prEN 12353

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.DChemische Desinfektionsmittel und Antiseptika - Aufbewahrung von Testorganismen für die Prüfung der bakteriziden (einschließlich Legionella), mykobakteriziden, sporiziden, fungiziden und viruziden (einschließlich Bakteriophagen) WirkungChemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposes07.100.99Drugi standardi v zvezi z mikrobiologijoOther standards related to microbiologyICS:Ta slovenski standard je istoveten z:prEN 12353oSIST prEN 12353:2019en,fr,de01-februar-2019oSIST prEN 12353:2019SLOVENSKI

Chemical disinfectants and antiseptics æ Preservation of test organisms used for the determination of bactericidal

Chemische Desinfektionsmittel und Antiseptika æ Aufbewahrung von Testorganismen für die Prüfung mykobakterizidená sporizidená fungiziden und This draft European Standard is submitted to CEN members for enquiryä It has been drawn up by the Technical Committee

If this draft becomes a European Standardá CEN members are bounwhich stipulate the conditions for giving this European Standard the status of a national standard without any alterationä

This draft European Standard was established by CEN in three ofer language made by 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 Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä

Recipients of this draft are invited to submitá with their commentsá notification of any relevant patent rights of which they are aware and to provide supporting documentationä

Warning ã This document is not a European Standardä It is distributed for review and commentsä It is subject to change without notice and shall not be referred to as a European Standardä

EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG

t r s { CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä prEN

prEN 12353:2019 (E) 2 Contents Page European foreword ....................................................................................................................................................... 5 Introduction .................................................................................................................................................................... 6 1 Scope .................................................................................................................................................................... 7 2 Normative references .................................................................................................................................... 7 3 Terms and definitions ................................................................................................................................... 7 4 Requirements ................................................................................................................................................... 7 5 Methods .............................................................................................................................................................. 8 5.1 Principle ............................................................................................................................................................. 8 5.2 Materials and reagents .................................................................................................................................. 8 5.2.1 Test organisms ................................................................................................................................................. 8 5.2.2 Culture media and reagents ........................................................................................................................ 8 5.2.3 Cell cultures .................................................................................................................................................... 16 5.2.4 Host strains for dairy bacteriophages (Lactococcus lactis) ........................................................... 18 5.3 Apparatus and glassware .......................................................................................................................... 18 5.3.1 General ............................................................................................................................................................. 18 5.3.2 Usual microbiological laboratory equipment .................................................................................... 18 5.4 Procedure for preservation of test organisms – General .............................................................. 20 5.4.1 Handling of freeze-dried / frozen test organisms from culture collections ........................... 20 5.4.2 Choice of incubation procedure, agar medium, cell culture/cell line ....................................... 20 5.5 Procedure for preservation of bacteria (incl. Legionella, spore-forming bacteria, excl. mycobacteria and bacterial spores) and yeasts ...................................................................... 20 5.5.1 Reconstitution of the freeze-dried test organisms .......................................................................... 20 5.5.2 Preparation for storage ............................................................................................................................. 20 5.5.3 Preparation of stock culture / working cultures .............................................................................. 21 5.6 Procedure for preservation of mycobacteria .................................................................................... 21 5.6.1 Reconstitution of the freeze-dried test organisms .......................................................................... 21 5.6.2 Preparation for storage ............................................................................................................................. 21 5.6.3 Preparation of working cultures ............................................................................................................ 22 5.7 Procedure for preservation of moulds (e.g. Aspergillus brasiliensis) ....................................... 22 5.7.1 Reconstitution of the freeze-dried test organism ............................................................................ 22 5.7.2 Preparation for storage ............................................................................................................................. 22 5.7.3 Preparation of stock culture / working cultures .............................................................................. 23 5.8 Procedure for preservation of viruses (except dairy bacteriophages) .................................... 23 5.8.1 Reconstitution of frozen virus ................................................................................................................. 23 5.8.2 Preparation for storage of stock virus suspension .......................................................................... 23 5.8.3 Preparation of test virus suspension .................................................................................................... 24 5.9 Procedure for preservation of bacteriophages ................................................................................. 24 5.9.1 Reconstitution of frozen bacteriophages ............................................................................................ 24 5.9.2 Preparation for storage ............................................................................................................................. 24 5.9.3 Preparation of bacteriophages working suspensions .................................................................... 25 5.10 Verification of the purity and identity of test organisms .............................................................. 25 5.10.1 General ............................................................................................................................................................. 25 5.10.2 Information on source of strains ............................................................................................................ 25 5.10.3 Purity ................................................................................................................................................................ 25 5.10.4 Identity ............................................................................................................................................................. 25 oSIST prEN 12353:2019

prEN 12353:2019 (E) 3 5.11 Documentation .............................................................................................................................................. 25 5.11.1 General ............................................................................................................................................................. 25 5.11.2 Freeze-dried test organism / frozen viruses ...................................................................................... 26 5.11.3 Cryovials of frozen test organism ........................................................................................................... 26 5.11.4 Stock culture ................................................................................................................................................... 26 5.11.5 Verification of purity and identity .......................................................................................................... 26 5.11.6 Storage of documentation ......................................................................................................................... 26 Annex A (informative)

Test organisms – Culture collection references and relation to CEN/TC 216 standards and prENs .......................................................................................................... 27 A.1 Bacteria (except mycobacteria and spore-forming bacteria) ...................................................... 27 A.1.1 Enterobacter cloacae ................................................................................................................................... 27 A.1.2 Enterococcus hirae ....................................................................................................................................... 27 A.1.3 Enterococcus faecium ................................................................................................................................. 27 A.1.4 Escherichia coli (1) ....................................................................................................................................... 27 A.1.5 Escherichia coli (2) K12 .............................................................................................................................. 27 A.1.6 Lactobacillus brevis ..................................................................................................................................... 27 A.1.7 Legionella pneumophila, subsp. Pneumophila .................................................................................... 27 A.1.8 Proteus hauseri ............................................................................................................................................. 27 A.1.9 Pseudomonas aeruginosa .......................................................................................................................... 28 A.1.10 Salmonella enterica subsp. enterica, Serotype typhimurium ........................................................ 28 A.1.11 Staphylococcus aureus subsp. aureus .................................................................................................... 28 A.2 Mycobacteria .................................................................................................................................................. 28 A.2.1 Mycobacterium avium subsp. avium ...................................................................................................... 28 A.2.2 Mycobacterium terrae ................................................................................................................................ 28 A.3 Spore-forming bacteria .............................................................................................................................. 28 A.3.1 Bacillus cereus ............................................................................................................................................... 28 A.3.2 Bacillus subtilis subsp. spizizenii ............................................................................................................. 28 A.3.3 Clostridium sporogenes ............................................................................................................................. 28 A.4 Fungi (moulds and yeasts) ........................................................................................................................ 29 A.4.1 Aspergillus brasiliensis (former “A. niger”) (mould) ...................................................................... 29 A.4.2 Candida albicans (yeast) ............................................................................................................................ 29 A.4.3 Saccharomyces cerevisiae (1) (yeast) ................................................................................................... 29 A.4.4 Saccharomyces cerevisiae (2) var. diastaticus (yeast) ................................................................... 29 A.5 Viruses .............................................................................................................................................................. 29 A.5.1 Adenovirus type 5, strain Adenoid 75 .................................................................................................... 29 A.5.2 Bovine Enterovirus Type 1(ECBO) ........................................................................................................... 29 A.5.3 Murine norovirus, strain S99 Berlin ....................................................................................................... 29 A.5.4 Murine Parvovirus, minute virus of mice, strain Crawford ............................................................ 29 A.5.5 Poliovirus type 1, Sabin strain LSc-2ab .................................................................................................. 29 oSIST prEN 12353:2019

prEN 12353:2019 (E) 4 A.5.6 Vaccinia virus, strain Modified Vaccinia Virus Ankara (MVA) ...................................................... 30 A.5.7 Vaccinia virus, strain Elstree .................................................................................................................... 30 A.6 Bacteriophages ............................................................................................................................................. 30 A.6.1 Lactococcus lactis subsp. lactis bacteriophage P008 ....................................................................... 30 A.6.2 Lactococcus lactis subsp. lactis bacteriophage P001 ....................................................................... 30 Annex B (informative)

Graphical representations........................................................................................ 31 Bibliography ................................................................................................................................................................. 36

prEN 12353:2019 (E) 5 European foreword This document (prEN 12353:2019) 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 document will supersede EN 12353:2013. The document was revised to adapt it to the latest state of science, to correct errors and ambiguities. The following are the significant technical changes since the last edition: — the methods of preservation of viruses are described more detailed (5.8.3); — the description of BCYE Agar for Legionella was added (5.2.2.24); — the information on source of strains was added (5.10.2); — the information of the storage of documentation was added (5.11.6); — the IP Number for fungi was deleted (see A.4); — the used virus strains are re-drafted (see A.5). The changes mentioned above have no impact on the test results obtained with reference to the previous version. Those results are still valid. oSIST prEN 12353:2019

prEN 12353:2019 (E) 6 Introduction Standardized tests for the determination of bactericidal (incl. Legionella pneumophila), mycobactericidal, sporicidal, fungicidal and virucidal (incl. bacteriophages) activity of chemical disinfectants and antiseptics necessitate the use of test organisms whose purity and identity have been verified and whose biological behaviour remains stable. Therefore it is essential to specify the storage requirements. This document aims to describe methods for preservation of test organisms used for such purposes. oSIST prEN 12353:2019

prEN 12353:2019 (E) 7 1 Scope This document specifies methods for keeping test organisms used and defined in European Standards for the determination of bactericidal (incl. Legionella pneumophila), mycobactericidal, sporicidal, fungicidal and virucidal (incl. bacteriophages) activity of chemical disinfectants and antiseptics drawn up by CEN/TC 216. These methods for keeping test organisms can only be carried out in connection with at least one of those standards where a reference to this document is established. NOTE 1 Annex A (informative) contains a non-exhaustive list of test organisms for which this document can be applied. NOTE 2 European Standards (EN and prEN) where this document is referenced are listed in the Bibliography. NOTE 3 A specific part on the preservation of bacterial spores could be added once the results of the ongoing ring trials are available. 2 Normative references The following documents are referred to in the text in such a way that some or all of their content constitutes requirements 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 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885 apply. ISO and IEC maintain terminological databases for use in standardization at the following addresses:

ISO Online browsing platform: available at http://www.iso.org/obp 4 Requirements Each test organism specified in a CEN/TC 216 European Standard and referred to in this document shall be handled as described in this document. The purity and identity of the preserved test organism shall be verified during the preparation and regularly during the storage, except for viruses where only the identity is checked before the stock virus suspension is stored. The preserved test organism – except viruses - should be checked at regular intervals (at least in the case of longer storage than 14 months) to ensure that its susceptibility to products has not changed. As long as CEN/TC 216 has not developed specific tests for this purpose any suitable method can be used e.g. EN 1040 for bacteria, EN 1275 for fungi, EN 14348 for mycobacteria, EN 13623 for Legionella pneumophila, EN 14476 for viruses or EN 13610 for dairy bacteriophages. oSIST prEN 12353:2019

prEN 12353:2019 (E) 8 5 Methods 5.1 Principle A sample of the test organism – in general in freeze-dried form - is obtained from a culture collection. This sample is cultured, prepared for storage, filled into storage vessels and placed in the deep freeze. From this sample a stock culture is prepared and subsequently used to prepare working cultures for the test procedure. In some cases the working cultures are directly prepared from the deep freeze samples. 5.2 Materials and reagents 5.2.1 Test organisms See Annex A for examples of test organisms. The documentation on the test organisms should follow 5.10.2 and 5.11.3. 5.2.2 Culture media and reagents 5.2.2.1 General The formulas of all media and reagents are given in case commercial ready-to-use material is not used. It is to be checked that each commercial supplier has established an appropriate quality control system. All weights of chemical substances given in this document refer to the anhydrous salts unless otherwise stated. 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 whenever possible, 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.

All specified pH values are measured at (20 ± 1) °C. For each culture medium, cell culture 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. If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. Sterilize in the autoclave (5.3.2.1a). Sterilization is not necessary if the water is used for e.g. preparation of culture media and subsequently sterilized. 5.2.2.3 Tryptone Soya Broth (TSB) for bacteria, except Legionella Tryptone soya broth, consisting of: Tryptone, pancreatic digest of casein 17,0 g Soya peptone, papaic digest of soybean meal 3,0 g Sodium chloride (NaCl) 5,0 g Water (5.2.2.2) 800,0 ml Dipotassium phosphate (K2HPO4) 2,5 g Glucose 2,5 g oSIST prEN 12353:2019

prEN 12353:2019 (E) 9 Water (5.2.2.2) to 1 000,0 ml Sterilize in the autoclave (5.3.2.1a). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2. 5.2.2.4 Malt Extract Broth (MEB) for fungi Malt extract broth, consisting of: Malt extract (food grade, e.g. Christomalt powder from Difal or equivalent that is not highly purified and not only based on maltose, e.g.

malt extract from OXOID)1 20,0 g Water (5.2.2.2) to 1 000,0 ml Sterilize in the autoclave (5.3.2.1a). After sterilization the pH of the medium shall be equivalent to 5,6 ± 0,2. 5.2.2.5 Cryoprotectant solution for bacteria, spore-forming bacteria, fungi Cryoprotectant solution, consisting of: Beef extract 3,0 g Tryptone, pancreatic digest of casein 5,0 g Glycerol (C3H8O3) [2] 150,0 g Water (5.2.2.2) to 1 000,0 ml Dissolve the constituents in boiling water. Sterilize in the autoclave (5.3.2.1a). After sterilization the pH of the solution shall be equivalent to 6,9 ± 0,2. Any commercially available cryoprotectant containing glycerol for preservation of test organisms equivalent to the solution described above may be used. If justified, any other equivalent cryoprotectant solution may be used, e.g. for Legionella (5.5.2). 5.2.2.6 Middlebrook 7 H 9 broth with 10 % ADC enrichment and glycerol as reconstituent and cryoprotectant solution for mycobacteria (MADC) Middlebrook 7 H 9 broth, consisting of: Middlebrook 7 H 9 broth powder 4,7 g Glycerol (C3H8O3) [2] 100,0 ml Water (5.2.2.2) 800,0 ml Treat in the autoclave (5.3.2.1a) for a holding time of only 10 min and cool to 45 °C. Add under aseptic conditions 100 ml Middlebrook ADC enrichment to obtain approximately 1 000,0 ml. The pH of the medium shall be equivalent to 6,6 ± 0,2.

1 This information is given for the information of users of this standard and does not constitute an endorsement of the products named. Corresponding products supplied by other manufacturers may be used if they can be shown to lead to the same results. oSIST prEN 12353:2019

prEN 12353:2019 (E) 10 5.2.2.7 Polysorbate 80 solution Polysorbate 80 solution, consisting of: Polysorbate 80 0,5 g Water (5.2.2.2) to 1 000,0 ml Sterilize in the autoclave (5.3.2.1a). 5.2.2.8 DMSO as cryoprotectant for cell culture freezing Dimethyl sulphoxide (DMSO) is used to help protect the cells from rupture by the formation of ice crystals. Since DMSO is toxic it should be handled with care. It can be absorbed through the skin and may cause irritation and/or burns. It is teratogenic and an allergen. Latex gloves should be worn when handling it. 5.2.2.9 Glutamine solution, 3 % Dissolve 12 g Glutamine in 400 ml of water (5.2.2.2) and sterilize by membrane filtration. The solution

± 1) °C. 5.2.2.10 TV (Trypsin-Versene) Dissolve 0,05 g Trypsin in 100 ml of 0,53 mM EDTA (Ethylene diamine tetra acetic acid) and sterilize by membrane filtration. Store at (4 ± 1) °C. 5.2.2.11 Antibiotic suspension Chemicals 50 million units Penicillin-G (eg Sigma PEN-K2) 50 g Streptomycin sulphate (approx. equal to 750 IU/mg) (eg

Preparation Dissolve vial contents of antibiotics in water (5.2.2.2) and fill up to 2,5 l. Dispense aseptically into 50 ml and 5 ml aliquots. Store at

t r °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

2 This information is given for the information of users of this standard and does not constitute an endorsement of the products named. Corresponding products supplied by other manufacturers may be used if they can be shown to lead to the same results. oSIST prEN 12353:2019

prEN 12353:2019 (E) 11 5.2.2.12 Phosphate-buffered saline solution (PBS) Sodium chloride (NaCl) 8,00 g Potassium chloride (KCl) 0,20 g Disodium hydrogen phosphate, 12-hydrate (Na2HPO4 x 12H2O) 2,89 g Potassium phosphate, monobasic (KH2PO4) 0,20 g Water (5.2.2.2) to 1 000,0 ml 5.2.2.13 Foetal calf serum (FCS) FCS has to be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may interfere with cell and virus growth resulting in false results. 5.2.2.14 Earle’s BSS Sodium chloride (NaCl) 68,0 g Potassium chloride (KCl) 4,0 g Calcium chloride (CaCl2) 2,0 g Magnesium sulphate, 7-hydrate (MgSO4 x 7H2O) 2,0 g Sodium hydrogenphosphate, 2-hydrate (NaH2PO2 x 2H2O) 1,4 g Glucose 10,0 g Phenol red, 1 % (5.2.2.15) 20,0 ml Water (5.2.2.2) to 1 000,0 ml 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 its final volume. The solution is 10-fold concentrated. It is sterilized by membrane filtration through a 0,22 µm Millipore or Seitz-type filter3 and can be stored at (4 ± 1) °C for 4 weeks. For use the solution is diluted 10-fold with water (5.2.2.2) and buffered by the addition of 2,5 % of an 8,8 % Sodium hydrogen carbonate (NaHCO3) solution. 5.2.2.15 Phenol red, 1 % solution a) A 1,0 N Sodium hydroxide (NaOH) solution is prepared. b) 10 g of alcohol soluble Phenol red, European Pharmacopeia [2] are placed in a 100 ml flask (5.3.2.12); 20 ml of the NaOH solution are added, mixed and allowed to stand for a few minutes. c) The dissolved dye is transferred in a 1 000 ml volumetric flask (5.3.2.12). d) Additional 10 ml amounts of the NaOH solution are added to the flask and the dissolved material is added to the volumetric flask. No more than a total of 70 ml of the NaOH solution should be used.

3 Millipore® and Seitz® are examples of suitable products 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. oSIST prEN 12353:2019

prEN 12353:2019 (E) 12 e) The solution is brought to a final volume of 1 000 ml with water (5.2.2.2) and stored at room temperature. 5.2.2.16 Sodium bicarbonate (8,8 % w/v solution) Dissolve 8,8 g sodium bicarbonate in water (5.2.2.2) to 100 ml and sterilize by autoclaving (5.3.2.1a).

Store at (4 ± 1) °C. 5.2.2.17 Eagle’s minimum essential medium (MEM) for cell cultures MEM is used for growth and maintenance of cell cultures. First prepare a stock solution. For use, the stock solution is diluted 10-fold with water (5.2.2.2). One % of the 3 % Glutamine solution (= 0,03 %) (5.2.2.9), Antibiotic suspension (5.2.2.11), and 2,5 % of a 8,8 % Sodium bicarbonate solution (5.2.2.16) are added. An appropriate concentration of foetal calf serum (FCS, (5.2.2.13); 10 % for growth, 2 % for maintenance) is added before use. The foll