SIST EN 1656:2010

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Antiseptika und Desinfektionsmittel - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)11.220VeterinarstvoVeterinary medicine11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 1656:2009SIST EN 1656:2010en,fr,de01-januar-2010SIST EN 1656:2010SLOVENSKI
STANDARDSIST EN 1656:20011DGRPHãþD



SIST EN 1656:2010



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 1656
November 2009 ICS 71.100.35 Supersedes EN 1656:2000English Version
Chemical disinfectants and antiseptics -Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase 2, étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 20 September 2009.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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 STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 1656:2009: ESIST EN 1656:2010



EN 1656:2009 (E) 2 Contents Page Foreword . 3Introduction . 41Scope . 52Normative references . 53Terms and definitions . 54Requirements . 55Test method . 6Annex A (informative)
Referenced strains in national collections . 25Annex B (informative)
Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids . 27Annex C (informative)
Graphical representations of dilution-neutralization method and membrane filtration method . 28Annex D (informative)
Example of a typical test report . 32Bibliography . 37 SIST EN 1656:2010



EN 1656:2009 (E) 3 Foreword This document (EN 1656:2009) 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 May 2010, and conflicting national standards shall be withdrawn at the latest by May 2010. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 1656:2000. This document was revised to include the results of a collaborative trial (ANDISTAND), to correct obvious errors and ambiguities, to harmonize the structure and wording with other quantitative suspension tests of CEN/TC 216 (existing or in preparation), and to improve the readability of the standard and thereby make it more understandable.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, 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 the United Kingdom.
SIST EN 1656:2010



EN 1656:2009 (E) 4 Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has or does not have a bactericidal activity in the fields described in the scope. This laboratory test takes into account practical conditions of application of the product, including contact time, temperature, test organisms and interfering substance, 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 utilization concentration of the chemical disinfectant or antiseptic 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 1656:2010



EN 1656:2009 (E) 5 1 Scope This European Standard specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant and antiseptic 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 European Standard applies to products that are used in the veterinary area – e.g. in the breeding, husbandry, transport and disposal of all animals except when in the food chain following death and entry to the processing industry. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. 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. 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 test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity EN 14885:2006, Chemical disinfectants and antiseptics — Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885:2006 apply. 4 Requirements The product shall demonstrate at least a five-decimal log (lg) reduction when diluted with hard water (5.2.2.7) or – in the case of ready-to-use products – with water (5.2.2.2) and tested in accordance with Clause 5 under simulated low-level soiling (3,0 g/l bovine albumin solution – 5.2.2.8.2) or simulated high-level soiling (10 g/l bovine albumin solution and 10 g/l yeast extract – 5.2.2.8.3) (or 10 g/l skimmed milk for teat disinfectants – 5.2.2.8.4) according to its practical applications and under the other obligatory test conditions four or three [for teat disinfectants] selected test organisms, 10 °C [30 ûC for teat disinfectants], 30 min [5 min for teat disinfectants]). The bactericidal activity shall be evaluated using the following organisms: a) Products for general disinfection: b) Teat disinfectants:  Enterococcus hirae;  Escherichia coli;  Proteus vulgaris;  Staphylococcus aureus;  Pseudomonas aeruginosa;  Streptococcus uberis. SIST EN 1656:2010



EN 1656:2009 (E) 6  Staphylococcus aureus.
Where indicated, additional specific bactericidal activity shall be determined applying other contact times, temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 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 sample of the product as delivered and/or diluted with hard water (or water for ready-to-use products) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture is maintained at (10 ± 1)
°C (or (30 ± 1) °C for teat disinfectants) for 30 min ± 10 s (5 min ± 10 s for teat disinfectants) (obligatory test conditions). At the end of this contact time, an aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving bacteria in each sample are determined and the reduction is calculated. 5.1.2 For general disinfectant products, the test is performed using Enterococcus hirae, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus as test organisms. For teat disinfectants the test is performed using Escherichia coli, Staphylococcus aureus and Streptococcus uberis as test organisms. 5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms 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):
a) General disinfection products:
 Enterococcus hirae ATCC 10541  Proteus vulgaris ATCC 13315
 Pseudomonas aeruginosa ATCC 15442
 Staphylococcus aureus ATCC 6538
b) Teat disinfectants:
 Escherichia coli ATCC 10536
 Staphylococcus aureus ATCC 6538
 Streptococcus uberis ATCC 19436
1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (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 1656:2010



EN 1656:2009 (E) 7 NOTE See Annex A for strain references in some other culture collections.
The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3). The same temperature (either 36 °C or 37 °C) 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 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 shelf life should be fixed (see ISO/IEC 17025). 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralized water. Sterilize in the autoclave (5.3.2.1 a). 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 [1] in the bibliography) 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 soya agar, consisting of: 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 ml Sterilize in the autoclave (5.3.2.1 a). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C. NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used. SIST EN 1656:2010



EN 1656:2009 (E) 8 5.2.2.4 Diluent Tryptone sodium chloride solution, consisting of: 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 (5.3.2.1 a). 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 5.5.1.2, 5.5.1.3 and 5.5.2. It shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.6 Rinsing liquid (for membrane filtration) The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane under the test conditions described in 5.5.3. NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.7 Hard water for dilution of products For the preparation of 1 000 ml of hard water, the procedure is as follows:  Prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave
(5.3.2.1 a). Autoclaving – if used – may cause a loss of liquid. In this case make up to 1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8) for no longer than one month;  Prepare 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 the refrigerator (5.3.2.8) for no longer than one week;  Place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9) 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 (5.3.2.4). 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 the 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. SIST EN 1656:2010



EN 1656:2009 (E) 9 The interfering substance shall be sterile and prepared at ten 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 and detergents) shall be defined. NOTE The term “interfering substance” is used even if it contains more than one substance. 5.2.2.8.2 Low-level soiling (bovine albumin solution) Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2). Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l. 5.2.2.8.3 High-level soiling (mixture of bovine albumin solution with yeast extract) Dissolve 50,0 g yeast extract powder in 150 ml of water (5.2.2.2) in a 250 ml volumetric flask (5.3.2.12) and allow foam to collapse. Make up to the mark with water (5.2.2.2). Transfer to a clean dry bottle and sterilize in an autoclave (5.3.2.1 a). Allow to cool to (20 ± 1) °C. Pipette 25 ml of this solution into a 50 ml volumetric flask (5.3.2.12) and add 10 ml of water (5.2.2.2). Dissolve 5,0 g of bovine albumin fraction V (suitable for microbiological purposes) in the solution with shaking and allow foam to collapse. Make up to the mark with water (5.2.2.2), sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration in the test procedure (5.5) is 10,0 g/l yeast extract and 10,0 g/l bovine albumin. 5.2.2.8.4 Milk for teat disinfectants Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder per litre of water (5.2.2.2), shall be prepared as follows: Prepare a solution of 100 g milk powder in 1 000 ml of water (5.2.2.2). Heat for 30 min at (105 ± 3) °C or 5 min at (121 ± 3) °C. The final concentration of reconstituted milk in the test procedure (5.5) is 10,0 g/l of reconstituted milk. 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 (30121+) °C for a minimum holding time of 15 min;
2) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 1656:2010



EN 1656:2009 (E) 10 b) for dry heat sterilization, a hot air oven capable of being maintained at (50180+) °C for a minimum holding time of 30 min, at (170 50+) °C for a minimum holding time of 1 h or at (50160+) °C for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (10 ± 1) °C and (30 ± 1) °C (for teat disinfection), at (45 ± 1) °C (to maintain melted TSA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled either at (36 ± 1) °C or (37 ± 1) °C (5.2.1). 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
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 5.3.2.6 Shaker 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. The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7), bovine albumin (5.2.2.8.2 and 5.2.2.8.3) and if the membrane filtration method (5.5.3) is used. The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the micro-organisms over the membrane and 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, or calibrated automatic pipettes. 5.3.2.10 Petri dishes (plates) of size 90 mm to 100 mm. 5.3.2.11 Glass beads, 3 mm to 4 mm in diameter. 5.3.2.12 Volumetric flasks 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.
3) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 1656:2010



EN 1656:2009 (E) 11 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 the test organisms (5.2.1), prepare a subculture from the stock culture (5.4.1.2) by streaking onto TSA slopes or plates (5.2.2.3) 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 (if produced) third subcultures are the working cultures. 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. For additional test organisms, any departure from this method of culturing the test organisms or preparing the suspensions shall be noted, giving the reasons in the test report. 5.4.1.4 Test suspension (“N”) a) Take 10 ml of diluent (5.2.2.4) and place it in a 100 ml vessel with 5 g of glass beads (5.3.2.11). Take the working culture (5.4.1.3) 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 (5.3.2.6 b). Aspirate the suspension from the glass beads and transfer to another tube. Adjust the number of cells in the suspension to (1,5 x 108) cfu/ml4) to (5 x 108) cfu/ml using diluent (5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the water bath at the test temperature θ (5.5.1.1 a) and use within 2 h. NOTE The use of spectrophotometer for adjusting the number of cells is highly recommended (approximately 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 generally found between 0,150 and 0,460. A colorimeter is a suitable alternative. b) For counting, prepare 10-6 and 10-7 dilutions of the test suspension using diluent (5.2.2.4). Mix (5.3.2.6 a). 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 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 ± 1) °C. 2) When using the spread plate technique, spread each 1,0 ml sample – divided into 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 (“Nv”) a) To prepare the validation suspension, dilute the test suspension (5.4.1.4) 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 10-5 dilution].
4) cfu/ml = colony forming unit(s) per millilitre. SIST EN 1656:2010



EN 1656:2009 (E) 12 b) For counting prepare a 10-1 dilution with diluent (5.2.2.4). Mix (5.3.2.6 a). Take a sample of 1,0 ml in duplicate and inoculate using the pour plate or the spread plate technique (5.4.1.4 c). For incubation and counting, see 5.4.1.6. 5.4.1.6 Incubation and counting of the test and the validation suspensions For incubation and counting of the test and validation suspension, the procedure is as follows: a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates that are not countable for any reason. Count the cfu on the plates to determine the total number of cfu. Incubate the plates for a further 20 h to 24 h. Do not recount plates that 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 methods given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7. 5.4.2 Product test solutions The concentration of a product test solution shall be 1,25 times the desired test concentration because it is diluted to 80 % during the test and the method validation (5.5.2 or 5.5.3). Product test solutions shall be prepared in hard water (5.2.2.7) at minimum three different concentrations to include one concentration in the active range and one concentration in the non-active range (5.8.2). The product as received may be used as one of the product test solutions, in this case the highest tested concentration is 80 %. Dilutions of ready-to-use products, i.e. products that are not diluted when applied, shall be prepared in water (5.2.2.2). For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (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 homogeneous preparation that is stable during the whole procedure. If during the procedure a visible inhomogeneity appears due to the formation of a precipitate or flocculent (for example, through the addition of the interfering substance), it shall be recorded in the test report. NOTE Counting micro-organisms embedded in a precipitate or flocculent is difficult and unreliable. The concentration of the product stated in the test
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