SIST EN 16438:2014
(Main)Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-porous surfaces without mechanical action - Test method and requirements (phase 2, step2)
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-porous surfaces without mechanical action - Test method and requirements (phase 2, step2)
EN 16438 specifies a test method and the minimum requirements for fungicidal or yeasticidal activity of chemical disinfectant and antiseptic products that form a homogeneous physically stable preparation in hard water or - in the case of ready-to-use products- with water. This European Standard applies 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".
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der fungiziden oder levuroziden 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 fungizide oder levurozide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die im Wasser standardisierter Härte oder — im Fall gebrauchsfertiger Produkte — im Wasser eine homogene, physikalisch stabile Zubereitung bilden.
Diese Europäische Norm gilt für Produkte für die Anwendung im 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 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.
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire sur des surfaces non poreuses sans action mécanique (phase 2, étape 2)
La présente Norme européenne spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité fongicide ou levuricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans l’eau dure ou — dans le cas de produits prêts à l’emploi — dans l’eau.
La présente Norme européenne s'applique aux produits destinés à être utilisés dans le domaine vétérinaire, à savoir la reproduction, l'élevage, la production, le transport et l’abattage de tous les animaux, sauf au cours hors 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.
Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah brez mehanskega delovanja za vrednotenje fungicidnega delovanja ali delovanja kemičnih razkužil in antiseptikov na kvasovke v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)
EN 16438 določa preskusno metodo in minimalne zahteve za fungicidno delovanje ali delovanje kemičnih razkužil in antiseptikov na kvasovke, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma, pri proizvodih, ki so pripravljeni za uporabo, z vodo. Ta evropski standard se uporablja za izdelke v veterini, 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.
General Information
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 fungiziden oder levuroziden 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 fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire sur des surfaces non poreuses sans action mécanique (phase 2, étape 2)Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-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 16438:2014SIST EN 16438:2014en,fr,de01-julij-2014SIST EN 16438:2014SLOVENSKI
STANDARD
SIST EN 16438:2014
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16438
February 2014 ICS 71.100.35 English Version
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of fungicidal or yeasticidal 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 fongicide ou levuricide 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 fungiziden oder levuroziden 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 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 16438:2014 ESIST EN 16438:2014
EN 16438:2014 (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 organism .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 suspensions and product test solutions ....................................... 11 5.4.1 Test organism suspension (test and validation suspension) ........................................................ 11 5.4.2 Product test solutions ........................................................................................................................ 15 5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product ............................ 16 5.5.1 General ................................................................................................................................................. 16 5.5.2 Test procedure (Dilution-neutralisation method) ............................................................................ 17 5.5.3 Observation of the test surface agar ................................................................................................ 20 5.6 Experimental data and calculation.................................................................................................... 20 5.6.1 Explanation of terms and abbreviations .......................................................................................... 20 5.6.2 Calculation ........................................................................................................................................... 21 5.7 Verification of methodology .............................................................................................................. 24 5.7.1 General ................................................................................................................................................. 24 5.7.2 Control of weighted mean counts ..................................................................................................... 24 5.7.3 Basic limits .......................................................................................................................................... 24 5.7.4 Microscopic observation ................................................................................................................... 24 5.8 Expression of results and precision ................................................................................................. 24 5.8.1 Reduction ............................................................................................................................................ 24 5.8.2 Control of active and non-active product test solution (5.4.2) ...................................................... 25 5.8.3 Fungicidal or yeasticidal concentration ........................................................................................... 25 5.8.4 Precision, repetitions ......................................................................................................................... 25 5.9 Interpretation of results - conclusion ............................................................................................... 25 5.9.1 General ................................................................................................................................................. 25 5.9.2 Fungicidal or yeasticidal activity for general purposes ................................................................. 26 5.9.3 Qualification for certain fields of application .................................................................................. 26 5.10 Test report ........................................................................................................................................... 26 Annex A (informative)
Referenced strains of national collections . 28 Annex B (informative)
Examples of neutralisers of the residual antimicrobial activity of chemical disinfectants and antiseptics . 29 Annex C (informative)
Graphical representations of dilution-neutralisation method . 31 Annex D (informative)
Example of a typical test report . 35 Bibliography . 39 SIST EN 16438:2014
EN 16438:2014 (E) 3
Foreword This document (EN 16438:2014) 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 16438:2014
EN 16438:2014 (E) 4 Introduction This European Standard specifies a surface test for establishing whether a chemical disinfectant or antiseptic has or does not have fungicidal or yeasticidal activity in the fields described in the scope. This laboratory test takes into account practical conditions of application of the product including contact time, temperature, test organisms and interfering substances, i.e. conditions which may influence its action in practical situations. The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each 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 may differ and therefore additional test conditions need to be used. SIST EN 16438:2014
EN 16438:2014 (E) 5 1 Scope This European Standard specifies a test method and the minimum requirements for fungicidal or yeasticidal activity of chemical disinfectant and antiseptic products that form a homogeneous physically stable preparation in hard water or – in the case of ready-to-use products– with water. This European Standard applies 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 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. 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 ISO 4793, Laboratory sintered (fritted) filters — Porosity grading, classification and designation 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 3 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 16438:2014
EN 16438:2014 (E) 6 Table 1 — Obligatory and additional test conditions Test Conditions
Fungicidal or yeasticidal activity on non-porous surfaces without mechanical action in the veterinary area Test organism Fungicidal activity a) obligatory Aspergillus brasiliensis (conidiospores) and Candida albicans (vegetative cells) Test organism Yeasticidal activity a) obligatory Candida albicans (vegetative cells) 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* 5 min ± 10 s; 30 min ± 10 s; 120 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 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 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 fungicidal 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 fungal conidiospores or yeast and interfering substance is inoculated onto the test surface and dried. After a drying time, an aliquot of the product under test is transferred to the surface, in a manner which covers the dried film. The surface is maintained at a specified temperature for a defined period of time specified in Clause 4 and 5.5.1.1. At the end of that contact time the surface is transferred to a neutraliser so that the action of the disinfectant is immediately neutralised. The numbers of surviving organisms which can be recovered from the surface is determined quantitatively. The number of fungi or yeast on a surface treated with water in place of the disinfectant is also determined and the reduction is calculated. The test is performed using the conidiospores of Aspergillus brasiliensis (formerly niger) and the vegetative cells of Candida albicans (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal SIST EN 16438:2014
EN 16438:2014 (E) 7 activity) as test organisms (Clause 4, Table 1). Additional and optional contact times and temperatures are specified (Clause 4, Table 1). Additional interfering substances and test organisms may be used. 5.2 Materials and reagents 5.2.1 Test organism The fungicidal activity shall be evaluated using the following strains as the test organisms1): Aspergillus brasiliensis (formerly Aspergillus niger) ATCC 16404 Candida albicans ATCC 10231 The yeasticidal activity shall be evaluated using only Candida albicans. NOTE See Annex A for strain references in some other culture collections. The required incubation temperature for these organisms is 30 °C ± 1 °C (5.3.2.3). The same temperature 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 organism. 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 medium may be used if it complies with the required specification. For each culture medium and reagent a limitation for use should be fixed.
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 16438:2014
EN 16438:2014 (E) 8 5.2.2.2 Water The water shall be freshly glass-distilled and not demineralised water. Sterilise in the autoclave [5.3.2.1 a)]. NOTE 1 Sterilisation is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilised. NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. NOTE 3 See 5.2.2.6 for the procedure to prepare hard water. 5.2.2.3 Malt extract agar (MEA) Malt extract agar, consisting of: Malt extract* 30,0 g Agar 15,0 g Water (5.2.2.2) to 1 000 ml *The malt extract should be food grade (e.g. Cristomalt powder from Difal) or equivalent that is not highly purified and not only based on maltose (Malt extract from OXOID)2). However if there are problems producing at least 75 % spiny spores see 5.4.1.4.2. Sterilise in the autoclave [5.3.2.1a)]. After sterilisation, the pH of the medium shall be equivalent to 5,6 ± 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 MEA. 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. It shall be sterile. NOTE Information on neutralisers that have been found to be suitable for some categories of products is given in Annex B.
2) 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. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16438:2014
EN 16438:2014 (E) 9 5.2.2.6 Hard water for dilution of products For the preparation of 1 000 ml 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 (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 (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 a different final water hardness in each test tube. In any case, the final hardness is lower than 375 mg/l of calcium carbonate (CaCO3) in the test tube. 5.2.2.7 Interfering substance 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. NOTE The term ‘interfering substance’ is used even if it contains more than one substance. 5.2.2.7.2 Low level soiling (bovine albumin solution) Dissolve 0,6 g of bovine albumin V (suitable for microbiological purposes) in 90 ml of water (5.2.2.2) in a 100 ml volumetric flask. Make up to the mark with water (5.2.2.2). Sterilise by membrane filtration (5.3.2.7) keep in the refrigerator (5.3.2.8) 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) 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 sterilise in an autoclave [5.3.2.1 a)]. Allow to cool to 20 °C ± 5 °C. 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 bovine albumin fraction V (suitable for microbiological purposes) in the solution with shaking and allow foam to SIST EN 16438:2014
EN 16438:2014 (E) 10 collapse. Make up to the mark with water (5.2.2.2) sterilise by membrane filtration and keep in a refrigerator (5.3.2.8) and use within one month. 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 % Decon®3) 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 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 equipment 4) 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 121 30+°C for a minimum holding time of 15 min; b) for dry heat sterilisation, a hot air oven capable of being maintained at 180 50+°C for a minimum holding time of 30 min, at 170 50+ C for a minimum holding time of 1 h or at 160 50+ C for a minimum holding time of 2 h; 5.3.2.2 Water bath, capable of being controlled at 10 °C ± 1 °C, 20 °C ± 1 °C, and 45 °C ± 1 °C (to maintain melted MEA. If pour plate technique is used), and at additional temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled at 30 °C ± 1 °C and 37 °C ± 1 °C (for drying surfaces) 5.3.2.4 pH-meter, having an accuracy of calibration of 0,1 pH units at 20 °C ± 1 °C.
3) Decon concentrate is obtained from Decon Laboratories Ltd, Conway Street, Hove, East Sussex, BN3 3LY, UK Tel. 01273 756598. Studies have shown that this method of cleaning is satisfactory. A suitable ‘Generic’ will be specified at a later stage. 4) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 16438:2014
EN 16438:2014 (E) 11 A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.2.2.3). 5.3.2.5 Stopwatch 5.3.2.6 Shakers a) Electromechanical agitator, e.g. Vortex® 5) mixer; 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 filtration of hard water (5.2.2.6) and bovine albumin (5.2.2.7.2, 5.2.2.7.3). 5.3.2.8 Refrigerator capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 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 Glass screw top container, with a base diameter of 4 cm - 5 cm. 5.3.2.15 Fritted filter, with porosity of 40 µm to 100 µm according to ISO 4793. 5.3.2.16 Flasks with vented caps. 5.3.2.17 Microscope capable of x 400 magnification. 5.3.2.18 Vacuum desicator capable of achieving a vacuum of 20-25 in. mercury. 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspension (test and validation suspension) NOTE Test and validation suspension are the same in this standard. 5.4.1.1 General For each test organism, one suspension shall be prepared: this is used as the fungal “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) Vortex ® is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 16438:2014
EN 16438:2014 (E) 12 5.4.1.3 Working culture of test organisms 5.4.1.3.1 Candida albicans (yeast) In order to prepare the working culture
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Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of
fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the
veterinary area on non-porous surfaces without mechanical action - (phase 2, step 2)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur
Bestimmung der fungiziden oder levuroziden 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 fongicide ou levuricide des antiseptiques et des désinfectants chimiques
utilisés dans le domaine vétérinaire sur des surfaces non poreuses sans action
mécanique (phase 2, étape 2)
Ta slovenski standard je istoveten z: prEN 16438
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
oSIST prEN 16438:2012 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 16438:2012
EUROPEAN STANDARD
DRAFT
prEN 16438
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2012
ICS 71.100.35
English Version
Chemical disinfectants and antiseptics - Quantitative surface
test for the evaluation of fungicidal or yeasticidal activity of
chemical disinfectants and antiseptics used in the veterinary
area on non-porous surfaces without mechanical action - (phase
2, step 2)
Antiseptiques et désinfectants chimiques - Essai quantitatif Chemische Desinfektionsmittel und Antiseptika -
de surface pour l'évaluation de l'activité fongicide ou Quantitativer Oberflächenversuch zur Bestimmung der
levuricide des antiseptiques et des désinfectants chimiques fungiziden oder levuroziden Wirkung chemischer
utilisés dans le domaine vétérinaire sur des surfaces non Desinfektionsmittel und Antiseptika für den
poreuses sans action mécanique - (phase 2, étape 2) Veterinärbereich auf nicht-porösen Oberflächen ohne
mechanische Wirkung - Prüfverfahren und Anforderungen
(Phase 2, Stufe 2)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 216.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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, 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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
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 Ref. No. prEN 16438:2012: E
worldwide for CEN national Members.
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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 organism .7
5.2.2 Culture media and reagents .7
5.2.3 Test surface .9
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 suspensions and product test solutions . 11
5.4.1 Test organism suspension (test and validation suspension) . 11
5.4.2 Product test solutions . 15
5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product . 15
5.5.1 General . 15
5.5.2 Test procedure (Dilution-neutralization method) . 16
5.5.3 Observation of the test surface agar . 19
5.6 Experimental data and calculation. 19
5.6.1 Explanation of terms and abbreviations . 19
5.6.2 Calculation . 20
5.7 Verification of methodology . 23
5.7.1 General . 23
5.7.2 Control of weighted mean counts . 23
5.7.3 Basic limits . 23
5.7.4 Microscopic observation . 23
5.8 Expression of results and precision . 24
5.8.1 Reduction . 24
5.8.2 Fungicidal or yeasticidal concentration . 24
5.8.3 Precision, repetitions . 24
5.9 Interpretation of results - conclusion . 24
5.9.1 General . 24
5.9.2 Fungicidal or yeasticidal activity for general purposes . 25
5.9.3 Qualification for certain fields of application . 25
5.10 Test report . 25
Annex A (informative) Referenced strains of national collections . 27
Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of chemical
disinfectants and antiseptics . 28
Annex C (informative) Graphical representations of dilution-neutralization method . 29
Annex D (informative) Example of a typical test report . 33
Bibliography . 37
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Foreword
This document (prEN 16438:2012) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
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Introduction
This European Standard specifies a surface test for establishing whether a chemical disinfectant or antiseptic
has fungicidal or yeasticidal activity in the fields described in the scope.
This laboratory test takes into account practical conditions of application of the product including contact time,
temperature, test organisms and interfering substances, i.e. conditions which may influence its action in
practical situations.
The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each utilization concentration of the chemical disinfectant or antiseptic, found by this test
corresponds to the chosen experimental conditions. However, for some applications the instructions of use of
a product may differ and therefore additional test conditions need to be used.
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1 Scope
This European Standard specifies a test method and the minimum requirements for fungicidal or yeasticidal
activity of chemical disinfectant and antiseptic products that form a homogeneous physically stable
preparation in hard water or – in the case of ready-to-use products– with water.
This European Standard applies 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 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.
2 Normative references
The following referenced documents are 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
EN 14885 Chemical disinfectants and antiseptics - Application of European Standards for chemical
disinfectants and antiseptics
ISO 4793 Laboratory sintered (fritted) filters – Porosity grading, classification and designation
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 3 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.
Table 1 Obligatory and additional test conditions
Test Conditions Fungicidal or yeasticidal activity on non-
porous surfaces without mechanical action
in the veterinary area
Test organism Fungicidal activity Aspergillus brasiliensis (formerly niger)
a) obligatory Candida albicans
Test organism Yeasticidal activity Candida albicans
a) obligatory
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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* 5 min + 10 s; 30 min + 10 s; 120 min + 10 s
Interfering substance
a) obligatory
low level soiling 3,0 g/l bovine albumin
high level soiling 10 g/l yeast extract plus 10 g/l bovine albumin
b) additional any relevant substance
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 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 fungicidal 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 fungal spores or yeast and interfering substance is inoculated onto the test surface and
dried. After a drying time, an aliquot of the product under test is transferred to the surface, in a manner which
covers the dried film. The surface is maintained at a specified temperature for a defined period of time
specified in Clause 4 and 5.5.1.1. At the end of that contact time the surface is transferred to a neutralizer so
that the action of the disinfectant is immediately neutralized. The numbers of surviving organisms which can
be recovered from the surface is determined quantitatively.
The number of fungi or yeast on a surface treated with water in place of the disinfectant is also determined
and the reduction is calculated.
The test is performed using Aspergillus brasiliensis and Candida albicans as test organisms (clause 4, table 1).
Additional and optional contact times and temperatures are specified (clause 4, table 1). Additional interfering
substances and test organisms may be used.
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5.2 Materials and reagents
5.2.1 Test organism
1)
The fungicidal activity shall be evaluated using the following strains as the test organisms :
Aspergillus brasiliensis (formerly niger) ATCC 16404
Candida albicans ATCC 10231
The yeasticidal activity shall be evaluated using only Candida albicans.
NOTE See annex A for strain references in some other culture collections.
The required incubation temperature for these organisms is (30 ± 1)°C (5.3.2.3). The same temperature 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 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 organism.
NOTE 1 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.
NOTE 2 Ready-to-use media may be used if it complies with the required specification.
NOTE 3 For each culture medium and reagent a limitation for use should be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled and not demineralised water.
Sterilize in the autoclave [5.3.2.1a)].
NOTE 1 Sterilization is not necessary if the water is used e. g. for preparation of culture media and subsequently
sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can
be used.
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.
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NOTE 3 See 5.2.2.6 for the procedure to prepare hard water.
5.2.2.3 Malt extract agar (MEA)
Malt extract agar, consisting of:
Malt extract* 30,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000 ml
*The malt extract should be food grade (Cristomalt powder from Difal is recommended) or equivalent that is
2)
not highly purified and not only based on maltose (Malt extract from OXOID is recommended) .
Sterilize in the autoclave [5.3.2.1a)]. After sterilization, the pH of the medium shall be equivalent to 5,6 ± 0,2
when measured at (20 ± 1) °C.
NOTE 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 MEA. 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.
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
Sterilize in the autoclave [5.3.2.1a)]. 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. it
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 1 000 ml of hard water, the procedure is as follows:
prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride (CaCl ) in
2 2
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.
2) 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. Equivalent products may be used if they can be shown to lead to the same
results.
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prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to 1 000
3
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 hours.
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 is lower than 375 mg/l of calcium carbonate
(CaCO ) in the test tube.
3
5.2.2.7 Interfering substance
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.
NOTE The term ‘interfering substance’ is used even if it contains more than one substance.
5.2.2.7.2 Low level soiling (bovine albumin solution)
Dissolve 0,6 g of bovine albumin V (suitable for microbiological purposes) in 90 ml of water (5.2.2.2) in a 100
ml volumetric flask. Make up to the mark with water (5.2.2.2).
Sterilize by membrane filtration (5.3.2.7) keep in the refrigerator (5.3.2.8) 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)
Dissolve 10 g yeast extract powder in 150 ml of water (5.2.2.2) in a 250 ml volumetric flask (5.3.2.13) 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
an autoclave [5.3.2.1a)]. Allow to cool to (20 ± 5) C.
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
bovine albumin fraction V (suitable for microbiological purposes) in the solution 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 a refrigerator
(5.3.2.8) and use within one month.
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.
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Prior to use the surfaces should be placed in a beaker (minimum size 50 ml) containing not less than 20 ml of
3)
5 % Decon® 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 2000 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 [5.3.2.1a)];
b) by dry heat, in a hot air oven [5.3.2.1b)].
4)
5.3.2 Usual microbiological laboratory equipment
and, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)
a) for moist heat sterilization, an autoclave capable of being maintained at 121 °C for a minimum holding
time of 15 min;
b) for dry heat sterilization, a hot air oven capable of being maintained at 180 °C for a minimum holding time
of 30 min, at 170 *C for a minimum holding time of 1 h or at 160 *C for a minimum holding time of 2 h;
5.3.2.2 Water bath, capable of being controlled at (4 ± 1) °C, (10 ± 1) °C, (20 ± 1)° C, (40 ± 1)°C and (45 ±
1) C (to maintain melted MEA, 5.5.2.2, 5.5.2.3, 5.5.2.4 and 5.5.2.5).
5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C
5.3.2.4 pH-meter, having an accuracy of calibration of 0,1 pH units at (20 °C ± 1) °C.
NOTE A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media
(5.2.2.3)
5.3.2.5 Stopwatch
5.3.2.6 Shakers
5)
a) Electromechanical agitator, e.g. Vortex® mixer .
3) Decon concentrate is obtained from Decon Laboratories Ltd, Conway Street, Hove, East Sussex, BN3 3LY, UK Tel.
01273 756598. Studies have shown that this method of cleaning is satisfactory. A suitable ‘Generic’ will be specified at a
later stage.
4) Disposable sterile equipment is an acceptable alternative to reusable glassware.
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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 filtration of hard water (5.2.2.6) and bovine albumin (5.2.2.7.2, 5.2.2.7.3).
5.3.2.8 Refrigerator capable of being controlled at 2ºC to 8ºC
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic
pipettes may be used.
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 Glass screw top container, with a base diameter of 4-5 cm.
5.3.2.15 Fritted filter, with porosity of 40 µm to 100 µm according to ISO 4793.
5.3.2.16 Flasks with vented caps.
5.3.2.17 Microscope capable of x 400 magnification.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspension (test and validation suspension)
Test and validation suspension are the same in this standard.
5.4.1.1 General
For each test organism, one suspension has to be prepared: this is used as the fungal “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
5.4.1.3.1 Candida albicans (yeast)
In order to prepare the working culture of Candida albicans (5.2.1), prepare a subculture f
...
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