Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal 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 virucidal 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.
NOTE 1   The method described is intended to determine the virucidal 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.

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden 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 viruzide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte oder - bei der Verwendung von gebrauchsfertigen Produkten - mit Wasser eine homogene, physikalisch stabile Zubereitung bilden. Die Produkte können nur bei einer Konzentration von 80 % oder darunter geprüft werden, da durch die Zugabe der Prüforganismen und der Belastungssubstanz immer eine Verdünnung hervorgerufen wird.
Diese Europäische Norm gilt für Produkte, die im Veterinärbereich eingesetzt werden, d. h. bei der Aufzucht, Haltung, Produktion, beim Transport und bei der Tierkörperbeseitigung aller Tierarten mit Ausnahme der Bereiche, in denen Tiere direkt nach der Tötung als Nahrungsmittel verwendet oder der weiterverarbeitenden Industrie zugeführt werden.
ANMERKUNG 1   Das beschriebene Verfahren ist zur Bestimmung der viruziden Wirkung von im Handel erhältlichen Zubereitungen oder aktiven Substanzen unter Anwendungsbedingungen vorgesehen.
ANMERKUNG 2   Das Verfahren entspricht der Phase 2, Stufe 1.

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

La présente Norme européenne décrit une méthode d'essai et les prescriptions minimales relatives à l'activité virucide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène et physiquement stable lorsqu’ils sont dilués avec de l'eau dure ou - dans le cas de produits prêts à l'emploi - avec de l'eau. Les produits ne peuvent être soumis à essai qu'à une concentration inférieure ou égale à 80 % car il se produit toujours une certaine dilution du fait de l’addition des organismes d'essai et de la substance interférente.
La présente Norme européenne s'applique aux produits utilisés dans le domaine vétérinaire, c'est-à-dire dans la sélection, l'élevage, la production, le transport et l'élimination de tous les animaux, sauf dans la chaîne alimentaire à la suite de leur mort et de leur entrée dans l’industrie de transformation.
NOTE 1   La méthode décrite a pour objet de déterminer l'activité virucide de formulations commerciales ou de substances actives dans leurs conditions d'emploi.
NOTE 2   Cette méthode correspond à un essai de phase 2, étape 1.

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

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Published
Publication Date
21-Apr-2015
Current Stage
9060 - Closure of 2 Year Review Enquiry - Review Enquiry
Due Date
02-Dec-2020
Completion Date
02-Dec-2020

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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 viruziden 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 virucide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthodes d'essai et prescriptions - (Phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal 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 14675:2015SIST EN 14675:2015en,fr,de01-julij-2015SIST EN 14675:2015SLOVENSKI

STANDARDSIST EN 14675:20061DGRPHãþD
SIST EN 14675:2015
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 14675
April 2015 ICS 71.100.35 Supersedes EN 14675:2006English Version

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal 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 de suspension pour l'évaluation de l'activité virucide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase 2, étape 1)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 14 February 2015.

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 © 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14675:2015 ESIST EN 14675:2015

EN 14675:2015 (E) 2 Contents Page Foreword ..............................................................................................................................................................4 Introduction .........................................................................................................................................................5 1 Scope ......................................................................................................................................................6 2 Normative references ............................................................................................................................6 3 Terms and definitions ...........................................................................................................................6 4 Requirements .........................................................................................................................................7 5 Test method ............................................................................................................................................8 5.1 Principle ..................................................................................................................................................8 5.2 Materials and reagents ..........................................................................................................................8 5.2.1 Test virus ................................................................................................................................................8 5.2.2 Culture media and reagents .................................................................................................................9 5.3 Apparatus and glassware .................................................................................................................. 15 5.3.1 General ................................................................................................................................................. 15 5.3.2 Usual microbiological laboratory equipment and, in particular, the following: ........................... 15 5.4 Product test solutions ........................................................................................................................ 16 5.5 Procedure for assessing the virucidal activity of the product ....................................................... 17 5.5.1 Experimental conditions (obligatory and additional)...................................................................... 17 5.5.2 Preparation of the test virus suspension ......................................................................................... 17 5.5.3 Preparation of cell line ....................................................................................................................... 18 5.6 Infectivity assay .................................................................................................................................. 18 5.6.1 Predilution of viral suspension ......................................................................................................... 18 5.6.2 Virus titration of cells in suspension on microtitre plates ............................................................. 18 5.6.3 Virus titration on monolayers of cells on microtiter plates ............................................................ 18 5.6.4 Plaque assay ....................................................................................................................................... 18 5.7 Virucidal test – preparation ............................................................................................................... 19 5.7.1 General ................................................................................................................................................. 19 5.7.2 Test method ......................................................................................................................................... 19 5.8 Control of efficiency for suppression of disinfectant activity ....................................................... 19 5.8.1 Dilution in ice-cold medium ............................................................................................................... 19 5.8.2 Filtration technique............................................................................................................................. 19 6 Calculation and expression of results .............................................................................................. 20 6.1 Protocol of the CPE result ................................................................................................................. 20 6.2 Calculation of infectivity titre (TCID50) .............................................................................................. 20 6.3 Calculation of PFU .............................................................................................................................. 20 6.4 Verification of the methodology ........................................................................................................ 20 6.5 Calculation of the virucidal activity of products ............................................................................. 20 6.6 Expression of results ......................................................................................................................... 20 7 Conclusion .......................................................................................................................................... 21 7.1 General ................................................................................................................................................. 21 7.2 Test report ........................................................................................................................................... 21 Annex A (informative)

Referenced strains of national collections ............................................................. 23 Annex B (normative)

Cytotoxicity, reference inactivation test, test virus titration and detoxification of test mixtures ........................................................................................................... 24 B.1 Cytotoxicity caused by product solutions ....................................................................................... 24 B.2 Reference inactivation test ................................................................................................................ 24 SIST EN 14675:2015

EN 14675:2015 (E) 3 B.3 Titration of test virus suspension ...................................................................................................... 25 B.4 Detoxification of test mixtures by molecular sieving ...................................................................... 25 Annex C (informative)

Calculation of the viral infectivity titre ..................................................................... 28 C.1 Quantal tests - Example of TCID50 determination by the Spearman-Kärber method .................. 28 C.2 Plaque test ........................................................................................................................................... 28 Annex D (informative)

Example of a typical test report ................................................................................ 30 Annex E (informative)

Presentation of test results of one active concentration ....................................... 32 Bibliography ...................................................................................................................................................... 34

SIST EN 14675:2015

EN 14675:2015 (E) 4 Foreword This document (EN 14675:2015) 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 2015 and conflicting national standards shall be withdrawn at the latest by October 2015. 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 14675:2006. 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 14675:2015

EN 14675:2015 (E) 5 Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has or does not have a virucidal activity in the areas 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 14675:2015

EN 14675:2015 (E) 6 1 Scope This European Standard specifies a test method and the minimum requirements for virucidal 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. NOTE 1 The method described is intended to determine the virucidal 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. 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 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885 and the following apply: 3.1 cytotoxicity morphological alteration of cells and/or their destruction or their reduced sensitivity to virus multiplication caused by the product 3.2 plaque forming units

PFU number of infectious virus particles per unit volume (ml) 3.3 reference test for virus inactivation test with a defined reagent (e.g. formalin) instead of a product for the internal control of the test Note 1 to entry: Results of reference virus inactivation test should be within limits for validating the method. 3.4 reference virus suspension virus suspension of a defined virus strain which is not passaged more than 10 times, is maintained in national culture collection centres and kept in small volumes (less than 1 ml) at a temperature of -70°C or preferably at about -196 °C under liquid nitrogen Note 1 to entry: Stock virus suspensions are prepared from reference virus suspensions. SIST EN 14675:2015

EN 14675:2015 (E) 7 3.5 stock virus suspension virus suspension of a defined strain that is multiplied in a suitable cell line which produces high virus titers, to obtain a virus suspension of the same characteristics as the reference virus suspension and kept in a small volume at a temperature of below 70 °C or preferably at about -196 °C over liquid nitrogen 3.6 test virus suspension virus suspension that is used in the virucidal testing of the disinfectant 3.7 tissue culture infectious dose

TCID50 viral dose that induces a cytopathic effect (CPE) (3.8) in 50 % of inoculated cell culture 3.8 viral cytopathic effect

CPE morphological alteration of cells and/or their destruction as a consequence of virus multiplication 3.9 viral plaque area of lysis formed in a cell monolayer under semisolid medium due to infection by and multiplication of a single infectious virus particle 3.10 virus titre amount of infectious virus per unit volume present in a cell culture lysate or in a solution 4 Requirements The product when diluted with hard water (5.2.2.3) 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 shall demonstrate at least a lg reduction in virus titre of 4. It is possible to test also the product as delivered (highest test concentration is 80 %). Table 1 — Obligatory and additional test conditions

Obligatory conditions Additional conditions Test organism Bovine enterovirus Type 1 (ECBO)

Test temperature a 10 °C 4 °C, 20 °C or 40 °C Contact time b 30 min 1 min, 5 min and 60 min Interfering substance–low level soiling c 3,0 g/l bovine albumin

Interfering substance-high level soiling c 10 g/l bovine albumin plus 10 g/l yeast extract

Allowed deviation ± 1 °C. b

The allowed deviation for each chosen contact time is ± 10 sf except for ≤1 min for which it is ± 5 s. c

To be chosen according to practical applications. SIST EN 14675:2015

EN 14675:2015 (E) 8 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 virus in a solution of an interfering substance. The mixture is maintained at 10 °C ± 1 °C for 30 min ± 10 s (obligatory test conditions). At the end of the contact time, 0,5 ml of virus/disinfectant mixture is taken. The virucidal activity is immediately suppressed by dilution in ice-cold diluent. A dilution series with a factor of ten is prepared in an ice-cold medium held in an ice bath for 10 min. Pipettes shall be changed after each dilution to avoid carry-over of virus. The dilutions are transferred into cell culture units (wells of microtitre plates) containing suspended cells. Eight series units shall be inoculated with each dilution. After incubation, the titre of infectivity is calculated. The titration results of quantal tests shall show dilution steps with the percentage of positive results (presence of CPE or plaques) lying between 100 % and 0 %. The values are calculated according to Spearman and Kärber (see Annex C). Values of virus inactivation are calculated from differences of virus titres before and after treatment with the product. 5.1.2 Additional and optional contact times and temperatures are specified. 5.2 Materials and reagents 5.2.1 Test virus The virucidal activity shall be evaluated using the following strain: — Bovine enterovirus Type 1 (Enteric Cytopathogenic Bovine Orphan Virus – ECBO) ATCC VR-2481). NOTE 1 Bovine enterovirus Type 1, strain ECBO, is selected as the model virus for the large Genus Picornavirus. The Genus Picornavirus includes many clinically important virus species, for example Coxsackie A and B, and enteric cytopathogenic human orphan (ECHO). Some of these viruses are of primary importance and therefore a constant risk for animals in the veterinary area. Moreover, they have a high resistance to chemicals, are acid-stable (except inter alia rhinovirus, aphtovirus) and are unaffected by lipid solvents such as ether, and most detergents or quaternary ammonium products. NOTE 2 It is the model virus for all applications namely for disinfection of instruments and surfaces and post-contamination treatment of post-mortem rooms, kennels and for animal accommodation. NOTE 3 Due to large differences of resistance against physical and chemical influences between and within different virus groups, the testing of all viruses against any particular chemical disinfectant or antiseptic is financially impossible. Therefore, in this European Standard, testing is restricted to only one so called ‘model virus’ that has been selected on the basis of the present knowledge as a representative example of virus tenacity and of important clinical relevance in the veterinary area. If a chemical disinfectant or antiseptic shows virucidal activity according to the requirements of this European Standard, it can be considered for a phase 2 step 2 test. If improvements in the methodology of virus multiplication, virus infectivity or cytoxicity reduction of products are elaborated, they may be used in parallel with the methodology described in this method to show the improvement.

1) ATCC VR-248, is a strain supplied by the American Type Culture Collections. 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. A corresponding strain supplied by other culture collections may be used if they can be shown to lead to the same results. SIST EN 14675:2015

EN 14675:2015 (E) 9 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 virological 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 shall be rigorously followed. For each culture medium and reagent a time limitation for use shall be fixed. Commercial ready-to-use products and culture media can be purchased, if they comply with the required specifications. 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. NOTE 2 See 5.2.2.3 for the procedure to prepare hard water. If distilled water of adequate quality is not available, water for injections (see bibliographic reference [3]) can be used. 5.2.2.3 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 [5.3.2.1 c)] 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 (5.3.2.16) 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 [5.3.2.1 c)]. Store the solution in the refrigerator (5.3.2.16) 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 (5.3.2.13) 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. SIST EN 14675:2015

EN 14675:2015 (E) 10 NOTE When preparing the product test solutions (5.4), 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. 5.2.2.4 Interfering substance 5.2.2.4.1 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. 5.2.2.4.2 Low level soiling (Bovine albumin solution) Dissolve 3 g of bovine albumin (Cohn fraction V for Dubos Medium) in 90 ml of water (5.2.2.2) in a 100 ml volumetric flask (5.3.2.13). Make up to the mark with water (5.2.2.2). Sterilize by membrane filtration [5.3.2.1 c)]. Keep in a refrigerator (5.3.2.16) and use within one month. The final concentration of bovine albumin in the test procedure (5.5) is 3 g/l. 5.2.2.4.3 High level soiling (mixture of bovine albumin solution with yeast extract) Dissolve 50 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 the 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 (5.3.2.13) and add 10 ml of water (5.2.2.2). Dissolve 5 g of the bovine albumin fraction V 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 [5.3.2.1 c)], keep in a refrigerator (at 2 °C to 8 °C) (5.3.2.16) and use within one month. The final concentration in the test procedure (5.5) is 10 g/l yeast extract and 10 g/l bovine albumin. 5.2.2.5 Antibiotic suspension Chemicals 50 million units penicillin-G (e.g. Sigma PEN-K 2)) 50 g streptomycin sulphate (approx. equal to 750 i.u./mg) (e.g. Sigma Cat: 56501 2)) 500 000 units mycostatin (e.g. Nystatin: E R Squibb 591502 2)) Water (5.2.2.2) to 2,5 l.

2) This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the products named. Corresponding products supplied by other manufacturers may be used if they can be shown to lead to the same results. SIST EN 14675:2015

EN 14675:2015 (E) 11 Preparation Dissolve vial contents in water (5.2.2.2) and make up to 2,5 l. Dispense aseptically into 50 ml and 5 ml aliquots. Store at -20 °C. Shake the bottle after thawing. Use 5 ml per litre of medium to give a final concentration of: Penicillin 100 units/ml Streptomycin 100 µg/ml Mycostatin 25 units/ml 5.2.2.6 Antibiotics-Trypsin-Versene® (ATV) 10 × Concentrate Chemicals Sodium chloride (NaCl) 80 g Potassium chloride (KCl) 4 g Glucose 10 g Trypsin 5 g (e.g. Difco 1:250 Cat No: 0152-15-9 2)) Versene® (EDTA) 2 g (e.g. Koch-Light Cat No: 0012424-/B3 2)) 0,2 % Phenol Red solution

in water (5.2.2.2) 1 000 ml Preparation Make up solution to 1 l with water (5.2.2.2) [omitting the sodium bicarbonate and antibiotics (5.2.2.5 and 5.2.2.6)]. Filter through a membrane filter (0,22 m pore size) using positive pressure. Add 76,2 ml 7,5 % sodium bicarbonate solution in water (5.2.2.2) and 5 ml antibiotics (5.2.2.5) to the sterile filtrate. Dispense aseptically into 50 ml aliquots and store at - 20 °C. For use add 50 ml of thawed ATV 10 × concentrate to 450 ml of water (5.2.2.2). The working strength solution contains trypsin 0,05 %, versene 0,02 %, pH = 7,8. 5.2.2.7 Dulbecco’s Phosphate Buffered Saline pH 7,2 – 7,4 (PBS) Sodium chloride (NaCl) 8,00 g Potassium dihydrogen phosphate (KH2PO4) 0,12 g Disodium hydrogen phosphate anhydrous (Na2HPO4) 0,91 g Potassium chloride (KCl) 0,2 g Water (5.2.2.2) to 1 000 ml SIST EN 14675:2015

EN 14675:2015 (E) 12 The solution may be sterilized by autoclaving at 121 °C for 15 min. 5.2.2.8 Earle’s balanced salt solution (BSS), 10 × concentrated Sodium chloride (NaCl) 68,0 g Magnesium sulphate heptahydrate (MgSO4 7H2O) 2,0 g Sodium dihydrogen orthophosphate monohydrate (NaH2PO4 H2O) 1,4 g Potassium chloride (KCl) 4,0 g Calcium chloride (CaCl2) 2,0 g Glucose 10,0 g The calcium chloride (CaCl2) should be dissolved separately in 100 ml of water (5.2.2.2) and added to the other dissolved reagents just before the solution is brought to a final volume of 1 000 ml with water (5.2.2.2). This 10x solution is sterilized by membrane filtration [5.3.2.1 c)] and shall be stored at 4 °C ± 1°C for 4 weeks. For use, the 10x solution is diluted 1:10 with sterile water (5.2.2.2) and buffered by the addition of 2,5 % of an 8,8 % sodium bicarbonate (NaHCO3) solution. 5.2.2.9 Phenol red, 1 % a) 10 g of alcohol-soluble phenol red is placed in a 100 ml beaker. Approximately 20 ml of 1N sodium hydroxide (NaOH) solution are added, mixed and allowed to stand for a few minutes; b) the dissolved dye is transferred into a 1 000 ml volumetric flask (5.3.2.13); c) additional 10 ml of 1N sodium hydroxide (NaOH) solution are added into the beaker. The dissolved material is transferred into the volumetric flask (5.3.2.13); d) the solution is brought to a final volume of 1 000 ml with water (5.2.2.2) and stored at room temperature. 5.2.2.10 Eagle’s minimum essential medium (MEM) The following protocol is given as an example of procedure for the preparation of this cell culture nutrient medium. It may be also obtained from commercial sources. The concentration of amino acids and vitamins can be doubled to give “fortified” MEM. The medium is prepared 10x concentrated and stored in the refrigerator (5.3.2.16). At the time of use glutamine and

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