SIST EN 16437:2014
(Main)Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary area on porous surfaces without mechanical action - Test method and requirements (phase 2, step2)
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary area on porous surfaces without mechanical action - Test method and requirements (phase 2, step2)
EN 16437 specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectants 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 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".
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf 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 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.
ANMERKUNG 3 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 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 poreuses sans action mécanique, à savoir la reproduction, l'élevage, la production, le transport et l’abattage de tous les animaux, sauf au cours de la chaîne alimentaire qui suit l’abattage et l’entrée dans l’industrie de transformation.
L'EN 14885 spécifie en détail la relation entre les différents essais et 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.
NOTE 3 Cette méthode ne peut pas être utilisée pour évaluer l'activité des produits vis-à-vis des mycobactéries ou des spores bactériennes.
Kemična razkužila in antiseptiki - Kvantitativni preskus na poroznih površinah brez mehanskega delovanja za vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)
Standard EN 16437 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 ali, pri proizvodih, ki so pripravljeni za uporabo, z vodo. Ta evropski standard se uporablja za izdelke v veterini na poroznih 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. EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo.
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf 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 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 porous surfaces without mechanical action - Test method and requirements (phase 2, step2)71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposes11.220VeterinarstvoVeterinary medicineICS:Ta slovenski standard je istoveten z:EN 16437:2014SIST EN 16437:2014en,fr,de01-julij-2014SIST EN 16437:2014SLOVENSKI
STANDARD
SIST EN 16437:2014
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16437
February 2014 ICS 71.100.35 English Version
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary area on 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 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 porösen Oberflächen ohne mechanische Wirkung - Prüfverfahren und Anforderungen (Phase 2, Stufe 2) This European Standard was approved by CEN on 30 November 2013.
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
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16437:2014 ESIST EN 16437:2014
EN 16437:2014 (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 .7 5.2.1 Test organisms .7 5.2.2 Culture media and reagents .7 5.2.3 Test surface . 10 5.3 Apparatus and glassware . 10 5.3.1 General . 10 5.3.2 Usual microbiological laboratory equipment . 10 5.4 Preparation of test organism suspension and product test solutions . 11 5.4.1 Test organism suspension (test and validation suspension) . 11 5.4.2 Product test solution . 13 5.5 Procedure for assessing the bactericidal activity of the product . 13 5.5.1 General . 13 5.5.2 Test procedure (Dilution-neutralisation method) . 14 5.6 Experimental data and calculation. 17 5.6.1 Explanation of terms and abbreviations . 17 5.6.2 Calculation . 18 5.7 Verification of methodology . 21 5.7.1 General . 21 5.7.2 Control of weighted mean counts . 21 5.7.3 Basic limits . 21 5.8 Expression of results and precision . 22 5.8.1 Reduction . 22 5.8.2 Control of active and non-active product test solution (5.4.2) . 22 5.8.3 Limiting test organism and bactericidal concentration . 22 5.8.4 Precision, repetitions . 22 5.9 Interpretation of results – conclusion . 22 5.9.1 General . 22 5.9.2 Bactericidal activity for general purposes . 23 5.9.3 Qualification for certain fields of application . 23 5.10 Test report . 23 Annex A (informative)
Referenced strains in national collections . 25 Annex B (informative)
Neutralisers - Examples of neutralisers of the residual antimicrobial activity of chemical disinfectants and antiseptics . 26 Annex C (informative)
Graphical representations of dilution-neutralisation method . 28 Annex D (informative)
Example of a typical test report . 32 Bibliography . 37 SIST EN 16437:2014
EN 16437:2014 (E) 3
Foreword This document (EN 16437:2014) 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 August 2014 and conflicting national standards shall be withdrawn at the latest by August 2014. 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. 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 16437:2014
EN 16437:2014 (E) 4 Introduction This European Standard specifies a surface test for establishing whether a chemical disinfectant or antiseptic, for use on porous surfaces without mechanical action, in the veterinary area. 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 laboratory test takes into account practical conditions of application of the product including pre-drying test organisms and interfering substances on a surface, contact time and temperature, 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 utilisation 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 can differ and therefore additional test conditions need to be used. SIST EN 16437:2014
EN 16437:2014 (E) 5 1 Scope This European Standard specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectants 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 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 or bacterial spores. 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 (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) 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 soiling (3,0 g/l bovine albumin). SIST EN 16437:2014
EN 16437:2014 (E) 6 Table 1 — Obligatory and additional test conditions Test Conditions
Bactericidal activity on 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 60 min ± 10 s b) additional 1 min ± 5 s, 5 min ± 10 s, 15 min + 10 s, 30 min ± 10 s; 120 min ± 10 s Interfering substance a) obligatory
3,0 g/l bovine albumin b) additional any relevant substance NOTE 1 The obligatory contact times 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 2 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. 5 Test method 5.1 Principle A test suspension of bacteria mixed with interfering substance is inoculated onto the test surface and dried. After the drying time the test surface is immersed into a sample of the product as delivered and/or diluted with hard water (for ready to use products: water) ensuring that the test surface is totally covered for one minute. The test surface is removed from the product solution and 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 test surface is transferred to a neutraliser so that the action of the disinfectant is immediately neutralised. The bacteria are removed from the surface by ultrasound treatment. The numbers of surviving bacteria 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). SIST EN 16437:2014
EN 16437:2014 (E) 7 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 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. 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 reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturer's 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.
1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collections (ATCC). 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 16437:2014
EN 16437:2014 (E) 8 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralised water. If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) may be used. Sterilise in the autoclave [5.3.2.1 a)]. Sterilisation is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilised. NOTE See 5.2.2.6 for the procedure to prepare hard water. 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 D(+)-Glucose 2,5 g Sodium chloride (NaCl) 5,0 g Agar 15,0 g Water (5.2.2.2) to 1 000 ml Sterilise in the autoclave [5.3.2.1 a)]. After sterilisation the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at 20 °C ± 1 °C. In case of encountering problems with neutralisation (5.5.1.2 and 5.5.1.3), it may be necessary to add neutraliser to the TSA. Annex B gives guidance on the neutralisers that may be used. It is recommended not to use a neutraliser that causes opalescence in the agar. 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) to 1 000 ml Sterilise in the autoclave [5.3.2.1 a)]. After sterilisation the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at 20 °C ± 1 °C. 5.2.2.5 Neutraliser The neutraliser shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. The neutraliser shall be sterile. The neutraliser is added to diluent (5.2.2.4) and TSB (5.2.2.8). NOTE Information on neutralisers 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. Sterilise by membrane filtration (5.3.2.7) or in the autoclave [5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to 1 000 ml with water SIST EN 16437:2014
EN 16437:2014 (E) 9 (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. Sterilise 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 °C ± 1 °C. 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 a different final water hardness in each test tube. In any case the final hardness expressed as calcium carbonate (CaC03) 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, detergents) shall be defined. NOTE The term “interfering substance” is used even if it contains more than one substance. 5.2.2.7.2 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). Sterilise 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. 5.2.2.8 Tryptone Soya Broth (TSB) with neutraliser Tryptone Soya broth, consisting of: Tryptone, pancreatic digest of casein 17,0 g Soya peptone, papaic digest of soybean meal 3,0 g Sodium chloride (NaCl) 5,0 g Dipotassiumhydrogenphosphate (K2HPO4) 2,5 g Water (5.2.2.2) to 1 000 ml Sterilise in the autoclave [5.3.2.1 a)]. After sterilisation the pH of the medium shall be equivalent to 7,3 ± 0,2 when measured at 20 °C ± 1 °C. SIST EN 16437:2014
EN 16437:2014 (E) 10 An adequate neutraliser shall be added according to its chemical properties before or after autoclaving (5.2.2.5). TSB with neutraliser should be filled into glass tubes in portions of 10 ml. 5.2.3 Test surface2) Poplar wood: Size: 10mm wide, 20 mm long and 0,6 mm −1,0 mm thick with visually smooth cut edges. Cut from sliced veneer, stored at least one year before use, from untreated wood of the European poplar tree. Prior to use put the surfaces into a glass Petri dish in a single layer and sterilise in the autoclave [5.3.2.1 a)] for 15 min. Test surfaces should be kept in a sterile vessel until use. The surfaces should be used only once. 5.3 Apparatus and glassware 5.3.1 General Sterilise 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)]. 5.3.2 Usual microbiological laboratory equipment3) and in particular, the following: 5.3.2.1 Apparatus for sterilisation (moist and dry heat) a) for moist heat sterilisation, an autoclave capable of being maintained at (30121+) °C for a minimum holding time of 15 min; b) for dry heat sterilisation, a hot air oven capable of being maintained at (50180+) °C for a minimum holding time of 30 min, at (50170+) °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 bath, capable of being controlled at 4 °C ± 1 °C, 10 °C ± 1 °C, 20 °C ± 1 °C, 40 °C ± 1 °C (5.5.1) and 45 °C ± 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 at 36 °C ± 1 °C or 37 °C ± 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 °C ± 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
2) DES-IN-TEST Supply Walbrunnenstrasse D-70599 Stuttgart Tel. ++49 (0) 711 45 54 06. This information is given for the convenience of the users of this European Standard and does not constitute an endorsement by CEN of this product. 3) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 16437:2014
EN 16437:2014 (E) 11 5.3.2.6 Shakers a) Electromechanical agitator, e.g. Vortex® mixer 4); b) Mechanical shaker. 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 with filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilisation of hard water (5.2.2.6) and bovine albumin (5.2.2.7). 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 °C ± 1 °C. 5.3.2.14 Ultrasonic bath, capable of operating at a frequency 30 kHz to 55 kHz, maximal output 1 000 W. 5.4 Preparation of test organism suspension and product test solutions 5.4.1 Test organism suspension (test and validation suspension) NOTE Test and validation suspension are the same in this European Standard. 5.4.1.1 General For each test organism, one suspension shall 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. 5.4.1.2 Preservation and stock cultures of test organisms The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.3 Working culture of test organisms In order to prepare the working culture of test organisms (5.2.1), prepare a subculture from the stock culture (5.4.1.2) 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 has been kept in the incubator (5.3.2.3) during the 48 h period. In these circumstances, prepare a further 24 h subculture before proceeding. Never produce and use a fourth subculture.
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 16437:2014
EN 16437:2014 (E) 12 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. 5.4.1.4 Test suspension (“N”) a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take the working culture (5.4.1.3) and transfer loopfuls of the cells into the diluent (5.2.2.4). The cells should be suspended in the diluent by rubbing the loop against the wet wall of the f
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