1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 12353:2013Chemical disinfectants andantiseptics Preservation oftest organisms used for thedetermination of bactericidal(including Legionella),mycobactericidal, sporicidal,f
2、ungicidal and virucidal(including bacteriophages)activityBS EN 12353:2013 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 12353:2013. Itsupersedes BS EN 12353:2006 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee CH/
3、216, Chemical disinfectants and antiseptics.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards
4、 Institution 2013. Published by BSI StandardsLimited 2013ISBN 978 0 580 74829 5ICS 11.080.20; 71.100.35Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 28 February
5、2013.Amendments issued since publicationDate Text affectedBS EN 12353:2013EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12353 February 2013 ICS 11.080.20; 71.100.35 Supersedes EN 12353:2006English Version Chemical disinfectants and antiseptics - Preservation of test organisms used for the det
6、ermination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity Antiseptiques et dsinfectants chimiques - Conservation des organismes test utiliss pour la dtermination de lactivit bactricide (Legionella inclus), mycobactri
7、cide, sporicide, fongicide et virucide (bacteriophages inclus) Chemische Desinfektionsmittel und Antiseptika - Aufbewahrung von Testorganismen fr die Prfung der bakteriziden (einschlielich Legionella), mykobakteriziden, sporiziden, fungiziden und viruziden (einschlilich Bakteriophagen) Wirkung This
8、European Standard was approved by CEN on 14 December 2012. 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 referen
9、ces 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 m
10、ember 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,
11、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 COMIT EUROPEN DE NORMALISATION EUROPISCHE
12、S KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12353:2013: EBS EN 12353:2013EN 12353:2013 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 No
13、rmative references 5 3 Terms and definitions .5 4 Requirements .5 5 Methods 5 5.1 Principle 5 5.2 Materials and reagents 6 5.3 Apparatus and glassware 15 5.4 Procedure for preservation of test organisms General . 16 5.5 Procedure for preservation of bacteria (incl. Legionella, spore-forming bacteria
14、, excl. mycobacteria and bacterial spores) and yeasts 17 5.6 Procedure for preservation of mycobacteria . 18 5.7 Procedure for preservation of moulds (e.g. Aspergillus brasiliensis) 19 5.8 Procedure for preservation of viruses (except dairy bacteriophages) . 20 5.9 Procedure for preservation of bact
15、eriophages . 20 5.10 Verification of the purity and identity of test organisms 21 5.11 Documentation 21 Annex A (informative) Test organisms Culture collection references and relation to CEN/TC 216 standards and prENs 23 A.1 Bacteria (except mycobacteria and spore-forming bacteria) . 23 A.2 Mycobact
16、eria . 24 A.3 Spore-forming bacteria 24 A.4 Fungi (moulds and yeasts) 25 A.5 Viruses . 25 A.6 Bacteriophages . 26 Annex B (informative) Graphical representations 27 Bibliography . 32 BS EN 12353:2013EN 12353:2013 (E) 3 Foreword This document (EN 12353:2013) has been prepared by Technical Committee C
17、EN/TC 216 “Chemical disinfectants and antiseptics”, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by August 2013, and conflicting national standards shall
18、 be withdrawn at the latest by August 2013. 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 12353:2006. Th
19、e 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: g127 The methods of preservation of Legionella, mycobacteria, bacteriophages and viruses are new and were added. Data obta
20、ined by using the former version of EN 12353 are still valid. According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estoni
21、a, 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. BS EN 12353:2013EN 12353:
22、2013 (E) 4 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 ide
23、ntity have been verified and whose biological behaviour remains stable. Therefore it is essential to specify the storage requirements. This European Standard aims at describing methods for preservation of test organisms used for such purposes. BS EN 12353:2013EN 12353:2013 (E) 5 1 Scope This Europea
24、n Standard 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
25、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 European Standard is established. NOTE 1 Annex A (informative) contains a non-exhaustive list of test organisms for which this standard can
26、be applied. NOTE 2 European Standards (EN and prEN) where this European Standard is referenced are listed in the Bibliography. NOTE 3 A specific part on the preservation of bacterial spores may be added once the results of the ongoing ring trials are available. 2 Normative references The following d
27、ocuments, 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 dis
28、infectants and antiseptics Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885 apply. 4 Requirements Each test organism specified in a CEN/TC 216 European Standard and refe
29、rred to in this European Standard shall be handled as described in this European Standard. 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 su
30、spension 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 s
31、uitable 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. 5 Methods 5.1 Principle A sample of the test organism in general in freeze dried form - is obtained from
32、 a culture collection. This sample is cultured, prepared for storage, filled into storage vessels and placed in the deep freeze. BS EN 12353:2013EN 12353:2013 (E) 6 From this sample a stock culture is prepared and subsequently used to prepare working cultures for the test procedure. In some cases th
33、e 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 origin (culture collection), taxonomic name and reference number, date of receipt and batch number of the freeze dried test organisms
34、 shall be recorded (5.11.2). 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
35、 chemical substances given in this European Standard 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 a
36、ppropriate 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 manufactu
37、rers 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
38、not demineralized water. If distilled water of adequate quality is not available, water for injections (see bibliographic reference g621g64) 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
39、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 gG
40、lucose 2,5 gWater (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. BS EN 12353:2013EN 12353:2013 (E) 7 5.2.2.4 Malt Extract Broth (MEB) for fungi Malt extract broth, consisting of: Malt extract (food grade, e.g. C
41、hristomalt powder from Difal or equivalent that is not highly purified and not only based on maltose, e.g. malt extract from OXOID)120,0 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 Cryoprotect
42、ant 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).
43、 After sterilization the pH of the solution shall be equivalent to 6,9 g114 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
44、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,
45、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. 5.2.2.7 Polysorbate 80 solution Polysorbate 80 solution,
46、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). 1This 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 b
47、e used if they can be shown to lead to the same results. BS EN 12353:2013EN 12353:2013 (E) 8 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 w
48、ith 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 sterilise by membrane filtration. The solutio
49、n is stored at (-20 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 sterilise 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 i.u./mg) (eg Sigma Cat : 565012) 25 g117 500,000 units Mycostatin (eg Nystatin : E R Squibb 591502) Water (5.2.2.2) to 2,5 l. Preparation Dissolve vial contents of antib
