Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in 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 is applicable to products for use in the veterinary area 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   Mycobacteria are the subject of a separate standard.
NOTE 2   This method corresponds to a Phase 2 Step 2 test.

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 hartem Wasser oder, im Fall gebrauchsfertiger Produkte, in Wasser eine homogene, physikalisch stabile Zubereitung bilden.
Diese Europäische Norm gilt für Produkte aus dem Veterinärbereich, 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 die Beziehungen der verschiedenen Verfahren untereinander und zu den Anwendungs¬empfehlungen im Detail fest.
ANMERKUNG 1   Mykobakterien sind Gegenstand einer eigenen Norm.
ANMERKUNG 2   Diese Methode entspricht einem Phase 2/Stufe 2-Versuch.

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 décrit une méthode d'essai et les exigences minimales relatives à l'activité bactéricide des produits désinfectants et antiseptiques chimiques qui forment une préparation homogène, physiquement stable dans l'eau dure ou dans l'eau (dans le cas de produits prêts à l'emploi).
La présente Norme européenne s’applique aux produits utilisés dans le domaine vétérinaire, c’est-à-dire pour la reproduction, l'élevage, la production, le transport et l'abattage de tous les animaux, sauf au cours de la chaîne alimentaire à la suite de leur mort et de leur entrée dans l'industrie de transformation.
EN 14885 spécifie de manière détaillée la relation entre les différents essais et les « recommandations d’utilisation ».
NOTE 1   Les mycobactéries font l'objet d'une norme distincte.
NOTE 2   La présente méthode correspond à un essai de type phase 2, étape 2.

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)

General Information

Status
Withdrawn
Publication Date
23-Oct-2007
Withdrawal Date
13-Nov-2012
Current Stage
9960 - Withdrawal effective - Withdrawal
Due Date
14-Nov-2012
Completion Date
14-Nov-2012

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfectionsmittel 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 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)Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in 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 14349:2007SIST EN 14349:2008en,fr,de01-marec-2008SIST EN 14349:2008SLOVENSKI

STANDARDSIST EN 14349:20041DGRPHãþD
SIST EN 14349:2008

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14349October 2007ICS 11.080.20Supersedes EN 14349:2004

English VersionChemical disinfectants and antiseptics - Quantitative surfacetest for the evaluation of bactericidal activity of chemicaldisinfectants and antiseptics used in veterinary area on non-porous surfaces without mechanical action - Test method andrequirements (phase 2, step 2)Antiseptiques et désinfectants chimiques - Essai quantitatifde surface pour l'évaluation de l'activité bactéricide desantiseptiques et des désinfectants chimiques utilisés dansle domaine vétérinaire sur des surfaces non poreuses sansaction mécanique - Méthode d'essai et prescriptions (phase2, étape 2)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Oberflächenversuch zur Bestimmung derbakteriziden Wirkung chemischer Desinfektionsmittel undAntiseptika für den Veterinärbereich auf nicht-porösenOberflächen ohne mechanische Wirkung - Prüfverfahrenund Anforderungen (Phase 2, Stufe 2)This European Standard was approved by CEN on 24 May 2007.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 CEN 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 CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36

B-1050 Brussels© 2007 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14349:2007: ESIST EN 14349:2008

EN 14349:2007 (E) 2 Contents Page Foreword..............................................................................................................................................................3 Introduction.........................................................................................................................................................4 1 Scope......................................................................................................................................................5 2 Normative references............................................................................................................................5 3 Terms and definitions...........................................................................................................................5 4 Requirements.........................................................................................................................................5 5 Test method............................................................................................................................................6 5.1 Principle..................................................................................................................................................6 5.2 Materials and reagents..........................................................................................................................6 5.2.1 Test organisms......................................................................................................................................6 5.2.2 Culture media and reagents.................................................................................................................6 5.2.3 Test surface............................................................................................................................................9 5.3 Apparatus and glassware.....................................................................................................................9 5.3.1 General....................................................................................................................................................9 5.3.2 Usual microbiological laboratory equipment and, in particular, the following:..............................9 5.4 Preparation of test organism suspensions and product test solutions........................................10 5.4.1 Test organism suspensions...............................................................................................................10 5.4.2 Microbial test suspension...................................................................................................................11 5.4.3 Product test solutions.........................................................................................................................11 5.5 Procedure for assessing the bactericidal activity of the product..................................................12 5.5.1 Experimental conditions (obligatory and additional).......................................................................12 5.5.2 Test procedure.....................................................................................................................................12 5.6 Calculation and expression of results...............................................................................................14 5.6.1 Calculation of viable counts (cfu/ml).................................................................................................14 5.6.2 Verification of methodology...............................................................................................................15 5.6.3 Expression of results..........................................................................................................................15 5.7 Conclusion...........................................................................................................................................15 5.8 Test report............................................................................................................................................16 Annex A (normative)

Validation of dilution neutralization..........................................................................18 A.1 Principle................................................................................................................................................18 A.2 Preparation of microbial suspension and test surface....................................................................18 A.3 Preparation of product test solution..................................................................................................18 A.4 Test for validation................................................................................................................................18 A.4.1 General..................................................................................................................................................18 A.4.2 Procedure.............................................................................................................................................18 A.4.3 Counting of the neutralizer toxicity and dilution neutralization controls......................................19 A.5 Validation..............................................................................................................................................19 Annex B (informative)

Suitable neutralizers.................................................................................................20 B.1 General..................................................................................................................................................20 B.2 Neutralizers..........................................................................................................................................20 Annex C (informative)

Example of a typical test report...............................................................................22 Annex D (informative)

Referenced strains in national collections.............................................................23 Bibliography......................................................................................................................................................24

SIST EN 14349:2008

EN 14349:2007 (E) 3 Foreword This document (EN 14349:2007) 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 April 2008, and conflicting national standards shall be withdrawn at the latest by April 2008. This document supersedes EN 14349:2004. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. SIST EN 14349:2008

EN 14349:2007 (E) 4 Introduction This European Standard specifies a surface test for establishing whether a chemical disinfectant or antiseptic in the veterinary area, on non-porous surfaces without mechanical action, has or does not have bactericidal activity under the laboratory conditions defined by this European Standard, which influence the action of disinfectants in practical use. The type and level of interfering substance can be selected as well as contact times and temperatures in addition to the levels specified in order to support recommendations for use under particular conditions. The method involves neutralization of the bactericidal activity at the moment of sampling by dilution into a previously validated neutralizer. The conditions that shall be tested are intended to cover general purposes and to allow reference between laboratories and product types. For some applications, however, the recommendations of use of a product can differ and therefore additional test conditions need to be used. SIST EN 14349:2008

EN 14349:2007 (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 is applicable to products for use in the veterinary area 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 Mycobacteria are the subject of a separate standard. NOTE 2 This method corresponds to a Phase 2 Step 2 test. 2 Normative references The following referenced document is indispensable for the application of this document. 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 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 product chemical agent or formulation used as chemical disinfectant or antiseptic 3.2 bactericide product that kills vegetative bacteria under defined conditions NOTE The adjective derived from 'bactericide' is 'bactericidal'. 3.3 bactericidal activity capability of a product to produce a reduction in the number of viable bacterial cells of relevant test organisms under defined conditions 4 Requirements The product diluted in hard water (5.2.2.6) or – in the case of ready-to-use products – with water (5.2.2.2) when tested, in accordance with Clause 5, shall demonstrate at least a lg 4 reduction in viable counts from a water control when the test organisms are Enterococcus hirae, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus. The test is carried out under simulated low level (3,0 g/l bovine albumin) or high level soiling conditions (10 g/l yeast extract and 10 g/l bovine albumin) according to its practical applications and under the required test conditions (10 °C, 30 min, 4 referenced strains). Where appropriate additional and SIST EN 14349:2008

EN 14349:2007 (E) 6 optional contact times and additional and optional temperatures in accordance with 5.2.1 and 5.5.1 are specified. 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. The surface is maintained at a specified temperature for a defined period of time. The surface is transferred to a previously validated neutralization medium so that the action of the disinfectant is immediately neutralized. The number 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 in viable counts calculated by difference. 5.2 Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains: Enterococcus hirae ATCC 105411); Proteus vulgaris ATCC 133151); Pseudomonas aeruginosa ATCC 154421); Staphylococcus aureus ATCC 65381). NOTE See Annex D for corresponding strain numbers in other culture collections. If required for specific applications, additional strains may be chosen and shall be noted in the test report. Their suitability for supplying inocula of sufficient concentration shall be verified. If the additional strains tested are not classified at a reference centre, identification characteristics shall be given. In addition they shall be held by the testing laboratory under a reference for 5 years (see EN 12353 for storage of strains). 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this European Standard refer to 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.

1) ATCC 10541, ATCC 13315, ATCC 15442 & ATCC 6538 are the collection numbers of strains supplied by the American Type Culture Collection. 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 14349:2008

EN 14349:2007 (E) 7 NOTE 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. 5.2.2.2 Water The water shall be freshly glass distilled and not demineralised water. Sterilize in the autoclave (5.3.2.1 a)). NOTE 1 If the water is sterilized during the sterilization of the reagents this is not necessary. NOTE 2 If distilled water of adequate quality is not available, water for injectable preparations (European Pharmacopoeia [1]) can be used. 5.2.2.3 Tryptone Soya Agar (TSA) For maintenance of bacterial strains and performance of viable counts: 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. 5.2.2.4 Diluent Tryptone Sodium Chloride solution: Tryptone, pancreatic digest of casein 1,0 g ; Sodium chloride (NaCl) 8,5 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 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 under test in accordance with Annex A. 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: SIST EN 14349:2008

EN 14349:2007 (E) 8  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 (5.3.2.1 c)) or in the autoclave (5.3.2.1 a)). Store the solution in the refrigerator (5.3.2.13) for no longer than one month; NOTE 1 In the case of loss of volume during sterilization by autoclave, make up solution to 1 000 ml with water (5.2.2.2) under aseptic conditions before storage.  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 (5.3.2.1 c)). Store the solution in the refrigerator (5.3.2.13) 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 (5.3.2.11) 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 2 When preparing the product test solutions (5.4.3), the addition of the product to the hard water produces different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube. 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. 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. 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 (Cohn fraction V for Dubos Medium) 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 (5.3.2.1 c)). Keep in a refrigerator (5.3.2.13) and use within one month. The final concentration of the bovine albumin in the test procedure (5.5.2) 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 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 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 and keep in 10 ml portions in a refrigerator (5.3.2.13) and use within one month. SIST EN 14349:2008

EN 14349:2007 (E) 9 The final concentration in the test procedure (5.5.2) 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 % Decon2) 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) moist heat, in an autoclave (5.3.2.1 a)); b) dry heat, in a hot air oven (5.3.2.1 b)). 5.3.2 Usual microbiological laboratory equipment3) and, in particular, the following: 5.3.2.1 Apparatus for sterilization a) For moist heat sterilization, an autoclave capable of being maintained at (121 30+) °C for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at (18050+) °C for a minimum holding time of 30 min, at (17050+) °C for a minimum holding time of 1 h or at (16050+) °C for a minimum holding time of 2 h; c) for media sterilization use suitable membrane filtration apparatus with filters of diameter 47 mm to 50 mm of 0,22 µm pore size. 5.3.2.2 Water baths, capable of being controlled at 4 °C ± 1 °C, 10 °C ± 1 °C, 20 °C ± 1 °C, 40 °C ± 1 °C and 45 °C ± 1 °C. 5.3.2.3 Incubator, capable of being controlled at 36 °C ± 1 °C or 37 °C ± 1 °C. An incubator at 37 °C ± 1 °C may be used if an incubator at 36 °C ± 1 °C is not available.

2) Decon® concentrate is obtained from Decon Laboratories Ltd, Convway St, Hovem BN3 3Lym UK Tel. 01273 756598. Studies have shown that this method of cleaning is satisfactory. A suitable ‘Generic’ will be specified at a later stage. 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. 3) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 14349:2008

EN 14349:2007 (E) 10 5.3.2.4 pH meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C. 5.3.2.5 Stopwatch. 5.3.2.6 Electromechanical agitator, e.g. Vortex ® mixer4). 5.3.2.7 Containers, test tubes flasks or bottles of suitable capacity. 5.3.2.8 Graduated pipettes, of nominal capacities 10 ml and 0,1 ml. Calibrated automatic pipettes may be used. 5.3.2.9 Petri dishes, (plates) of size 90 mm to 100 mm. 5.3.2.10 Glass beads, diameter: 3 mm to 4 mm. 5.3.2.11 Volumetric flasks. 5.3.2.12 Mechanical shaker. 5.3.2.13 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.3.2.14 Temperature controlled cabinet, capable of being controlled at 10 °C ± 1 °C. 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions 5.4.1.1 Preservation and stock cultures of test organisms The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.2 Working culture of test organisms In order to prepare the working culture of strains (5.2.1), prepare a subculture from the stock culture (5.4.1.1) by streaking onto TSA slopes (5.2.2.3) 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. NOTE The second and/or third cultures are the working culture(s). If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used for subsequent sub-culturing, provided that the subculture has been kept in the incubator during the 48 h period. In these circumstances, prepare a further 24 h subculture before proceeding. Do not take a fourth subculture. For additional strains, any departure from this method of culturing the bacteria or preparing the suspensions shall be noted, giving the reasons in the test report. 5.4.1.3 Preparation of bacterial test suspension Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.10). Take the working cultures (5.4.1.2) and transfer loopfuls of the cells into the diluent. The cells should be suspended in the diluent by rubbing the loop against the wet wall of the flask to dislodge the cells before immersing in the diluent. Shake the flask for 3 min using a mechanical shaker (5.3.2.12). Aspirate the suspension from the

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 14349:2008

EN 14349:2007 (E) 11 glass beads and transfer to a tube. Adjust the number of cells in the suspension to 1,5 × 109 cfu/ml5) to 5 × 109

cfu/ml using the diluent, estimating the number of cfu/ml by any suitable means. Maintain this suspension in the water bath at 20 °C ± 1 °C and use within 2 h. 5.4.1.4 Counting of bacterial test suspensions For counting of the bacterial test suspension prepare 10-7 and 10-8 dilutions of the test suspensions (5.4.1.3) using diluent (5.2.2.4). Mix with an electromechanical agitator (5.3.2.6). Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or spread plate technique. When using: a) pour plate technique, pipette each 1,0 ml sample in separate Petri dishes and add 12 ml to 15 ml melted TSA (5.2.2.3), cooled to (45 ± 1) °C ; b) spread plate technique, spread each 1,0 ml sample on an appropriate number of surface dried plates containing TSA (5.2.2.3). Incubate (5.3.2.3) the plates for 24 h. Discard any plates which are not countable. Count the plates and determine the number of colony forming units. Incubate the plates for a further 24 h. Do not recount plates which no longer show well-separated colonies. Recount the remaining plates. Determine the higher number of colonies Vc for each plate. Calculate N, the lg10

number of cfu per 0,05 ml of the test suspension using the equation given in 5.6.1.1. 5.4.2 Microbial test suspension 2 min before the commencement of the test (5.5.2), add 1 ml of the bacterial suspension (5.4.1.3) to 1 ml of the interfering substance (5.2.2.7) to be used in the test procedure. Immediately before addition, the microbial suspension should be well mixed to fully re-suspend the organisms. 5.4.3 Product test solutions Product test solutions shall be prepared in hard water (5.2.2.6) at minimum three different concentrations to include one concentration in the active range and one concentration in the non-active range (5.8). The product as received may be used as one of the product test solutions. Dilutions of ready-to-use products, i.e. products that are not diluted when applied, shall be prepared in water (5.2.2.2). For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in a volumetric flask and filling up with hard water (5.2.2.6). Subsequent dilutions (lower concentrations) shall be prepared in volumetric flasks (5.3.2.11) on a volume/volume basis in hard water (5.2.2.6). For liquid products, dilutions of the product shall be prepared with hard wa

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