SIST EN 14204:2013
(Main)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of mycobactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of mycobactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)
This European Standard specifies a test method and the minimum requirements for mycobactericidal 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 - i.e. in the breeding, husbandry, production, 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.
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der mykobakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Diese Europäische Norm legt ein Prüfverfahren und die Mindestanforderungen an die mykobakterizide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei der Verdünnung mit Wasser standardisierter Härte oder - im Fall von gebrauchsfertigen Produkten - mit Wasser als homogene, physikalisch stabile Zubereitung vorliegen.
Produkte können nur bei einer Konzentration von 80 % oder weniger geprüft werden, da immer eine bestimmte Verdünnung durch Zugabe des Prüfkeims und der Belastungssubstanz erfolgt.
Diese Europäische Norm gilt für Produkte für den Veterinärbereich, d. h. bei deren Verwendung bei der Aufzucht, Haltung, Produktion und beim Transport von Tieren sowie bei der Tierkörperbeseitigung, außer wenn die Tiere nach der Tötung durch Zuführung in die weiterverarbeitende Industrie in die Nahrungsmittel¬kette eintreten.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den "Anwendungsempfehlungen" fest.
ANMERKUNG 1 Das beschriebene Verfahren ist für die Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen unter den Bedingungen, unter denen sie verwendet werden, bestimmt.
ANMERKUNG 2 Das Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité mycobactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (Phase 2, étape 1)
La présente Norme européenne spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité mycobactéricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l'eau dure ou - dans le cas de produits prêts à l’emploi - dans l’eau.
Les produits ne peuvent être soumis à l’essai qu’à la concentration de 80 % ou à des concentrations inférieures, car l’ajout des microorganismes d’essai et de la substance interférente s’accompagne forcément d’une dilution.
La présente Norme européenne s'applique aux produits utilisés dans le domaine vétérinaire, à savoir la reproduction, l'élevage, la production, le transport et l’abattage de tous les animaux, hors de la chaîne alimentaire qui suit l’abattage et l’entrée dans l’industrie de transformation.
L'EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».
NOTE 1 La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives dans les conditions dans lesquelles elles sont utilisées.
NOTE 2 Cette méthode correspond à un essai de phase 2, étape 1.
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje mikobaktericidnega delovanja kemičnih razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 1)
Ta evropski standard določa preskusno metodo in minimalne zahteve za mikobaktericidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma z vodo pri proizvodih, ki so pripravljeni za uporabo. Proizvode je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji, ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje. Ta evropski standard se uporablja za izdelke v veterini, tj. pri vzreji, živinoreji, proizvodnji, prevozu in odstranjevanju vseh živali, razen če so v prehrambeni verigi po smrti in so del predelovalne industrije. Standard EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo. OPOMBA 1 Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo. OPOMBA 2 Ta metoda ustreza preskusu faze 2, stopnje 1.
General Information
Relations
Standards Content (Sample)
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 und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité mycobactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - 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 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 14204:2012SIST EN 14204:2013en,fr,de01-januar-2013SIST EN 14204:2013SLOVENSKI
STANDARDSIST EN 14204:20041DGRPHãþD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 14204
November 2012 ICS 71.100.35 Supersedes EN 14204:2004English Version
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of mycobactericidal 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 mycobactéricide 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 mykobakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 22 September 2012.
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-CENELEC 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-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14204:2012: ESIST EN 14204:2013
Page Foreword .3Introduction .41Scope .52Normative references .53Terms and definitions .54Requirements .55Test method .65.1Principle .65.2Materials and reagents .75.2.1Test organisms .75.2.2Culture media and reagents .75.3Apparatus and glassware . 105.3.1General . 105.3.2Usual microbiological laboratory equipment and, in particular, the following: . 105.4Preparation of mycobacterial test suspension and product test solutions . 115.4.1Mycobacterial test suspensions (test and validation suspension) . 115.4.2Product test solutions . 135.5Procedure for assessing the mycobactericidal activity of the product . 145.5.1General . 145.5.2Test procedure - Dilution-neutralization method . 155.5.3Test procedure - Membrane filtration method . 175.6Experimental data and calculation. 195.6.1Explanation of terms and abbreviations . 195.6.2Calculation . 195.7Verification of methodology . 235.7.1General . 235.7.2Control of weighted mean counts . 235.7.3Basic limits . 235.8Expression of results . 245.8.1Reduction . 245.8.2Control of active and non-active product test solution (5.4.2) . 245.8.3Mycobactericidal concentration . 245.9Interpretation of results - conclusion . 245.9.1General . 245.9.2Mycobactericidal activity for general purposes . 245.9.3Qualification for certain fields of application . 245.10Test report . 25Annex A (informative)
Referenced strain in national collections . 27Annex B (informative)
Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids . 28Annex C (informative)
Graphical representations of dilution-neutralization method . 30Annex D (informative)
Example of a typical test report . 32Bibliography . 36 SIST EN 14204:2013
Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has mycobactericidal activity in the area and 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 substances, i.e. conditions which may influence its action in practical situations. The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each utilization concentration of the chemical disinfectant or antiseptic, 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 14204:2013
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 – i.e. in the breeding, husbandry, production, 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 documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. 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, 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 apply. 4 Requirements The product shall demonstrate at least a 4 decimal log (lg) reduction when tested in accordance with Table 1 and Clause 5 under simulated low level (3,0 g/l bovine albumin) or high level soiling (10 g/l yeast extract and 10 g/l bovine albumin). SIST EN 14204:2013
Mycobacterium avium b) additional any relevant test organism Test temperature a) obligatory
10°C ± 1°C b) additional 4°C ± 1°C; 20°C ± 1°C; 40°C ± 1°C Contact time a) obligatory
60 min ± 10 sa b) additional 1 min ± 5 s; 5 min ± 10 s; 10 min ± 10 s, 15 min ± 10 s, 30 min ± 10 s, 120 min ± 10 s Interfering substance a) obligatory low level soiling high level soiling
3,0 g/l bovine albumin
10 g/l yeast extract plus 10 g/l bovine albumin b) additional any relevant substance NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions. a The obligatory contact time for disinfectants stated in Table 1 was chosen to enable comparison of standard conditions. The referenced test conditions are by no means intended as requirements for the use of a product.
The recommended contact time for the use of the product is within the responsibility of the manufacturer.
Any additional specific mycobactericidal activity shall be determined in accordance with 5.2.1 and 5.5.1.1 in order to take into account intended specific use conditions. 5 Test method 5.1 Principle A sample of the product as delivered and/or diluted in hard water (or water for ready to use products) is added to a test suspension of mycobacteria in a solution of an interfering substance. The mixture is maintained at (10 ± 1) °C for 60 min ± 10 s. At the end of this contact time, an aliquot is taken and the mycobactericidal action in this portion is immediately neutralized or suppressed by a validated method. The number of surviving mycobacteria in each sample is determined and the reduction in viable counts calculated. SIST EN 14204:2013
NOTE See Annex A for corresponding strain numbers in some other culture collections. The required incubation temperature for this test organism is (36 ± 1) °C or (37 ± 1) °C. 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 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. If additional strains do not grow on the media (5.2.2.3) or cannot be used with diluent (5.2.2.4) additional media shall be used and shall be reported as well as additional incubation conditions. To improve the 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. Ready-to-use media may be used if it complies with the required specification. 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 and not demineralised water. If distilled water of adequate quality is not available, water for injectable preparations (see bibliographic reference [1] Pharmacopoeia) can be used.
1) ATCC 15769 is the collection number of strain supplied by the American Type Culture Collections. 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 14204:2013
100 ml Middlebrook OADC enrichment under aseptic conditions. Final pH = 6,6 ± 0,2 at 25 °C. In case of encountering problems with neutralization (5.5.1.2), it may be necessary to add neutralizer to the 7H10. Annex B gives guidance on the neutralizers that may be used. 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 (see 5.2.2.2) to 1 000 ml. Sterilize in the autoclave [see 5.3.2.1a)]. After sterilization the pH of the medium 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 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 Annex B. Sterilize in the autoclave. 5.2.2.6 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 [5.3.2.1a)]. Store the solution at 2 °C to 8°C for no longer than one month. NOTE 1 In the case of loss of volume during sterilization by autoclave, make up to 1 000 ml with water (5.2.2.2) under aseptic conditions before storage. SIST EN 14204:2013
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; 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 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 C ± 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 2 When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces different final water hardness in each test tube. In any case the final hardness is lower than 375 mg/l of calcium carbonate (CaCO3) in the test tube. 5.2.2.7 Interfering substances 5.2.2.7.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product. The interfering substance shall be sterile and prepared at 10 times its final concentration in the test. For all additional interfering substances 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). 5.2.2.7.2 Low-level soiling (Bovine albumin solution) Dissolve 3 g of bovine albumin (Cohn fraction V for Dubos Medium) in 90 ml of water (5.2.2.2) in a 100 ml volumetric flask (5.3.2.12). 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 of the bovine albumin in the test procedure (5.5.2) is 3 g/l. 5.2.2.7.3 High-level soiling (mixture of bovine albumin solution with yeast extract) a) dissolve 50 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 [5.3.2.1a)]. Allow to cool to 20 °C ± 5 °C. b) pipette 25 ml of this solution into a 50 ml volumetric flask and add 10 ml of water (5.2.2.2). Dissolve 5 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 and keep in a refrigerator (5.3.2.8) and use within one month. The final concentration in the test procedure (5.5) is 10 g/l yeast extract and 10 g/l bovine albumin. 5.2.2.8 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. SIST EN 14204:2013
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 1213+0°C for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at 180+50°C for a minimum holding time of 30 min, at 170+50°C for a minimum holding time of 1 h or at 160+50°C for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (4 ±1) °C, (10 ± 1) °C, (20 ± 1) °C, (40 ± 1) °C,
(45 ± 1) °C and 50 °C to 55 °C. 5.3.2.3 CO2 Incubator, capable of being controlled at either (36 ±1) °C or (37 ± 1) °C. An incubator at
(36 ± 1) °C or (37 ± 1)°C may be used if a CO2 incubator is not available. 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C. 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
2) Disposable Sterile Equipment is an acceptable alternative to reusable glassware. 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 14204:2013
5.3.2.14 Containers, test tubes, flasks or bottles of suitable capacity. 5.3.2.15 Coned bottom screw cap tube 5.4 Preparation of mycobacterial test suspension and product test solutions 5.4.1 Mycobacterial test suspensions (test and validation suspension) 5.4.1.1 General 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 of test organism The test organism and stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.3 Working culture of test organism In order to prepare a working culture of the test organism (5.2.1) subculture from the stock culture (5.4.1.2) by streaking on to at least two plates of 7H10 medium (5.2.2.3) and incubate (5.3.2.3). If a CO2 incubator is not used, plates shall be placed into polyethylene bags or sealed with insulating tape to prevent drying of the medium. After 21 days prepare a second subculture from the first subculture in the same way and incubate for 21 days. The first and/or the second subculture is the working culture(s). Never produce and use a third subculture.
4) Potter Apparatus 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 14204:2013
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 colony forming units per millilitre 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 3,0 x 108 cfu/ml5)
to 8,0 x 108 cfu/ml using water (5.2.2.2). Maintain this test suspension in the water bath at (20 ± 1) °C and use at the day of preparation. Adjust the temperature according to 5.5.1.1a) and 5.5.1.4 only immediately before the start of the test. 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 0,200 and 0,400. To achieve reproducible results of this measurement it may be necessary to dilute the test suspension, e.g. 1+9.
NOTE A colorimeter is a suitable alternative. c) For counting prepare 10-6 and 10-7 dilutions of the test suspension using diluent (5.2.2.4). Mix [(5.3.2.6a)]. d) Take a sample of 1,0 ml of each dilution in duplicate. Divide each sample in two nearly equal amounts and spread onto separate surface dried plates (5.3.2.10) containing 18 ml to 20 ml of 7H10 medium (5.2.2.3), i.e. four plates per duplicate 1,0 ml samples. For incubation and counting see 5.4.1.6.
5) cfu/ml = colony forming unit(s) per millilitre SIST EN 14204:2013
Note for each plate the exact number of colonies but record >330 for any counts higher than 330 and determine VC values according to 5.6.2.2.
c) Calculate the number of cfu/ml in the test suspension N and in the validation suspension NV using the method given in 5.6.2.3 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).
Product test solutions shall be prepared in hard water (5.2.2.6) at a 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 (5.2.2.6) or water (5.2.2.2) for ready to use products. Subsequent dilutions (lower concentrations) shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.6). For liquid products, dilutions of the product shall be prepared with hard water (5.2.2.6) or water (5.2.2.2) for ready to use products 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 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 14204:2013
°C; the obligatory and additional temperatures to be tested are specified in Clause 4, Table 1; the allowed deviation for each chosen temperature is ± 1 °C; b) contact time: t in min; the obligatory and additional contact times to be tested are specified in Clause 4, Table 1; the allowed deviation for each chosen contact time is ± 10 s, (except for 1 min ± 5 s); c) interfering substance; the obligatory interfering substance to be tested is 3,0 g/l bovine albumin (5.2.2.7.2) low level soiling or 10,0 g/l albumin/yeast extract mixture (5.2.2.7.3) for high level soiling conditions according to Clause 4, Table 1 and practical applications. Additional interfering substances may be tested according to specific fields of application. d) test organism; Mycobacterium avium (Clause 4, Table 1). Additional test organisms may be tested. 5.5.1.2 Choice of test method (dilution-neutralization or membrane filtration) The method of choice is the dilution-neutralization method (5.5.2). To determine a suitable neutralizer, carry out the validation of the dilution neutralization method (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 taking into account the information given in Annex B. In special circumstances it may be necessary to add neutralizer to 7H10 medium (5.2.2.3). If neutralizer is added to 7H10 medium, the same amount shall be added to 7H10 medium used in the test procedure. 5.5.1.3 General instructions for validation and control procedures 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 or filtration control and method validation) shall be performed at the same time with the test and with the same neutralizer – or rinsing liquid – used in the test. SIST EN 14204:2013
[5.5.1.1a)] 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 [5.3.2.6a)] and place the tube in a water bath controlled at
for the chosen contact time t [5.5.1.1b)]. Just before the end of t, mix [5.3.2.6a)] again. b) At the end of t, take a 1,0 ml 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 [5.3.2.6a)] and place in a water bath controlled at (20°C ± 1)°C. After a neutralization time of 5 min ± 10 s, immediately take a sample of 1,0 ml of the neutralized mixture "Na" (containing neutralizer, product test solution, interfering substance and test suspension) and transfer to a tube containing 9,0 ml diluent (5.2.2.4). Take a sample of 1,0 ml of the neutralized and ten-fold diluted test mixture "Na" in duplicate. Divide each sample in two portions of approximately equal size and inoculate 7H10 plates (5.2.2.3) using the spread plate technique. For incubation and counting see 5.5.2.7. c) Perform the procedures a) and b) using the other product test solutions at the same time. d) Perform the procedures a) to c) applying the other obligatory and – if appropriate – other additional experimental conditions (5.5.1.1).
6) For a graphical representation of this method, see Annex C SIST EN 14204:2013
for 2 min ± 10 s. At the end of this time, add 8,0 ml of hard water (5.2.2.6). 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
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 divide each sample in two portions of approximately equal size and inoculate 7H10 plates (5.2.2.3) using the spread plate technique. For incubation and counting, see 5.5.2.7. 5.5.2.5 Neutralizer control "B" – Verification of the absence of toxicity of the neutralizer To verify the absence of toxicity of the neutralizer, the procedure is as follows. 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 (5.4.1.5). Start the stopwatch at the beginning of the addition, mix [5.3.2.6a)], and place the tube in a water bath controlled at (20 ± 1)°C for 5 min ± 10 s. Just before the end of this time, mix [5.3.2.6a)]. b) At the end of this time, take a sample of 1,0 ml of this mixture "B" in duplicate divide each sample in two portions of approximately equal size and inoculate 7H10 plates (5.2.2.3) using the spread plate technique. For incubation and counting, see 5.5.2.7. 5.5.2.6 Method validation "C" – Dilution-neutralization validation To validate the dilution neutralization method, the procedure is as follows. a) Pipette 1,0 ml of the interfering substance used in the test into a tube. Add 1,0 ml of the diluent (5.2.2.4) and then, starting a stopwatch, add 8,0 ml of the product test solution only of the highest concentration used in the test. Mix [5.3.2.6a)] and place the tube in a water bath controlled at
for t. Just before the end of t, mix [5.3.2.6a)] again. b) At the end of t transfer 1,0 ml of the mixture into a tube containing 8,0 ml of neutralizer (5.2.2.5). Restart the stopwatch immediately at the beginning of the addition. Mix [5.3.2.6a)] and place the tube in a water bath controlled at (20 ± 1) C for 5 min ± 10 s. Add 1,0 ml of the validation suspension (5.4.1.5). Start a stopwatch (5.3.2.5) at the beginning of the addition and mix [5.3.2.6a)]. Place the tube in a water bath controlled at (20 ± 1) °C for the contact time t. 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 divide each sample in two portions of approximately equal size and inoculate 7H10 plates (5.2.2.3) using the spread plate technique. For incubation and counting, see 5.5.2.7. 5.5.2.7 Incubation and counting of the test mixture and the control and validation mixtures For incubation and counting of the test mixture and the control and validation mixtures, the procedure is as follows. SIST EN 14204:2013
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.