SIST EN 13704:2018
(Main)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)
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.
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Standards Content (Sample)
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
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
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
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
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
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
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
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 stored for a maximum of 5 years if periodically checked. Use glutaraldehyde and peracetic acid at set concentrations. Perform the tests
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 13704:2018
· 3 lg. — 0,001 % (v/v) – 30 min: Peracetic acid solution should achieve a lg reduction of < 3 lg. — 0,05 % (v/v) – 30 min: Peracetic acid solution should achieve a lg reduction of
· 3 lg With Bacillus cereus without interfering substance: — 0,5 % (v/v) – 15 min: Glutaraldehyde solution should achieve a lg reduction of < 3 lg — 3,0 % (v/v) – 15 min: Glutaraldehyde solution should achieve a lg reduction of
· 3 lg. — 0,05 % (v/v) –30 min: Peracetic acid solution should achieve a lg reduction of < 3 lg. — 0,50 % (v/v) – 30 min: Peracetic acid solution should achieve a lg reduction of
· 3 lg Glutaraldehyde 50 % shall be used with pH between 3,1 and 4,5. For the validation a specific Standard-Biocide should be used. Appropriate substances are e.g.: Glutaraldehyd – Product name: BIOBANTM GA 50 Antimicrobial4, DOW Chemical Company Ltd, Diamond House, Lotus Park, Kingsbury Crascent, TW18 3 AG Staines, Middlesex, United Kingdom. Peracetic acid – Product name: PES 5/25 (mixture of 5 % peracetic acid and 25 % hydrogen peroxide), Stockmeier Chemie Eilenburg GmbH and Co. KG, Gustav-Adolf-Ring 5, D-04838 Eilenburg. Stored at 2 °C to 8 °C. For the preparation of the stock spore suspensions of additional strains (see 5.2.1) refer to : — Annex A for Bacillus cereus; CIP 105.151 — Annex C for Clostridium sporogenes ATCC 19404, CIP 79.3. For Clostridium sporogenes no susceptibility check shall be done as there are no data available in order to define a susceptibility range. The spore suspension may be stored for a maximum of 12 months. 5.4.1.2 Spore test suspension To prepare the spore test suspension, dilute the spore stock suspension (see 5.4.1.1) with water (see 5.2.2.2). The number of spores in the test suspension shall be adjusted to 1,5 × 106 to 5 × 106 cfu/ml, estimating the number of units by any suitable mean. Maintain the suspension test in the water bath at (20 ± 1) °C and use within 2 h. Microscopic examination under 400 × magnification shall be carried out immediately after the preparation of the spore test suspension and just before the test, to show the absence of vegetative cells and germinative spores. If there is any evidence of spore germination, the suspension shall be discarded.
4 to be obtained through: DOW Chemical Company Ltd, Diamond House SIST EN 13704:2018
5) cfu/ml = Colony forming unit per ml. SIST EN 13704:2018
(in °C): The temperatures to be tested are specified in Clause 4, Table 1. The allowed deviation for each chosen temperature is ± 1 °C. b) contact time t (in min): The contact times to be tested are specified in Clause 4, Table 1. The allowed deviation for each chosen contact time is ± 10 s (±5 s when the contact time is 1 min). c)
interfering substance: The interfering substance to be tested is 0,30 g/l bovine albumin (5.2.2.7.2) under clean conditions or 3,0 g/l bovine albumin (5.2.2.7.3) under dirty conditions – according to Clause 4, Table 1 and practical applications. Additional interfering substance may be tested according to the specific intended uses of the product. The product shall not cause the formation of any precipitate in the experimental conditions used. Each selected experimental condition (, t, strains) shall be validated in accordance with Annex B. The longest contact time and the highest concentration shall be validated. 5.5.2 Test procedure for assessing the sporicidal effect of the product 5.5.2.1 General The method of choice is the dilution-neutralization method. To determine a suitable neutralizer the following procedure shall be adopted. Carry out the validation of the dilution neutralization method (B.4.1) using a suitable neutralizer, chosen according to laboratory experience and published data. If this neutralizer is unsuitable, repeat the validation test with another neutralizer taking into account the information given in Annex D. If neither of the two neutralizers is considered valid, the membrane filtration method (5.5.2.3) may be used. The inactivation of the sporicidal activity of the product shall be validated for each of the tested strains and for each of the chosen experimental conditions (see 5.5.1). 5.5.2.2 Dilution-neutralization method 5.5.2.2.1 General a) if the test temperature is lower or equal to 40 °C ± 1 °C: Prior to testing, equilibrate all reagents (product test solutions, spore test suspension, interfering substance) to the test temperature of
°C ± 1 °C using the water bath (see 5.3.2.2) controlled at
°C ± 1 °C. Check that the temperature of the reagents is stabilized at
°C ± 1 °C. The neutralizer and water (see 5.2.2.2) shall be equilibrated at a temperature of 20 °C ± 1 °C. SIST EN 13704:2018
°C ± 1 °C using the water bath (see 5.3.2.2) controlled at
°C ± 1 °C. Check that the temperature of the reagents is stabilized at
°C ± 1 °C. The neutralizer, the spore test suspension, the interfering substance and the water (see 5.2.2.2) shall be equilibrated at a temperature of 20 °C ± 1 °C. 5.5.2.2.2 Test procedure for sporicidal activity of products Pipette 1,0 ml of interfering substance (see 5.2.2.7) into a test tube. Add 1,0 ml of the spore test suspension containing 1,5 x 106 to 5 x 106 cfu/ml 6) (see 5.4.1.2). Start the stopwatch immediately, mix (see 5.3.2.6) and place the test tube in the water bath at
°C ± 1 °C for 2 min ± 10 s. At the end of the contact time, add 8,0 ml of each of the product test solutions. Restart the stopwatch immediately, mix (see 5.3.2.6) and place the test tube in a water bath controlled at
°C ± 1°C for the appropriate contact time (t ± 10) s (±5 s when the contact time is 1 min). When adding spore suspension, care should be taken to avoid touching the upper part of the test tube sides. Just before the end of the contact time, mix (see 5.3.2.6). At the end of the contact time pipette 1,0 ml of the test mixture into a tube containing 8,0 ml neutralizer (see 5.2.2.4) and 1,0 ml water (see 5.2.2.2). Mix (see 5.3.2.6) and place in a water bath controlled at (20 ± 1) °C. After a neutralization time of 5 min ± 10 s, immediately take a sample of 1,0 ml of neutralized mixture (neutralizer, product test solution, interfering substance, spore test suspension) in duplicate and transfer each 1,0 ml sample into separate Petri dishes (see 5.3.2.10) and quickly add 12 ml to 15 ml melted TSA (see 5.2.2.3), cooled to (45 ± 1) °C. 5.5.2.2.3 Incubation and counting of the test mixture Incubate the Petri dishes at (30 ± 1) °C (see 5.3.2.3) for 3 days. Determine the highest number of colonies Vc for each plate. Calculate the number of cfu/ml in the test mixture (Na) using the method given in 5.6.2. For calculating the viable count of the test mixture, the dilution factor is 1:10. The text mixture Na and two 1:10 dilutions are performed and counted. 5.5.2.3 Membrane filtration method 5.5.2.3.1 General Prior to testing, equilibrate all reagents (product test solutions, spore test suspension, interfering substance) to the test temperature of ( ± 1) °C using the water bath (see 5.3.2.2) controlled at ( ± 1) °C. Check that the temperature of the reagents is stabilized at ( ± 1) °C. The rinsing liquid (see 5.2.2.5) and water (5.2.2.2) shall be equilibrated at a temperature of (20 ± 1) °C. a) if the test temperature is lower or equal to 40 °C ± 1 °C: Prior to testing, equilibrate all reagents (product test solutions, spore test suspension, interfering substance) to the test temperature of ( ± 1) °C using the water bath (see 5.3.2.2) controlled at ( ± 1) °C. Check that the temperature of the reagents is stabilized at ( ± 1) °C. The rinsing liquid (see 5.2.2.5) and water (5.2.2.2) shall be equilibrated at a temperature of (20 ± 1) °C. b) if the test temperature is higher than (40 ± 1) °C:
6) cfu/ml = Colony forming unit per ml. SIST EN 13704:2018
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