SIST EN 14348:2005

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der mykobakteriziden Wirkung chemischer Desinfektionsmittel im humanmedizinischen Bereich einschließlich der Instrumentendesinfektionsmittel - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif en suspension pour l'evaluation de l'activité mycobactéricide des désinfectants chimiques utilisés en médecine humaine et incluant les désinfectants pour dispositifs médicaux - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of mycobactericidal activity of chemical disinfectants in the medical area including instrument disinfectants - Test methods and requirements (phase 2, step 1)11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 14348:2005SIST EN 14348:2005en,fr,de01-junij-2005SIST EN 14348:2005SLOVENSKI
STANDARD



SIST EN 14348:2005



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14348January 2005ICS 11.080.20English versionChemical disinfectants and antiseptics - Quantitative suspensiontest for the evaluation of mycobactericidal activity of chemicaldisinfectants in the medical area including instrumentdisinfectants - Test methods and requirements (phase 2, step 1)Désinfectants chimiques - Essai quantitatif de suspensionpour l'evaluation de l'activité mycobactéricide desdésinfectants chimiques utilisés en médecine y compris lesdésinfectants pour instruments - Méthode d'essai etprescriptions (phase 2, étape 1)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Suspensionsversuch zur Bestimmung dermykobakteriziden Wirkung chemischer Desinfektionsmittelim humanmedizinischen Bereich einschließlich derInstrumentendesinfektionsmittel - Prüfverfahren undAnforderungen (Phase 2, Stufe 1)This European Standard was approved by CEN on 22 November 2004.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, 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© 2005 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14348:2005: ESIST EN 14348:2005



EN 14348:2005 (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 methods.7 5.1 Principle.7 5.2 Materials and reagents.7 5.3 Apparatus and glassware.10 5.4 Preparation of test organism suspensions and product test solutions.11 5.5 Procedure for assessing the mycobactericidal activity of the product.14 5.6 Experimental data and calculation.16 5.7 Verification of methodology.22 5.8 Expression of results and precision.23 5.9 Interpretation of results - conclusion.23 5.10 Test report.24 Annex A (informative)
Referenced strains in national collections.26 Annex B (informative)
Suitable neutralizers.27 Annex C (informative)
Graphical representations of the test method.29 Annex D (informative)
Example of a typical test report.31 Annex E (informative)
Information on the application and interpretation of European standards on chemical disinfectants and antiseptics.33 Annex ZA (informative)
Clauses of this document addressing essential requirements or other provisions of EU Directives.35 Bibliography.36
SIST EN 14348:2005



EN 14348:2005 (E) 3 Foreword This document (EN 14348:2005) 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 July 2005, and conflicting national standards shall be withdrawn at the latest by July 2005.
This document 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 Medical Devices Directive 93/42. For relationship with EU Directive, see informative Annex ZA, which is an integral part of this document. A collaborative trial has been undertaken and will be evaluated to provide a precision annex to this document. Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different applications in the medical area are in preparation. 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, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. SIST EN 14348:2005



EN 14348:2005 (E) 4 Introduction This document describes a suspension test for establishing whether a chemical disinfectant has or does not have a mycobactericidal activity in the area defined in the scope. This laboratory test takes into account practical conditions of application of the product ,including contact time, temperature, test organisms and interfering substances, i.e. conditions which may influence its action in practical situations.
The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each concentration of the chemical disinfectant found by this test corresponds to the chosen experimental conditions. However, for some applications the instructions of use of a product may differ and therefore additional test conditions need to be used. SIST EN 14348:2005



EN 14348:2005 (E) 5 1 Scope This document specifies a test method and the minimum requirements for mycobactericidal (or tuberculocidal) activity of chemical disinfectant products that form a homogeneous, physically stable preparation when diluted with hard water - or in the case of ready-to-use products - with water. Products can only be tested at a concentration of 80 % or less as some dilution is always produced by adding the test organisms and interfering substance. This document applies to products that are used in the medical area including those that are covered by the EEC/93/42 Directive on Medical Devices. This document 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 kindergartens 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 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2, step 1 test (see Annex E). 2 Normative references The following referenced documents are indispensable for the application 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 12353, Chemical disinfectants and antiseptics — Preservation of microbial strains used for the determination of bactericidal and fungicidal activity. EN 14820, Single-use containers for human venous blood specimen collection. 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 product chemical agent or formulation used as a chemical disinfectant or antiseptic 3.2 mycobactericide product which kills mycobacteria under defined conditions NOTE The adjective derived from “mycobactericide” is “mycobactericidal”. 3.3 mycobactericidal activity
capability of a product to produce a reduction in the number of viable mycobacterial cells of relevant test organisms under defined conditions SIST EN 14348:2005



EN 14348:2005 (E) 6 3.4 mycobacteriostatic activity capability of a product to inhibit the growth of mycobacteria under defined conditions 3.5 tuberculocide product which kills Mycobacterium tuberculosis under defined conditions NOTE The adjective derived from “tuberculocide” is “tuberculocidal”. 3.6 tuberculocidal activity capability of a product to kill Mycobacterium tuberculosis, demonstrated by the capability to produce a reduction in the number of viable cells of Mycobacterium terrae under defined conditions 3.7 clean conditions conditions representative of surfaces which have received a satisfactory cleaning programme and/or are known to contain minimal levels of organic and/or inorganic substances 3.8 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 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 (one or two selected test organisms, 20 °C, 60 min), shall demonstrate at least a decimal log (lg) reduction in counts of 4. It is possible to test also the product as delivered (highest test concentration is 80 %). The mycobactericidal activity shall be evaluated using the following two test organisms: Mycobacterium avium and Mycobacterium terrae. The tuberculocidal activity shall be evaluated using the following test organism:
Mycobacterium terrae. Where indicated, additional specific mycobactericidal or tuberculocidal activity shall be determined applying other contact times, temperatures 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. SIST EN 14348:2005



EN 14348:2005 (E) 7 5 Test methods 5.1 Principle 5.1.1 A test suspension of mycobacteria in a solution of an interfering substance is added to a sample of the product as delivered and/or diluted with hard water (for ready to use products: water). The mixture is maintained at 20 °C ± 1 °C for 60 min ± 10 s (obligatory test conditions). At the end of this contact time, an aliquot is taken; the mycobactericidal and/or the mycobacteriostatic activity in this portion is immediately neutralized or suppressed by a validated method. The numbers of surviving mycobacteria in each sample are determined and the reduction is calculated. 5.1.2 The test is performed using Mycobacterium avium and Mycobacterium terrae or only Mycobacterium terrae as test organisms (obligatory test conditions). 5.1.3 Additional and optional contact times and temperatures are specified. Additional interfering substances can be used. 5.2 Materials and reagents 5.2.1 Test organisms The mycobactericidal activity shall be evaluated using the following two test-organisms1):
Mycobacterium avium ATCC
15769
Mycobacterium terrae ATCC
15755 The tuberculocidal activity shall be evaluated using only Mycobacterium terrae. NOTE See annex A for corresponding strain reference in some other culture collections. 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this 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).
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 standard and does not constitute an endorsement by CEN of the product named.
SIST EN 14348:2005



EN 14348:2005 (E) 8 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. 5.2.2.3 Middlebrook and Cohn 7 H 10 medium with 10 % OADC enrichment (MCO) Middlebrook 7 H 10 agar-powder
19,0 g Glycerol (C3H8O3) (see [1])
5,0 ml Water (5.2.2.2)
to 900,0 ml Heat to boiling to dissolve completely. Sterilize in the autoclave (5.3.1) and cool to 50 ºC to 55 ºC. Add 100 ml Middlebrook OADC enrichment under aseptic conditions. Fill 18 ml to 20 ml per plate (5.3.2.10). The pH of the medium shall be equivalent to 6,6 ± 0,2 when measured at 25 °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 MCO (see annex B.3). Do not 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 (see 5.3.1). After sterilization the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at 20 °C. NOTE There is scientific experience that sodium chloride (NaCl) may inhibit the growth of mycobacteria. But the presence of NaCl in products and in the interfering substance (5.2.2.8) is unavoidable and therefore diluent might be used for the preparation of neutralizer (5.5.1.2). 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 Middlebrook 7 H 9 broth with 10 % ADC enrichment (MADC-broth) MADC-broth will be used for deep freeze storage Middlebrook 7H9 broth-powder
4,7 g Glycerol (C3H8O3) (see [1])
100,0 ml Water (5.2.2.2)
750,0 ml
Sterilize in the autoclave (5.3.1) and cool to 45 ºC. Add under aseptic conditions 100 ml Middlebrook ADC enrichment and 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 25 ºC (5.3.2.4).
5.2.2.7 Hard water for dilution of products Prepare: SIST EN 14348:2005



EN 14348:2005 (E) 9  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 at 2 °C to 8 °C for no longer than one month.  Solution B: Dissolve 35,02 g sodium hydrogencarbonate (NaHCO³) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution at 2 °C to 8 °C for no longer than one week. Hard water: For the preparation of 1 litre, place 600 ml to 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. 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. NOTE When preparing the product test solutions (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 (at 2 °C to 8 °C) 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) 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 (5.2.2.9) or purchase such blood from a commercial supplier (5.2.2.9). Centrifuge the erythrocytes at 800 g 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 later 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 (5.5) shall be 3 g/l and 3 ml/l respectively. SIST EN 14348:2005



EN 14348:2005 (E) 10 5.2.2.9 Sterile defibrinated sheep blood The sterile defibrinated sheep blood can be acquired from a commercial supplier or prepared according to EN 14820. 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)]. 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 ) 121(3 0°+ for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at (18050+) °C for a minimum holding time of 30 min, at (17050+) °C for a minimum holding time of 1 h or at (16050+) °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 50° C to 55° C (to prepare the MCO, see 5.2.2.3) and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled at either 36 °C ± 1 °C or at 37 °C ± 1°C.
NOTE 1 The same temperature should be used for all incubations performed during a test and its controls and validation.
NOTE 2 A CO2 – incubator and a temperature of 36 ºC ± 1 ºC are better suited for the test organisms. 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. The additional inaccuracy of the electrodes shall not be more than ± 0,1 pH units. NOTE A puncture electrode or a flat membrane electrode should be used for measuring the PH of the agar media (5.2.2.3). 5.3.2.5 Stopwatch
2) Disposable equipment is an acceptable alternative to reusable glassware. SIST EN 14348:2005



EN 14348:2005 (E) 11 5.3.2.6 Electromechanical agitator e.g. Vortex® mixer 3). 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the product, with a filter holder of at least 50 ml volume, and suitable for use of filters with diameter 47 mm to 50 mm and 0,22 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 prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml in 20 s to 40 s. 5.3.2.8 Containers: Test tubes, culture bottles or flasks of suitable capacity. 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 Plastic disposable 10µµµµl loops 5.3.2.14 Coned bottom screw cap tubes, contents of 50 ml (diameter about 28 mm). 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions (Test and validation suspension) Two different test organism suspensions have to be prepared: the “test suspension” to perform the test and the “validation suspension” to perform the controls and method validation. 5.4.1.1 Preservation of test organisms Test organisms shall be prepared and kept in accordance with the requirements of EN 12353, after having been supplemented by specific procedures for mycobacteria. Cultures are prepared by subcultivating lyophilized test organisms (5.2.1) dissolved according to the culture collection’s instructions on plates containing MCO (5.2.2.3). Incubate Mycobacterium terrae and Mycobacterium avium for 21 days (5.3.2.3). The test organisms are recovered from the plates using 10 ml MADC broth (5.2.2.6) and diluted up to 100 ml with MADC broth. Pipette from this solution, 0,5 ml portions (minimum amount) in cryovials and store them at minimum – 70 °C for prolonged storage.
NOTE It is possible to prepare the test organisms for prolonged storage directly from the dissolved lyophilized test organisms by diluting the dissolution broth with 100 ml to 120 ml of MADC broth (5.2.2.6). Immediately afterwards the cryovials are filled (up to 2,0 ml) according to the amount of working cultures to be prepared. 5.4.1.2 Working culture of test organisms In order to prepare the working culture of test organisms (5.2.1), subculture directly from the defrosted cryovials (5.4.1.1) by streaking onto at least two plates containing MCO (5.2.2.3.) and incubate (5.3.2.3). After
3) Vortex® in 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 14348:2005



EN 14348:2005 (E) 12 21 days prepare a second subculture from the first subculture in the same way and incubate for 21 days. The first and/or second subculture is/are the working culture(s). Never produce and use a third subculture. 5.4.1.3 Test suspension (N) a) Prepare a suitable homogeneous suspension from the working cultures (5.4.1.2) using either  the homogenization by glass beads: with the aid of a plastic disposable loop (5.3.2.13) transfer the test organisms from at least two plates of the working culture (5.4.1.2) into a coned bottom screw cap tube (5.3.2.14) containing 6 g to 7 g of dry glass beads (5.3.2.11). The test organisms are homogenized by mixing (see 5.3.2.6) for at least 5 min to distribute them homogeneously on the beads and on most of the parts of the screw cap tube’s internal surface. Add 10 ml water (5.2.2.2) drop by drop and resuspend them by mixing (5.3.2.6). After 20 min sedimentation time the supernatant is transferred to a tube.
 or the homogenization by Potter S 1 apparatus: pipette 5,0 ml water (5.2.2.2) on each of the plates (at least two) of the working culture (5.4.1.2) and recover with a glass spatula the test organisms. Pipette all of the liquid from the plates into a 25 ml centrifugation tube. Add up to 18 ml with water (5.2.2.2). Wash with water (5.2.2.2), centrifuging 3 times at approximately 2 000 gN for 15 min. After each centrifuging, discard the supernatant, resuspend by mixing (5.3.2.6) and fill up to the original volume with water (5.2.2.2).
After the last centrifuging discard the supernatant and transfer the sediment into a 15 ml glass vessel of the Potter S 1 apparatus. Fill up to 15 ml with water (5.2.2.2). Mix, cool with ice and homogenize for 15 min. After 20 min sedimentation time, during which enough ice for cooling should be present, the supernatant is transferred to a tube. NOTE 1 Other methods of homogenization are allowed provided that the number of cfu/ml4) obtained is appropriate and stable during the time of the test and microscopic examination shows that the suspension is as homogeneous as after the two procedures described above. NOTE 2 Do not use tensio-active substances (surfactants). b) Adjust the number of cells in the (supernatant) suspension to 1,5 × 109 cfu/ml to 5,0 × 109 cfu/ml using water. Maintain this test suspension at 20 °C and use at the day of preparation. Adjust the temperature to
[5.5.1.1. a) and 5.5.1.4] about 30 min before the test starts. NOTE 3 The use of spectrophotometer for adjusting the number of cells is highly recommended (about 620 nm wavelength - cuvette 10 mm path length). Each laboratory should therefore produce calibration data for each test organism knowing that suitable values of optical density are found between 2 200 and 2 400. 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 10-7 and 10-8 dilutions of the test suspension using water (5.2.2.2). Mix
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