Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-porous surfaces without mechanical action - Test method and requirements (phase 2, step 2)

This European Standard specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant and antiseptic products that form a homogeneous physically stable preparation when diluted with hard water, or - in the case of ready-to-use-products - with water.
This European Standard applies to products that are used in the veterinary area on non-porous surfaces without mechanical action i.e. in the breeding, husbandry, production, transport and disposal of all animals except when in the food chain following death and entry to the processing industry.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendations".
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 2 test.
NOTE 3   This method cannot be used to evaluate the activity of products against mycobacteria.

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf nicht-porösen Oberflächen ohne mechanische Wirkung - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Diese Europäische Norm legt ein Prüfverfahren und die Mindestanforderungen an die bakterizide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte oder — im Fall gebrauchsfertiger Produkte — mit Wasser eine homogene, physikalisch stabile Zubereitung bilden.
Diese Europäische Norm gilt für Produkte, die im Veterinärbereich auf nicht-porösen Oberflächen ohne mechanische Wirkung angewendet werden, d. h. bei der Aufzucht, Haltung, Produktion und beim Transport von Tieren sowie bei der Tierkörperbeseitigung, außer wenn die Tiere nach der Tötung durch Zuführung in die weiterverarbeitende Industrie in die Nahrungsmittelkette eintreten.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwendungsempfehlungen“ fest.
ANMERKUNG 1   Das beschriebene Verfahren ist für die Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen unter den Bedingungen, unter denen sie verwendet werden, bestimmt.
ANMERKUNG 2   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2.
Dieses Verfahren kann nicht angewendet werden, um die Wirkung von Produkten gegen Mykobakterien zu bewerten.

Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire sur des surfaces non poreuses sans action mécanique - Méthode d'essai et prescriptions (phase 2, étape 2)

La présente Norme européenne spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité bactéricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l'eau dure ou – dans le cas de produits prêts à l’emploi – dans l’eau.
La présente Norme européenne s'applique aux produits utilisés dans le domaine vétérinaire sur des surfaces non poreuses sans action mécanique, à savoir la reproduction, l'élevage, la production, le transport et l’abattage de tous les animaux, hors de la chaîne alimentaire qui suit l’abattage et l’entrée dans l’industrie de transformation.
L'EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».
NOTE 1   La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives dans les conditions dans lesquelles elles sont utilisées.
NOTE 2   Cette méthode correspond à un essai de phase 2, étape 2.
Cette méthode ne peut pas être utilisée pour évaluer l'activité des produits vis-à-vis des mycobactéries.

Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah brez mehanskega delovanja za vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)

Ta evropski standard določa preskusno metodo in minimalne zahteve za baktericidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma z vodo pri proizvodih, ki so pripravljeni za uporabo.  Ta evropski standard se uporablja za izdelke v veterini na neporoznih površinah brez mehanskega delovanja, tj. pri vzreji, živinoreji, proizvodnji, prevozu in odstranjevanju vseh živali, razen če so v prehrambeni verigi po smrti in so del predelovalne industrije. Standard EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo. OPOMBA 1 Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo. OPOMBA 2 Ta metoda ustreza preskusu faze 2, stopnje 2. OPOMBA 3 Te metode ni mogoče uporabiti za vrednotenje delovanja izdelkov proti mikobakterijam.

General Information

Status
Published
Publication Date
13-Nov-2012
Current Stage
9093 - Decision to confirm - Review Enquiry
Due Date
04-Jun-2018
Completion Date
31-Jan-2019

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf nicht-porösen Oberflächen ohne mechanische Wirkung - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation de l'activité bactéricide des antiseptique et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescritions (phase 2, étape 2)Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-porous surfaces without mechanical action - Test method and requirements (phase 2, step 2)11.220VeterinarstvoVeterinary medicine11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 14349:2012SIST EN 14349:2013en,fr,de01-januar-2013SIST EN 14349:2013SLOVENSKI

STANDARDSIST EN 14349:20081DGRPHãþD
SIST EN 14349:2013
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 14349
November 2012 ICS 71.100.35 Supersedes EN 14349:2007English Version

Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-porous surfaces without mechanical action - Test method and requirements (phase 2, step 2)

Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire sur des surfaces non poreuses sans action mécanique - Méthode d'essai et prescriptions (phase 2, étape 2)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf nicht-porösen Oberflächen ohne mechanische Wirkung - Prüfverfahren und Anforderungen (Phase 2, Stufe 2) This European Standard was approved by CEN on 22 September 2012.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which 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 CEN-CENELEC 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 translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,

B-1000 Brussels © 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14349:2012: ESIST EN 14349:2013

EN 14349:2012 (E) 2 Contents Page Foreword ..............................................................................................................................................................3Introduction .........................................................................................................................................................41Scope ......................................................................................................................................................52Normative references ............................................................................................................................53Terms and definitions ...........................................................................................................................54Requirements .........................................................................................................................................55Test method ............................................................................................................................................75.1Principle ..................................................................................................................................................75.2Materials and reagents ..........................................................................................................................75.2.1Test organisms ......................................................................................................................................75.2.2Culture media and reagents .................................................................................................................85.2.3Test surface ......................................................................................................................................... 105.3Apparatus and glassware .................................................................................................................. 105.3.1General ................................................................................................................................................. 105.3.2Usual microbiological laboratory equipment .................................................................................. 115.4Preparation of test organism suspensions and product test solutions ....................................... 125.4.1Test organism suspensions (test and validation suspension) ...................................................... 125.4.2Product test solutions ........................................................................................................................ 145.5Procedure for assessing the bactericidal activity of the product ................................................. 145.5.1General ................................................................................................................................................. 145.5.2Test procedure (Dilution-neutralization method)) ........................................................................... 155.5.3Observation of test surface agar ....................................................................................................... 185.6Experimental data and calculation.................................................................................................... 185.6.1Explanation of terms and abbreviations .......................................................................................... 185.6.2Calculation ........................................................................................................................................... 195.7Verification of methodology .............................................................................................................. 225.7.1General ................................................................................................................................................. 225.7.2Control of weighted mean counts ..................................................................................................... 225.7.3Basic limits .......................................................................................................................................... 225.8Expression of results and precision ................................................................................................. 225.8.1Reduction ............................................................................................................................................ 225.8.2Control of active and non-active product test solution (5.4.2) ...................................................... 235.8.3Limiting test organism and bactericidal concentration .................................................................. 235.8.4Precision, repetitions ......................................................................................................................... 235.9Interpretation of results – conclusion .............................................................................................. 235.9.1General ................................................................................................................................................. 235.9.2Bactericidal activity for general purposes ....................................................................................... 235.9.3Qualification for certain fields of application .................................................................................. 245.10Test report ........................................................................................................................................... 24Annex A (informative)

Referenced strains in national collections .............................................................. 26Annex B (informative)

Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics ............................................................................................................ 27Annex C (informative)

Graphical representation of the method ................................................................. 29Annex D (informative)

Example of a typical test report ................................................................................ 33Bibliography ..................................................................................................................................................... 36 SIST EN 14349:2013

EN 14349:2012 (E) 3 Foreword This document (EN 14349:2012) has been prepared by Technical Committee CEN/TC 216 “Chemical disinfectants 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 identical text or by endorsement, at the latest by May 2013, and conflicting national standards shall be withdrawn at the latest by May 2013. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 14349:2007. Data obtained using the former version of EN 14349 may still be used. It was revised to correct obvious errors and ambiguities, to harmonize the structure and wording with other tests of CEN/TC 216 (existing or in preparation), and to improve the readability of the standard and thereby make it more understandable. According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 14349:2013

EN 14349:2012 (E) 4 Introduction This European Standard specifies a surface test for establishing whether a chemical disinfectant or antiseptic has bactericidal activity in the area and fields described in the scope. This laboratory test takes into account practical conditions of application of the product including contact time, temperature, test organisms and interfering substances, i.e. conditions which may influence its action in practical situations. The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each utilization concentration of the chemical disinfectant or antiseptic, found by this test corresponds to the chosen experimental conditions. However, for some applications, the instructions of use of a product may differ and therefore additional test conditions need to be used. SIST EN 14349:2013

EN 14349:2012 (E) 5 1 Scope This European Standard specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant and antiseptic products that form a homogeneous physically stable preparation when diluted with hard water, or – in the case of ready-to-use-products – with water.

This European Standard applies to products that are used in the veterinary area on non-porous surfaces without mechanical action i.e. in the breeding, husbandry, production, transport and disposal of all animals except when in the food chain following death and entry to the processing industry. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. 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 2 test. This method cannot be used to evaluate the activity of products against mycobacteria. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity EN 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885 apply. 4 Requirements The product shall demonstrate at least a 4 decimal log (lg) reduction from a water control, when tested in accordance with Table 1 and Clause 5 under simulated low level (3,0 g/l bovine albumin) or high level soiling (10 g/l yeast extract and 10 g/l bovine albumin) on a surface. SIST EN 14349:2013

EN 14349:2012 (E) 6 Table 1 - Obligatory and additional test conditions Test conditions

Bactericidal activity on non-porous surfaces without mechanical action in the veterinary area Test organism a) obligatory Enterococcus hirae Proteus vulgaris

Pseudomonas aeruginosa Staphylococcus aureus b) additional any relevant test organism Test temperature a) obligatory

10 °C + 1 °C b) additional 4 °C + 1 °C; 20 °C + 1 °C; 40 °C + 1 °C Contact time a) obligatory

30 min + 10 s b) additional 1 min + 5 s; 5 min + 10 s; 60 min + 10 s Interfering substance a) obligatory low level soiling

high level soiling
3,0 g/l bovine albumin

10 g/l yeast extract plus 10 g/l bovine albumin b) additional any relevant substance The obligatory contact time for surface disinfectants stated in Table 1 were chosen to enable comparison of standard conditions.

The recommended contact time for the use of the product is within the responsibility of the manufacturer.

NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions.

Any additional specific bactericidal activity shall be determined in accordance with 5.2.1 and 5.5.1.1 in order to take into account intended specific use conditions. SIST EN 14349:2013

EN 14349:2012 (E) 7 5 Test method 5.1 Principle A test suspension of bacteria mixed with interfering substance is inoculated onto the test surface and dried. After a drying time, 0,1 ml of the product is transferred to the surface, in a manner which covers the dried film. The surface is maintained at a specified temperature for a defined period of time specified in Clause 4 and 5.5.1.1. At the end of that contact time the surface is transferred to a neutralizer so that the action of the disinfectant is immediately neutralized. The numbers of surviving organisms which can be recovered from the surface is determined quantitatively. The number of bacteria on a surface treated with water in place of the disinfectant is also determined and the reduction is calculated. The test is performed using Enterococcus hirae, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus as test organisms (Clause 4, Table 1). Additional and optional contact times and temperatures are specified (Clause 4, Table 1). Additional interfering substances and test organisms may be used. 5.2 Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains1)

: Enterococcus hirae ATCC 10541; Proteus vulgaris ATCC 13315; Pseudomonas aeruginosa ATCC 15442; Staphylococcus aureus ATCC 6538. NOTE See Annex A for strain references in some other culture collections. The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3). The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its control and validation. If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture collection under a reference for five years.

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 European Standard and does not constitute an endorsement by CEN of the product named. SIST EN 14349:2013

EN 14349:2012 (E) 8 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this European Standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. If additional strains do not grow on the media (5.2.2.3) or cannot be used with diluent (5.2.2.4) additional media shall be used and shall be reported as well as additional incubation conditions. To improve the reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturers’ instructions relating to the preparation of these products should be rigorously followed. Ready-to-use media may be used if it complies with the required specification. For each culture medium and reagent, a time limitation for use should be fixed. 5.2.2.2 Water The water shall be freshly glass-distilled and not demineralised water. If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) may be used. Sterilize in the autoclave [5.3.2.1a)]. Sterilization is not necessary if the water is used e. g. for preparation of culture media and subsequently sterilized. NOTE The procedure to prepare hard water is described in 5.2.2.6. 5.2.2.3 Tryptone Soya Agar (TSA) Tryptone soya agar, consisting of: Tryptone, pancreatic digest of casein 15,0 g; Soya peptone, papaic digest of soybean meal 5,0 g; Sodium chloride (NaCl)

5,0 g; Agar
15,0 g; Water (5.2.2.2)

to 1 000 ml. Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ±1) °C. In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use a neutralizer that causes opalescence in the agar. SIST EN 14349:2013

EN 14349:2012 (E) 9 5.2.2.4 Diluent Tryptone sodium chloride solution, consisting of: Tryptone, pancreatic digest of casein

1,0 g; Sodium chloride (NaCl)
8,5 g; Water (5.2.2.2)

1 000 ml. Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the diluent shall be equivalent to 7,0 ± 0,2, when measured at (20 ±1) °C. 5.2.2.5 Neutralizer The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. The neutralizer shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.6 Hard water for dilution of products For the preparation of 1 l of hard water, the procedure is as follows: - prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave [5.3.2.1a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to 1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8) for no longer than one month. - prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for no longer than one week. Place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (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 (5.2.2.2). The pH of the hard water shall be

7,0 ± 0,2, when measured at (20 ±1) °C (5.3.2.4). 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 (HCl). The hard water shall be freshly prepared under aseptic conditions and used within 12 h. NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces different final water hardness in each test tube. In any case the final hardness expressed as calcium carbonate (CaCO3) is in the test tube lower than 375 mg/l. 5.2.2.7 Interfering substances 5.2.2.7.1 General 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 2 times its final concentration in the test. For the additional interfering substances, the ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids and detergents) shall be defined. SIST EN 14349:2013

EN 14349:2012 (E) 10 NOTE The term ‘interfering substance’ is used even if it contains more than one substance. 5.2.2.7.2 Low level soiling (bovine albumin solution) Dissolve 0,6 g of bovine albumin V (suitable for microbiological purposes) in 90 ml of water (5.2.2.2) in a

100 ml volumetric flask. Make up to the mark with water (5.2.2.2). Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month. The final concentration of the bovine albumin in the test procedure (5.5) is 3 g/l. 5.2.2.7.3 High level soiling (mixture of bovine albumin solution with yeast extract) a) Dissolve 10 g yeast extract powder in 150 ml of water (5.2.2.2) in a 250 ml volumetric flask (5.3.2.12) and allow foam to collapse. Make up to the mark with water (5.2.2.2). Transfer to a clean dry bottle and sterilize in the autoclave [5.3.2.1 a)]. Allow to cool to 20 °C ± 5 °C; b) pipette 25 ml of this solution into a 50 ml volumetric flask (5.3.2.12) and add 10 ml of water (5.2.2.2). Dissolve 1 g of the bovine albumin in the solution in the flask with shaking and allow foam to collapse. Make up to the mark with water (5.2.2.2), sterilize by membrane filtration (5.3.2.7) and keep in 10 ml portions in a refrigerator (5.3.2.8) and use within one month. The final concentration in the test procedure (5.5) is 10 g/l yeast extract and 10 g/l bovine albumin. 5.2.3 Test surface Stainless steel discs (2 cm diameter discs) 304 with grade 2 finish on both sides. The surfaces should be flat. The surfaces should be used only once. Prior to use the surfaces should be placed in a beaker (minimum size 50 ml) containing not less than 20 ml of 5 % Decon®2) for 60 min. Immediately rinse the discs with running freshly distilled water for 10 s. The surface shall not be allowed to dry to any extent. The discs shall only be handled with forceps. Rinse the discs with flowing water for a further 10 s to ensure complete removal of the surfactant. To supply a satisfactory flow of water, a fluid dispensing pressure vessel with suitable hose and connectors or other suitable method can be used and regulated to supply approximately 2 000 ml per min. Place the clean discs in a bath containing 95 % 2-propanol for 15 min. Remove the discs and dry by evaporation. 5.3 Apparatus and glassware 5.3.1 General Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods: a) by moist heat, in an autoclave [5.3.2.1 a)] b) by dry heat, in a hot air oven [5.3.2.1 b)]

2) Decon concentrate is obtained from Decon Laboratories Ltd, Conway Street, Hove, East Sussex, BN3 3LY UK Tel. 01273 756598. Studies have shown that this method of cleaning is satisfactory. A suitable ‘Generic’ will be specified at a later stage. SIST EN 14349:2013

EN 14349:2012 (E) 11 5.3.2 Usual microbiological laboratory equipment3) and, in particular, the following: 5.3.2.1 Apparatus for sterilization (moist and dry heat) a) for moist heat sterilization, an autoclave capable of being maintained at (30121+) °C for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at (50180+) °C for a minimum holding time of 30 min, at (170 50+) °C for a minimum holding time of 1 h or at (50160+) °C for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (4 ± 1) °C, (10 ± 1) °C, (20 ± 1) °C, (40 ± 1) °C and (45 ± 1) °C (to maintain melted TSA, 5.2.2.3, 5.5.2.2 and 5.5.2.3). 5.3.2.3 Incubator, capable of being controlled either at (36 ± 1) °C or (37 ± 1) °C (5.2.1). The same temperature shall be used for incubation performed during a test and its control and validation. 5.3.2.4 pH meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C. A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.2.2.3). 5.3.2.5 Stopwatch. 5.3.2.6 Shakers

a) Electromechanical agitator, e.g. Vortex®4) mixer ; b) Mechanical shaker.

3) Disposable sterile equipment is an acceptable alternative to reusable glassware. 4) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 14349:2013

EN 14349:2012 (E) 12 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm to 50 mm and 0,45 µm pore size for filtration of hard water (5.2.2.6) and bovine albumin (5.2.2.7.2 and 5.2.2.7.3). 5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 0,1 ml and 0,05 ml, or calibrated automatic pipettes. 5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm. 5.3.2.11 Glass beads (diameter 3 mm to 4 mm). 5.3.2.12 Volumetric flasks. 5.3.2.13 Temperature controlled cabinet, capable of being controlled at (10 ±1) °C. 5.3.2.14 Glass screw top container, with a base diameter of 4-5 cm. 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions (test and validation suspension) 5.4.1.1 Introduction Test and validation suspension are the same in this standard. 5.4.1.2 General For each test organism, one suspension has to be prepared: this is used as the bacterial “test suspension” to perform the test and the “validation suspension” to perform the controls and method validation. 5.4.1.3 Preservation and stock cultures of test organisms The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.4 Working culture of test organisms In order to prepare the working culture of the test organisms (5.2.1), prepare a subculture from the stock culture (5.4.1.3) by streaking onto TSA (5.2.2.3) slopes or plates and incubate (5.3.2.3). After 18 h to 24 h prepare a second subculture from the first subculture in the same way and incubate for 18 h to 24 h. From this second subculture, a third subculture may be produced in the same way. The second and (if produced) third subculture are the working cultures. If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used for subsequent subculturing, provided that the subculture

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