SIST EN 14561:2006

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Keimträgerversuch zur Prüfung der bakteriziden Wirkung für Instrumente im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)Désinfectants chimiques et antiseptiques - Essai quantitatif de porte germe pour l'évaluation de l'activité bactéricide 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 bactericidal 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 14561:2006SIST EN 14561:2006en,fr,de01-september-2006SIST EN 14561:2006SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14561May 2006ICS 11.080.20 English VersionChemical disinfectants and antiseptics - Quantitative carrier testfor the evaluation of bactericidal activity for instruments used inthe medical area - Test method and requirements (phase 2, step2)Désinfectants et antiseptiques chimiques - Essai quantitatifde porte germe pour l'évaluation de l'activité bactéricidepour instruments utilisés en médecine humaine - Méthoded'essai et prescriptions (phase 2, étape 2)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Keimträgerversuch zur Prüfung derbakteriziden 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 14561:2006: ESIST EN 14561:2006

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.34 Annex ZA (informative)
Relationship between this European Standard and the Essential Requirements of EU Directive 93/42/EEC.36 Bibliography.37
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 bactericidal activity in the fields described in the scope. The laboratory test closely simulates practical conditions of application including pre-drying bacteria 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 of use of a product may differ and therefore additional test conditions need to be used. SIST EN 14561:2006

1 Scope This European Standard specifies a test method and the minimum requirements for bactericidal 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.
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 test organisms used for the determination of bactericidal, sporicidal 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 bactericide product that kills vegetative bacteria under defined conditions NOTE The adjective derived from "bactericide" is "bactericidal". 3.3 bactericidal activity capability of a product to produce a reduction in the number of viable bacterial cells of relevant test organisms under defined conditions 3.4 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 SIST EN 14561:2006

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 bacteria 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 bacteria are to be severed from the surface by shaking. The numbers of surviving bacteria in each sample are determined and the reduction is calculated. 5.1.2 The test is performed using Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus hirae 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 bactericidal activity shall be evaluated using the following strains as test organisms1):  Pseudomonas aeruginosa ATCC
15442  Staphylococcus aureus ATCC
6538  Enterococcus hirae ATCC
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 14561:2006

±
1 ° (5.3.2.3). The same temperature (either 36 °C or 37 °) shall be used for all incubations performed during a test and its control and validation. 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. 5.2.2.3 Tryptone Soya Agar (TSA)  Tryptone, pancreatic digest of casein 15,0 g  Soya peptone, papaic digest of Soybean meal 5,0 g  Sodium Chloride (NaCl) 5,0 g  Agar 15,0 g  Water (5.2.2.2) to 1 000,0 mlSterilize in the autoclave (5.3.1). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1)°C (5.3.2.4). SIST EN 14561:2006

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 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, 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 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. 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 substances 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product.
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 (5.5) is 0,3 g/l. 5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solutions – high concentration with sheep erythrocytes (see 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 (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)]. 5.3.2 Usual microbiological laboratory equipment2)
and in particular, the following: 5.3.2.1 Apparatus for sterilization: a) for moist heat sterilization, an autoclave capable of being maintained at ()C12130°+ for a minimum holding time of 15 min;
2) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 14561:2006

+) °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 – see 5.5.1.7) 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. The same temperature shall be used for all incubations performed during a test and its controls and validation.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no 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 an 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) 5.3.2.14 Centrifuge (800 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
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 14561:2006

Key 1 Inoculation square Figure 1 — Frosted glass carrier with markings
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 In order to prepare the working culture of test organisms (5.2.1), subculture from the stock culture (5.4.1.2) by streaking onto TSA (5.2.2.3) slopes or plates and incubate (5.3.2.3). After 18 h to 24 h prepare a second subculture from the first subculture in the same way and incubate for 18 h to 24 h. From this second subculture, a third subculture may be produced in the same way. The second and/or the third subculture are the working culture(s). If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used for subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 48 h period.
Never produce and use a fourth subculture. 5.4.1.4 Test suspension 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) and transfer loopfuls (5.3.2.16) 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 SIST EN 14561:2006

b) Adjust the number of cells in the suspension to 1,5 x 109 cfu/ml4) to 5,0 x 109 cfu/ml using the diluent, estimating the numbers of units 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 for each test organism, knowing that suitable values of optical density are generally found between 0,150 and 0,460. 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 diluent (5.2.2.4). Mix [5.3.2.6a)].
d) Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread plate technique.
1) When using the pour plate technique, transfer about half of each 1 ml sample into separate Petri dishes (i.e. in duplicate = four plates) and add 15 ml to 20 ml melted TSA (5.2.2.3), cooled to 45 °C ± 1 °C.
2) 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 TSA (5.2.2.3). For incubation and counting see 5.4.1.6. 5.4.1.5 Validation suspension a) To prepare the validation suspension, dilute the test suspension (5.4.1.3) with the diluent (5.2.2.4) to obtain 3,0 x 102 cfu/ml to 1,6 x 103 cfu/ml (about one fourth (1+3) of the 10-6 dilution). b) For counting prepare a 10-1 dilution with the diluent (5.2.2.4). Mix [5.3.2.6a)]. Take a sample of 1,0 ml in duplicate and inoculate using the pour plate or the spread plate technique [5.4.1.3 d1) or d2)]. For incubation and counting see 5.4.1.6. 5.4.1.6 Incubation and counting of the test and the validation suspensions a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any reason). Count the plates and determine the number of colony forming units. Incubate the plates for a further 20 h to 24 h. Do not recount plates which no longer show well separated colonies. Recount the remaining plates. If the number has increased use only the higher number for further evaluation. b) Note for each plate the exact number of colonies but record > 330 for any counts higher than 330 and determine the VC-values according to 5.6.2.2.
c) Calculate the numbers of cfu/ml in the test suspension N and in the validation suspension NV using the method given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7.
4) cfu/ml = colony forming unit per millilitre. SIST EN 14561:2006

Dilutions of ready-to-use products, i.e. products which are not diluted when applied, shall be prepared in water (5.2.2.2) instead of hard water. For solid products, dissolve the product as received by weighing at least 1 g ± 10 mg of the product in a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (i.e. lower concentrations) shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water on a volume/volume basis using volumetric flasks (5.3.2.12). The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a physically homogenous preparation, stable during the whole procedure. If during the procedure a visible inhomogeneity appears due to the formation of a precipitate or flocculant (for example through the addition of the interfering substances), it shall be recorded in the test report. NOTE Counting micro-organisms embedded in a precipitate or flocculant is difficult and unreliable. Record the test concentration in terms of mass per volume or volume per volume and details of the product sample as received. 5.5 Procedure for assessing the bactericidal activity of the product 5.5.1 General 5.5.1.1 Experimental conditions (obligatory and additional) Besides the obligatory temperature, contact time, interfering substances and test organisms, additional experimental conditions may be selected according to the practical use considered for the product (Clause 4): a) temperature θ
(in °C):  the obligatory temperature to be tested is θ
= 20 °C;  additional temperatures (Clause 4) may be chosen according to the manufacturer’s recommendation, but not higher than 60 °C;  the allowed deviation for each chosen temperature is ± 1 °C . b) contact time t (in min):  the obligatory contact time to be tested is t = 60 min;  additional contact times (Clause 4) may be chosen from 5 min; 15 min and 30 min;
 the allowed deviation for each chosen contact time is ± 10 s. c) interfering substance:  the obligatory interfering substance to be tested is 0,30 g/l bovine albumin (5.2.2.8.2) under clean or a mixture of 3 ml/l sheep erythrocytes and 3 g/l bovine albumin (5.2.2.8.3) under dirty conditions – according to practical applications.
 Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae (5.2.1). 5.5.1.2 Selection of neutralizer To determine a suitable neutralizer carry out the validation of the dilution neutralization method (see 5.5.2.3, 5.5.2.4 and 5.5.2.5 in connection with 5.5.2.6) using a neutralizer, chosen according to laboratory experience and published data. If this neutralizer is not valid, repeat the validation test using an alternative neutralizer containing a combination of polysorbate 80 (30 g/l), saponin (30 g/l), L-histidine (1 g/l), lecithin (3 g/l), sodium thiosulphate (5 g/l) in either diluent (5.2.2.4) or in phosphate buffer 0,0025 mol/l (see B.2).
NOTE In special circumstances it may be necessary to add neutralizer to TSA (5.2.2.3). If neutralizer is added to TSA the same amount should be added to TSA used in the test procedure. 5.5.1.3 Validation and control procedures – General instruction The neutralization and/or removal of the bactericidal and/or bacteriostatic activity of the product shall be controlled and validated – only for the highest product test concentration – for each of the used test organisms and for each experimental condition (interfering substance, temperature, contact time). These procedures (experimental condition control, neutralizer control and method validation) shall be performed at the same time with the test and with the same neutralizer used in the test. In the case of ready-to-use-products use water (5.2.2.2) instead of hard water. If because of problems with neutralization, a neutralizer has been added to TSA (5.5.1.2) used for the validation and control procedures, the TSA used for the test shall contain the same amount of this neutralizer as well. 5.5.1.4 Equilibration of temperature Prior to testing, equilibrate all reagents (product test solutions (5.4.2), test suspension (5.4.1.4), validation suspension (5.4.1.5), diluent (5.2.2.4), hard water (5.2.2.7) and interfering substance (5.2.2.8)) to the test temperature of θ using the water bath (5.3.2.2) controlled at θ. Observe the provisions laid down in 5.4.1.4 b). Check that the temperature of the reagents is stabilized at θ. The neutralizer (5.2.2.5) and water (5.2.2.2) shall be equilibrated at a temperature of 20 °C ± 1 °C. In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to the test temperature of θ. 5.5.1.5 Precautions for manipulation of test organisms Do not touch the upper part of the test tube sides when adding the test or the validation suspensions. 5.5.1.6 Inoculation of the carriers
Pipette 1,0 ml of interfering substances (5.2.2.8) into a tube. Add 9,0 ml of the test suspension (5.4.1.4). Mix [5.3.2.6a)] and pipette 0,05 ml of this mixture on the “inoculation square” of a carrier (5.3.2.17) and distribute equally inside the square, e.g. with the tip of the pipette. Let the inoculum dry in the incubator (5.3.2.3) until visible dryness, maximum 60 min. Use the carrier immediately after the end of the drying time. Note the drying time in the test report.
b) At the end of t transfer the carrier into a second cylindrical screw tube (5.3.2.15), placed in a water bath controlled at 20 ºC and filled with 10 ml of neutralizer (5.2.2.5) and approximately 1 ml of glass beads (5.3.2.13). Restart the stopwatch and mix [5.3.2.6a)] for 15 s. After a neutralization time of 5 min ± 10 s, mix and immediately take a sample of 1,0 ml of the neutralized test mixture Na (containing neutralizer, product test solution, interfering substance, test suspension) in duplicate and inoculate using the pour plate or the spread plate technique. Additionally transfer 0,5 ml of the test mixture Na into a tube containing 4,5 ml of neutralizer (10-1 dilution of Na), mix [5.3.2.6a)] and dilute accordingly to produce
10-2 and 10-3 dilutions of Na with neutralizer. Take samples of 1,0 ml from each dilution tube in duplicate and inoculate each 1,0 ml sample into separate Petri dishes using the pour plate or the spread plate technique. The number of 1,0 ml samples of Na shall be 8 altogether . When using the pour plate technique, pipette each 1,0 ml sample into separate Petri dishes and add 15 ml to 20 ml melted TSA (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 TSA (5.2.2.3).
For incubation and counting see 5.5.2.6. c) Perform the procedure a) to b) using the other product test solutions at the same time. d) Water control NW: Perform the procedure a) to b), but instead of the product test solution, pipette 10 ml of hard water (5.2.2.7) or – in the case of ready-to-use products – water (5.2.2.2). Deviating from b) produce 10-5 dilutions from the neutralized test mixture NW for incubation and counting. e) Perform the procedure a) to d) applying the other obligatory and – if appropriate – other additional experimental conditions (5.5.1.1). 5.5.2.3 Experimental condition control “A” (Validation of the selected experimental conditions or verification of the absence of any lethal effect in the test conditions) a) Pipette 1,0 ml of the interfering substance used in the test (5.5.2.2) into a tube. Add 1,0 ml of the validation suspension (5.4.1.5). Start the stopwatch immediately, mix [5.3.2.6a)] and place the tube in a water bath controlled at θ
for 2 min ± 10 s. At the end of this time add 8,0 ml of hard water (5.2.2.7). (In the case of ready-to-use products: water (5.2.2.2) instead of hard water). Restart the stopwatch at the beginning of the addition. Mix [5.3.2.6a)] and place the tube in a water bath controlled at θ
for t. Just before the end of t, mix [5.3.2.6a)] again. b) At the end of t, take a sample of 1,0 ml of this mixture A in duplicate and inoculate using the pour plate or the spread plate technique [5.5.2.2b)].
5 For a graphical representation of this method see Annex C. SIST EN 14561:2006

for t. Just before the end of t, mix [5.3.2.6a)] again. NOTE It is not necessary to prepare the highest concentration of the product test solution 1,25 times higher than the derived (actually tested) concentration though it is diluted during the method validation by interfering substance and diluent (8+1+1). In the test Na the amount of neutralizer in relation to the product test solution is much higher. b) At the end of t transfer 1,0 ml of the mixture into a tube containing 8,0 ml of neutralizer (used in 5.5.2.2). Restart the stopwatch at the beginning of the addition, mix [5.3.2.6a)] and place the tube in a water bath controlled at 20 °C ± 1 °C for 5 min ± 10 s. Add 1,0 ml of the validation suspension (5.4.1.5). Start a stopwatch at the beginning of the addition and mix [5.3.2.6a)]. Place the tube in a water bath controlled at 20 °C ± 1 °C for 30 min ± 1 min. Just before the end of this time, mix [5.3.2.6a)] again. At the end of this time take a sample of 1,0 ml of the mixture C in duplicate and inoculate using the pour plate or the spread plate technique [5.5.2.2b)]. For incubation and counting see 5.5.2.6. 5.5.2.6 Incubation and counting of the test mixture and the control and validation mixtures a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any reason). Count the plates and determine the number of colony forming units. Incubate the plates for a further 20 h to 24 h. Do not recount plates which no longer show well separated colonies. Recount the remaining plates. If the number has increased use only the higher number for further evaluation. b) Note for each plate the exact number of colonies but record > 330 for any counts higher than 330 and determine the VC-values according to 5.6.2.2.
c) Calculate the numbers of cfu/ml in the test mixtures Na and NW and in the validation mixtures A, B and C using the methods given in 5.6.2.3b), 5.6.2.4 and 5.6.2.6. Verify according to 5.7. 5.6 Experimental data and calculation 5.6.1 Explanation of terms and abbreviations 5.6.1.1 Overview of the different suspensions / test mixtures  N and NV represent the bacterial suspensions, Na represents the bactericidal test mixture, NW represents the test mixture in the water control, A (experimental conditions control), B (neutralizer control), C (method validation) represent the different control test mixtures. SIST EN 14561:2006

Number of cells per ml in the bacterial suspensions Number of cells per ml in the test mixtures at the beginning of the contact time (time 0) Number of survivors per ml in the test mixtures at the end of the contact-time t (A) or of
5 min (B) or of 30 min (C) Test N Test suspension N/20 (= theoretical number on the carrier) Na, NW
(before neutralization) Controls NV Validation suspension Nvo (NV0 = NV /10) A, B, C
5.6.1.2 VC-values All experimental data are reported as VC-values : VC-value is the number of cfu counted per 1,0 ml sample. 5.6.2 Calculation 5.6.2.1 General The first step is the determination of the VC-values, the second the calculation of N, Na, NW, NV, NV0, A, B and C. The third step is the calculation of the reduction R (5.8). 5.6.2.2 Determination of VC-values a) The usual limits for counting bacteria on agar plates are between 15 and 300 colonies. In this European Standard a deviation of 10 % is accepted, so the limits are 14 and 330. NOTE 1 The lower limit (14) is based on the fact that the variability increases the smaller the number counted in the sample (1 ml) is and therefore subsequent calculations may lead to wrong results. The lower limit refers only to the sample (and not necessarily to the counting on one plate), e.g. two plates per 1 ml sample with 11 cfu and 5 cfu give a VC-value of 16. The upper limit (330) reflects the imprecision of counting confluent colonies and growth inhibition due to nutriment depletion. It refers only to the counting on one plate, and not necessarily to the sample. b) According to the number of plates used per 1 ml sample (5.6.1.2), determine and record the VC-values.
NOTE 2 If more than one plate per 1 ml sample has been used to determine the VC-value the countings per plate should be noted. If the count on one plate is higher than 330 report the number as “> 330”. If more than one plate per 1 ml sample has been used and at least one of them shows a number higher than 330 report the number of this VC-value as “more than sum of the counts”, e.g. for “> 330, 280, 305” report “> 915”. If a VC-value is lower than 14 report the number (but substitute by “<14” for further calculations in the case of Na). c) Only VC-values within the counting limits are taken into account for further calculation, except in the case of Na (5.6.2.4). SIST EN 14561:2006
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