Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)

This document specifies a test method and the minimum requirements for fungicidal or yeasticidal 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 document applies to products that are used in food, industrial, domestic and institutional areas excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in the above considered areas. The following areas are at least included:
a) processing, distribution and retailing of:
1) food of animal origin:                    
- milk and milk products;
- meat and meat products;
- fish, seafood, and related products;
- eggs and egg products;
- animal feeds;
- etc.
2) food of vegetable origin:
- beverages;
- fruits, vegetables and derivatives (including sugar, distillery ...);
-  flour, milling and baking;
- animal feeds;
- etc.
b) institutional and domestic areas:
- catering establishments;
-public areas;
- public transports;
- schools;
- nurseries;
- shops;
- sports rooms;
- waste containers (bins ...);
- hotels;
- dwellings;
- clinically non-sensitive areas of hospitals;
- offices;
- etc.
c) other industrial areas:
- packaging material;
- biotechnology (yeast, proteins, enzymes, ...);
- pharmaceutical;
- cosmetics and toiletries;
- textiles;
- space industry, computer industry;
- etc.
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 fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Dieses Dokument legt ein Prüfverfahren und die Mindestanforderungen an die fungizide oder levurozide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die in hartem Wasser oder im Fall gebrauchs¬fertiger Lösungen in Wasser eine homogene, physikalisch stabile Zubereitung bilden. Produkte können nur bis zu einer Konzentration von 80 % geprüft werden, da immer eine bestimmte Verdünnung durch Zugabe der Impfkultur oder der Belastungssubstanz erfolgt.
Dieses Dokument gilt für Produkte, die im Lebensmittelbereich, in der Industrie, im Haushalt und in öffentlichen Einrichtungen benutzt werden. Ausgenommen sind Bereiche und Situationen, in denen die Desinfektion medizinisch indiziert ist und Produkte, die auf lebenden Geweben benutzt werden, außer solchen, die zur Handhygiene in den oben genannten Bereichen verwendet werden. Die folgenden Bereiche sind mindestens eingeschlossen:
a)   Verarbeitung, Vertrieb und Verkauf von:
1)   Lebensmitteln tierischer Herkunft:
   Milch und Milchprodukte;
   Fleisch und Fleischprodukte;
   Fisch, Meerestiere und daraus hergestellte Zubereitungen;
   Eier und Eiprodukte;
   Tiernahrung;
   usw.
2)   Lebensmitteln pflanzlicher Herkunft:
   Getränke;
   Früchte, Gemüse und daraus hergestellte Zubereitungen (einschließlich Zucker, Destillationsprodukte usw.);
   Mehl, gemahlene und gebackene Produkte;
   Tiernahrung;
   usw.
b)   öffentliche Einrichtungen und Haushaltsbereiche:
   Verpflegungsbetriebe;
   Öffentliche Bereiche;
   Öffentliche Verkehrsmittel;
   Schulen;
   Kindergärten;
   Geschäfte;
   Sportstätten;
   Abfallbehälter (Mülltonnen usw.);
   Hotels;
   Wohngebäude;
   klinisch nicht relevante Bereiche in Krankenhäusern;
   Büroräume;
   usw.
c)   andere industrielle Bereiche:
   Packmaterial;
   Biotechnologie (Hefe, Proteine, Enzyme usw.);
   Hersteller von Pharmazeutika;
   Hersteller von Kosmetika und Toilettenartikel;
   Hersteller von Textilien;
   Weltraumforschungsindustrie, Computerindustrie;
   usw.
EN 14885 legt im Einzelnen die Beziehungen der verschiedenen Prüfungen untereinander und zu den „Anwendungsempfehlungen“ fest.
ANMERKUNG 1   Das beschriebene Verfahren dient dazu, die Aktivität handelsüblicher Zubereitungen oder Wirksubstanzen unter deren Anwendungsbedingungen zu bestimmen.
ANMERKUNG 2   Diese Methode entspricht einem Phase 2/Stufe 1-Verfahren.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)

Le présent document spécifie une méthode d'essai et les prescriptions minimales relatives à l'activité
fongicide ou levuricide des produits antiseptiques et désinfectants chimiques qui forment une préparation
homogène, physiquement stable, lorsqu'ils sont dilués dans 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.
Le présent document s'applique aux produits qui sont utilisés dans les domaines alimentaire, industriel,
domestique et en collectivités à l'exclusion des domaines et des situations où la désinfection est
médicalement indiquée et à l'exclusion des produits utilisés sur les tissus vivants à l'exception de ceux utilisés
pour l'hygiène des mains dans les domaines considérés ci-dessus. Sont inclus au moins les domaines
suivants :

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje fungicidnega delovanja ali delovanja kemičnih razkužil in antiseptikov na kvasovke v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in zahteve (faza 2, stopnja 1) (vključno z dopolnilom A1)

Ta dokument določa preskusno metodo in minimalne zahteve za fungicidno delovanje ali delovanje kemičnih razkužil in antiseptikov na kvasovke, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma, pri proizvodih, ki so pripravljeni za uporabo, z vodo. Proizvode je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji, ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje. Ta dokument velja za izdelke, ki se uporabljajo v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah, izključeni pa so področja in primeri, ko obstajajo zdravstvene indikacije za dezinfekcijo ter izdelki, ki se uporabljajo na živih tkivih, razen tistih, ki se uporabljajo za higieno rok v zgoraj navedenih področjih. Naslednja področja so najmanj vključena:
a) predelava, distribucija in prodaja:
1) hrane živalskega izvora:
– mleko in mlečni izdelki;– meso in mesni izdelki;
– ribe, morski sadeži in podobno;
– jajca in jajčni izdelki;
– živalska krma;
– itn.
2) hrane rastlinskega izvora:
– napitki;
– sadje, zelenjava in derivati (vključno s sladkorjem, destilati ...);
– moka, mletje in peka;
– živalska krma;
– itn.
b) javne ustanove in gospodinjstva:
– priprava in dostava hrane;
– javne ustanove;
– javni prevozi;
– šole;
– vrtci;
– trgovine;
– športni objekti;
– zbiralniki odpadkov (koši ...);
– hoteli;
– bivališča;
– klinično neobčutljivi prostori bolnišnic;
– pisarne;
– itn.
c) druga industrijska področja:
– embalaža;
– biotehnologija (kvasovke, proteini, encimi ...);
– farmacevtski izdelki;
– kozmetični in toaletni izdelki;
– tkanine;
– vesoljska in računalniška industrija;
– itn.
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 stopnje 1 faze 2.

General Information

Status
Withdrawn
Publication Date
05-Jun-2013
Withdrawal Date
08-Oct-2019
Current Stage
9900 - Withdrawal (Adopted Project)
Start Date
13-Sep-2019
Due Date
06-Oct-2019
Completion Date
09-Oct-2019

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposesICS:Ta slovenski standard je istoveten z:EN 1650:2008+A1:2013SIST EN 1650:2008+A1:2013en,fr,de01-julij-2013SIST EN 1650:2008+A1:2013SLOVENSKI
STANDARDSIST EN 1650:20081DGRPHãþD



SIST EN 1650:2008+A1:2013



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 1650:2008+A1
May 2013 ICS 71.100.35 Supersedes EN 1650:2008
English Version
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 5 April 2008 and includes Amendment 1 approved by CEN on 28 March 2013.
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 © 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 1650:2008+A1:2013: E SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 2 Contents Page Foreword .3 Introduction .4 1 Scope .5 2 Normative references .6 3 Terms and definitions .6 4 Requirements .6 5 Test method .7 5.1 Principle .7 5.2 Materials and reagents .7 5.3 Apparatus and glassware . 11 5.4 Preparation of test organism suspensions and product test solutions . 13 5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product . 18 5.6 Experimental data and calculation. 24 5.7 Verification of methodology . 27 5.8 Expression of results and precision . 28 5.9 Interpretation of results - conclusion . 29 5.10 Test report . 29 Annex A (informative)
Referenced strains in national collections . 31 Annex B (informative)
Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids . 32 Annex C (informative)
Graphical representations of dilution-neutralization method and membrane filtration method . 34 Annex D (informative)
Example of a typical test report . 38 Annex E (informative)
Precision of the test result . 44 Bibliography . 47
SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 3 Foreword This document (EN 1650:2008+A1:2013) 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 November 2013, and conflicting national standards shall be withdrawn at the latest by November 2013. 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 includes Amendment 1, approved by CEN on 2013-03-28. This document supersedes !EN 1650:2008". The start and finish of text introduced or altered by amendment is indicated in the text by tags
!". This European Standard was revised to include the results of a collaborative trial (ANDISTAND), to correct obvious errors and ambiguities, to harmonize the structure and wording with other quantitative suspension tests of CEN/TC 216 (existing or in preparation), and to improve the readability of the standard and thereby make it more understandable. According to the CEN/CENELEC Internal Regulations, the national standards organisations 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 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 4 Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has or does not have a fungicidal or yeasticidal activity in the fields described in the scope. This laboratory test takes into account practical conditions of application of the product including contact time, temperature, test organisms and interfering substance, i.e. conditions which may influence its action in practical situations.
The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test corresponds to defined experimental conditions. However, for some applications the recommendations of use of a product may differ and therefore additional test conditions should be used. SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 5 1 Scope This document specifies a test method and the minimum requirements for fungicidal or yeasticidal 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 document applies to products that are used in food, industrial, domestic and institutional areas excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in the above considered areas. The following areas are at least included:
a) processing, distribution and retailing of: 1) food of animal origin: 2) food of vegetable origin:  milk and milk products;  beverages;  meat and meat products;  fruits, vegetables and derivatives (including sugar, distillery .);  fish, seafood, and related products;  flour, milling and baking;  eggs and egg products;  animal feeds;  animal feeds;  etc.  etc.
b) institutional and domestic areas:  catering establishments;  public areas;  public transports;  schools;  nurseries;  shops;  sports rooms;  waste containers (bins .);  hotels;  dwellings;  clinically non-sensitive areas of hospitals;  offices;  etc. SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 6 c) other industrial areas:  packaging material;  biotechnology (yeast, proteins, enzymes, .);  pharmaceutical;  cosmetics and toiletries;  textiles;  space industry, computer industry;  etc. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1 test. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 12353, Chemical disinfectants and antiseptics – Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity EN 14885, Chemical disinfectants and antiseptics – Application of European Standards for chemical disinfectants and antiseptics ISO 4793, Laboratory sintered (fritted) filters – Porosity grading, classification and designation 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 a reduction of at least a 4 decimal log (lg) when diluted with hard water (5.2.2.7) or - in the case of ready-to-use products - with water (5.2.2.2) and tested in accordance with Clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution - 5.2.2.8.2) or simulated dirty conditions (3 g/l bovine albumin solution - 5.2.2.8.3) according to its practical applications and under the other obligatory test conditions (one or two selected test organisms, 20 °C, 15 min).
The fungicidal activity shall be evaluated using the following two test organisms:
 Candida albicans (vegetative cells); SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 7  Aspergillus niger (spores). The yeasticidal activity shall be evaluated using the following test organism:  Candida albicans (vegetative cells); Where indicated, additional specific fungicidal or yeasticidal activity shall be determined applying other contact times, temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in order to take into account intended specific use conditions.
NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions. 5 Test method 5.1 Principle 5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use products) is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an interfering substance. The mixture is maintained at (20 ± 1) °C for 15 min ± 10 s (obligatory test conditions). At the end of this contact time, an aliquot is taken, and the fungicidal and/or the fungistatic activity in this portion is immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving fungi in each sample are determined and the reduction is calculated. 5.1.2 The test is performed using the vegetative cells of Candida albicans and the spores of Aspergillus niger (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as test organisms (obligatory test conditions). 5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can be used. 5.2 Materials and reagents 5.2.1 Test organisms The fungicidal activity shall be evaluated using the following strains as test organisms:1)
 Candida albicans ATCC 10231  Aspergillus niger ATCC 16404
The yeasticidal activity shall be evaluated using only Candida albicans. NOTE See annex A for strain references in some other culture collections. The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3). If required for specific applications, additional strains may be chosen from, e.g. for breweries:
1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC). This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named.
SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 8  Saccharomyces cerevisiae DSM 1333  Saccharomyces cerevisiae var. diastaticus DSM 70487 If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture collection under a reference for five years.
5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. NOTE 1 To improve reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be rigorously followed.
NOTE 2 For each culture medium and reagent, a limitation for use should be fixed. SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 9 5.2.2.2 Water The water shall be freshly glass-distilled water and not 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 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Malt extract agar (MEA) !Malt extract agar, consisting of:
Malt extract
30,0 g
Agar
15,0 g
Water (5.2.2.2)
to 1 000,0 ml The malt extract should be food grade (e.g. Cristomalt powder from Difal) or equivalent that is not highly purified and not only based on maltose (e.g. Malt extract from OXOID) 1). However if there are problems producing at least 75 % spiny spores see 5.4.1.4.2.
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. NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add neutralizer to the MEA. 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 (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. 5.2.2.5 Neutralizer The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. It shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in Annex B.
1) 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. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 10 5.2.2.6 Rinsing liquid (for membrane filtration) The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane under the test conditions described in 5.5.3. NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.7 Hard water for dilution of products For the preparation of 1 000 ml of hard water, the procedure is as follows:  prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave [5.3.2.1a)]. 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) for no longer than one month;  prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for no longer than one week;  place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The pH of the hard water shall be 7,0 ± 0,2, when measured at (20 ± 1)°C (5.3.2.4). If necessary, adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h. NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces a different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube. 5.2.2.8 Interfering substance 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product. 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 and detergents) shall be defined. NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration) Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration of bovine albumin in the test procedure (5.5) is 0,3 g/l. SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 11 5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration) Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2). Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l. 5.2.2.8.4 Milk (dairies .) Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder per litre of water (5.2.2.2), shall be prepared as follows :  prepare a solution of 100 g milk-powder in 1 000 ml water (5.2.2.2). Heat for 30 min at (105 ± 3) °C [or 5 min at (121 ± 3 °C)]. The final concentration of reconstituted milk in the test procedure (5.5) is 10,0 g/l of reconstituted milk. 5.2.2.8.5 Yeast extract (breweries .) Dehydrated yeast extract for bacteriology, shall be prepared as follows :  prepare a 100 g/l solution in water (5.2.2.2), adjust to pH 7,0 ± 0,2 with sodium hydroxide (NaOH);  sterilize in the autoclave [5.3.2.1a)]. The final concentration of yeast extract in the test procedure (5.5) is 10,0 g/l. 5.2.2.8.6 Sucrose (beverage, soft drink industries) Prepare a 100 g/l solution of sucrose in water (5.2.2.2), sterilize by membrane filtration (5.3.2.7). The final concentration of sucrose in the test procedure (5.5) is 10,0 g/l. 5.2.2.8.7 pH 5,0 and pH 9,0 buffer solutions (cleaning in place .) The buffer solution used shall be described in the test report and pH values shall be recorded. The final pH in the test tubes (together with test organisms and product) shall be controlled and found equal to 5,0 ± 0,2 or 9,0 ± 0,2. 5.2.2.8.8 Sodium dodecyl sulphate (cosmetic area .) Prepare a 50 g/l solution of sodium dodecyl sulphate (C12H25NaO4S) in water (5.2.2.2). Sterilize in the autoclave [5.3.2.1a)]. The final concentration of sodium dodecyl sulphate in the test procedure (5.5) is 5,0 g/l. 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 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 12 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 (C) 1213 0°+ for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at (50180+)°C for a minimum holding time of 30 min, at (50170+) °C for a minimum holding time of 1 h or at (50160+)°C for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted MEA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C. 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® mixer 3). b) Mechanical shaker 5.3.2.7 Membrane filtration apparatus constructed of a material compatible with the substances to be filtered. The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of 5.2.2.7), bovine albumin (5.2.2.8.2 and 5.2.2.8.3) and sucrose (5.2.2.8.6), and if the membrane filtration method is used (5.5.3). The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s. 5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml, or calibrated automatic pipettes. 5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
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 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 13 5.3.2.11 Glass beads, 3 mm to 4 mm in diameter. 5.3.2.12 Volumetric flasks 5.3.2.13 Fritted filter 5.3.2.14 Flasks with ventilated caps."
5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions (test and validation suspension) 5.4.1.1 General For each test organism, two different suspensions have to be prepared: the “test suspension” to perform the test and the “validation suspension” to perform the controls and method validation. 5.4.1.2 Preservation and stock cultures of test organisms The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.3 Working culture of test organisms 5.4.1.3.1 Candida albicans (yeast) In order to prepare the working culture of Candida albicans (5.2.1), prepare a subculture from the stock culture (5.4.1.2) by streaking onto MEA (5.2.2.3) slopes or plates (5.3.2.10) and incubate (5.3.2.3). After 42 h to 48 h, prepare a second subculture from the first subculture in the same way and incubate for 42 h to 48 h. From this second subculture, a third subculture may be produced in the same way. The second and (if produced) third subcultures are the working cultures. If it is not possible to prepare the second subculture on a particular day, a 72 h subculture may be used for subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 72 h period. Never produce and use a fourth subculture. ! 5.4.1.3.2 Aspergillus brasiliensis (previously A. niger) (mould) For Aspergillus brasiliensis (previously A. niger) (5.2.1), use only the first subculture grown on MEA (5.2.2.3) in Petri dishes or flasks with ventilated caps (5.3.2.14) and incubate for 7 d to 9 d. No further subculturing is needed. Do not stack the Petri dishes during the incubation to improve the temperature homogenization. At the end of incubation, all the cultures have to show a dark brown or black surface. Cultures with appearance of rare and small white or grey areas might be kept (see Figure 1).
SIST EN 1650:2008+A1:2013



EN 1650:2008+A1:2013 (E) 14
Figure 1 — Photos N°1: A. brasiliensis ATCC 16404 after 7 d of incubation at 30 °C (right cultures)
Figure 2 — Photo N°2: A. brasiliensis ATCC 16404 after 7 d of incubation at 30 °C (wrong culture)" 5.4.1.3.3 Other test organisms (yeasts or moulds) For additional test organisms, any departure from this method of culturing the yea
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