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

This European Standard specifies a test method and the minimum requirements for bactericidal 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, 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.
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 bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1); Deutsche und Englische Fassung prEN 1276:2016

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die bakterizide Wirkung von chemischen Desinfektionsprodukten und antiseptischen Produkten fest, die bei Verdünnung in Wasser standardisierter Härte oder – im Falle gebrauchsfertiger Produkte – in Wasser als homogenes und physikalisch stabiles Präparat vorliegen. Die Produkte können nur bei einer Konzentration von höchstens 80 % geprüft werden, da durch die Zugabe der Prüforganismen und der Belastungssubstanz immer eine bestimmte Verdünnung auftritt.
Dieses Dokument gilt für Produkte, die im Lebensmittelbereich, in der Industrie, im Haushalt und in öffentlichen Einrichtungen genutzt werden; ausgenommen sind Bereiche und Situationen, in denen die Desinfektion medizinisch angezeigt ist, und Produkte, die auf lebenden Geweben verwendet werden, hier wiederum außer denen, die zur Handhygiene in den vorstehend genannten Bereichen verwendet werden. Es werden mindestens folgende Bereiche abgedeckt:
a)   Verarbeitung, Vertrieb und Verkauf von:
1)   Lebensmitteln tierischer Herkunft:
-   Milch und Milcherzeugnisse;
-   Fleisch und Fleischprodukte;
-   Fisch, Meerestiere und zugehörige Erzeugnisse;
-   Eier und Eierprodukte;
-   Tiernahrung;
-   usw.
2)   Lebensmitteln pflanzlicher Herkunft:
-   Getränke;
-   Obst, Gemüse und zugehörige Erzeugnisse (einschließlich Zucker, Destillationsprodukte usw.);
-   Mehl, gemahlene und gebackene Produkte;
-   Tiernahrung;
-   usw.
b)   öffentliche Einrichtungen und Haushalt:
-   Verpflegungseinrichtungen;
-   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:
-   Verpackungsmaterial;
-   Biotechnologie (Hefe, Proteine, Enzyme usw.);
-   Pharmazeutika;
-   Kosmetika und Toilettenartikel;
-   Textilien;
-   Luft- und Raumfahrtindustrie, Computerindustrie;
-   usw.
EN 14885 legt im Einzelnen den Zusammenhang zwischen den verschiedenen Prüfungen sowie zu den „Anwendungsempfehlungen“ fest.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l’évaluation de l’activité bactéricide 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)

La présente Norme européenne spécifie une méthode d’essai et les exigences minimales relatives à
l’activité bactéricide des produits antiseptiques et désinfectants chimiques qui forment une préparation
homogène, physiquement stable, lorsqu’ils sont dilués dans de l’eau dure ou ‐ dans le cas de produits
prêts à l’emploi ‐ dans l’eau. Les produits ne peuvent être soumis à l’essai qu’à une concentration
inférieure ou égale à 80 %, car l’addition des microorganismes d’essai et de la substance interférente
s’accompagne nécessairement d’une dilution.
Le présent document s’applique aux produits utilisés dans le domaine de l’agroalimentaire, dans
l’industrie, dans les domaines domestiques et les collectivités, à l’exclusion des domaines et des situations
où la désinfection est médicalement prescrite et à l’exclusion des produits utilisés sur les tissus vivants
(excepté ceux destinés à l’hygiène des mains dans les domaines indiqués ci‐dessus). Les domaines
concernés sont notamment :
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 ;
 alimentation animale ;
 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 ;
 alimentation animale ;
 etc. ;
b) les domaines domestiques et les collectivités :
 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. ;
prEN1276:2018 (F)
7
c) les autres secteurs industriels :
 matériaux d’emballage ;
 biotechnologie (levures, protéines, enzymes, etc.) ;
 industrie pharmaceutique ;
 cosmétiques et produits d’hygiène corporelle ;
 textiles ;
 industrie spatiale, secteur informatique ;
 etc.
L’EN 14885 spécifie de façon détaillée larelation entre les différents essais et les « recommandations
d’utilisation ».
NOTE 1 La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives
dans les conditions dans lesquelles elles sont utilisées.
NOTE 2 Cette méthode correspond à un essai de phase 2, étape 1.

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

Ta evropski standard določa preskusno metodo in minimalne zahteve za baktericidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma z vodo pri izdelkih, ki so pripravljeni za uporabo. Izdelke je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji, ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje.
Ta dokument se uporablja 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 itd.);
– 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 itd.);
– hoteli;
– bivališča;
– klinično neobčutljivi prostori bolnišnic;
– pisarne;
– itn.
c) druga industrijska področja:
– embalaža;
– biotehnologija (kvasovke, proteini, encimi itd.);
– 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
Published
Public Enquiry End Date
02-Jul-2018
Publication Date
08-Oct-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
13-Sep-2019
Due Date
18-Nov-2019
Completion Date
09-Oct-2019

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SLOVENSKI STANDARD
SIST EN 1276:2019
01-november-2019
Nadomešča:
SIST EN 1276:2010
SIST EN 1276:2010/AC:2010
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za

vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v živilski in

drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in
zahteve (faza 2, stopnja 1)

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation

of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial,

domestic and institutional areas - Test method and requirements (phase 2, step 1)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur

Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in

den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen -

Prüfverfahren und Anforderungen (Phase 2, Stufe 1); Deutsche und Englische Fassung

prEN 1276:2016
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour

lévaluation de lactivité bactéricide des antiseptiques et des désinfectants chimiques

utilisés dans le domaine de lagro-alimentaire, dans lindustrie, dans les domaines

domestiques et en collectivité - Méthode dessai et prescriptions (phase 2, étape 1)

Ta slovenski standard je istoveten z: EN 1276:2019
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
SIST EN 1276:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN 1276:2019
---------------------- Page: 2 ----------------------
SIST EN 1276:2019
EN 1276
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2019
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 1276:2009
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of bactericidal 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 Chemische Desinfektionsmittel und Antiseptika -

quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der

bactéricide des antiseptiques et des désinfectants bakteriziden Wirkung chemischer Desinfektionsmittel

chimiques utilisés dans le domaine de l'agro- und Antiseptika in den Bereichen Lebensmittel,

alimentaire, dans l'industrie, dans les domaines Industrie, Haushalt und öffentliche Einrichtungen -

domestiques et en collectivité - Méthode d'essai et Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

prescriptions (phase 2, étape 1)
This European Standard was approved by CEN on 17 June 2019.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 1276:2019 E

worldwide for CEN national Members.
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SIST EN 1276:2019
EN 1276:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Requirements ................................................................................................................................................... 7

5 Test method ...................................................................................................................................................... 8

5.1 Principle ............................................................................................................................................................. 8

5.2 Materials and reagents .................................................................................................................................. 9

5.3 Apparatus and glassware .......................................................................................................................... 12

5.4 Preparation of test organism suspensions and product test solutions .................................... 13

5.5 Procedure for assessing the bactericidal activity of the product ............................................... 15

5.6 Experimental data and calculation ........................................................................................................ 21

5.7 Verification of methodology ..................................................................................................................... 24

5.8 Expression of results and precision ...................................................................................................... 25

5.9 Interpretation of results - conclusion ................................................................................................... 25

5.10 Test report ...................................................................................................................................................... 26

Annex A (informative) Referenced strains in national collections ......................................................... 28

Annex B (informative) Neutralizers and rinsing liquids............................................................................. 29

Annex C (informative) Graphical representations of dilution neutralization method and

membrane filtration method ................................................................................................................... 31

Annex D (informative) Example of a typical test report ............................................................................. 35

Annex E (informative) Precision of the test result ........................................................................................ 39

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SIST EN 1276:2019
EN 1276:2019 (E)
European foreword

This document (EN 1276:2019) has been prepared by Technical Committee CEN/TC 216 “Chemical

disinfectants and antiseptics”, the secretariat of which is held by AFNOR.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by February 2020 and conflicting national standards shall

be withdrawn at the latest by February 2020.

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 1276:2009.
Data obtained by using the latest version of EN 1276 are still valid.
The main changes in relation to EN 1276:2009 are:

— handrub and handwash test conditions and test requirements have been harmonized with

EN 13727;

— interfering substance for breweries, soft drinks, cosmetics and cleaning in place have been deleted.

A sentence to allow additional interfering substance for specific applications has been added;

— the obligatory conditions (temperature and contact time) have been deleted. The text has been

harmonized with EN 13727 keeping specified time intervals and temperature steps;
— test conditions for temperatures ≥ 40 °C have been added.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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SIST EN 1276:2019
EN 1276:2019 (E)
Introduction

This document describes a suspension test for establishing whether a chemical disinfectant or

antiseptic has or does not have bactericidal 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 test conditions. However, for some applications, the recommendations of use of

a product can differ and therefore additional test conditions need to be used.
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SIST EN 1276:2019
EN 1276:2019 (E)
1 Scope

This document specifies a test method and the minimum requirements for bactericidal 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, 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.);
---------------------- Page: 7 ----------------------
SIST EN 1276:2019
EN 1276:2019 (E)
— 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.

EN 14885 specifies in detail the relationship of the various tests to one another and to “use

recommendations”.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal

(including bacteriophages) activity

EN 14885:2018, 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 14885:2018 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
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SIST EN 1276:2019
EN 1276:2019 (E)
4 Requirements

The product shall demonstrate at least a 5 decimal logarithm (lg) reduction (3 lg for handwashes) 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 suitable test conditions as described in 5.5.1.1, Tables 1 and 2 here

below.
Table 1 — Test conditions for general purpose disinfection
Test Conditions Bactericidal activity
Enterococcus hirae
Escherichia coli
Test organism
(see 5.2.1)
Pseudomonas aeruginosa
obligatory
Staphylococcus aureus
E. faecium (for temperatures ≥ 40 °C)
Example of Salmonella Typhimurium
additional test Lactobacillus brevis
microorganisms Enterobacter cloacae
Test temperature
in a range from 4 °C to 60 °C
Contact time
in a range from 1 min to 60 min (from 1 min to 5 min at intervals of 1
min and from 5 min to 60 min at intervals of 5 min)
0,3 g/l Bovine Albumin for Staphylococcus aureus, Enterococcus hirae,
Escherichia coli and
Clean conditions
Pseudomonas aeruginosa
3,0 g/l Bovine Albumin for Staphylococcus aureus, Enterococcus hirae,
Dirty conditions
Pseudomonas aeruginosa and Escherichia coli
additional any relevant substance
Log reduction (decimal lg) ≥ 5 lg

The recommended contact time for the use of the product is within the responsibility of the

manufacturer.
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SIST EN 1276:2019
EN 1276:2019 (E)
Table 2 — Test conditions for hand hygiene
Test Conditions Bactericidal activity
Enterococcus hirae
Test organisms
Escherichia coli K12 (NCTC 10538)
(see 5.2.1)
Pseudomonas aeruginosa
obligatory
Staphylococcus aureus
Test temperature
20 °C
Contact time
30 s or 60 s
clean conditions
0,3 g/l Bovine Albumin
(for hygienic handrubs)
Dirty conditions
3,0 g/l Bovine Albumin
(for hygienic handwashes)
≥ 5 lg for handrubs
Log reduction (decimal lg)
≥ 3 lg for handwashes

Where indicated, additional specific bactericidal activity shall be determined applying other 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.
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 with the exception of handwash products whose first dilution is done in hard water (5.4.2)) is

added to a test suspension of bacteria in a solution of an interfering substance. The mixture is

maintained at the chosen test temperature for the adopted contact time. At the end of this contact time,

an aliquot is taken, and the bactericidal and/or the bacteriostatic 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 bacteria in

each sample are determined and the reduction is calculated.

5.1.2 The test is performed using Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus

and Enterococcus hirae as test organisms. For temperatures ≥ 40 °C only Enterococcus faecium shall be

used.
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SIST EN 1276:2019
EN 1276:2019 (E)

For testing of hand hygiene products, Pseudomonas aeruginosa, Escherichia coli K12, Staphylococcus

aureus and Enterococcus hirae are used as test organisms.
5.1.3 Additional test organisms can be used.
5.2 Materials and reagents
5.2.1 Test organisms

The bactericidal activity shall be evaluated using the following strains as test organisms:

— Pseudomonas aeruginosa ATCC 15442;
— Escherichia coli ATCC 10536;
— Staphylococcus aureus ATCC 6538;
— Enterococcus hirae ATCC 10541;
— Escherichia coli K12 NCTC 10538;
— Enterococcus faecium ATCC 6057.

If required for specific applications, additional strains may be chosen, for example from:

— Salmonella Typhimurium ATCC 13311;
— Lactobacillus brevis DSM 6235;
— Enterobacter cloacae DSM 6234.
Refer to Annex A for strain references in some other culture collections.

The required temperature for growing these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3). The

same temperature (either 36 °C or 37 °C) shall be used for all incubations for growing microorganisms

performed during a test and its control and validation.

If additional test organisms are used, they shall be incubated under optimum growth conditions

(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms

selected do not correspond to the specified strains, their suitability for supplying the required inocula

shall be verified. If these additional test organisms are not classified at a reference centre, their

identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or

national culture collection under a reference for five years.
5.2.2 Culture media and reagents
5.2.2.1 General

All weights of chemical substances given in this European Standard refer to the anhydrous salts.

Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for

consequent molecular weight differences.
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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.

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 are to be rigorously followed.
NOTE For each culture medium and reagent, a limitation for use is to be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave (see 5.3.2.1 a).

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 can be used.

See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Tryptone Soya Agar (TSA)
Tryptone soya agar, consisting of:
Tryptone, pancreatic digest of casein 15,0 g
Soya peptone, papaic digest of soybean meal 5,0 g
Sodium chloride (NaCl) 5,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave (5.3.2.1 a). After sterilization the pH of the medium shall be equivalent to

7,2 ± 0,2 when measured at (20 ± 1) °C.

NOTE In the case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it can be necessary to

add neutralizer to the TSA. Annex B gives guidance on the neutralizers that can 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 (5.3.2.1 a). 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.
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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 (MgCl ) and 46,24 g calcium chloride

(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the

autoclave (5.3.2.1 a). 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 (NaHCO ) 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.

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

375 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.

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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, etc.)

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 10,0 % (v/v) in water (5.2.2.2) by adding 10 parts of reconstituted milk to 90

parts of water. 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 1,0 % (v/v) 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) by moist heat, in the autoclave (5.3.2.1 a);
b) by dry heat, in the hot air oven (5.3.2.1 b).

5.3.2 Usual microbiological laboratory equipment and, in particular, the following

5.3.2.1 Apparatus for sterilization:

a) for moist heat sterilization, an autoclave capable of being maintained at for a minimum

()121 °C
holding time of 15 min;

b) for dry heat sterilization, a hot air oven capable of being maintained at ()180 °C for a minimum

+5 +5

holding time of 30 min, at ()170 °C for a minimum holding time of 1 h or at for a

()160 °C
0 0
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

TSA 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 either at (36 ± 1) °C or (37 ± 1) °C (5.2.1).

5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.

NOTE A puncture electrode or a flat membrane electrode are to be used for measuring the pH of the agar

media (5.2.2.3).

1) Disposable sterile equipment is an acceptable alternative to reusable glassware.

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5.3.2.5 Stopwatch
5.3.2.6 Shaker
® 2)
a) Electromechanical agitator, e.g. Vortex mixer .
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 diameter 47 mm to 50 mm and 0,45 μm pore size for sterilization of hard water (5.2.2.7), bovine

albumin (5.2.2.8.2 and 5.2.2.8.3), 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 microorganisms over the membrane and to prevent overlong filtration, the device shall be set so

as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.

5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml, or calibrated

automatic pipettes.
5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads, 3 mm to 4 mm in diameter.
5.3.2.12 Volumetric flasks
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 shall 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

In order to prepare the working culture of the test organisms (5.2.1), prepare a subculture from the

stock culture (5.4.1.2) by streaking onto TSA slopes (5.2.2.3) or plates (5.3.2.10) and incubate (5.3.2.3).

After 18 h to 24 h prepare a second subculture from the first subculture in the same way and incubate

for 18 h to 24 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.

2) 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.

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If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used

for subsequent subculturing, provided
...

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