SIST EN 14562:2006
(Main)Chemical disinfectants and antiseptics - Quantitative carrier test for the evaluation of fungicidal or yeasticidal activity for instruments used in the medical area - Test method and requirements (phase 2, step 2)
Chemical disinfectants and antiseptics - Quantitative carrier test for the evaluation of fungicidal or yeasticidal activity for instruments used in the medical area - Test method and requirements (phase 2, step 2)
This European Standard specifies a test method and the minimum requirements for fungicidal or yeasticidal activity of chemical disinfectant products for instruments that form a homogeneous, physically stable preparation when diluted with hard water - or in the case of ready-to-use products - with water.
This European Standard applies to products that are used in the medical area for disinfecting instruments by immersion - even if they are not covered by the EEC/93/42 Directive on Medical Devices.
This European Standard applies to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example:
- in hospitals, in community medical facilities and in dental institutions;
- in clinics of schools, of kindergardens and of nursing homes;
and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients.
NOTE This method corresponds to a phase 2, step 2 test (see Annex E).
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Keimträgerversuch zur Prüfung der fungiziden oder levuroziden Wirkung für Instrumente im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Diese Europäische Norm legt ein Prüfverfahren und die Mindestanforderungen an die fungizide oder levurozide Wirkung von chemischen Desinfektionsmitteln fest, die in Wasser standardisierter Härte als homogenes physikalisch stabiles Präparat vorliegen bzw. bei gebrauchsfertigen Produkten in Wasser.
Diese Europäische Norm gilt für Produkte, die zur Instrumentendesinfektion im humanmedizinischen Bereich durch Untertauchen verwendet werden, selbst wenn diese nicht durch die Richtlinie 93/42/EWG über Medizinprodukte erfasst sind.
Diese Europäische Norm gilt für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist, z. B. bei der Patientenbetreuung in
- Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
medizinischen Einrichtungen in Schulen, Kindergärten und Heimen;
und können auch am Arbeitsplatz oder im häuslichen Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung von Patienten dienen.
ANMERKUNG Das Verfahren entspricht einer Prüfung der Phase 2, Stufe 2 (Anhang E).
Désinfectants et antiseptiques chimiques - Essai quantitatif de porte germe pour l'évaluation de l'activité fongicide ou levuricide pour instruments utilisés en médecine humaine - Méthode d'essai et prescriptions (phase 2, étape 2)
Le présent document spécifie une méthode d'essai et les prescriptions minimales relatives a l'activité fongicide ou levuricide des désinfectants chimiques pour instruments qui forment une préparation homogene, physiquement stable, lorsqu’ils sont dilués dans l'eau dure ou dans le cas de produits prets a l’emploi dans l’eau.
Le présent document s’applique aux produits utilisés en médecine humaine pour désinfecter des instruments par immersion, y compris ceux qui sont couverts par la Directive européenne 93/42/CEE relative aux dispositifs médicaux.
Le présent document s’applique dans les zones et les situations ou la désinfection est médicalement préconisée. De telles indications se rencontrent dans le cadre des soins apportés aux patients, par exemple :
- dans des hôpitaux, centres de soins médicaux et cabinets dentaires ;
- dans des infirmeries d’écoles, de jardins d’enfants et de maisons de retraite ;
et peuvent aussi se rencontrer sur les lieux de travail ou a domicile. Elles peuvent également concerner des services tels que des blanchisseries et des cuisines qui fournissent des produits directement aux patients.
NOTE Cette méthode correspond a la phase 2, étape 2 (voir Annexe E).
Kemična razkužila in antiseptiki - Kvantitativni preskus s steklenim nosilcem za vrednotenje fungicidnega delovanja ali delovanja kemičnih razkužil in antiseptikov na kvasovke za instrumente, ki se uporabljajo v humani medicini - Preskusna metoda in zahteve (faza 2, stopnja 2)
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Keimträgerversuch zur Prüfung der fungiziden oder levuroziden Wirkung für Instrumente im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)Désinfectants et antiseptiques chimiques - Essai quantitatif de porte germe pour l'évaluation de l'activité fongicide ou levuricide pour instruments utilisés en médecine humaine - Méthode d'essai et prescriptions (phase 2, étape 2)Chemical disinfectants and antiseptics - Quantitative carrier test for the evaluation of fungicidal or yeasticidal activity for instruments used in the medical area - Test method and requirements (phase 2, step 2)11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 14562:2006SIST EN 14562:2006en,fr,de01-september-2006SIST EN 14562:2006SLOVENSKI
STANDARD
SIST EN 14562:2006
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14562May 2006ICS 11.080.20 English VersionChemical disinfectants and antiseptics - Quantitative carrier testfor the evaluation of fungicidal or yeasticidal activity forinstruments used in the medical area - Test method andrequirements (phase 2, step 2)Désinfectants et antiseptiques chimiques - Essai quantitatifde porte germe pour l'évaluation de l'activité fongicide oulevuricide pour instruments utilisés en médecine humaine -Méthode d'essai et prescriptions (phase 2, étape 2)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Keimträgerversuch zur Prüfung der fungizidenoder levuroziden Wirkung für Instrumente imhumanmedizinischen Bereich - Prüfverfahren undAnforderungen (Phase 2, Stufe 2)This European Standard was approved by CEN on 29 August 2005.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 14562:2006: ESIST EN 14562:2006
EN 14562:2006 (E) 2 Contents page Foreword.3 Introduction.4 1 Scope.5 2 Normative references.5 3 Terms and definitions.5 4 Requirements.6 5 Test method.6 5.1 Principle.6 5.2 Materials and reagents.7 5.3 Apparatus and glassware.9 5.5 Procedure for assessing the fungicidal / yeasticidal activity of the product.14 5.6 Experimental data and calculation.18 5.7 Verification of methodology.23 5.8 Expression of results and precision.24 5.9 Interpretation of results – conclusion.25 5.10 Test report.26 Annex A (informative)
Referenced strains in national collections.28 Annex B (informative)
Suitable neutralizers.29 Annex C (informative)
Graphical representations of the test method.31 Annex D (informative)
Example of a typical test report.33 Annex E (informative)
Information on the application and interpretation of European Standards on chemical disinfectants and antiseptics.37 Annex ZA (informative)
Relationship between this European Standard and the Essential Requirements of EU Directive 93/42/EEC.39 Bibliography.40
SIST EN 14562:2006
EN 14562:2006 (E) 3 Foreword This European Standard (EN 14562:2006) 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 November 2006, and conflicting national standards shall be withdrawn at the latest by November 2006. Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different applications in the medical field are in preparation. A collaborative trial will be undertaken to provide a precision annex to this standard. This European Standard has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association and supports essential requirements of EU Directive(s). For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this standard. 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 14562:2006
EN 14562:2006 (E) 4 Introduction This European Standard specifies a carrier test for establishing whether a chemical disinfectant for use on instruments (surgical instruments, anaesthesia material, endoscopes etc.) has a fungicidal or yeasticidal activity in the fields described in the scope. The laboratory test closely simulates practical conditions of application including pre-drying fungi on a carrier, contact time, temperature, test organisms and interfering substances i.e. conditions which may influence the action of chemical disinfectants in practical situations.
The obligatory conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each utilization concentration of the chemical disinfectant found by this test corresponds to defined experimental conditions. However, for some applications the recommendations and/or instructions of use of a product may differ and therefore additional test conditions need to be used. SIST EN 14562:2006
EN 14562:2006 (E) 5 1 Scope This European Standard specifies a test method and the minimum requirements for fungicidal or yeasticidal activity of chemical disinfectant products for instruments that form a homogeneous, physically stable preparation when diluted with hard water – or in the case of ready-to-use products – with water.
This European Standard applies to products that are used in the medical area for disinfecting instruments by immersion – even if they are not covered by the EEC/93/42 Directive on Medical Devices. This European Standard applies to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example: in hospitals, in community medical facilities and in dental institutions; in clinics of schools, of kindergardens and of nursing homes; and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients. NOTE This method corresponds to a phase 2, step 2 test (see Annex E). 2 Normative references The following referenced documents are indispensable for the application of this European Standard. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 12353, Chemical disinfectants and antiseptics – Preservation of microbial strains used for the determination of bactericidal and fungicidal activity 3 Terms and definitions For the purposes of this European Standard, the following terms and definitions apply. 3.1 product chemical agent or formulation used as a chemical disinfectant or antiseptic 3.2 fungicide product that kills fungi (moulds and yeasts) and their spores under defined conditions NOTE The adjective derived from "fungicide" is "fungicidal". 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 yeasticide product that kills yeasts under defined conditions NOTE The adjective derived from "yeasticide" is "yeasticidal". SIST EN 14562:2006
EN 14562:2006 (E) 6 3.5 yeasticidal activity capability of a product to produce a reduction in the number of viable yeast cells of relevant test organisms under defined conditions 3.6 clean conditions conditions representative of surfaces which have been cleaned satisfactorily and/or are known to contain minimal levels of organic and/or inorganic substances 3.7 dirty conditions conditions representative of surfaces which are known to or may contain organic and/or inorganic substances 4 Requirements The product, when diluted with hard water or – in the case of ready-to-use products – with water (5.2.2.2), and tested in accordance with Clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution) or simulated dirty conditions (3 g/l bovine albumin solution, plus 3 ml/l washed sheep erythrocytes) according to its practical applications and under the obligatory test conditions (two selected test organisms, 20 °C, 60 min), shall demonstrate at least a decimal log (lg) reduction in counts of 4. The fungicidal activity shall be evaluated using the following two test organisms: Candida albicans (vegetative cells) and Aspergillus niger (spores). The yeasticidal activity shall be evaluated using the following test organism: Candida albicans (vegetative cells). Where indicated, additional specific fungicidal or yeasticidal activity shall be determined applying other contact times, temperatures, test organisms and interfering substances (5.5.1.1) in order to take into account intended specific use conditions. NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions. 5 Test method 5.1 Principle 5.1.1 A test suspension of fungi (yeast cells or mould spores) in a solution of interfering substances is spread on a glass carrier. After drying the carrier is immersed into a sample of the product as delivered and/or diluted with hard water (for ready to use products: water). The carrier is maintained at 20 °C ± 1 °C for 60 min ± 10 s (obligatory test conditions). At the end of this contact time, the carrier is transferred into a neutralizer containing glass beads. The yeast cells or mould spores are to be severed from the surface by shaking. The numbers of surviving yeast cells or mould spores in each sample are determined and the reduction is calculated. 5.1.2 The test is performed using Candida albicans and Aspergillus niger or only Candida albicans as test organisms (obligatory test conditions). 5.1.3 Additional and optional contact times and temperatures are specified. Additional interfering substances can be used. SIST EN 14562:2006
EN 14562:2006 (E) 7 5.2 Materials and reagents 5.2.1 Test organisms The fungicidal activity shall be evaluated using the following strains as test or organisms1): Candida albicans
ATCC
10231
Aspergillus niger
ATCC
16404 The yeasticidal activity shall be evaluated using only Candida albicans ATCC 10231. NOTE See Annex A for strain references in some other culture collections. The required incubation temperature for these test organisms is 30 °C ± 1 °C (5.3.2.3) If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition. They shall be held by the testing laboratory or national culture collection under a reference for five years.
5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this European Standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms.
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 (5.3.1). NOTE 1 Sterilization is not necessary if the water is used – e.g. for preparation of culture media – and subsequently sterilized. NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. NOTE 3 See 5.2.2.7 for the procedure to prepare hard water.
1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collections (ATCC). This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. SIST EN 14562:2006
EN 14562:2006 (E) 8 5.2.2.3 Malt extract agar (MEA) Malt extract 30,0 g Soya peptone, papaic digest of Soybean Meal 3,0 g Agar 15,0 g Water (5.2.2.2) to 1 000,0 ml Sterilize in the autoclave (5.3.1). After sterilization, the pH of the medium shall be equivalent to 5,6 ± 0,2 when measured at (20 ± 1)°C (5.3.2.4). NOTE In special circumstances (problems with neutralization, see 5.5.1.2 and 5.5.1.3) it may be necessary to add neutralizer to MEA (see B.3). It is recommended not to use neutralizer that causes opalescence in the agar. 5.2.2.4 Diluent Tryptone Sodium Chloride Solution: Tryptone, pancreatic digest of casein 1,0 g Sodium chloride (NaCl) 8,5 g Water (5.2.2.2) to 1 000,0 ml Sterilize in the autoclave (5.3.1). After sterilization the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at (20 ± 1)°C. 5.2.2.5 Neutralizer The neutralizer shall be validated for the product being tested in accordance with 5.5.1 and 5.5.2. The neutralizer shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in B.2. 5.2.2.6 Sterile defibrinated sheep blood The sterile defibrinated sheep blood can be acquired from a commercial supplier or prepared according to EN 14820. 5.2.2.7 Hard water for dilution of products Prepare: Solution A: Dissolve 19,84 g anhydrous magnesium chloride (MgCl2) or an equivalent of hydrated magnesium chloride and 46,24 g anhydrous calcium chloride (CaCl2) or an equivalent of hydrated calcium chloride in water (5.2.2.2) and dilute to 1 000 ml. Sterilize in the autoclave (5.3.1). Store the solution in a refrigerator (5.3.2.8) for no longer than one month.
Solution B: Dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in a refrigerator (5.3.2.8) for no longer than one week. Hard water: For the preparation of 1 l hard water, place 600 ml – 700 ml water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) 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 ± 1) °C. 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 14562:2006
EN 14562:2006 (E) 9 NOTE When preparing the product test solutions (see 5.4.2) the addition of the product to this hard water produces a different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube 5.2.2.8 Interfering substance 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test. The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids, detergents) shall be defined.
NOTE In the following, the term “interfering substance” is used even if it contains more than one substance. 5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration) Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent (5.2.2.4).
Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within 1 month. The final concentration of the bovine albumin in the test procedure (see 5.5) is 0,3 g/l . 5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solutions – high concentration with sheep erythrocytes (5.2.2.6)) Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent (5.2.2.4). Sterilize by membrane filtration (5.3.2.7). Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (5.2.2.6). Centrifuge the sheep blood at 800 gN for 10 min. After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4). Repeat this procedure at least 3 times, until the supernatant is colourless. Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see above). To avoid contamination this mixture should be split in portions probably needed per day and kept in separate containers for a maximum of 7 days in a refrigerator at 2 °C to 8 °C. The final concentration of bovine albumin and sheep erythrocytes in the test procedure (see 5.5) shall be 3 g/l and 3 ml/l respectively. 5.3 Apparatus and glassware 5.3.1 General Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods: a) by moist heat, in the autoclave [5.3.2.1 a)]; b) by dry heat, in the hot air oven [5.3.2.1 b)]. SIST EN 14562:2006
EN 14562:2006 (E) 10 5.3.2 Usual microbiological laboratory equipment 2) 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 (1805 0
+) °C for a minimum holding time of 30 min, at (1705 0
+) °C for a minimum holding time of 1 h or at (1605 0
+) °C for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C, at 45 °C ± 1 °C (if pour plate technique is used) and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled at 30 °C ± 1 °C.
5.3.2.4 pH-meter, having an inaccuracy of calibration of not more than ± 0,1 pH units at (20 ± 1)°C.
NOTE For measuring the pH of the agar-media (5.2.2.3) a puncture electrode or a flat membrane electrode should be used. 5.3.2.5 Stopwatch 5.3.2.6 Shakers a) Electromechanical agitator, e.g. Vortex® mixer3). b) Mechanical shaker 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin (5.2.2.8). The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the microorganisms over the membrane and in order to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s. 5.3.2.8 Refrigerator capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes of nominal capacities 10 ml 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 beads (Diameter: 3 mm to 4 mm). 5.3.2.12 Volumetric flasks. 5.3.2.13
Glass beads (Diameter: 0,25 mm to 0,5 mm).
2) Disposable sterile equipment is an acceptable alternative to reusable glassware. 3) Vortex® in an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 14562:2006
EN 14562:2006 (E) 11 5.3.2.14 Centrifuge (800 gN and 2 000 gN).
5.3.2.15
Cylindrical plastic screw cap tubes, contents of about 15 ml, diameter about 18 mm (for the carrier). 5.3.2.16 Loop (metal or plastic) 5.3.2.17 Frosted glass carriers, 15 mm x 60 mm x 1 mm, one surface sandblasted. For preparation the glass carrier is boiled 10 min in a suitable detergent, cleaned minimum 3 times with water (5.2.2.2) and at the end once with ethanol (70 Vol.%). Mark a square (lateral length: 10 mm) at one end of the dried carrier on its sandblasted surface, about 2 mm off the three edges. Sterilize in the heat oven [5.3.1b)]. After the sterilization process the markings of the “inoculation square” shall be clearly visible.
Key 1 Inoculation square Figure 1 — Frosted glass carrier with markings 5.3.2.18 Roux bottles or similar flasks. 5.3.2.19 Frittered filters: Porosity of 40 µm to 100 µm (see ISO 4793).
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (Test and validation suspension) 5.4.1.1 General For each test organism two different suspensions have to be prepared: the “test suspension” to perform the test and the “validation suspension” to perform the controls and method validation. 5.4.1.2 Preservation and stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.3 Working culture of test organisms 5.4.1.3.1 Candida albicans (yeast) In order to prepare the working culture of Candida albicans (5.2.1), subculture from the stock culture (5.4.1.2) by streaking onto MEA (5.2.2.3) slopes or plates and incubate (5.3.2.3). After 42 h to 48 h prepare a second subculture from the first subculture in the same way and incubate for 42 h to 48 h. From this second subculture a third subculture may be produced in the same way. The second and/or the third subculture is/are the working culture(s). SIST EN 14562:2006
EN 14562:2006 (E) 12 If it is not possible to prepare the second subculture on a particular day, a 72 h subculture may be used for subsequent sub-culturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 72 h period.
Never produce and use a fourth subculture.
5.4.1.3.2 Aspergillus niger (mould) For Aspergillus niger use only the first subculture grown on MEA (5.2.2.3) in Roux bottles (5.3.2.18) and incubate for 9 days to11 days. No further sub-culturing is needed. 5.4.1.4 Test suspension (N) 5.4.1.4.1 Candida albicans
a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take the working culture (5.4.1.2.1) and transfer loopfuls of the cells into the diluent (5.2.2.4).The cells should be suspended in the diluent by rubbing the loop against the wet wall of the flask to dislodge the cells before immersing in the diluent. Shake the flask for 3 min using a mechanical shaker [5.3.2.6b)]. Aspirate the suspension from the glass beads and transfer to a tube.
b) Adjust the number of cells in the suspension to 1,5 x 108 cfu/ml 4) to 5,0 x 108 cfu/ml using the diluent (5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the water bath at 20 °C and use within 2 h. Adjust the temperature according to 5.5.1.1a) and 5.5.1.4 only immediately before the start of the test. NOTE The use of a spectrophotometer for adjusting the number of cells is highly recommended (about 620 nm wavelength – cuvette 10 mm path length). Each laboratory should therefore produce a calibration curve knowing that suitable values of optical density are generally found between 0,200 and 0,350. To achieve reproducible results of this measurement it may be necessary to dilute the test suspension, e.g. 1+9. A colorimeter is a suitable alternative. c) For counting prepare 10-6 and 10-7 dilutions of the test suspension using diluent (5.2.2.4). Mix [5.3.2.6a)]. Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread plate technique. When using the pour plate technique, transfer about half of each 1,0 ml sample into separate Petri dishes (i.e. in duplicate = four plates) and add 15 ml to 20 ml melted MEA (5.2.2.3), cooled to 45 °C ± 1 °C. When using the spread plate technique spread each 1,0 ml sample – divided in portions of approximately equal size – on an appropriate number (at least two) of surface dried plates containing MEA (5.2.2.3). For incubation and counting see 5.4.1.6. 5.4.1.4.2 Aspergillus niger a) Take the working culture (5.4.1.3.2) and suspend the spores in 10 ml of sterile 0,05 % w/v polysorbate 80 solution in water (5.2.2.2). Using a sterile glass spatula detach the conidiospores from the culture surface. The suspension is transferred into a flask and gently shaken by hand for one minute together with 5 g of glass beads (5.3.2.11). The suspension is filtered through a fritted filter (5.3.2.19). b) Carry out a microscopic examination under x 400 magnification immediately after the preparation and just before the test, to show the absence of mycelia fragments and spore germination (check at least ten fields of view for absence of both). If germinated spores are present, discard the suspension. If mycelia are present, set up a washing process (centrifugation) as follows: Transfer the filtered suspension to centrifuge tubes.
4) cfu/ml = colony forming unit(s) per milliliter SIST EN 14562:2006
EN 14562:2006 (E) 13 The filtered suspension is centrifuged (5.3.2.14) at 2 000 gN for 20 min. The conidiospores are washed at least twice by resuspension in diluent (5.2.2.4) and subsequent centrifugation. If mycelia are still present, repeat the washing process.
c) Adjust the number of spores in the suspension to 1,5 x 108 cfu/ml to 5,0 x 108 cfu/ml using the diluent (5.2.2.4), estimating the number of units by any suitable means. Use the suspension within 4 h. It can be stored up to 2 d in the refrigerator and shall then be checked just before the t
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