this, 8 parts of water are added to 1 part of stock solution. Sterilize in the autoclave (see5.3.1).5.2.2.10 Phosphate-buffered salinePrepare first a 10 mmol/l sodium phosphate buffer (pH 7,2):¾ solution A: 1,42 g anhydrous Na2HP04 are dissolved in water (see 5.2.2.2) and diluted to 1 000 ml with water;¾ solution B: 1,20 g anhydrous NaH2P04 are dissolved in water (see 5.2.2.2) and diluted to 1 000 ml with water.Mix solutions A and B under constant stirring to obtain a final solution with a pH of 7,2.Dissolve 8,5 g NaCl in 10 mmol/l sodium phosphate buffer (pH 7,2) and dilute to 1 000 ml with this buffer. Sterilizein the autoclave (see 5.3.1).5.2.2.11 Sephadex® 2) G-25 gel for molecular sieving (i.e. gel filtration)Resuspend 22 g of Sephadex®2) G-25 powder in 100 ml phosphate-buffered saline (see 5.2.2.10). Sterilize in theautoclave (see 5.3.1). After cooling down to room temperature, fill 20 ml of the gel suspension into sterile plasticsyringes placed in a sterile centrifuge bottle. Remove excess of buffer by centrifugation in a bench-top centrifuge(see 5.3.2.13) at 1 000 x g for 10 min under aseptic conditions. Store these ready-to-use units at 4 °C to 8 °C. Theyshall be used within a 4 h-period.NOTEAlternatively, commercially available, disposable ready-to-use columns of suitable capacity can be used.5.2.2.12 NeutralizerThe neutralizer shall be validated for the product under test in accordance with Annex A. The neutralizer shall besterile.NOTEInformation on neutralizers that have been found to be suitable for some categories of products is given in Annex B.

2)Analytical quality of cross-linked dextran beads for molecular sieving (i.e. gel filtration). Sephadex® G-25 is an example of asuitable product available commercially. This information is given for the convenience of users of this standard and does notconstitute an endorsement by CEN of this product.SIST EN 13610:2003

EN 13610:2002 (E)115.2.2.13 Hard water for dilution of productsHard water shall be prepared as follows:¾ solution A: dissolve 19,84 g anhydrous magnesium chloride (MgCl2) or an equivalent of hydrated magnesiumchloride and 46,24 g anhydrous calcium chloride (CaCl2) or an equivalent of hydrated calcium chloride in water(see 5.2.2.2) and dilute to 1 000 ml.Sterilize in the autoclave (see 5.3.1). Store the solution at 2 °C to 8 °C for no longer than one month;¾ solution B: dissolve 35,02 sodium bicarbonate (NaHCO3 ) in water (see 5.2.2.2) and dilute to 1 000 ml. Sterilizeby membrane filtration (see 5.3.2.7). Store the solution at 2 °C to 8 °C for no longer than one week.Hard Water: For the preparation of 1 litre, place at least 600 ml water (see 5.2.2.2) in a 1 000 ml volumetric flask(see 5.3.2.12) and add 6,0 ml of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (see5.2.2.2). The pH of the hard water shall be 7,0 ± 0,2.If necessary adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH)or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCI).The hard water shall be freshly prepared under aseptic conditions and used within 12 h.NOTEWhen preparing the working culture (see 5.4.2) the addition of the product to this hard water produces a differentfinal water hardness in each test tube.In any case the final hardness is lower than 300 mg/kg of calcium carbonate (CaCO3) in the test tube.5.2.2.14 Interfering substances5.2.2.14.1 GeneralThe ionic composition (pH, calcium and/or magnesium hardness) and chemical composition (mineral substances,protein, glycosides, lipids, detergents ...) shall be fully defined.The 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 of its final concentration in the test.The method of preparation and sterilization together with the composition shall be noted in the test report (see 5.9).5.2.2.14.2 Whey solutionPrepare acidic whey solution from pasteurized low fat milk (1,5 % fat content) for the test conditions as follows:¾ add 0,3 ml of a volume fraction of 10 % lactic acid solution (see 5.2.2.9) to 10 ml milk, mix (see 5.3.2.6) andkeep the sample for 30 min at room temperature. Mix (see 5.3.2.6) occasionally during this 30 min period.Subsequently sediment the precipitated milk proteins in a bench top centrifuge (see 5.3.2.13) at maximumspeed (4 000 x g at minimum) for 30 min. Sterilize the supernatant (whey) by membrane filtration (0,45 µmpore size) (see 5.3.2.7) and store at 4 °C to 8 °C;¾ to obtain a volume fraction of 10 % working solution which is required as the obligatory interfering substancefor the phage suspension test (see 5.7.2), dilute 1 part of acidic whey broth with 9 parts of water (see 5.2.2.2).Store the volume fraction of 10 % whey solution at 4 °C to 8 °C.The whey solutions shall be stored for up to 1 month at 4 °C to 8 °C. For longer storage periods, they shall be keptfrozen at - 18 °C to - 20 °C or lower.SIST EN 13610:2003

EN 13610:2002 (E)12The final concentration of the whey solution in the test procedure (see 5.7.1) shall be a volume fraction of

1,0 %.5.2.2.14.3 Skim milkPrepare reconstituted skim milk (1,5 % fat content) for the test conditions as follows:¾ reconstitute skim milk powder, guaranteed free of antibiotics or additives, at a rate of 100 g/l of water (see5.2.2.2);¾ sterilize by steaming at 100 °C on 3 successive days (30 min each) and leave between steamings at roomtemperature.Do not leave between subsequent steamings in the refrigerator !NOTEUndiluted skim milk is used for maintenance of the bacterial host strain (see 5.4.1).Alternatively, sterilize at (3 0

115+) °C for 15 min.To obtain a volume fraction of 10 % working solution, dilute 1 part of skim milk with 9 parts of sterile water(see 5.2.2.2) which is required as an optional interfering substance for the phage suspension test (see 5.7.2).Store the volume fraction of 10 % skim milk at 4 °C to 8 °C.The final concentration of the skim milk in the test procedure (see 5.7.1) shall be a volume fraction of 1 %.5.3 Apparatus and glassware5.3.1 GeneralSterilize all glassware and parts of apparatus that will come into contact with the culture media and reagents or thesample, except those which are supplied sterile, by one of the following methods:a) in the autoclave (see 5.3.2.1) by maintaining it at (3 0

121+) °C for a minimum holding time of 15 min;b) in the dry heat sterilizer (see 5.3.2.1) by maintaining it at 180 °C for a minimum holding time of 30 min, at170 °C for a minimum holding time of 1 h or at 160 °C for a minimum holding time of 2 h.5.3.2Usual microbiological laboratory equipment 3) - in particular the following:5.3.2.1Apparatus for sterilization:a) for moist heat sterilization, an autoclave capable of being maintained at (3 0

121+) °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 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.5.3.2.2Water baths capable of being controlled at 20 °C ± 1 °C, at additional test temperatures ± 1 °C (see5.7.1), and at 4 °C ± 1 °C for cooling down melted M17 top agar (see 5.5.2 and 5.5.3).5.3.2.3Incubator, capable of being controlled at 30 °C ± 1 °C.

3)Disposable equipment is an acceptable alternative to reusable glassware.SIST EN 13610:2003

EN 13610:2002 (E)135.3.2.4pH-meter, having an accuracy of calibration of ± 0,1 pH units at 25 °C.5.3.2.5Stopwatch.5.3.2.6Electromechanical agitator, ( i.e. Vortexâ mixer 4)).5.3.2.7Membrane filtration apparatus with a filter holder and suitable for use with filters of 0,45 µm poresize.NOTEDisposable equipment is strongly recommended.5.3.2.8Containers: Test tubes, culture bottles or flasks of suitable capacity.5.3.2.9Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml.Calibrated automatic pipettes may be used.NOTECare should be taken to avoid contamination of the automatic pipettes with virus (i.e. bacteriophage) aerosols.5.3.2.10Petri dishes of size 90 mm to 100 mm.5.3.2.11Volumetric flasks.5.3.2.12Mechanical shaker / stirrer.5.3.2.13Benchtop centrifuge, capable to achieve a minimum centrifugation force of 4 000 x g.5.3.2.14Microwave oven.Extreme care shall be taken to avoid uncontrolled overheating in the oven which may cause uncontrolledoverboiling of liquefied agar. Lids shall be unscrewed loosely covered on the containers to avoid the hazard ofexplosion.5.3.2.15Visible-light spectrophotometer (optional) equipped with suitable optical filters. Optionally withsuitable sample holders for glass test tubes to allow a direct reading of the optical density (e.g. at 620 nm) ofbacterial suspensions.5.4 Preparation of host bacteria suspensions5.4.1 Stock culture of host bacteriaInoculate reconstituted skim milk (see 5.2.2.14.3) with a volume fraction of 1 % liquid culture or with a loop ofbacteria from a M17 slope or agar plate, incubate for 2 h at 30 °C ± 1 °C (see 5.3.2.3) and maintain this stockculture of the host strain in reconstituted skim milk in a refrigerator at 4 °C to 8 °C. In 2-weeks-intervals, let thesestock cultures grow overnight at 30 °C ± 1 °C and repeat the method to obtain a fresh stock culture.If prolonged storage is necessary, freeze the skim milk cultures at - 18 °C to -20 °C or lower.Alternatively use lyophilized cultures.

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