EN 14349:2012

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: Withdrawn
• Publication Date: 13-Nov-2012
• Withdrawal Date: 20-May-2025
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: CEN/TC 216 - Chemical disinfectants and antiseptics
• Drafting Committee: CEN/TC 216/WG 2 - Veterinary use
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 31-Jan-2019
• Completion Date: 21-May-2025
• Ref Project: SIST EN 14349:2013 - 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)

Relations

• Replaces: EN 14349:2007 - 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)
• Effective Date: 11-Dec-2010
• Replaced By: EN 14349:2025 - 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)
• Effective Date: 19-Jan-2023

Standards Content (Sample)

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

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

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

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

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

: 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

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

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 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

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. [phone]. Studies have shown that this method of cleaning is satisfactory. A suitable ‘Generic’ will be specified at a later stage. SIST EN 14349:2013

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

Never produce and use 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. SIST EN 14349:2013

to 5,0 x 109 cfu/ml5) using diluent (5.2.2.4), estimating the number of cfu/ml by any suitable means. Maintain this suspension in the water bath at (20 ± 1) °C and use within 2 h. Adjust the temperature according to [5.5.1.1 a)] and 5.5.1.4 only immediately before the start of the test. The use of spectrophotometer for adjusting the number of cells is highly recommended (about 620 nm wavelength – cuvette 10 mm path length). Each laboratory should therefore produce calibration data for each test organism knowing that suitable values of optical density are generally found between 0,150 and 0,460. To achieve reproducible results of this measurement it may be necessary to dilute the test suspension, e.g. 1+9.
NOTE A colorimeter is a suitable alternative. c) For counting of the bacterial test suspension prepare 10-7 and 10-8 dilutions of the test suspension using diluent (5.2.2.4). Mix [5.3.2.6 a)].
Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or spread plate technique. 1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add 15 ml to 20 ml melted TSA (5.2.2.3), cooled to (45 ± 1) °C; 2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of approximately equal size – on an appropriate number (at least two) of surface dried plates containing TSA (5.2.2.3). For incubation and counting see 5.4.1.6. 5.4.1.6 Incubation and counting of the test and the validation suspensions a) Incubate (5.3.2.3) the plates for 20 h - 24 h. Discard any plates that are not countable for any reason. Count the plates and determine the total number of cfu. Incubate the plates for a further 20 h - 24 h. Do not recount plates that no longer show well-separated colonies. Recount the remaining plates. If the number has increased, use only the higher number for further evaluation. b) Note for each plate the exact number of colonies but record >330 for any counts higher than 330 and determine the VC values according to 5.6.2.2. c) Calculate the numbers of cfu per 0,025 ml in the test suspension N and the validation suspension Nv using the methods given in 5.6.2.3. Verify according to 5.7. NOTE 0,05 ml of an equal parts mixture of the test suspension and interfering substance is added to the surface therefore 0,025 ml of the suspension is added.
5) cfu/ml = colony forming unit(s) per millilitre SIST EN 14349:2013

The concentration of the product stated in the test report shall be the desired test concentration. Record the test concentration in terms of mass per volume or volume per volume and details of the product sample as received. 5.5 Procedure for assessing the bactericidal activity of the product 5.5.1 General 5.5.1.1 Experimental conditions (obligatory and additional) Besides the obligatory temperature, contact time, interfering substances and test organisms additional experimental conditions (including test organisms) may be selected according to the practical use considered for the product (Clause 4): a)
temperature (in °C): The obligatory and additional temperatures to be tested are specified in Clause 4, Table 1. The allowed deviation for each chosen temperature is ± 1 °C. b) contact time t (in min): The obligatory and additional contact times to be tested are specified in Clause 4, Table 1. The allowed deviation for each chosen time is ± 10 s, except for 1 min for which it is ± 5 s. c) interfering substance: The obligatory interfering substance to be tested is 3,0 g/l bovine albumin (5.2.2.7.2) for low level soiling or 10 g/l bovine albumin plus 10 g/l yeast extract (5.2.2.7.3) for high level soiling according to Clause 4, Table 1 and practical applications. Additional interfering substances may be tested according to specific fields of application. d) test organisms: Enterococcus hirae, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus (Clause 4, Table 1 and 5.2.1). Additional test organisms may be tested. SIST EN 14349:2013

To determine a suitable neutralizer carry out the validation of the dilution-neutralization method (5.5.2.4 and 5.5.2.5 in connection with 5.5.2.6) using a suitable neutralizer, chosen according to laboratory experience and published data. If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into account the information given in Annex B. In special circumstances it may be necessary to add neutralizer to TSA (5.2.2.3). 5.5.1.3 General instruction for validation and control procedures The neutralization and/or removal of the bactericidal and/or bacteriostatic activity of the product shall be controlled and validated - only for the highest product test concentration - for each of the used test organisms and for each experimental condition (interfering substance, temperature, contact time). These procedures (water control, neutralizer control and method validation) shall be performed at the same time with the test and with the same neutralizer used in the test. If because of problems with neutralization a neutralizer has been added to TSA (5.5.1.2) used for the validation and control procedures the TSA used for the test shall contain the same amount of this neutralizer as well. 5.5.1.4 Equilibration of temperature Prior to testing, equilibrate the inoculated and dried stainless steel discs and all reagents (product test solutions (5.4.2), hard water (5.2.2.6) to the test temperature of
[5.5.1.1 a)] using the water bath (5.3.2.2) and/or the temperature controlled cabinet (5.3.2.13) controlled at . Check that the temperature of the reagents is stabilized at . The neutralizer (5.2.2.5) and diluent (5.2.2.4) shall be equilibrated at a temperature of (20 ± 1) °C. 5.5.2 Test procedure (Dilution-neutralization method)6)
5.5.2.1 General The test and the control and validation procedures (5.5.2.2, 5.5.2.3, 5.5.2.4 and 5.5.2.5) shall be carried out at the same time. 5.5.2.2 Test “Na” – Determination of bactericidal concentrations
The procedure for determining bactericidal concentrations is as follows: a) To prepare the microbial test suspension pipette 1,0 ml of the interfering substance (5.2.2.7) into a tube. Add 1,0 ml of the test suspension (5.4.1.5). Start the stopwatch (5.3.2.5) immediately, mix [5.3.2.6 a)] and place the tube in a water bath or temperature controlled cabinet at the chosen test temperature
[5.5.1.1 a)] for 2 min ± 10 s. Immediately before addition, the test suspension should be well mixed to fully re-suspend the organisms. b) Place the test surfaces (5.2.3) in a sterile Petri dish (5.3.2.10) and ensure that the dish is in a horizontal position. Prepare the test surfaces by inoculating 0,05 ml of the microbial test suspension [5.5.2.2 a)] on to each test surface. The inoculum may not be spread over the surface. Dry the surfaces at 37 °C until they are visibly dry.
6) For a graphical representation of this method see C.1 SIST EN 14349:2013

c) For the disinfectant test (Na) pipette 0,1 ml of each product test solution (5.4.2) to be tested on to separate dried surfaces ensuring that the dried inoculum is totally covered by the test product. Place the surfaces in a temperature controlled cabinet (5.3.2.13) at the chosen test temperature
and contact time t. d) At the end of t, transfer each of the surfaces (Na) to a separate container (5.3.2.14) containing 10 ml of neutralizer (5.2.2.5) together with sufficient glass beads (5.3.2.11) to support the surface. The surfaces should be placed with the inoculated surface downwards in contact with the beads. Place the containers in a horizontal shaking device [5.3.2.6 b)] or place on a horizontal surface and shake in a horizontal manner by hand for 5 min ± 10 s. The shaking should be sufficiently vigorous to ensure that the test surface moves constantly over the beads. Ensure that the beads are able to move freely. After the neutralization time of 5 min ± 10 s prepare a series of ten-fold dilutions from 10-1 to 10-2 of the neutralized mixture in the diluent (5.2.2.4). Take a 1,0 ml sample of the neutralized mixture and each of the dilutions in duplicate and inoculate using pour plate or spread plate technique. 1) When using the pour plate technique, pipette each 1,0 ml sample into separate Petri dishes and add 15 ml to 20 ml of melted TSA (5.2.2.3), cooled to (45 ± 1) °C. 2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of approximately equal size – on an appropriate number (at least two) of surface dried plates containing TSA (5.2.2.3). For incubation and counting, see 5.5.2.6. e) Recover the test surface (“Na”), let the neutralizer drain off and rinse with 10 ml of water (5.2.2.2). Transfer to a Petri dish (5.3.2.10) containing 10 ml of solidified TSA (5.2.2.3) and place on top of the agar test side uppermost. Add 10 ml of TSA (5.2.2.3) melted and cooled to 45 °C. f) Perform the procedure a) to e) using the other product test solutions at the same time. g) Perform the procedure a) to f) applying the other obligatory and – if appropriate – other additional experimental conditions (5.5.1.1). 5.5.2.3 Water control “Nw” The procedure for determining the water control is as follows: NOTE The control A - Experimental conditions control A (Validation of the selected experimental conditions or verification of the absence of any lethal effect in the test conditions) differs from other standard due to the fact it is directly determined in the water control “Nw”. a) Place one test surface (5.2.3) in a sterile Petri dish (5.3.2.10) and ensure that the dish is in a horizontal position. Inoculate 0,05 ml of the microbial test suspension [5.5.2.2 a)] on to the test surface. The inoculum may not be spread over the surface. Dry the surface at 37 °C until it is visibly dry [5.5.2.2 b)]. b) For the water control (Nw), pipette 0,1 ml of hard water (5.2.2.6) or water (5.2.2.2) in the case of ready-to-use products on to the test surface ensuring that the dried inoculum is totally covered by the water.
Place the surface in a temperature controlled cabinet at the chosen test temperature of (10 ± 1) °C (5.3.2.13). SIST EN 14349:2013

(20 ± 1) °C. c) Transfer the inoculated and dried test surface into the container and place the inoculated surface downwards in contact with the beads (5.3.2.11). Place the container in a horizontal shaking device [5.3.2.6 b)] or place on a horizontal surface and shake in a horizontal manner by hand for 5 min ± 10 s. The shaking should be sufficiently vigorous to ensure that the test surface moves constantly over the beads. Ensure that the beads are able to move freely and that there are sufficient beads to support the surface. d) After a neutralization time of 5 min ± 10 s prepare a s
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