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
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

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
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

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

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

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

Mycobacterium avium ATCC
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.
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

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

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

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

 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 (5.3.2.6). Take a sample of 1,0 ml of each dilution in duplicate and transfer divided in nearly equal amounts onto two separate plates (5.3.2.10) containing MCO (5.2.2.3), i.e. two plates per 1,0 ml sample, four plates per duplicate 1,0 ml samples. For incubation and counting see 5.4.1.5. 5.4.1.4 Validation suspension (Nv) a) To prepare the validation suspension, dilute the test suspension (5.4.1.3) with water (5.2.2.2) to obtain 3,0 × 10² cfu/ml to 1,6 ×10³ cfu/ml (about one fourth (1 + 3) of the 10-6 dilution). Maintain and adjust the validation suspension, the same way as the test suspension (5.4.1.3 b)
4) cfu/ml = colony forming unit(s) per millilitre SIST EN 14348:2005

a) Incubate (5.3.2.3) the plates for 21 days. Discard any plates which are not countable (for any reason). Count the plates and determine the number of colony forming units (cfu) for each plate.
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 5.6.2.1. c) Calculate the number of cfu/ml in the test suspension N and in the validation suspension Nv using the method given in 5.6.2.2 or 5.6.2.4. 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). 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 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 in volumetric flasks (5.3.2.12) on a volume/volume basis. The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a physically homogenous preparation. 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. The concentration of the product stated in the test report shall be the (desired) test concentration. Record the test concentration in terms of mass per volume or volume per volume and details of the product sample as received. SIST EN 14348:2005

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 albumine (5.2.2.8.2) under clean or a mixture of 3 ml/l sheep erythrocytes and 3,0 g/l bovine albumine (5.2.2.8.3) under dirty conditions – according to practical applications.
 Additional interfering substances may be tested according to specific field of application.
d) test organisms:  the obligatory test organisms are:
Mycobacterium avium and Mycobacterium terrae or only Mycobacterium terrae (5.2.1) 5.5.1.2 Selection of neutralizer To determine a suitable neutralizer carry out the validation of the dilution neutralization method (5.5.2.2, 5.5.2.3 and 5.5.2.4 in connection with 5.5.2.5) 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 MCO (5.2.2.3). If neutralizer is added to MCO the same amount should be added to MCO used in the test procedure. 5.5.1.3 Validation and control procedures - General instruction The neutralization and/or removal of the mycobactericidal and/or mycobacteriostatic 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 always 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. SIST EN 14348:2005

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. 5.5.2 Method5) (Dilution – neutralization) The test and the control and validation procedures (5.5.2.1 till 5.5.2.4) shall be carried out at the same time – separately for each experimental condition (5.5.1.1). 5.5.2.1 Test Na (Determination of mycobactericidal concentrations) a) Pipette 1,0 ml of the interfering substance (5.2.2.8) into a tube. Add 1,0 ml of the test suspension (5.4.1.3). Start the stopwatch (5.3.2.5) immediately, mix (5.3.2.6) and place the tube in a water bath controlled at the chosen temperature θ (5.5.1.1 a) for 2 min ± 10 s. At the end of this time, add 8,0 ml of one of the product test solutions (5.4.2). Restart the stopwatch at the beginning of the addition, mix (5.3.2.6) and place the tube in a water bath controlled at θ for the chosen contact time t (5.5.1.1 b). Just before the end of t, mix (5.3.2.6) again. b) At the end of t take a 1,0 ml sample of the test mixture Na and transfer into a tube containing 8,0 ml neutralizer (5.2.2.5) and 1,0 ml water (5.2.2.2). Mix (5.3.2.6) and place in a water bath controlled at 20 °C. After a neutralization time of 5 min ± 10 s, 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 transfer divided in nearly equal amounts onto two separate plates (5.3.2.10) containing MCO (5.2.2.3). 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 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 transfer divided in nearly equal amounts onto two separate plates (5.3.2.10) containing MCO (5.2.2.3).
For incubation and counting see 5.5.2.5. c) Perform the procedure a) to b) using the other product test solutions at the same time. d) Perform the procedure a) to c) applying the other obligatory and - if appropriate - other additional experimental conditions (5.5.1.1) 5.5.2.2 Experimental condition control A (Validation of the selected experimental conditions or verification of the absence of any lethal effect in the test conditions) a) Pipette1,0 ml of the interfering substance used in the test (5.5.2.1) into a tube. Add 1,0 ml of the validation suspension (5.4.1.4). Start the stopwatch immediately, mix (5.3.2.6) and place the tube in a water bath controlled at
for 2 min ± 10 s. At the end of this time add 8,0 ml of hard water (5.2.2.7). (In
5) For a graphical representation of the method see annex C SIST EN 14348:2005

for t. Just before the end of t, mix (5.3.2.6) again.
b) At the end of t, take a sample of 1,0 ml of this mixture A in duplicate and transfer divided in nearly equal amounts onto two separate plates (5.3.2.10) containing MCO (5.2.2.3). For incubation and counting see 5.5.2.5. 5.5.2.3 Neutralizer control B (Verification of the absence of toxicity of the neutralizer) a) Pipette 8,0 ml of the neutralizer – used in the test (5.5.2.1) - and 1,0 ml of water (5.2.2.2) into a tube. Add 1,0 ml of the validation suspension (5.4.1.4). Start the stopwatch at the beginning of the addition, mix (5.3.2.6), and place the tube in a water bath controlled at 20 °C
± 1°C for 5 min ± 10 s. Just before the end of this time, mix (5.3.2.6).
b) At the end of this time take a sample of 1,0 ml of this mixture B in duplicate and transfer divided in nearly equal amounts onto two separate plates (5.3.2.10) containing MCO (5.2.2.3). For incubation and counting see 5.5.2.5.
5.5.2.4 Method validation C (Dilution-neutralization validation)
a) Pipette 1,0 ml of the interfering substance used in the test (5.5.2.1) into a tube. Add 1,0 ml of water (5.2.2.2) and then, starting a stopwatch, 8,0 ml of the product test solution only of the highest concentration used in the test (5.5.2.1). Mix (5.3.2.6) and place the tube in a water bath controlled at θ for t. Just before the end of t, mix (5.3.2.6) again. b) At the end of t transfer 1,0 ml of the mixture into a tube containing 8,0 ml of neutralizer (used in 5.5.2.1). Restart the stopwatch immediately, mix (5.3.2.6) 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 (5.4.1.4). Start a stopwatch at the beginning of the addition and mix (5.3.2.6). 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 (5.3.2.6) again. At the end of this time take a sample of 1,0 ml of the mixture C in duplicate and transfer
divided in nearly equal amounts onto two separate plates (5.3.2.10) containing MCO (5.2.2.3) For incubation and counting see 5.5.2.5. 5.5.2.5 Incubation and counting of the test mixture and the control and validation mixtures a) Incubate (5.3.2.3) the plates for 21 days. Discard any plates which are not countable (for any reason). Count the plates and determine the number of cfu for each plate.
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 5.6.2.1. Calculate the numbers of cfu/ml in the test mixture Na and in the validation mixtures A, B and C using the method given in 5.6.2.3 or 5.6.2.5. Verify according to 5.7. 5.6 Experimental data and calculation 5.6.1 Explanation of terms and abbreviations a) Overview of the different suspensions / test mixtures:  N and Nv represent the mycobacterial suspensions, Na represents the mycobactericidal test mixture, A (experimental conditions control), B (neutralizer control), C (method validation) represent the different control test mixtures. SIST EN 14348:2005

Number of cells per ml in the mycobacterial 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
No (N0 = N/10)
Na (before neutralisation)
Controls
Nv Validation suspension
Nv0
(Nv0 = Nv/10)
A, B, C
b) 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 The first step is the determination of the Vc-values, the second the calculation of N, N0, Na, Nv, Nv0, A, B and C. The third step is the calculation of the reduction R (5.8). 5.6.2.1 Determination of Vc-values a) The usual limits for counting mycobacteria on agar plates are between 15 and 300 colonies. In this document 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 is increasing the smaller 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 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.b), determine and record the Vc-values. NOTE 2 Countings per plate should be noted. If the count on one plate is higher than 330 report the number “> 330”. If at least one of the plates per 1 ml sample shows a
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