Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)

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 when diluted with hard water or - in the case of ready-to-use-products - with water. Products can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance.
This European Standard applies to products that are used 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 1 test (Annex F).

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Diese Europäische Norm legt ein Prüfverfahren und die Mindestanforderungen an die fungizide oder levurozide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die in Wasser standardisierter Härte oder im Fall gebrauchsfertiger Lösungen in Wasser eine homogene, physikalisch stabile Zubereitung bilden. Die Produkte können nur bis zu einer Konzentration von 80 % geprüft werden, da immer eine bestimmte Verdünnung durch Zugabe der Prüforganismen oder der Belastungssubstanz erfolgt.
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 gelangen.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwendungsempfehlungen“ fest.
ANMERKUNG 1   Das beschriebene Verfahren ist zur Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen unter deren Anwendungsbedingungen vorgesehen.
ANMERKUNG 2   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase 2, étape 1)

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. Les produits ne peuvent être soumis à l’essai qu’à la concentration de 80 % ou à des concentrations inférieures, car l’ajout des microorganismes d’essai et de la substance interférente s’accompagne forcément d’une dilution.
La présente Norme européenne s’applique aux produits utilisés dans le domaine vétérinaire, 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 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 d’utilisation.
NOTE 2   Cette méthode correspond à un essai de phase 2, étape 1 (voir Annexe F).

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje fungicidnega delovanja ali delovanja kemičnih razkužil in antiseptikov na kvasovke v veterini - Preskusna metoda in zahteve (faza 2, stopnja 1)

Ta evropski standard določa preskusno metodo in minimalne zahteve za fungicidno delovanje ali delovanje na kvasovke kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo ali, pri izdelkih, ki so pripravljeni za uporabo, z vodo. Izdelke je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji, ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje.
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«.
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 stopnje 1 faze 2 (dodatek F).

General Information

Status
Published
Public Enquiry End Date
05-Oct-2014
Publication Date
11-Oct-2016
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
08-Sep-2016
Due Date
13-Nov-2016
Completion Date
12-Oct-2016

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)11.220VeterinarstvoVeterinary medicine11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 1657:2016SIST EN 1657:2016en,fr,de01-november-2016SIST EN 1657:2016SLOVENSKI
STANDARDSIST EN 1657:2006/AC:2007SIST EN 1657:20061DGRPHãþD



SIST EN 1657:2016



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 1657
April
t r s x ICS
y sä s r rä u w Supersedes EN
s x w yã t r r wEnglish Version
Chemical disinfectants and antiseptics æ Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area æ Test method and Antiseptiques et désinfectants chimiques æ Essai quantitatif de suspension pour l 5évaluation de l 5activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine
Chemische Desinfektionsmittel und Antiseptika æ Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich æ Prüfverfahren und Anforderungen This European Standard was approved by CEN on
t u January
t r s xä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey andUnited Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels
9
t r s x CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x w yã t r s x ESIST EN 1657:2016



EN 1657:2016 (E) 2 Contents European foreword . 4 Introduction . 5 1 Scope . 6 2 Normative references . 6 3 Terms and definitions . 6 4 Requirements . 6 5 Test method . 7 5.1 Principle . 7 5.2 Materials and reagents . 8 5.2.1 Test organisms . 8 5.2.2 Culture media and reagents . 8 5.3 Apparatus and glassware . 11 5.3.1 General . 11 5.3.2 Usual microbiological laboratory equipment and, in particular, the following . 11 5.4 Preparation of test organism suspensions and product test solutions . 12 5.4.1 Test organism suspensions (test and validation suspension) . 12 5.4.2 Product test solutions . 17 5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product . 18 5.5.1 General . 18 5.5.2 Dilution-neutralization method . 19 5.5.3 Membrane filtration method . 21 5.6 Experimental data and calculation . 23 5.6.1 Explanation of terms and abbreviations . 23 5.6.2 Calculation . 24 5.7 Verification of methodology . 26 5.7.1 General . 26 5.7.2 Control of weighted mean counts . 27 5.7.3 Basic limits . 27 5.7.4 Additional limits for Aspergillus brasiliensis . 27 5.8 Expression of results and precision . 27 5.8.1 Reduction . 27 5.8.2 Control of active and non-active product test solution (5.4.2) . 27 5.8.3 Limiting test organism and fungicidal/yeasticidal concentration . 28 5.8.4 Precision, replicates . 28 5.9 Interpretation of results – conclusion . 28 5.9.1 General . 28 5.9.2 Fungicidal activity for general purposes . 28 5.9.3 Fungicidal activity for specific purposes . 28 5.9.4 Yeasticidal activity for general purposes . 28 5.9.5 Yeasticidal activity for specific purposes . 29 5.9.6 Yeasticidal activity for teat disinfectants . 29 5.10 Test report . 29 Annex A (informative) Referenced strains in national collections . 31 SIST EN 1657:2016



EN 1657:2016 (E) 3 Annex B (informative) Suitable neutralizers and rinsing liquids . 32 Annex C (informative) Graphical representation of test procedures . 34 C.1 Dilution-neutralization method . 34 C.2 Membrane filtration method . 36 Annex D (informative) Example of a typical test report . 38 Annex E (informative) Precision of the test result . 42 Bibliography . 45
SIST EN 1657:2016



EN 1657:2016 (E) 4 European foreword This document (EN 1657:2016) 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 October 2016, and conflicting national standards shall be withdrawn at the latest by October 2016. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 1657:2005. This European Standard was revised to harmonize the preparation of the fungal spore suspension with other fungicidal tests of CEN/TC 216 and to incorporate amendments applicable to all European Standards. An additional requirement has been added for the Aspergillus spore suspension and therefore results obtained using EN 1657:2005 and not fulfilling this additional requirement will need to be confirmed by repeating the tests using EN 1657:2015. The test conditions for teat disinfectants have been added. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, 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 1657:2016



EN 1657:2016 (E) 5 Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has a 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 defined experimental conditions. However, for some applications the recommendations of use of a product may differ and therefore additional test conditions need to be used. SIST EN 1657:2016



EN 1657:2016 (E) 6 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 when diluted with hard water or — in the case of ready-to-use-products — with water. Products can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance. This European Standard applies to products that are used 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 1 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 4 decimal log (lg) reduction when diluted with hard water (5.2.2.7) or – in the case of ready-to-use products – with water (5.2.2.2) and tested in accordance with Table 1 and Clause 5 under simulated low level soiling (3,0 g/l bovine albumin) or high level soiling (10 g/l yeast extract and 10 g/l bovine albumin) or 10 g/l skimmed milk for teat disinfectants or in additional test conditions. SIST EN 1657:2016



EN 1657:2016 (E) 7 Table 1 — Obligatory and additional test conditions Test conditions Fungicidal activity Yeasticidal activity Yeasticidal activity for teat disinfectants Test organisms obligatory Aspergillus brasiliensis Candida albicans Candida albicans Candida albicans additional any relevant test organism any relevant test organism any relevant test organism Test temperature obligatory 10°C ± 1°C 10°C ± 1°C 30°C ± 1°C additional 4°C ± 1°C; 20°C ± 1°C; 40°C ± 1°C; 4°C ± 1°C; 20°C ± 1°C; 40°C ± 1°C 20°C ± 1°C Contact time obligatory 30 min ± 10 s 30 min ± 10 s 5 min ± 10 s for post-milking teat disinfectants 30 s ± 5 s for pre-milking teat disinfectants additional 5 min ± 10 s; 60 min ± 10 s; 120 min ± 10 sa 5 min ± 10 s; 60 min ± 10 s; 120 min ± 10 sa 1 min ± 5 s Interfering substance obligatory
low level soiling high level soiling 3,0 g/l bovine albumin 10 g/l yeast extract plus 10 g/l bovine albumin 3,0 g/l bovine albumin 10 g/l yeast extract plus 10 g/l bovine albumin 10,0 g/l of reconstituted skimmed milk additional any relevant substance any relevant substance any relevant substance The obligatory contact times for disinfectants stated in Table 1 were chosen to enable comparison of standard conditions. NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions. a The recommended contact time for the use of the product is within the responsibility of the manufacturer. 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 5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use products) is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an interfering substance. The mixture is maintained at 10°C ± 1 °C for 30 min ± 10 s or 30°C ± 1 °C for 5 min ± 10 s or 30 s± 5 s for teat disinfectants (obligatory test conditions). At the end of this contact time, an aliquot is taken, and the fungicidal/yeasticidal and/or the fungistatic/yeastistatic activity in this portion is immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving fungi in each sample are determined and the reduction is calculated. SIST EN 1657:2016



EN 1657:2016 (E) 8 5.1.2 The test is performed using the vegetative cells of Candida albicans and the spores of Aspergillus brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as test organisms (obligatory test conditions). 5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can be used. 5.2 Materials and reagents 5.2.1 Test organisms The fungicidal activity shall be evaluated using the following strains as test organisms: 1) — Candida albicans ATCC 10231; — Aspergillus brasiliensis ATCC 16404.
(formerly A.niger) 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 test organisms is (30 ± 1) °C (see 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 organisms. 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. 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 water and not demineralized water. Sterilize in the autoclave [5.3.2.1 a)].
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 1657:2016



EN 1657:2016 (E) 9 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. NOTE 3 See 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Malt extract agar (MEA) Malt extract agar, consisting of: Malt extract a 30,0 g Agar 15,0 g Water (5.2.2.2) to 1 000,0 ml a The malt extract should be of food grade (Cristomalt poudre from Difal is recommended) or equivalent that is not highly purified and not only based on maltose (Malt extract from OXOID is recommended 2)). Sterilize in the autoclave [5.3.2.1 a)]. After sterilization, 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 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. 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 1 000,0 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 °C ± 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 Rinsing liquid (for membrane filtration) The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane under the test conditions described in 5.5.3. NOTE Information on rinsing liquids 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 1657:2016



EN 1657:2016 (E) 10 5.2.2.7 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 [5.3.2.1 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. — 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 (5.3.2.9) 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 300 mg/l of calcium carbonate (CaCO3) in the test tube. 5.2.2.8 Interfering substance 5.2.2.8.1 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 10 times its final concentration in the test. The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids and detergents) shall be defined. NOTE The term “interfering substance” is used even if it contains more than one substance. 5.2.2.8.2 Low-level soiling (bovine albumin solution) Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of 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 bovine albumin in the test procedure (5.5) is 3,0 g/l. 5.2.2.8.3 High-level soiling (mixture of bovine albumin solution with yeast extract) Dissolve 50,0 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 an autoclave [5.3.2.1 a)]. Allow to cool to 20 °C ± 1 °C. 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 5,0 g of bovine albumin fraction V (suitable for microbiological purposes) in the solution with SIST EN 1657:2016



EN 1657:2016 (E) 11 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), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration in the test procedure (5.5) is 10,0 g/l yeast extract and 10,0 g/l bovine albumin. 5.2.2.8.4 Milk for teat disinfectants Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder per litre of water (5.2.2.2), shall be prepared as follows: Prepare a solution of 100 g milk powder in 1 000 ml water (5.2.2.2). Heat for 30 min at 105°C ±3 °C or 5 min at 121 °C ±3 °C. The final concentration of reconstituted milk in the test procedure (5.5) is 10,0 g/l. 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 the autoclave [5.3.2.1 a)]; b) by dry heat, in the hot air oven [5.3.2.1 b)]. 5.3.2 Usual microbiological laboratory equipment3) and, in particular, the following 5.3.2.1 Apparatus for sterilization a) for moist heat sterilization, an autoclave capable of being maintained at (+30121) °C for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at (+50180) °C for a minimum holding time of 30 min, at (+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 baths, capable of being controlled at 4°C ± 1 °C, 10 °C ± 1 °C, at 20 °C ± 1 °C, at 30°C ± 1 °C, 40°C ± 1 °C at 45 °C ± 1 °C (to maintain melted MEA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled at 30 °C ± 1 °C. 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
3) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 1657:2016



EN 1657:2016 (E) 12 5.3.2.6 Shakers a) Electromechanical agitator, e.g. Vortex® mixer4) b) Mechanical shaker 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered. The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin (5.2.2.8), and if the membrane filtration method is used (5.5.3). The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the microorganisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s. 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 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, 3 mm to 4 mm in diameter. 5.3.2.12 Volumetric flasks. 5.3.2.13 Fritted filter,
...

SLOVENSKI STANDARD
oSIST prEN 1657:2014
01-september-2014
.HPLþQDUD]NXåLODLQDQWLVHSWLNL.YDQWLWDWLYQLVXVSHQ]LMVNLSUHVNXV]D
YUHGQRWHQMHIXQJLFLGQHJDGHORYDQMDDOLGHORYDQMDNHPLþQLKUD]NXåLOLQDQWLVHSWLNRY
QDNYDVRYNHYYHWHULQL3UHVNXVQDPHWRGDLQ]DKWHYH ID]DVWRSQMD
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the
veterinary area - Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel
und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2,
Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants
chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase
2, étape 1)
Ta slovenski standard je istoveten z: prEN 1657 rev
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
oSIST prEN 1657:2014 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 1657:2014

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oSIST prEN 1657:2014

EUROPEAN STANDARD
DRAFT
prEN 1657 rev
NORME EUROPÉENNE

EUROPÄISCHE NORM

April 2014
ICS 71.100.35 Will supersede EN 1657:2005
English Version
Chemical disinfectants and antiseptics - Quantitative suspension
test for the evaluation of fungicidal or yeasticidal activity of
chemical disinfectants and antiseptics used in the veterinary
area - Test method and requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif Chemische Desinfektionsmittel und Antiseptika -
de suspension pour l'évaluation de l'activité fongicide ou Quantitativer Suspensionsversuch zur Bestimmung der
levuricide des antiseptiques et des désinfectants chimiques fungiziden oder levuroziden Wirkung chemischer
utilisés dans le domaine vétérinaire - Méthode d'essai et Desinfektionsmittel und Antiseptika für den Veterinärbereich
prescriptions (phase 2, étape 1) - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
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, 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.

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

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 1657 rev:2014 E
worldwide for CEN national Members.

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prEN 1657:2014 (E)
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
5.3 Apparatus and glassware . 10
5.4 Preparation of test organism suspensions and product test solutions . 11
5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product . 16
5.6 Experimental data and calculation. 22
5.7 Verification of methodology . 25
5.8 Expression of results and precision . 26
5.9 Interpretation of results – conclusion . 27
5.10 Test report . 28
Annex A (informative) Referenced strains in national collections . 30
Annex B (informative) Suitable neutralizers and rinsing liquids . 31
Annex C (informative) Graphical representation of test procedures . 33
Annex D (informative) Example of a typical test report . 37
Annex E (informative) Precision of the test result . 43
Bibliography . 45

2

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Foreword
This document (prEN 1657:2014) 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.
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, and conflicting national standards shall be withdrawn at the latest by
May 2006.
This European Standard will supersede EN 1657:2005.
This European Standard was revised to harmonize the preparation of the fungal spore suspension with other
fungicidal tests of CEN/TC 216 and to incorporate amendments applicable to all EN standards.
An additional requirement has been added for the Aspergillus spore suspension (to use a spore suspension
containing at least 75 % spiny spores) and therefore results obtained using the previous versions of EN 1657
and not fulfilling this additional requirement will need to be confirmed by repeating the tests using
EN 1657:2014.
3

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Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant or
antiseptic has a 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 defined experimental conditions. However, for some applications the recommendations of use
of a product may differ and therefore additional test conditions need to be used.
4

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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 when diluted with hard water or — in the case of ready-to-use-products — with water. Products
can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test
organisms and interfering substance.
This European Standard applies to products that are used 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 1 test (Annex F).
2 Normative references
The following referenced documents are indispensable for the application of this European Standard. 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 an definitions given in EN 14885 apply.
4 Requirements
The product shall demonstrate at least a 4 decimal log (lg) reduction when diluted with hard water (5.2.2.7) or
– in the case of ready-to-use products – with water (5.2.2.2) and 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).
5

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Table 1 Obligatory and additional test conditions
Test Conditions Fungicidal or yeasticidal activity in the
veterinary area

Test organism Fungicidal activity Aspergillus brasilliensis
Candida albicans
a) obligatory
Candida albicans
Test organism Yeasticidal activity
a) obligatory
b) additional any relevant test organism
Test temperature 10°C ± 1°C
a) obligatory
b) additional 4°C ± 1°C; 20°C ± 1°C; 40°C ± 1°C
Contact time 30 min ± 10 s
a) obligatory
b) additional 5 min ± 10 s; 60 min ± 10 s; 120 min ± 10 s*
Interfering substance 3,0 g/l bovine albumin
a) obligatory 10 g/l yeast extract plus 10 g/l bovine albumin
low level soiling
high level soiling
b) additional any relevant substance
The obligatory contact times for 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
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use products)
is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an interfering substance.
The mixture is maintained at (10 ± 1) °C for 30 min ± 10 s (obligatory test conditions). At the end of this
contact time, an aliquot is taken, and the fungicidal and/or the fungistatic activity in this portion is immediately
neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable
neutralizer cannot be found, membrane filtration is used. The numbers of surviving fungi in each sample are
determined and the reduction is calculated.
5.1.2 The test is performed using the vegetative cells of Candida albicans and the spores of Aspergillus
brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as test
organisms (obligatory test conditions).
6

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5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can be
used.
5.2 Materials and reagents
5.2.1 Test organisms
1)
The fungicidal activity shall be evaluated using the following strains as test organisms:
— Candida albicans ATCC 10231;
— Aspergillus brasiliensis ATCC 16404.
(formerly A.niger)
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 test 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 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.
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.
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 water and not demineralized water.
Sterilize in the autoclave [5.3.2.1 a)].
NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently
sterilized.

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.

7

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NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can
be used.
NOTE 3 See 5.2.2.7 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 1 000,0 ml
* The malt extract should be of food grade (Cristomalt poudre 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 [5.3.2.1 a)]. After sterilization, 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 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.
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
Tryptone, pancreatic digest of 1,0 g
casein
Sodium chloride (NaCl) 8,5 g
Water (5.2.2.2) to 1 000,0 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 °C ± 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 Rinsing liquid (for membrane filtration)
The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3.
It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane
under the test conditions described in 5.5.3.
NOTE Information on rinsing liquids 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 y CEN of
b
the product named. Equivalent products may be used if they can be shown to lead to the same results.”

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5.2.2.7 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 (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 [5.3.2.1
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;
— 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 (5.3.2.9)
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 a
different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of calcium carbonate
(CaCO ) in the test tube.
3
5.2.2.8 Interfering substance
5.2.2.8.1 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 10 times its final concentration in the test.
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral
substances, protein, carbohydrates, lipids and detergents) shall be defined.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Low-level soiling (bovine albumin solution)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of 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 bovine albumin in the test procedure (5.5) is 3,0 g/l.
5.2.2.8.3 High-level soiling (mixture of bovine albumin solution with yeast extract)
Dissolve 50,0 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
an autoclave [5.3.2.1 a)]. Allow to cool to 0,20 °C ± 1 °C.
9

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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
5,0 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 (5.3.2.7), keep in
the refrigerator (5.3.2.8) and use within one month.
The final concentration in the test procedure (5.5) is 10,0 g/l yeast extract and 10,0 g/l bovine albumin.
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 the autoclave [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
3)
5.3.2 Usual microbiological laboratory equipment and, in particular, the following:
5.3.2.1 Apparatus for sterilization:
+3
a) for moist heat sterilization, an autoclave capable of being maintained at ( 121 ) °C for a minimum
0
holding time of 15 min;
+5
b) for dry heat sterilization, a hot air oven capable of being maintained at (180 ) °C for a minimum holding
0
+5
+5
time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a minimum holding
0
0
time of 2 h.
5.3.2.2 Water baths, capable of being controlled at 10 °C ± 1 °C, at 20 °C ± 1 °C, at 45 °C ± 1 °C (to
maintain melted MEA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1).
5.3.2.3 Incubator, capable of being controlled at 30 °C ± 1 °C.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (±1) °C. A
puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media
(5.2.2.3).
5.3.2.5 Stopwatch
5.3.2.6 Shakers
4)
a) Electromechanical agitator, e.g. Vortex® 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.
10

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5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be
filtered.
The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of
diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin
(5.2.2.8), and if the membrane filtration method is used (5.5.3).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the
microorganisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain
the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
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 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, 3 mm to 4 mm in diameter.
5.3.2.12 Volumetric flasks.
5.3.2.13 Fritted filter, with porosity of 40 µm to 100 µm according to ISO 4793.
5.3.2.14 Centrifuge (2 000 gN).
5.3.2.15 Flasks with ventilated caps.
5.3.2.16 Microscope capable of x 400 magnification
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
5.4.1.1 General
For each test organism, two different suspensions have to be prepared: the “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 from the stock culture
(5.4.1.2) by streaking onto MEA (5.2.2.3) slopes or plates (5.3.2.10) and incubate (5.3.2.3). After 42 h to 48 h,
prepare a second subculture from the first subculture in the same way and incubate for 42 h to 48 h. From this
second subculture, a third subculture may be produced in the same way. The second and (if produced) third
subcultures are the working cultures.
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If it is not possible to prepare the second subculture on a particular day, a 72 h subculture may be used for
subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 72 h
period.
Never produce and use a fourth subculture.
5.4.1.3.2 Aspergillus brasilliensis (mould)
For Aspergillus brasilliensis (5.2.1), use only the first subculture grown on MEA (5.2.2.3) in Petri dishes or
flasks with ventilated caps (5.3.2.15) and incubate for 7 days to 9 days at 30 °C ± 1°C. No further subculturing
is needed. Do not stack the Petri dishes during the incubation to improve the temperature homogenization. At
the end of incubation, all the cultures have to show a dark brown or black surface. Cultures with rare and
small white or grey areas may be used (see Figure 1).
Figure 1 - Photo No 1: A. brasiliensis
ATCC 16404 after 7 d of incubation
at 30°C

Figure 1 - Photo No 1: A. brasiliensis ATCC 16404 after 7 d of incubation at 30°C
Figure 2- Photo No.2: Example of
inappropriate (not usable) culture of
A. brasiliensis ATCC 16404 after 7 d
of incubation at 30°C

Figure 2- Photo No.2: Example of inappropriate (not usable) culture of A. brasiliensis ATCC 16404
after 7 d of incubation at 30°C
5.4.1.3.3 Other test organisms (yeasts or moulds)
For additional test organisms, any departure from this method of culturing the yeast or the mould or of
preparing the suspensions shall be noted, giving the reasons in the test report.
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oSIST prEN 1657:2014
prEN 1657:2014 (E)
5.4.1.4 Test suspension (“N”)
5.4.1.4.1 Candida albicans
The procedure for preparing the Candida albicans test suspension is as follows:
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 (5.4.1.3.1) and transfer loopfuls of the cells into the diluent (5.2.2.4). The cells should be
suspended in the diluent rubbing the loop against the wet wall of the flask to dislodge the cells before
immersing in the dilutent. Shake the flask for 3 min using a mechanical shaker [5.3.2.6b)]. Aspirate the
suspension from the glass beads and transfer to a tube;
7 7
5)
b) adjust the number of cells in the suspension to 1,5 × 10 cfu/ml ) to 5,0 × 10 cfu/ml using diluent
(5.2.2.4), estimating the number of cfu by any suitable means. Ma
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