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

This document specifies a test method (phase 2/step 1) and the minimum requirements for sporicidal activity of chemical disinfectant products that form a homogeneous, physically stable preparation in hard water and 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.
This European Standard applies at least to the following:
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, etc.);
-   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, etc.);
-   hotels;
-   dwellings;
-   clinically non sensitive areas of hospitals;
-   offices;
-   etc.;
c)   other industrial areas:
-   packaging material;
-   biotechnology (yeast, proteins, enzymes, etc.);
-   pharmaceutical;
-   cosmetics and toiletries;
-   textiles;
-   space industry, computer industry;
-   etc.
Using this European Standard, it is not possible to determine the sporicidal activity of undiluted product as some dilution is always produced by adding the inoculum and interfering substance. Products can only be tested at a concentration of 80 % or less.
NOTE   The method described is intended to determine the activity of commercial formulations or active substances on spores in the conditions in which they are used.

Chemische Desinfektionsmittel - Quantitativer Suspensionversuch zur Bestimmung der sporiziden Wirkung chemischer Desinfektionsmittel in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Diese Europäische Norm legt ein Prüfverfahren (Phase 2, Stufe 1) und die Mindestanforderungen an die sporizide Wirkung von chemischen Desinfektionsmitteln in den Bereichen Lebensmittel, Industrie, Haushalt und in öffentlichen Einrichtungen fest, die mit Wasser standardisierter Härte eine homogene, physikalisch stabile Lösung ergeben. Ausgenommen davon sind Bereiche und Situationen mit medizinisch indizierter Desinfektion und Produkte, die auf lebenden Geweben benutzt werden, mit Ausnahme derjenigen für die Handhygiene in den oben angegebenen Bereichen.
Diese Europäische Norm gilt mindestens für den folgenden Anwendungsbereich:
a)   Verarbeitung, Vertrieb und Verkauf von:
1)   Lebensmitteln tierischer Herkunft:
-   Milch und Milchprodukte;
-   Fleisch und Fleischprodukte;
-   Fisch, Meerestiere und daraus hergestellte Erzeugnisse;
-   Eier und Eiprodukte;
-   Tiernahrung;
-   usw.;
2)   Lebensmitteln pflanzlicher Herkunft:
-   Getränke;
-   Früchte, Gemüse und daraus hergestellte Erzeugnisse (einschließlich Zucker, Destillationsprodukte usw.);
-   Mehl, gemahlene und gebackene Produkte;
-   Tiernahrung;
-   usw.;
b)   öffentliche Einrichtungen und Haushaltsbereiche:
-   Verpflegungseinrichtungen;
-   öffentliche Bereiche;
-   öffentliche Verkehrsmittel;
-   Schulen;
-   Kindergärten;
-   Geschäfte;
-   Sportstätten;
-   Abfallbehälter (Mülltonnen usw.);
-   Hotels;
-   Wohngebäude;
-   klinisch nicht relevante Bereiche von Krankenhäusern;
-   Büroräume;
-   usw.;
c)   andere industrielle Bereiche:
-   Verpackungsmaterial;
-   Biotechnologie (Hefe, Proteine, Enzyme usw.);
-   Hersteller von Pharmazeutika;
-   Hersteller von Kosmetika und Toilettenartikeln;
-   Hersteller von Textilien;
-   Weltraumforschungsindustrie und Computerindustrie;
-   usw.
Bei Anwendung dieser Europäischen Norm ist die Bestimmung der sporiziden Wirkung des unverdünnten Produktes nicht möglich, da immer eine bestimmte Verdünnung durch Zugabe der Impfkultur und der Belastungssubstanz auftritt. Produkte können nur bei einer Konzentration von 80 % oder darunter geprüft werden.
ANMERKUNG   Das beschriebene Verfahren bestimmt die Wirksamkeit gewerblich hergestellter Rezepturen oder anderer Wirkstoffe auf Sporen unter Anwendungsbedingungen.

Désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité sporicide 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 (phase 2, étape 1) et les exigences minimales relatives à l'activité sporicide des produits désinfectants chimiques qui forment une préparation homogène, physiquement stable dans l'eau dure et qui sont utilisés dans le domaine de l'agro‐alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité, à l'exclusion des domaines et des situations où la désinfection est médicalement prescrite et à l'exclusion des produits utilisés sur des tissus vivants
(excepté ceux destinés à l'hygiène des mains dans les domaines indiqués ci‐dessus). La présente Norme européenne s'applique au moins aux domaines suivants :
a) la transformation, la distribution et le commerce de détail des :
1) aliments d'origine animale :
- lait et produits laitiers ;
- viande et produits carnés ;
- poisson, fruits de mer et leurs dérivés ;
- oeufs et produits dérivés ;
- aliments pour animaux ;
- etc. ;
2) aliments d'origine végétale :
- boissons ;
- fruits, légumes et leurs dérivés (y compris le sucre, et les produits de distillation, etc.) ;
- farine, minoterie et boulangerie ;
- aliments pour animaux ;
- etc. ;
b) les domaines domestiques et la collectivité :
- établissements de restauration ;
- lieux publics ;
- transports publics ;
- écoles ;
- crèches ;
- magasins ;
- salles de sport ;
- conteneurs pour déchets (poubelles, etc.) ;
- hôtels ;
- locaux d'habitation ;
- zones cliniquement non sensibles des hôpitaux ;
- bureaux ;
- etc. ;
c) les autres domaines industriels :
- matériaux d’emballage ;
- biotechnologie (levure, protéines, enzymes, etc.) ;
- industrie pharmaceutique ;
- cosmétiques et produits d'hygiène corporelle ;
- textiles ;
- industrie spatiale, secteur informatique ;
- etc.
Il n'est pas possible d'utiliser la présente Norme européenne pour déterminer l'activité sporicide des produits purs, car l'ajout de l'inoculum et de la substance interférente s'accompagne forcément d'une dilution. Les produits ne peuvent être soumis à l'essai qu'à la concentration de 80 % ou à des concentrations inférieures.
NOTE La méthode décrite vise à déterminer l'activité des formulations commerciales ou des substances actives sur les spores dans les conditions dans lesquelles elles sont utilisées.

Kemična razkužila - Kvantitativni suspenzijski preskus za vrednotenje sporocidnega delovanja kemičnih razkužil v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in zahteve (faza 2, stopnja 1)

Ta evropski standard določa preskusno metodo (faza 2/stopnja 1) (glej dodatek H) in minimalne zahteve za sporocidno delovanje kemičnih razkužil, ki tvorijo homogen, fizikalno stabilen pripravek v trdi vodi ter se uporabljajo v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah, izključeni pa so področja in primeri, ko obstajajo zdravstvene indikacije za dezinfekcijo ter proizvodi, ki se uporabljajo na živih tkivih, razen tistih, ki se uporabljajo za higieno rok na zgoraj navedenih področjih. Ta evropski standard se uporablja vsaj za: a) predelavo, distribucijo in prodajo: 1) hrane živalskega izvora: 2) hrane rastlinskega izvora: b) gospodinjstvo in javne ustanove: c) druge industrije: Če se uporablja ta evropski standard, ni mogoče določiti sporocidnega delovanja nerazredčenega proizvoda, ker dodajanje inokuluma in moteče snovi vedno povzroči nekaj razredčenja. Proizvode je mogoče preskusiti le pri največ 80-odstotni koncentraciji. OPOMBA: Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi na sporah pod pogoji, v katerih se uporabljajo.

General Information

Status
Published
Publication Date
17-Jul-2018
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
18-Jul-2018
Completion Date
18-Jul-2018

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.HQMHXVWULMDKChemische Desinfektionsmittel - Quantitativer Suspensionversuch zur Bestimmung der sporiziden Wirkung chemischer Desinfektionsmittel in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité sporicide 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 - Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants 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 13704:2018SIST EN 13704:2018en,fr,de01-oktober-2018SIST EN 13704:2018SLOVENSKI

STANDARDSIST EN 13704:20021DGRPHãþD
SIST EN 13704:2018
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 13704
July
t r s z ICS
y sä s r rä u w Supersedes EN
s u y r vã t r r tEnglish Version

Chemical disinfectants æ Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants used in foodá industrialá domestic and Désinfectants chimiques æ Essai quantitatif de suspension pour l 5évaluation de l 5activité sporicide des désinfectants chimiques utilisés dans le domaine de l 5agroæalimentaireá dans l 5industrieá dans les domaines domestiques et en collectivité æ Méthode d 5essai et

Chemische Desinfektionsmittel æ Quantitativer Suspensionversuch zur Bestimmung der sporiziden Wirkung chemischer Desinfektionsmittel in den Bereichen Lebensmittelá Industrieá Haushalt und öffentliche Einrichtungen æ Prüfverfahren und This European Standard was approved by CEN on

s x March
t r s zä

egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä

translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä

CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä

EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre:
Rue de la Science 23,
B-1040 Brussels

t r s z CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN

s u y r vã t r s z ESIST EN 13704:2018
EN 13704:2018 (E) 2 Contents

Page European foreword ................................................................................................................................................................ 4 Introduction ............................................................................................................................................................................. 5 1 Scope ............................................................................................................................................................................. 6 2 Normative references ............................................................................................................................................. 7 3 Terms and definitions ............................................................................................................................................ 7 4 Requirements ............................................................................................................................................................ 8 5 Test method ............................................................................................................................................................... 8 5.1 Principle ...................................................................................................................................................................... 8 5.2 Materials and reagents ........................................................................................................................................... 9 5.2.1 Test organisms .......................................................................................................................................................... 9 5.2.2 Culture media and reagents ................................................................................................................................. 9 5.3 Apparatus and glassware ................................................................................................................................... 11 5.3.1 General ...................................................................................................................................................................... 11 5.3.2 Usual microbiological laboratory equipment and, in particular, the following ............................. 11 5.4 Preparation of spore test suspension and test solutions ....................................................................... 12 5.4.1 Spore suspensions ................................................................................................................................................ 12 5.4.2 Product test solution ............................................................................................................................................ 14 5.5 Procedure................................................................................................................................................................. 15 5.5.1 Choice of experimental conditions ................................................................................................................. 15 5.5.2 Test procedure for assessing the sporicidal effect of the product ...................................................... 15 5.5.3 Validation of dilution neutralization and membrane filtration method .......................................... 17 5.6 Calculation and expression of results ............................................................................................................ 17 5.6.1 Overview of the different suspensions and test mixtures ...................................................................... 17 5.6.2 Calculation ............................................................................................................................................................... 18 5.7 Verification of methodology .............................................................................................................................. 23 5.7.1 General ...................................................................................................................................................................... 23 5.7.2 Control of weighted mean counts .................................................................................................................... 23 5.7.3 Basic limits .............................................................................................................................................................. 23 5.7.4 Expression of results ............................................................................................................................................ 24 5.8 Conclusion................................................................................................................................................................ 24 5.9 Test report ............................................................................................................................................................... 24 Annex A (normative)

Preparation of Bacillus subtilis and Bacillus cereus spore stock suspensions ............................................................................................................................................................. 26 A.1 Material and reagents .......................................................................................................................................... 26 A.2 Preparation of Bacillus spore stock suspensions ...................................................................................... 26 Annex B (normative)

Validation of dilution-neutralization and membrane filtration methods ......... 28 B.1 Principle ................................................................................................................................................................... 28 B.2 Preparation of spore suspension .................................................................................................................... 28 B.3 Preparation of product test solution.............................................................................................................. 28 B.4 Test for validation ................................................................................................................................................. 28 B.4.1 Dilution-neutralization method....................................................................................................................... 28 SIST EN 13704:2018

EN 13704:2018 (E) 3 B.4.1.1 General ...................................................................................................................................................................... 28 B.4.1.2 Validation ................................................................................................................................................................. 29 B.4.2 Membrane filtration method ............................................................................................................................. 30 B.4.2.1 General ...................................................................................................................................................................... 30 B.4.2.2 Validation ................................................................................................................................................................. 30 B.5 Validation ................................................................................................................................................................. 32 Annex C (informative)

Preparation of Clostridium sporogenes spore stock suspension .......................... 33 C.1 Culture media and reagents ............................................................................................................................... 33 C.2 Apparatus and glassware.................................................................................................................................... 34 C.3 Preparation of regenerated media and incubation conditions ............................................................ 34 C.4 Preparation of Clostridium spore stock suspension ................................................................................. 34 Annex D (informative)

Neutralizers and rinsing liquids ..................................................................................... 36 Annex E (informative)

Example of a typical test report ...................................................................................... 38 Annex F (informative)

Referenced strains in national collections .................................................................. 40 F.1 Bacillus subtilis ....................................................................................................................................................... 40 F.2 Bacillus cereus ......................................................................................................................................................... 40 F.3 Clostridium sporogenes ........................................................................................................................................ 40 Bibliography .......................................................................................................................................................................... 41

SIST EN 13704:2018

EN 13704:2018 (E) 4 European foreword This document (EN 13704:2018) 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 January 201, and conflicting national standards shall be withdrawn at the latest by January 201. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 13704:2002. It was revised in order to include the spore susceptibility controls, to better detail spore preparation procedures and to harmonize counting and elaboration of the results with the other CEN/TC 216 current standards. EN 13704:2018 includes the following changes compared to EN 13704:2002: — inclusion of spore suspension susceptibility controls; — modification of spore preparation procedures; — inclusion of spore maturation time; — harmonization of counting and elaboration of the results with the other CEN/TC 216 current standards; — deletion of obligatory conditions for contact time and temperature; — inclusion of minimum and maximum conditions for contact time and temperature; — inclusion of dirty conditions. Results obtained with the previous version are not valid. 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 13704:2018

EN 13704:2018 (E) 5 Introduction This European Standard describes a suspension test method for establishing whether a chemical disinfectant has or does not have a sporicidal activity in the fields described in Clause 1. The laboratory test closely simulates practical conditions of application. Chosen conditions (contact time, temperature, in suspension, etc.) reflect parameters which are found in practical situations including conditions which can influence the action of disinfectants. Each utilization concentration found from this test corresponds to defined experimental conditions. The conditions are intended to cover general purposes and to allow reference between laboratories and product types. However for some applications the recommendations of use of a product can differ and therefore additional test conditions need to be used. SIST EN 13704:2018

EN 13704:2018 (E) 6 1 Scope This document specifies a test method (phase 2/step 1) and the minimum requirements for sporicidal activity of chemical disinfectant products that form a homogeneous, physically stable preparation in hard water and 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. This European Standard applies at least to the following: 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, etc.); — 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, etc.); SIST EN 13704:2018

EN 13704:2018 (E) 7 — hotels; — dwellings; — clinically non sensitive areas of hospitals; — offices; — etc.; c) other industrial areas: — packaging material; — biotechnology (yeast, proteins, enzymes, etc.); — pharmaceutical; — cosmetics and toiletries; — textiles; — space industry, computer industry; — etc. Using this European Standard, it is not possible to determine the sporicidal activity of undiluted product as some dilution is always produced by adding the inoculum and interfering substance. Products can only be tested at a concentration of 80 % or less. NOTE The method described is intended to determine the activity of commercial formulations or active substances on spores in the conditions in which they are used. 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 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. SIST EN 13704:2018

EN 13704:2018 (E) 8 4 Requirements The product shall demonstrate at least 3 decimal log (lg) reduction, when tested in accordance with Table 1 here below and Clause 5. Table 1 — Minimum and additional test conditions Test Conditions for Surface disinfection Minimum spectrum of test organisms Bacillus subtilis Additional sporicidal activity vs anaerobes for specific uses Clostridium sporogenes Additional sporicidal activity vs aerobes for specific uses Bacillus cereus

Required reduction

· 3 lg Test temperature according to the manufacturer’s recommendation, but between 4 °C and 75 °C Contact time (in minutes) according to the manufacturer’s recommendation, but between 1 min and 60 min (only contact times of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min are allowed in this range) Interfering substance Clean conditions: 0,3 g/l bovine albumin solution or Dirty conditions: 3,0 g/l bovine albumin solution Additional interfering substance for dairies 10,0 g / l of reconstituted milk Other additional strains and additional test conditions may be tested according to product claim. 5 Test method 5.1 Principle A test suspension of bacterial spores in a solution of interfering substance, simulating clean and/or dirty conditions, is added to a prepared sample of the product under test diluted in hard water (in water for ready-to-use products). The mixture is maintained at specific test temperature ± 1 °C for the specific test contact (time ± 10) s (required test conditions). In case the contact time is 1 min, the tolerance allowed shall be ± 5 s At this contact time, an aliquot is taken; the sporicidal action 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 number of surviving bacterial spores in each sample are determined and the reduction in viable counts is calculated. SIST EN 13704:2018

EN 13704:2018 (E) 9 5.2 Materials and reagents 5.2.1 Test organisms The sporicidal activity shall be evaluated by using spores of the following strain : — Bacillus subtilis ATCC 6633 1). If required for specific applications or products, additional strains may be chosen from, for example : — Bacillus cereus CIP 105151; — Clostridium sporogenes ATCC 19404, CIP 79.3 1). NOTE 1 See Annex F for corresponding strain numbers in some other culture collections. NOTE 2 See Annex C for particular culture and handling conditions for Clostridium sporogenes. NOTE 3

It has been noted that different sources of Bacillus cereus strain can lead to different sporulation behaviour, in particular CIP 105151 strain seems to sporulate better. If additional strains are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere) and noted in the test report. If the additional strains selected do not correspond to the specified strains, their suitability for supplying inocula of sufficient concentration shall be verified. If the additional strains tested 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 under a reference for 5 years. 5.2.2 Culture media and reagents 5.2.2.1 General The reagents shall be of analytical grade and/or appropriate for microbiological purposes. 5.2.2.2 Water The water shall be free from substances that are toxic or inhibiting to the bacterial spores or to the bacteria. It shall be freshly glass distilled water and not demineralized water. Sterilize in the autoclave (5.3.2.1). Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilized. If distilled water of adequate quality is not available, water for injectable preparation can be used. See 5.2.2.6 for preparation of hard water. 5.2.2.3 Tryptone Soja Agar (TSA) For counting of viable Bacillus spores : Tryptone, pancreatic digest of casein 15,0 g Soya peptone, papaic digest of Soybean meal 5,0 g Sodium Chloride (NaCl) 5,0 g

1) ATCC 6633 and ATCC 19404 are the collection numbers of strains supplied by the American Type Culture Collections. CIP 79.3 is the collection number of spores supplied by the Collection de l'Institut Pasteur. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named. Corresponding strains supplied by other culture collections may be used if they can be shown to lead to the same results. SIST EN 13704:2018

EN 13704:2018 (E) 10 Agar 15,0 g Water (see 5.2.2.2) 1 000,0 ml Sterilize in the autoclave (5.3.2.1). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 measured at (20 ± 1) °C 5.2.2.4 Neutralizer The neutralizer shall be validated for the product under test in accordance with Annex D. 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 D. 5.2.2.5 Rinsing liquid (for membrane filtration) The liquid shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane under the test conditions described in Annex B. NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in Annex D. 5.2.2.6 Hard water for dilution of products 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 1000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave (5.3.2.1 a)). Autoclaving – if used - may cause a loss of liquid. In this case make up to 1000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator at 5 °C ± 3 °C (according 5.3.2.15) for no longer than four weeks; — prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator at 5 °C ± 3 °C (according 5.3.2.15) for no longer than one week; — place 600 ml to 700 ml of water (5.2.2.2) in a 1000 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 1000 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 expressed as calcium carbonate (CaCO3) is in the test tube lower than 375 mg/l. SIST EN 13704:2018

EN 13704:2018 (E) 11 5.2.2.7 Interfering substance 5.2.2.7.1 General The interfering substance shall be sterile and prepared at 10 times its final concentration in the test. 5.2.2.7.2 Clean conditions Bovine albumin solution for the test conditions shall be prepared as follows: — dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (see 5.2.2.2) ; — sterilize by membrane filtration (5.3.2.7). The final concentration of the bovine albumin in the test procedure (5.5.2) is 0,3 g/l. 5.2.2.7.3 Dirty conditions Dissolve 3,00 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). The final concentration of bovine albumin in the test procedure (5.5.2) shall be 3,0 g/l. 5.2.2.7.4 Additional interfering substance for 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 (5.5.1,c)) is 10,0 g/l of reconstituted milk. 5.3 Apparatus and glassware 5.3.1 General Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods : a) in the autoclave (see 5.3.2.1); b) in the dry heat sterilizer (see 5.3.2.1). 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 (+3

0121) °C for a minimum holding time of 15 min ; b) for dry heat sterilization, a hot air oven capable of being maintained at 180 °C for a minimum holding time of 30 min, at 170 °C for a minimum holding time of 1 h, or at 160 °C a minimum holding time of 2 h.

2) Disposable equipment is an acceptable alternative to reusable glassware. SIST EN 13704:2018

EN 13704:2018 (E) 12 5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, (45 ± 1) °C, (75 ± 1) °C and at test temperatures ± 1 °C (see 5.5.1). 5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C and (36 ± 1) °C or (37 ± 1) °C. 5.3.2.4 pH-meter, having an accuracy of calibration of ± 0,1 pH units at 20 °C or equivalent. 5.3.2.5 Stopwatch 5.3.2.6 Vortex mixer (electromechanical agitator, i.e. Vortex® mixer 3)) 5.3.2.7 Membrane filtration apparatus (if this method is used), constructed of a material compatible with the product under test, with a filter holder which shall have a usable volume 50 ml minimum, and suitable for use with filters of diameter 47 mm to 50 mm, of 0,45 µm pore size. The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the microorganisms over the membrane and in order 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 Containers : Test tubes or flasks of suitable capacity. 5.3.2.9 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic pipettes may be used. 5.3.2.10 Petri dishes of size 90 mm to 100 mm. 5.3.2.11 Glass beads (Diameter : 3 mm to 4 mm). 5.3.2.12 Volumetric flasks. 5.3.2.13 Glass Roux bottles with straight neck. 5.3.2.14 Microscope, preferably, a phase-contrast type, with magnification of at least x 400. 5.3.2.15

Fridge, capable of being controlled at (5 ± 3) °C. 5.3.2.16 Jars for anaerobiosis with oxygen removal system or any other system suitable for generating anaerobiosis. 5.3.2.17 Centrifuge capable of 10 000 g acceleration. 5.4 Preparation of spore test suspension and test solutions 5.4.1 Spore suspensions 5.4.1.1 Stock spore suspension of test organism The Bacillus subtilis ATCC 6633 CIP 52.62 spore stock suspension shall be prepared according to Annex A. Check the viability and the susceptibility of each spore batch after at least 4 weeks storage at 2°C to 8°C for Bacillus spores. Check the viability and the susceptibility of each spore batch after at least 12 month storage at 2°C to 8°C for Bacillus spores. The Bacillus spore suspensions may be st

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