Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test method and requirements (phase 2, step 2)

This European Standard specifies a test method and the minimum requirements for the microbicidal activity of a defined disinfection process for the treatment of contaminated linen. This procedure is carried out by using a washing machine as defined in Chapter 5.3.2.17 and refers to the disinfection step without prewash.
This European Standard applies to areas and situations where disinfection is indicated. Such indications occur in patient care, for example:
-   in hospitals, in community medical facilities and in dental institutions;
-   in schools, kindergartens and nursing homes;
- institutions where patients are accommodated, which could suffer from transmissible diseases;
- other applications where hygienic treatment of linen is necessary (e.g. food processing, hotels, workwear e. g. from the pharmaceutical industry, laboratories, foodstuffs area or similar institutions).
The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used.
NOTE This method corresponds to a phase 2 step 2 test (Annex F).

Chemisches Desinfektionsmittel und Antiseptika - Chemothermische Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Diese Europäische Norm legt ein Prüfverfahren für und die Mindestanforderungen an die mikrobizide Wirkung eines festgelegten Desinfektionsprozesses zur Behandlung von kontaminierten Textilien fest. Dieses Verfahren wird unter Einsatz einer in 5.3.2.18 festgelegten Waschmaschine durchgeführt und bezieht sich auf den Desinfektionsschritt ohne Vorwaschen. Dieses Verfahren ist nicht auf bestimmte Arten von Textilien beschränkt. Die Anweisungen des Lieferanten müssen ausreichen, um die vollständige Durchführung (z. B. Dosierung des Desinfektionsmittels in einer beliebigen Waschphase, z. B. Spülen, Desinfektion bei 40 °C) des in der Norm beschriebenen Verfahrens zu ermöglichen.
Diese Europäische Norm gilt für Bereiche und unter Bedingungen, in denen eine Desinfektion angezeigt ist. Indikationen dieser Art liegen bei der Patientenbetreuung vor, z. B.:
   in Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
   in Schulen, Kindergärten und Heimen;
   Einrichtungen, in denen Patienten untergebracht sind, die an übertragbaren Krankheiten leiden könnten;
   andere Anwendungsarten, bei denen eine hygienische Behandlung von Textilien notwendig ist (z. B. Lebensmittelverarbeitung, Hotels, Arbeitsbekleidung z. B. aus der pharmazeutischen Industrie, Laboratorien, aus dem Lebensmittelbereich oder ähnlichen Einrichtungen).
Das beschriebene Verfahren dient der Bestimmung der Wirksamkeit eines Produkts oder einer Kombination von Produkten unter den Bedingungen, unter denen es/sie angewendet wird/werden. Das ist ein Laborversuch der Phase 2, Stufe 2, der die Bedingungen der Produktanwendung simuliert.
ANMERKUNG   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2 (siehe EN 14885).

Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile - Méthode d'essai et prescriptions (phase 2, étape 2)

La présente Norme européenne spécifie une méthode d'essai et les prescriptions minimales relatives à l'activité microbicide d’un processus défini de désinfection destiné au traitement de textile contaminé. Cette procédure consiste à utiliser une machine à laver comme défini en 5.3.2.18 et se rapporte à l’étape de désinfection sans prélavage. Cette procédure ne se limite pas à certains types de textile. Les instructions du fournisseur doivent être suffisantes pour permettre de réaliser complètement la méthode indiquée dans la norme (par exemple, le dosage du désinfectant quelle que soit l’étape de lavage, par exemple le rinçage, la désinfection à 40 °C).
La présente Norme européenne s’applique aux secteurs et situations pour lesquels la désinfection est préconisée. Ces préconisations touchent les soins délivrés aux patients, par exemple :
-   dans les hôpitaux, les cabinets médicaux et dentaires ;
-   dans les écoles, les écoles maternelles et les maisons de repos ;
-   dans les établissements recevant des patients susceptibles d’être atteints de maladies contagieuses ;
-   dans d’autres applications pour lesquelles un traitement hygiénique de textile est nécessaire (par exemple, transformation alimentaire, hôtels, tenues de travail notamment celles utilisées dans l’industrie pharmaceutique, les laboratoires, le secteur des denrées alimentaires ou des établissements similaires).
La méthode décrite a pour objet de déterminer l'activité d’un produit ou d’une combinaison de produits dans leurs conditions d'emploi. Il s'agit d'un essai en laboratoire de phase 2, étape 2 qui simule les conditions d'application du produit.
NOTE   Cette méthode correspond à un essai de phase 2, étape 2 (voir EN 14885).

Kemična razkužila in antiseptiki - Termokemično razkuževanje tekstila - Preskusna metoda in zahteve (faza 2, stopnja 2)

Ta evropski standard določa preskusno metodo in minimalne zahteve za mikrobicidno delovanje definiranega postopka razkuževanja pri obdelavi kontaminiranega lanu. Ta postopek se izvaja z uporabo pralnega stroja, kot je določeno v poglavju 5.3.2.17, in se nanaša na korak razkuževanja brez predpranja.  Ta evropski standard se uporablja za področja in primere, ko obstajajo indikacije za razkuževanje. Te indikacije se pojavljajo pri negi bolnikov, na primer:
– v bolnišnicah, javnih zdravstvenih in zobozdravstvenih ustanovah;
– v ambulantah šol, vrtcev in domov za starejše;
– v institucijah, kjer so lahko nastanjeni pacienti, ki trpijo za prenosljivimi boleznimi;
– pri drugih uporabah, kjer je zahtevana higienska obdelava lanu (npr. predelava hrane, hoteli, delovne obleke, na primer v farmacevtski industriji, laboratorijih, področjih z živili ali podobnih institucijah). Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo. OPOMBA: Ta metoda ustreza preskusu stopnje 2 faze 2 (dodatek F).

General Information

Status
Published
Public Enquiry End Date
31-Jul-2013
Publication Date
09-Sep-2015
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Sep-2015
Due Date
07-Nov-2015
Completion Date
10-Sep-2015

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemisches Desinfektionsmittel und Antiseptika - Chemothermische Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile - Méthode d'essai et prescriptions (phase 2, étape 2)Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test method and requirements (phase 2, step 2)71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposes11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 16616:2015SIST EN 16616:2015en,fr,de01-oktober-2015SIST EN 16616:2015SLOVENSKI

STANDARD
SIST EN 16616:2015
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16616
August 2015 ICS 11.080.20 English Version

Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test method and requirements (phase 2, step 2)

Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile - Méthode d'essai et prescriptions (phase 2, étape 2)

Chemisches Desinfektionsmittel und Antiseptika - Chemothermische Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2) This European Standard was approved by CEN on 3 July 2015.

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
CEN-CENELEC Management Centre:
Avenue Marnix 17,

B-1000 Brussels © 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16616:2015 ESIST EN 16616:2015

EN 16616:2015 (E) 2 Contents Page European foreword .............................................................................................................................................3 Introduction .........................................................................................................................................................4 1 Scope ......................................................................................................................................................5 2 Normative references ............................................................................................................................5 3 Terms and definitions ...........................................................................................................................5 4 Requirements .........................................................................................................................................6 5 Test methods ..........................................................................................................................................7 5.1 Principle ..................................................................................................................................................7 5.2 Materials and reagents ..........................................................................................................................7 5.2.1 Test organisms ......................................................................................................................................7 5.2.2 Culture media and reagents .................................................................................................................8 5.3 Apparatus and glassware .................................................................................................................. 10 5.3.1 General ................................................................................................................................................. 10 5.3.2 Usual microbiological laboratory equipment .................................................................................. 11 5.4 Preparation of test organism suspensions and product test solutions ....................................... 14 5.4.1 Test organism suspensions (test and validation suspension) ...................................................... 14 5.4.2 Product test solutions for validation tests ....................................................................................... 19 5.5 Procedure for assessing the microbicidal activity of the product ................................................ 19 5.5.1 General ................................................................................................................................................. 19 5.5.2 Method ................................................................................................................................................. 21 5.6 Experimental data and calculation.................................................................................................... 24 5.6.1 Explanation of terms and abbreviations .......................................................................................... 24 5.6.2 Calculation ........................................................................................................................................... 24 5.7 Verification of methodology .............................................................................................................. 26 5.7.1 General ................................................................................................................................................. 26 5.7.2 Control of weighted mean counts ..................................................................................................... 26 5.7.3 Basic limits .......................................................................................................................................... 27 5.8 Expression of results and precision ................................................................................................. 27 5.8.1 Reduction ............................................................................................................................................ 27 5.8.2 Repetitions .......................................................................................................................................... 28 5.9 Interpretation of results – conclusion .............................................................................................. 28 5.9.1 General ................................................................................................................................................. 28 5.9.2 Microbicidal activity ........................................................................................................................... 28 5.10 Test report ........................................................................................................................................... 29 Annex A (informative)

Referenced strains in national collections .............................................................. 30 Annex B (informative)

Suitable neutralizers and rinsing liquids ................................................................. 32 B.1 General ................................................................................................................................................. 32 B.2 Neutralizers ......................................................................................................................................... 32 B.3 Neutralizer added to the agar for counting ...................................................................................... 33 Annex C (informative)

Graphical representations of the test method ........................................................ 34 Annex ZA (informative)

Relationship between this European Standard and the Essential Requirements of EU Directive 93/42/EEC concerning medical devices ....................................... 35 Bibliography ..................................................................................................................................................... 36

SIST EN 16616:2015

EN 16616:2015 (E) 3 European foreword This document (EN 16616:2015) 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 February 2016, and conflicting national standards shall be withdrawn at the latest by February 2016. 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 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 Directive(s). For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this document. 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, 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 the United Kingdom. SIST EN 16616:2015

EN 16616:2015 (E) 4 Introduction This European Standard specifies a carrier test for establishing whether a single-wash disinfecting product or combination of products for the treatment of contaminated textile has or does not have necessary microbicidal activity. The standard only intends to validate the disinfection part of the laundry process. 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 practice. The conditions are intended to cover general purposes and to allow reference between microbiological laboratories and types of detergents and disinfectants. Each effective dosage of the chemical disinfectant found by this test corresponds only to the chosen experimental conditions. Where actual conditions vary additional testing in microbiological laboratories shall be needed to determine the effective dosage. Instructions for use are the responsibility of manufactures of detergents or disinfectants. SIST EN 16616:2015

EN 16616:2015 (E) 5 1 Scope This European Standard specifies a test method and the minimum requirements for the microbicidal activity of a defined disinfection process for the treatment of contaminated textile. This procedure is carried out by using a washing machine as defined in 5.3.2.18 and refers to the disinfection step without prewash. This procedure is not limited to certain types of textile. The suppliers instructions shall be sufficient to allow the method in the standard to be carried out fully (e.g. dosing disinfectant in whatever washing phase e.g. rinsing, disinfecting at 40 °C). This European Standard applies to areas and situations where disinfection is indicated. Such indications occur in patient care, for example: — in hospitals, in community medical facilities and in dental institutions; — in schools, kindergartens and nursing homes; — institutions where patients are accommodated, which could suffer from transmissible diseases; — other applications where hygienic treatment of textile is necessary (e.g. food processing, hotels, workwear e.g. from the pharmaceutical industry, laboratories, foodstuffs area or similar institutions). The method described is intended to determine the activity of a product or product combination under the conditions in which they are used. This is a phase 2, step 2 laboratory test that simulates the conditions of application of the product. NOTE This method corresponds to a phase 2, step 2 test (see EN 14885). 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 (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity EN 13624, Chemical disinfectants and antiseptics

Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity in the medical area

Test method and requirements (phase 2, step 1) EN 13727, bactericidal
EN 14348, 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) 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 and the following apply. SIST EN 16616:2015

EN 16616:2015 (E) 6 3.1 liquor ratio ratio of the weight of dry textile in kilogram and volume of wash liquor in litre (w/v) 3.2 disinfection process process taking into account practical conditions of application of the product including contact time, temperature, test organisms and interfering substances to disinfect the textile 3.3 treatment of contaminated textile handling the textile according the disinfection process to obtain disinfected textile 4 Requirements The test results shall fulfil the basic limits (see 5.7.3). The following phase 2, step 1 test shall be passed in addition to this test: EN 13727, EN 13624 and EN 14348 under the following test conditions: — temperature as recommended by the manufacturer; — contact time recommended by the manufacturer; — dirty conditions and — reduction as recommended for instrument disinfection. For products used > 60 °C EN 13624 and EN 14348 should be passed with Aspergillus brasiliensis and M. avium. a) Processes with temperatures < 60°C A chemical-thermal textile disinfection process, when tested in accordance with Clause 5, is considered effective when in three test runs (within a given time, with a suggested dosage, at a suggested temperature and liquor ratio) a reduction of bacteria in and on germ carriers of more than 7 log-units, a reduction of Candida albicans and Aspergillus brasiliensis of 6 lg-units, a reduction of mycobacteria (M. avium and/or M. terrae) of 7 lg-units is reached. NOTE The implementation of spores and viruses was discussed. Further development is necessary to make it technically feasible. Further, no test organisms are to be detected in 100 ml washing/disinfection liquid. As a minimum requirement the bactericidal and yeasticidal activity shall be evaluated using the following test organisms: for bactericidal activity Pseudomonas aeruginosa, Escherichia coli (K12), Staphylococcus aureus, Enterococcus hirae and for yeasticidal activity Candida albicans as test organism. If an additional fungicidal activity is claimed, also Aspergillus brasiliensis shall be used as test organism. In case of an additional proof of tuberculocidal activity, Mycobacterium terrae is to be tested. If a mycobactericidal effect should be confirmed, it is necessary to perform the test also with Mycobacterium terrae and Mycobacterium avium. SIST EN 16616:2015

EN 16616:2015 (E) 7 b) Processes with temperatures ≥ 60°C A chemical-thermal textile disinfection process, when tested in accordance with Clause 5, is considered as effective when in three test runs (within a given time, with a suggested dosage, at a process temperature

≥ 60°C and a defined liquor ratio) a reduction of Enterococcus faecium in and on germ carriers of more than

7 lg-units will be achieved. Further, no test organisms are to be detected in 100 ml washing/disinfection liquid. This includes fungicidal, mycobactericidal / tuberculocidal activity. 5 Test methods 5.1 Principle Germ carriers made of cotton fabric are contaminated with a test suspension of microorganisms in defibrinated sheep blood. After drying the carriers are transferred into cotton bags and then the disinfection process in the washing machine is performed at test temperatures either t < 60°C or ≥ 60°C. The process refers to the disinfection step without prewash. At the end of the disinfection step of the procedure, the bags with the carriers have to be taken out (see 5.3.2.18). Each carrier is transferred into a separate tube containing neutralizer and glass-beads. The bacteria should be recovered from the carriers by shaking. The number of surviving bacteria in each sample is determined and the reduction rate is calculated. 5.2 Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains as test organisms1) — Pseudomonas aeruginosa ATCC 15442 — Escherichia coli (K12) ATCC 10538 — Staphylococcus aureus ATCC 6538 — Enterococcus hirae ATCC 10541 — Enterococcus faecium

ATCC 6057

The yeasticidal/fungicidal activity shall be evaluated using the following test organisms: — Candida albicans ATCC 10231 — Aspergillus brasiliensis (formerly Aspergillus Niger ATCC 16404)

ATCC 16404

The mycobactericidal/tuberculocidal activity shall be evaluated using the following test organisms: — Mycobacterium avium ATCC 15769 — Mycobacterium terrae

ATCC 15755
NOTE See Annex A for strain reference in some other culture collections.

1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collections (ATCC). This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. SIST EN 16616:2015

EN 16616:2015 (E) 8 The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3) [C. albicans and A. brasiliensis: (30 ± 1) °C]. The same temperature 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, and media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains/species, 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 stored 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 growth of test organisms. 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. For each culture medium and reagent, a limitation for use should be fixed. 5.2.2.2 Water used for preparation of media a) The water shall be fresh distilled water and not just demineralized water. Sterilize in the autoclave [5.3.2.1a)]. NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilized. NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. NOTE 3 See 5.2.2.3 for the procedure to prepare hard water. 5.2.2.3 Hard water for dilution of products for validation tests For the preparation of 1 l 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)]. 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) at 2 °C to 8 °C 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) at 2 °C to 8 °C 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 with the use of a pipette (5.3.2.9) 6,0 ml of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water SIST EN 16616:2015

EN 16616:2015 (E) 9 (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 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. 5.2.2.4 Tryptone Soy Agar (TSA) Tryptone, pancreatic digest of casein 15,0 g Soy 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 [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C. 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. 5.2.2.5 Tryptone Soy Broth (TSB) Tryptone, pancreatic digest of casein 15,0 g Soy peptone, papaic digest of soybean meal 5,0 g Sodium chloride (NaCl) 5,0 g Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C. 5.2.2.6 Brain Heart Infusion Agar (BHI) Example: Brain heart infusion 12,5 g Beef heart infusion 5,0 g Proteose-Peptone 10,0 g Glucose 2,0 g Sodium chloride (NaCl) 5,0 g Dinatriumhydrogenphosphate 2,5 g Agar 10,0 g Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C. SIST EN 16616:2015

EN 16616:2015 (E) 10 5.2.2.7 Malt Extract Agar (MEA) Malt extract 30,0 g Soy peptone, papaic digest of soybean meal 3,0 g Agar 15,0 g Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to 5,6 ± 0,2 when measured at (20 ± 1) °C. 5.2.2.8 Middlebrook and Cohn 7H10 medium incl. 10 % OADC (7H10) Middlebrook 7H10 agar 19,0 g Glycerol 5,0 ml Water (5.2.2.2) To 900,0 ml

Heat to boiling to dissolve completely. Sterilize for 10 min in the autoclave [5.3.2.1a)] and cool to 50 °C to 55 °C. Add 100 ml Middlebrook OADC enrichment under aseptic conditions. The final pH of the medium shall be equivalent 6,6 ± 0,2 when measured at (25 ± 1) °C. 5.2.2.9 Diluent Tryptone, pancreatic digest of casein 1,0 g Sodium chloride (NaCl) 8,5 ml Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization, the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at (20 ± 1) °C. 5.2.2.10 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. 5.2.2.11 Sterile defibrinated sheep blood The sterile defibrinated sheep blood can be acquired from a commercial supplier. 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 [5.3.2.1a)]; b) by dry heat, in the hot air oven [5.3.2.1b)]. SIST EN 16616:2015

EN 16616:2015 (E) 11 5.3.2 Usual microbiological laboratory equipment2) and, in particular, the following: 5.3.2.1 Apparatus for sterilization (moist and dry heat) a) for moist heat sterilization, an autoclave capable of being maintained at (30121+)°C for a minimum contact time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at (50180+)°C for a minimum contact time of 30 min, at (170 50+)°C for a minimum contact time of 1 h or at (50160+)°C for a minimum contact time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (36 ± 1) °C or (37 ± 1) °C, at (45 ± 1) °C (to maintain melted medium in case of pour plate technique) and at additional test temperatures ± 1 °C, temperatures til 70 °C ± 1°C shall be adjustable. 5.3.2.3 Incubator, capable of being controlled either at (30 ± 1) °C or (36 ± 1) °C. The same temperature shall be used for incubations performed during a test and its control and validation. 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C. A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.2.2.4 to 5.2.2.8). 5.3.2.5 Stopwatch, a digital stopwatch is recommended 5.3.2.6 Shakers a) Electromechanical agitator, e.g. Vortex® mixer3) b) Orbital shaker 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.3). The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of microorganisms 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 (diameter 3 mm to 4 mm)

2) Disposable sterile equipment is an acceptable alternative to reusable glassware. 3) Vortex® in an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 16616:2015

EN 16616:2015 (E) 12 5.3.2.12 Volumetric flasks 5.3.2.13 Centrifuge (3 000 gN) 5.3.2.14 Coned bottom screw cap tubes (contents of 50 ml, diameter: about 28 mm) 5.3.2.15 Cylindrical screw vial (contents about 15 ml to 50 ml) for recovery of the test organisms from the carriers 5.3.2.16 Cotton carriers, 1 cm2: The germ carriers are prepared by using standard cotton fabric thoroughly cooked in double-distilled water three times. The fabric is cut into 1 cm2 cotton carriers and autoclaved [see 5.3.2.1a)]. For practical reasons the 1 cm2 carriers are put in the pockets of a bigger cotton towel. NOTE 1 The incorporation of the test suspension can be improved if the carriers are not dried after autoclaving. Mass per unit area: (170 ± 10) g/m2 (real 160 g/m2) Fibrous material (wrap and weft): cotton, double corded Fibre length (wrap and weft): at least 27 mm Yarn linear density (wrap and weft): (295 ± 10) dtex Yarn twist (wrap and weft): Z-twist (700 ± 25) t/m Weave: plain weave 1 Threads per unit length:

270 threads/dm each

The cotton control cloth shall be bleached, unfinished and not brightened. After three washes the maximum tensile strength, wet, in the warp should be (63 ± 5) daN. NOTE 2 Cotton proofed in accordance with DIN 53919 [6] fulfils the requirements. 5.3.2.17 Machine ballast load and preparation of

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