SIST EN 12353:2006
Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity
Chemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity
This European Standard specifies methods for keeping test organisms used and defined in European Standards for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal 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 standard is established.
NOTE Annex A (informative) contains a non-exhaustive list of test organisms for which this standard can be applied.
Chemische Desinfektionsmittel und Antiseptika - Aufbewahrung von Testorganismen für die Prüfung der bakteriziden, mykobakteriziden, sporiziden und fungiziden Wirkung
Diese Europäische Norm legt ein Verfahren zur Haltung von Prüfkeimen fest, wie sie in den Europäischen Normen des CEN/TC 216 zur Bestim¬mung der bakteriziden, mykobakteriziden, sporiziden und fungiziden Wirkung chemischer Desinfektionsmittel und Antiseptika verwendet und festgelegt werden. Dieses Verfahren zur Haltung von Prüfkeimen kann nur in Verbindung mit mindestens einer dieser Normen erfolgen, wenn dort auf diese Norm verwiesen wurde.
ANMERKUNG 1 Anhang A (informativ) enthält eine nicht abschließende Liste von Prüfkeimen für welche diese Norm angewendet werden kann.
ANMERKUNG 2 Europäische Normen (EN und prEN), die auf diese Europäischen Normen verweisen, sind im Anhang Literaturhinweise aufgelistet.
Antiseptiques et désinfectants chimiques - Conservation des microorganismes d'essai utilisés pour la détermination de l'activité bactéricide, mycobactéricide, sporicide et fongicide
La présente Norme européenne spécifie les méthodes de conservation des microorganismes d’essai utilisées et définies dans les Normes européennes relatives a la détermination de l’activité bactéricide, mycobactéricide, sporicide et fongicide des désinfectants et antiseptiques chimiques établies par le CEN/TC 216. Ces méthodes peuvent etre mises en oeuvre uniquement en association avec au moins une des normes citant en référence la présente norme.
NOTE 1 L’Annexe A (informative) dresse une liste non exhaustive des microorganismes d’essai auxquels s’applique la présente norme.
NOTE 2 Les Normes européennes (EN et prEN) faisant référence a la présente Norme européenne sont indiquées dans la Bibliographie.
Kemična razkužila in antiseptiki – Shranjevanje preskusnih organizmov za določanje baktericidnega, sporocidnega in fungicidnega delovanja
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Aufbewahrung von Testorganismen für die Prüfung der bakteriziden, mykobakteriziden, sporiziden und fungiziden WirkungAntiseptiques et désinfectants chimiques - Conservation des microorganismes d'essai utilisés pour la détermination de l'activité bactéricide, mycobactéricide, sporicide et fongicideChemical disinfectants and antiseptics - Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal 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:EN 12353:2006SIST EN 12353:2006en,fr,de01-september-2006SIST EN 12353:2006SLOVENSKI
STANDARDSIST EN 12353:20011DGRPHãþD
SIST EN 12353:2006
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 12353June 2006ICS 07.100.99; 11.080.20; 71.100.35Supersedes EN 12353:1999
English VersionChemical disinfectants and antiseptics - Preservation of testorganisms used for the determination of bactericidal,mycobactericidal, sporicidal and fungicidal activityAntiseptiques et désinfectants chimiques - Conservationdes microorganismes d'essai utilisés pour la déterminationde l'activité bactéricide, mycobactéricide, sporicide etfongicideChemische Desinfektionsmittel und Antiseptika -Aufbewahrung von Testorganismen für die Prüfung derbakteriziden, mykobakteriziden, sporiziden und fungizidenWirkungThis European Standard was approved by CEN on 23 February 2006.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2006 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 12353:2006: ESIST EN 12353:2006
EN 12353:2006 (E) 2 Contents Page Foreword.3 Introduction.4 1 Scope.5 2 Normative references.5 3 Terms and definitions.5 4 Requirements.6 5 Methods.6 5.1 Principle.6 5.2 Materials and reagents.6 5.3 Apparatus and glassware.8 5.4 Procedure for preservation of test organisms – General.10 5.5 Procedure for preservation of bacteria (incl. spore-forming bacteria, excl. bacterial spores) and yeasts.10 5.6 Procedure for preservation of mycobacteria.11 5.7 Procedure for preservation of moulds (e.g. Aspergillus niger).12 5.8 Verification of the purity and identity of test organisms.13 5.9 Documentation.13 Annex A (informative)
Test organisms – Culture collection references and relation to CEN/TC 216 standards and prENs.15 Annex B (informative)
Graphical representations.18 Bibliography.22
SIST EN 12353:2006
EN 12353:2006 (E) 3 Foreword This European Standard (EN 12353:2006) 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 December 2006, and conflicting national standards shall be withdrawn at the latest by December 2006. This European Standard supersedes EN 12353:1999. It was revised to include mycobacteria and spore-forming bacteria and to harmonize the structure and wording with other CEN/TC 216 standards. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. SIST EN 12353:2006
EN 12353:2006 (E) 4 Introduction Standardized tests for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal 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 European Standard aims at describing methods for preservation of test organisms used for such purposes. SIST EN 12353:2006
EN 12353:2006 (E) 5 1 Scope This European Standard specifies methods for keeping test organisms used and defined in European Standards for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal 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 standard is established. NOTE 1 Annex A (informative) contains a non-exhaustive list of test organisms for which this standard can be applied. NOTE 2 European Standards (EN and prEN) where this European Standard is referenced are listed in the Bibliography. 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. ISO 4793, Laboratory sintered (fritted) filters – Porosity grading, classification and designation. 3 Terms and definitions For the purposes of this European Standard, the following terms and definitions apply. 3.1 bactericidal activity capability of a product to produce a reduction in the number of viable bacterial cells of relevant test organisms under defined conditions 3.2 conidium asexual fungal spore, produced exogenously from a hyphal tip NOTE Conidiospore is a synonym for conidium. 3.3 fungicidal activity capability of a product to produce a reduction in the number of viable vegetative yeast cells and mould spores of relevant test organisms under defined conditions 3.4 mycobactericidal activity capability of a product to produce a reduction in the number of viable mycobacterial cells of relevant test organisms under defined conditions 3.5 product chemical agent or formulation used as a chemical disinfectant or antiseptic 3.6 sporicidal activity capability of a product to produce a reduction in the number of viable bacterial spores of relevant test organisms under defined conditions SIST EN 12353:2006
EN 12353:2006 (E) 6 3.7 test organism strain of a micro-organism selected for testing products within a standardized test NOTE For the purpose of this European Standard the term micro-organism includes vegetative bacteria, bacterial spores, fungi, fungal spores and viruses. 3.8 yeasticidal activity capability of a product to produce a reduction in the number of viable yeast cells of relevant test organisms under defined conditions 4 Requirements Each test organism specified in a CEN/TC 216 European Standard and referred to in this standard shall be handled as described in this standard. The purity and identity of the preserved test organism shall be verified during the preparation and regularly during the storage. The preserved test organism 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 or EN 14348 for mycobacteria. 5 Methods
5.1 Principle A freeze dried sample of the test organism is obtained from a culture collection. This sample is cultured, prepared for storage, filled into storage vessels and placed in the deep freeze.
From the deep freeze samples 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 origin (culture collection), taxonomic name and reference number, date of receipt and batch number of the freeze dried test organisms shall be recorded (5.9.2). 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. SIST EN 12353:2006
EN 12353:2006 (E) 7 NOTE 1 To improve reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be rigorously followed.
NOTE 2 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 [5.3.2.1a)]. NOTE 1 Sterilization is not necessary if the water is used for e.g. 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. 5.2.2.3 Tryptone Soya Broth (TSB) for bacteria 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 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 when measured at 20 °C ± 1 °C. 5.2.2.4 Malt Extract Broth (MEB) for fungi Malt extract broth, consisting of: Malt extract
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 6,9 ± 0,2 when measured at 20 °C ± 1 °C
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 SIST EN 12353:2006
EN 12353:2006 (E) 8 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 when measured at 20 °C ± 1 °C. NOTE 1 Any commercially available cryoprotectant containing glycerol for preservation of test organisms equivalent to the solution described above may be used. NOTE 2 If justified, any other equivalent cryoprotectant solution may be used. 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)
750,0 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 and then sterilized water (5.2.2.2) to 1 000,0 ml. The pH of the medium shall be equivalent to 6,6 ± 0,2 when measured at 20 °C ± 1 °C.
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)]. 5.3 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 [5.3.2.1a)]; b) by dry heat, in the hot air oven [5.3.2.1b)]. SIST EN 12353:2006
EN 12353:2006 (E) 9 5.3.2 Usual microbiological laboratory equipment1) and in particular, the following: 5.3.2.1 Apparatus for sterilization: a) for moist heat sterilization, an autoclave capable of being maintained at ()C
1213 0°+ for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at ()C 1805 0°+ for a minimum holding time of 30 min, at ()C1705 0°+ for a minimum holding time of 1 h or at ()C1605 0°+for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C and at 45 °C ± 1 °C if pour plate technique is used. 5.3.2.3 Incubator, capable of being controlled at either 36 °C ± 1 °C or at 37 °C ± 1 °C (for bacteria and mycobacteria) or at 30 °C ± 1 °C (for fungi). NOTE For mycobacteria a CO2 – incubator and a temperature of 36 ºC ± 1 ºC is better suited. If a CO2 – incubator is not used, the inoculated plates should be protected from drying by sealing with insulating tape or packing them into polyethylene bags. 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.
NOTE A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.4.2).
5.3.2.5 Fritted filter: porosity of 40 µm to 100 µm (ISO 4793). 5.3.2.6 Electromechanical agitator e.g. Vortex® mixer2). 5.3.2.7 Forceps or wire. 5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic pipettes may be used. 5.3.2.10 Petri dishes (plates), of size 90 mm to 100 mm. 5.3.2.11 Glass or ceramic beads, (3 mm to 4 mm in diameter). 5.3.2.12 Volumetric flasks. 5.3.2.13 Equipment for deep freezing test organisms, at a temperature of –70 °C or less – including cryovials of nominal capacity of 0,5 ml (min) to 2,0 ml (max). 5.3.2.14 Centrifuge (2 000 gN).
1) Disposable equipment is an acceptable alternative to reusable glassware. 2) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 12353:2006
EN 12353:2006 (E) 10 5.4 Procedure for preservation of test organisms – General
5.4.1 Handling of freeze dried test organisms from culture collections Follow for reconstitution of the freeze dried samples of the test organisms the procedures described in section 5.5. If in special cases it is not possible or appropriate, follow the supplier's recommendations. 5.4.2 Choice of incubation procedure and agar medium Apply always the same incubation procedure (temperature, time and type of incubator) and use the same agar as prescribed for the preparation of the working cultures / test suspension (e.g. “N”) of test organisms in the corresponding European Standard. NOTE In most of the CEN/TC 216 standards you will find this informa
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