EN 13697:2023

Chemical disinfectants and antiseptics - Quantitative non-porous surface test for the evaluation of bactericidal and yeasticidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas without mechanical action - Test method and requirements without mechanical action (phase 2, step 2)

This document specifies a test method (phase 2/step 2) and the minimum requirements for bactericidal and yeasticidal and/or fungicidal activity of chemical disinfectants that form a homogeneous physically stable preparation in hard water or – in the case of ready-to-use products – with water in food, industrial, domestic and institutional areas, excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues.
This document applies to products that are used for disinfecting non-porous surfaces without mechanical action.
This document is applicable at least to the following:
a)   processing, distribution and retailing of:
1)   food of animal origin:
i)   milk and milk products;
ii)   meat and meat products;
iii)   fish, seafood and products;
iv)   eggs and egg products;
v)   animal feeds;
vi)   etc.
2)   food of vegetable origin:
i)   beverages;
ii)   fruits, vegetables and derivatives (including distilleries and sugar refineries);
iii)   flour, milling and baking;
iv)   animal feeds;
v)   etc.
b)   institutional and domestic areas:
1)   catering establishments;
2)   public areas;
3)   public transports;
4)   schools;
5)   nurseries;
6)   shops;
7)   sports rooms;
8)   waste containers (bins);
9)   hotels;
10)   dwellings;
11)   clinically non sensitive areas of hospitals;
12)   offices;
13)   etc.
c)   other industrial areas:
1)   packaging material;
2)   biotechnology (yeast, proteins, enzymes...);
3)   pharmaceutical;
4)   cosmetics and toiletries;
5)   textiles;
6)   space industry, computer industry;
7)   etc.
This document does not apply when the product is applied via an automatic airborne disinfection method; in such cases, see EN 17272.
Using this document, it is possible to determine the bactericidal and/or yeasticidal and/or fungicidal activity of the undiluted product. As three concentrations are tested, in the active to non-active range, dilution of the product is required and, therefore, the product forms a homogeneous stable preparation in hard water.
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 on bacteria and/or fungi in the conditions in which they are used.
NOTE 2   This method cannot be used to evaluate the activity of products against mycobacteria.

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächen-Versuch zur Bestimmung der bakteriziden und levuroziden und/oder fungiziden Wirkung chemischer Desinfektionsmittel auf nicht porösen Oberflächen in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen ohne mechanische Behandlung - Prüfverfahren und Anforderungen ohne mechanische Behandlung (Phase 2, Stufe 2)

Dieses Dokument legt ein Prüfverfahren (Phase 2, Stufe 2) und die Mindestanforderungen an die bakterizide und levurozide und/oder fungizide Wirkung von chemischen Desinfektionsmitteln in den Bereichen Lebensmittel, Industrie, Haushalt und in öffentlichen Einrichtungen fest, die mit Wasser standardisierter Härte oder – im Fall von gebrauchsfertigen Produkten – mit Wasser eine homogene, physikalisch stabile Zubereitung ergeben; ausgenommen davon sind Bereiche und Situationen mit medizinisch indizierter Desinfektion und Produkte, die auf lebenden Geweben benutzt werden.
Dieses Dokument ist anwendbar für Produkte, die zur Desinfektion auf nicht porösen Oberflächen ohne mechanische Behandlung verwendet werden.
Dieses Dokument ist mindestens anwendbar für:
a)   Verarbeitung, Vertrieb und Verkauf von:
1)   Lebensmitteln tierischer Herkunft:
i)   Milch und Milchprodukte;
ii)   Fleisch und Fleischprodukte;
iii)   Fisch, Meerestiere und daraus hergestellte Erzeugnisse;
iv)   Eier und Eiprodukte;
v)   Tiernahrung;
vi)   usw.
2)   Lebensmitteln pflanzlicher Herkunft:
i)   Getränke;
ii)   Früchte, Gemüse und daraus hergestellte Erzeugnisse (einschließlich Brennereien und Zuckerraffinerien);
iii)   Mehl, gemahlene und gebackene Produkte;
iv)   Tiernahrung;
v)   usw.
b)   gewerbliche Einrichtungen und Haushaltsbereiche:
1)   Verpflegungseinrichtungen;
2)   öffentliche Bereiche;
3)   öffentliche Verkehrsmittel;
4)   Schulen;
5)   Kindergärten;
6)   Geschäfte;
7)   Sportstätten;
8)   Abfallbehälter (Mülltonnen);
9)   Hotels;
10)   Wohngebäude;
11)   klinisch nicht relevante Bereiche von Krankenhäusern;
12)   Büroräume;
13)   usw.
c)   andere industrielle Bereiche:
1)   Verpackungsmaterial;
2)   Biotechnologie (Hefe, Proteine, Enzyme usw.);
3)   Pharmazeutika;
4)   Kosmetika und Toilettenartikel;
5)   Textilien;
6)   Raumfahrtindustrie und Computerindustrie;
7)   usw.
Dieses Dokument ist nicht anwendbar, wenn das Produkt über ein automatisches Verfahren zur luftübertragenen Desinfektion angewendet wird; siehe in solchen Fällen EN 17272.
Bei Anwendung dieses Dokuments ist die Bestimmung der bakteriziden und levuroziden und/oder fungiziden Wirkung des unverdünnten Produkts möglich. Da vom aktiven bis zum nicht aktiven Bereich drei Konzentrationen geprüft werden, bedarf es einer Verdünnung des Produkts, deshalb bildet das Produkt eine homogene stabile Zubereitung mit Wasser standardisierter Härte.
In EN 14885 ist die Beziehung zwischen den einzelnen Prüfungen untereinander und zu den Anwendungsempfehlungen detailliert festgelegt.
ANMERKUNG 1   Das beschriebene Verfahren ist dazu vorgesehen, die Wirkung gewerblich hergestellter Zubereitungen oder anderer aktiver Substanzen auf Bakterien und/oder Pilze unter Anwendungsbedingungen zu bestimmen.
ANMERKUNG 2   Dieses Verfahren kann nicht zur Bestimmung der Wirkung der Produkte gegenüber Mykobakterien angewendet werden.

Antiseptiques et désinfectants chimiques - Essai quantitatif de surface non poreuse pour l'évaluation de l'activité bactéricide, levuricide et/ou fongicide des désinfectants chimiques utilisés sans action mécanique dans le domaine de l'agro alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai sans action mécanique et prescriptions (phase 2, étape 2)

Le présent document spécifie une méthode d’essai (phase 2, étape 2) ainsi que les prescriptions minimales relatives à l’activité bactéricide, levuricide et/ou fongicide des désinfectants chimiques qui forment une préparation homogène, physiquement stable dans l’eau dure ou, dans le cas des produits prêts à l’emploi, ceux qui contiennent de l’eau. Ces désinfectants sont utilisés dans le domaine de l’agro-alimentaire, dans l’industrie, dans les domaines domestiques et en collectivité, à l’exception des domaines et des situations dans lesquels la désinfection est médicalement prescrite et à l’exclusion des produits utilisés sur les tissus vivants.
Le présent document s’applique aux produits utilisés pour la désinfection des surfaces non poreuses sans action mécanique.
Le présent document est applicable, sans y être limité, aux secteurs suivants :
a)   la fabrication, distribution et vente au détail de :
1)   produits alimentaires d’origine animale :
i)   lait et produits laitiers ;
ii)   viande et produits carnés ;
iii)   poisson, fruits de mer et leurs produits dérivés ;
iv)   œufs et produits dérivés ;
v)   alimentation animale ;
vi)   etc. ;
2)   produits alimentaires d’origine végétale :
i)   boissons ;
ii)   fruits, légumes et leurs dérivés (y compris les produits de distillation et de raffinerie du sucre) ;
iii)   farine, produits de minoterie et de boulangerie ;
iv)   alimentation animale ;
v)   etc. ;
b)   les domaines domestiques et en collectivité :
1)   établissements de restauration ;
2)   lieux publics ;
3)   transports publics ;
4)   écoles ;
5)   crèches ;
6)   magasins ;
7)   salles de sport ;
8)   conteneurs à déchets (poubelles) ;
9)   hôtels ;
10)   locaux d’habitation ;
11)   zones cliniquement non sensibles des hôpitaux ;
12)   bureaux ;
13)   etc. ;
c)   les autres secteurs industriels :
1)   industrie des matériaux d’emballage ;
2)   biotechnologie (levure, protéines, enzymes...) ;
3)   industrie pharmaceutique ;
4)   industrie des cosmétiques et produits d’hygiène corporelle ;
5)   industrie textile ;
6)   industrie spatiale, industrie informatique ;
7)   etc.
Le présent document ne s’applique pas lorsqu’une méthode de désinfection des surfaces par voie aérienne est utilisée pour appliquer le produit ; dans un tel cas, voir l’EN 17272.
Il est possible d’utiliser le présent document pour déterminer l’activité bactéricide, levuricide et/ou fongicide du produit non dilué. Trois concentrations, dont au moins une concentration efficace et une inefficace, étant soumises à essai, une dilution du produit est nécessaire. Le produit forme donc une préparation homogène stable dans l’eau dure.
L’EN 14885 spécifie en détail la relation des différents essais entre eux et par rapport aux recommandations d’usage.
NOTE 1   La méthode décrite est destinée à déterminer l’activité des formulations commerciales ou des substances actives vis-à-vis des bactéries et/ou des champignons et levures dans les conditions dans lesquelles elles doivent être utilisées.
NOTE 2   La présente méthode ne peut pas être utilisée pour évaluer l’activité des produits vis-à-vis des mycobactéries.

Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah brez mehanskega delovanja za vrednotenje baktericidnega in/ali fungicidnega delovanja ter delovanja kemičnih razkužil na kvasovke v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in zahteve brez mehanskega delovanja (faza 2, stopnja 2)

Ta dokument določa preskusno metodo (faza 2/stopnja 2) in minimalne zahteve za baktericidno in/ali fungicidno delovanje ali delovanje kemičnih razkužil na kvasovke, ki tvorijo homogen, fizikalno stabilen pripravek v trdi vodi ali – pri izdelkih, ki so pripravljeni za uporabo – se z vodo uporabljajo v živilski in drugih industrijah, gospodinjstvih in javnih ustanovah, pri čemer so izključena področja in primeri, ko obstajajo zdravstvene indikacije za dezinfekcijo, ter izdelki, ki se uporabljajo na živih tkivih.
Področje uporabe tega dokumenta se nanaša vsaj na:
a) predelavo, distribucijo in prodajo:
1) hrane živalskega izvora:
i) mleka in mlečnih izdelkov;
ii) meso in mesne izdelke;
iii) ribe, morske sadeže in izdelke;
iv) jajca in jajčne izdelke;
v) živalsko krmo;
vi) itd.
2) hrano rastlinskega izvora:
i) pijače;
ii) sadje, zelenjavo in derivate (vključno z destilati sladkorja);
iii) moko, mletje in peko;
iv) živalsko krmo;
v) itd.
b) javne ustanove in gospodinjstva:
1) pripravo in dostavo hrane;
2) javne površine;
3) javni prevoz;
4) šole;
5) vrtce;
6) trgovine;
7) športne dvorane;
8) zbiralnike odpadkov (koši);
9) hotele;
10) bivališča;
11) klinično neobčutljive prostore bolnišnic;
12) pisarne;
13) itd.
c) druga industrijska področja:
1) embalažo;
2) biotehnologijo (kvasovke, proteini, encimi ipd.);
3) farmacevtske izdelke;
4) kozmetične in toaletne izdelke;
5) tkanine;
6) vesoljsko in računalniško industrijo;
7) itd.
S tem dokumentom je mogoče določiti baktericidno ali fungicidno delovanje ali delovanje kemičnih razkužil na kvasovke pri nerazredčenem izdelku. Ker so preskušene tri koncentracije od aktivnega do neaktivnega razpona, je potrebno razredčevanje izdelka in zato tvorijo homogen in stabilen pripravek v trdi vodi.
Standard 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 na bakterijah in/ali glivah pod pogoji, v katerih se uporabljajo.
OPOMBA 2: Te metode ni mogoče uporabiti za vrednotenje delovanja izdelkov proti mikobakterijam.

General Information

• Status: Published
• Publication Date: 31-Oct-2023
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: CEN/TC 216 - Chemical disinfectants and antiseptics
• Drafting Committee: CEN/TC 216/WG 3 - Food hygiene and domestic and institutional use
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 01-Nov-2023
• Due Date: 20-Sep-2023
• Completion Date: 01-Nov-2023
• Ref Project: SIST EN 13697:2023 - Chemical disinfectants and antiseptics - Quantitative non-porous surface test for the evaluation of bactericidal and yeasticidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas without mechanical action - Test method and requirements without mechanical action (phase 2, step 2)

Relations

• Replaces: EN 13697:2015+A1:2019 - Chemical disinfectants and antiseptics - Quantitative non-porous surface test for the evaluation of bactericidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas - Test method and requirements without mechanical action (phase 2, step 2)
• Effective Date: 28-Jul-2021

Overview

EN 13697:2023 is a CEN standard specifying a quantitative surface test (phase 2, step 2) for evaluating the bactericidal, yeasticidal and/or fungicidal activity of chemical disinfectants on non-porous surfaces without mechanical action. Intended for products used in food, industrial, domestic and institutional areas, the standard defines test conditions, minimum efficacy requirements and reporting rules for disinfectants that form a homogeneous, physically stable preparation in hard water (or ready-to-use products in water). It excludes medical indications, products for living tissues and airborne disinfection methods.

Key topics and technical requirements

• Test scope: Non‑porous surfaces under realistic laboratory conditions (contact time, temperature range approx. 4 ±1 °C to 40 ±1 °C).
• Minimum performance: ≥ 4 log10 reduction for bacteria and ≥ 3 log10 reduction for fungi/yeasts when tested per the standard.
• Organisms covered: Core bacterial species (e.g., Enterococcus hirae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium) and fungal/yeast species (e.g., Candida albicans, Aspergillus brasiliensis, Saccharomyces cerevisiae). Listeria monocytogenes is included as an additional test organism in the 2023 revision.
• Concentration range: Three concentrations are tested (active → non‑active range); dilution of products is required to determine effective in‑use concentrations.
• Method details & annexes: The standard covers materials, test surfaces, preparation of inocula, neutralizers (Annex B), drying monitoring (Annex F), precision data (Annex E) and calculation/counting rules.
• Limitations: Not suitable for assessing mycobactericidal activity; airborne application methods are covered by EN 17272.

Applications and who uses it

• Disinfectant manufacturers - for efficacy validation and labeling of surface disinfectants for food processing, catering, hospitality, institutional cleaning and industrial uses.
• Independent testing laboratories / QC labs - to perform standardized efficacy testing and comparative assessments.
• Regulatory authorities & procurement specialists - to verify claims and approve products for non‑medical surface disinfection.
• Food processors, hospitality and facility managers - to select products and set contact times/concentrations that meet harmonized European test criteria.

Related standards

• EN 14885 - relationship of tests and use recommendations (application framework).
• EN 17272 - automatic airborne disinfection methods (if applicable).
• EN 12353 and ISO 4793 - referenced for organism preservation and filter specifications.

Keywords: EN 13697:2023, surface test, non-porous surfaces, bactericidal, yeasticidal, fungicidal, chemical disinfectants, phase 2 step 2, CEN standard, disinfectant validation.

Standards Content (Sample)

SLOVENSKI STANDARD
01-december-2023
Nadomešča:
SIST EN 13697:2015+A1:2019
Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah
brez mehanskega delovanja za vrednotenje baktericidnega in/ali fungicidnega
delovanja ter delovanja kemičnih razkužil na kvasovke v živilski in drugih
industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in zahteve brez
mehanskega delovanja (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Quantitative non-porous surface test for the
evaluation of bactericidal and yeasticidal and/or fungicidal activity of chemical
disinfectants used in food, industrial, domestic and institutional areas without mechanical
action - Test method and requirements without mechanical action (phase 2, step 2)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächen-Versuch zur
Bestimmung der bakteriziden und levuroziden und/oder fungiziden Wirkung chemischer
Desinfektionsmittel auf nicht porösen Oberflächen in den Bereichen Lebensmittel,
Industrie, Haushalt und öffentliche Einrichtungen ohne mechanische Behandlung -
Prüfverfahren und Anforderungen ohne mechanische Behandlung (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface non poreuse pour
l'évaluation de l'activité bactéricide, levuricide et/ou fongicide des désinfectants
chimiques utilisés sans action mécanique dans le domaine de l'agro alimentaire, dans
l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai sans
action mécanique et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 13697:2023
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 13697
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2023
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 13697:2015+A1:2019
English Version
Chemical disinfectants and antiseptics - Quantitative non-porous
surface test for the evaluation of bactericidal and yeasticidal
and/or fungicidal activity of chemical disinfectants used in food,
industrial, domestic and institutional areas without mechanical
action - Test method and requirements without mechanical action
(phase 2, step 2)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de surface non poreuse pour l'évaluation de Quantitativer Oberflächen-Versuch zur Bestimmung
l'activité bactéricide, levuricide et/ou fongicide des der bakteriziden und levuroziden und/oder fungiziden
désinfectants chimiques utilisés sans action mécanique Wirkung chemischer Desinfektionsmittel auf nicht
dans le domaine de l'agro alimentaire, dans l'industrie, porösen Oberflächen in den Bereichen Lebensmittel,
dans les domaines domestiques et en collectivité - Industrie, Haushalt und öffentliche Einrichtungen ohne
Méthode d'essai sans action mécanique et mechanische Behandlung - Prüfverfahren und
prescriptions (phase 2, étape 2) Anforderungen ohne mechanische Behandlung (Phase
2, Stufe 2)
This European Standard was approved by CEN on 4 September 2023.

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, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13697:2023 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 8
4 Requirements . 8
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents . 10
5.2.1 Test organisms . 10
5.2.2 Culture media and reagents . 11
5.2.3 Test surface . 14
5.3 Apparatus and glassware . 14
5.4 Preparation of test organism suspensions and product test solutions . 15
5.4.1 Test organism suspensions . 15
5.4.2 Product test solutions . 19
5.5 Procedure. 19
5.5.1 Choice of experimental conditions . 19
5.5.2 Test procedure . 20
5.5.3 Quantifying of the test mixtures . 24
5.6 Calculation and expression of results . 25
5.6.1 Elaboration of data: counting of weighed average values . 25
5.6.2 Verification of methodology . 26
5.6.3 Expression of results . 26
5.6.4 Conclusion. 26
5.7 Test report . 27
Annex A (informative) Corresponding referenced strains . 30
Annex B (informative) Neutralizers . 32
Annex C (informative) Expression of results with the dilution-neutralization method. 34
Annex D (informative) Bactericidal activity in surfaces in general use conditions (for clean
conditions) . 36
Annex E (informative) Precision of the test result . 38
Annex F (informative) Alternative drying end point . 39
Bibliography . 41

European foreword
This document (EN 13697:2023) 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 May 2024, and conflicting national standards shall be
withdrawn at the latest by May 2024.
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 13697:2015+A1:2019.
The main changes compared to the previous version EN 13697:2015+A1:2019 amending edition
EN 13697:2015 are listed below:
— the inoculum under clean conditions for Pseudomonas aeruginosa and Candida albicans has been
reduced to the previous level as in EN 13697:2015;
— a more exact monitoring of the drying process has been included as Annex F (informative) in order
to support achieving sufficient levels of surviving cells for valid results;
— inclusion of yeasticidal activity in the title of the document;
— clarification that 1 % reconstituted milk can be used as sole soiling in dairy industry (no need to test
BSA);
— the designation of variables Nd, Nc, NC and NT have been renamed to Na, A, B and C for harmonization
with other CEN TC 216 standards;
— clarification of the counting procedure in 5.5.3;
— correction of the Formulae (2) and (3) in 5.5.3;
— correction of the calculation error for counts 0/0, resulting now in “lg = 0,7”;
— Listeria monocytogenes has been included as additional test organism;
— several editorial corrections and clarifications have been made.
The following changes in version EN 13697:2015+A1:2019 amending edition EN 13697:2015 were
maintained:
— deletion of obligatory and additional conditions (see Table 1 and 5.5.1);
— update of Bovine albumin and skimmed milk solutions preparations (see 5.2.2.8.2 and 5.2.2.8.3
respectively);
— add of instruction for using vacuum desiccator;
— clarification to the determination of microbicidal concentrations by updating 5.5.2.1 b) and adding
pictures of carriers.
Data obtained from EN 13697:2015 and from EN 13697:2015+A1:2019 are still valid. Data obtained from
EN 13697:2001 are still valid with the exception of Aspergillus brasiliensis (due to modified spore
preparation).
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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, 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, Türkiye and the United
Kingdom.
Introduction
This document describes a surface test method for establishing whether a product proposed as a
disinfectant in the fields described in Clause 1 has or does not have bactericidal and yeasticidal and/or
fungicidal activity on non-porous surfaces.
This document has been revised in order to modify the test parameters under “clean conditions” adopted
for P. aeruginosa and C. albicans; in order to harmonize the designation of test variables N, B, C, A, Na with
the other recent CEN TC 216 standards and to clarify and/or correct details in colony counting and
calculation.
The laboratory test closely simulates practical conditions of application. Chosen conditions (contact time,
temperature, organisms on surfaces.) reflect parameters which are found in practical situations
including conditions which can influence the action of disinfectants. Each use concentration found from
this test corresponds to specified 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.

1 Scope
This document specifies a test method (phase 2/step 2) and the minimum requirements for bactericidal
and yeasticidal and/or fungicidal activity of chemical disinfectants that form a homogeneous physically
stable preparation in hard water or – in the case of ready-to-use products – with water in food, industrial,
domestic and institutional areas, excluding areas and situations where disinfection is medically indicated
and excluding products used on living tissues.
This document applies to products that are used for disinfecting non-porous surfaces without mechanical
action.
This document is applicable at least to the following:
a) processing, distribution and retailing of:
1) food of animal origin:
i) milk and milk products;
ii) meat and meat products;
iii) fish, seafood and products;
iv) eggs and egg products;
v) animal feeds;
vi) etc.
2) food of vegetable origin:
i) beverages;
ii) fruits, vegetables and derivatives (including distilleries and sugar refineries);
iii) flour, milling and baking;
iv) animal feeds;
v) etc.
b) institutional and domestic areas:
1) catering establishments;
2) public areas;
3) public transports;
4) schools;
5) nurseries;
6) shops;
7) sports rooms;
8) waste containers (bins);
9) hotels;
10) dwellings;
11) clinically non sensitive areas of hospitals;
12) offices;
13) etc.
c) other industrial areas:
1) packaging material;
2) biotechnology (yeast, proteins, enzymes.);
3) pharmaceutical;
4) cosmetics and toiletries;
5) textiles;
6) space industry, computer industry;
7) etc.
This document does not apply when the product is applied via an automatic airborne disinfection
method; in such cases, see EN 17272.
Using this document, it is possible to determine the bactericidal and yeasticidal and/or fungicidal activity
of the undiluted product. As three concentrations are tested, in the active to non-active range, dilution of
the product is needed and, therefore, the product forms a homogeneous stable preparation in hard water.
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 on bacteria and/or fungi in the conditions in which they are used.
NOTE 2 This method cannot be used to evaluate the activity of products against mycobacteria.
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, Chemical disinfectants and antiseptics — Application of European Standards for chemical
disinfectants and antiseptics
ISO 4793, Laboratory sintered (fritted) filters — Porosity grading, classification and designation
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online Browsing Platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Requirements
The product shall demonstrate at least a 4 decimal log (lg) reduction for bacteria and at least a 3 decimal
log (lg) reduction for fungi, when tested in accordance with Table 1 and 5.5.1.
Table 1 — Experimental conditions
Bactericidal activity on Yeasticidal activity on Fungicidal activity on
non-porous surfaces non-porous surfaces non-porous surfaces
Test conditions
without mechanical without mechanical without mechanical
action action action
Test organisms
Enterococcus hirae
(see 5.2.1)
Escherichia coli Candida albicans
minimum
Candida albicans
spectrum of Pseudomonas aeruginosa Aspergillus brasiliensis
test
Staphylococcus aureus
organisms
Saccharomyces
cerevisiae (for
Salmonella typhimurium
breweries)
Test organisms
any relevant test
additional Lactobacillus brevis
Saccharomyces
organism
(examples)
cerevisiae var.
Enterobacter cloacae
diastaticus (for
breweries)
In a range from (4 ± 1) °C In a range from (4 ± 1) In a range from (4 ± 1) °C
to (40 ± 1) °C °C to (40 ± 1) °C to (40 ± 1) °C
Test
For tests performed at For tests performed at For tests performed at
temperature
room temperature, the room temperature, the room temperature, the
range shall be between range shall be between range shall be between
18 °C and 25 °C 18 °C and 25 °C 18 °C and 25 °C
Bactericidal activity on Yeasticidal activity on Fungicidal activity on
non-porous surfaces non-porous surfaces non-porous surfaces
Test conditions
without mechanical without mechanical without mechanical
action action action
in a range from 1 min to
in a range from 1 min to in a range from 1 min to
60 min (from 1 min to 5
60 min (from 1 min to 5 60 min (from 1 min to 5
min at intervals of 1 min
Contact time min at intervals of 1 min min at intervals of 1 min
and from 5 min to 60
and from 5 min to 60 min and from 5 min to 60 min
min at intervals of 5
at intervals of 5 min) at intervals of 5 min)
min)
0,3 g/l bovine albumin for
Interfering
Staphylococcus aureus, 0,3 g/l bovine albumin
0,3 g/l bovine albumin
substance
Enterococcus hirae, for C. albicans and A.
for C. albicans
Escherichia coli and brasiliensis
clean conditions
Pseudomonas aeruginosa
3,0 g/l bovine albumin for
Interfering
Staphylococcus aureus, 3,0 g/l bovine albumin
3,0 g/l bovine albumin
substance
Enterococcus hirae, for C. albicans and A.
for C. albicans
Pseudomonas aeruginosa brasiliensis
dirty conditions
and Escherichia coli
Interfering
1,0 % reconstituted milk 1,0 % reconstituted milk 1,0 % reconstituted milk
substance
(= 1,0 g/l milk powder) (= 1,0 g/l milk powder) (= 1,0 g/l milk powder)
in dairy industry
Interfering
substance
any relevant substance any relevant substance any relevant substance
additional
Log reduction
from a water
≥ 4 lg ≥ 3 lg ≥ 3 lg
control (decimal
lg)
The referenced test conditions (General purposes) are by no means intended as requirements for the
use of a product, nor as requirements for the evaluation and acceptance of products by regulatory
authorities.
The application time for the product is specified by the manufacturer.
If specific applications are considered, the bactericidal/yeasticidal/fungicidal activity shall be
determined additionally under relevant conditions concerning application time, temperature, strains
and interfering substances.
5 Test methods
5.1 Principle
An inoculum test suspension consisting of bacteria or fungi in a solution of interfering substances is
inoculated onto a test stainless steel surface and dried. A prepared sample of the product under test is
applied in a manner which covers the dried film. The surface is maintained at a specified temperature for
a specified period of time. The surface is transferred to a previously validated neutralization medium so
that the action of the disinfectant is immediately neutralized. The number of surviving organisms which
can be recovered from the surface is determined quantitatively.
The number of bacteria or fungi on a surface treated with hard water in place of the disinfectant is also
determined and the reduction in viable counts attributed to the product is calculated by difference.
5.2 Materials and reagents
5.2.1 Test organisms
The bactericidal activity shall be evaluated using the following four strains:
— Pseudomonas aeruginosa
ATCC® 15442™ ;
— Staphylococcus aureus ATCC® 6538™;
— Enterococcus hirae ATCC® 10541™;
— Escherichia coli ATCC® 10536™.
The fungicidal or yeasticidal activity shall be evaluated using the following two strains:
— Candida albicans ATCC® 10231™;
— Aspergillus brasiliensis (ex A. niger) ATCC® 16404™.
If required for specific applications, additional strains may be chosen from, for example:
— Salmonella typhimurium ATCC® 13311™;
— Lactobacillus brevis DSM 6235;
— Enterobacter cloacae DSM 6234;
— Listeria monocytogenes ATCC® 15313™ or ATCC® 19117™
— Saccharomyces cerevisiae (for breweries) or ATCC® 9763™ or DSM 1333;
— Saccharomyces cerevisiae var. diastaticus (for DSM 70487.
breweries)
NOTE See Annex A for corresponding strain numbers in some other culture collections.
If additional strains are used, they shall be incubated under optimum growth conditions (temperature,
time, atmosphere) and noted in the test report.

ATCC® 15442™, ATCC® 6538™, ATCC® 10541™, ATCC® 10536™, ATCC® 10231™, ATCC® 16404™, ATCC® 13311™,
ATCC® 15313™, ATCC® 19117™ and ATCC® 9763™ are the trademarks of products supplied by the American Type Culture
Collections. This information is given for the convenience of users of this document and does not constitute an endorsement by
CEN of the product named. Equivalent products can be used if they can be shown to lead to the same results.
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 collection 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 bacteria and fungi. It shall be freshly
glass distilled or of equal quality/purity and not demineralized water.
Sterilize in the autoclave (see 5.3.2.1).
NOTE 1 If the water is sterilized during sterilization of the reagents, this is not necessary.
NOTE 2 If distilled water of adequate quality is not available, water for injectable preparation (see European
Pharmacopoeia) can be used.
5.2.2.3 Tryptone Soya Agar (TSA)
For maintenance of bacterial strains and performance of viable counts.
Tryptone, pancreatic digest of casein 15,0 g
Soya peptone, papaic digest of Soybean meal 5,0 g
NaCl 5,0 g
Agar 15,0 g
Water (see 5.2.2.2) 1 000,0 ml
Sterilize in the autoclave (see 5.3.2.1). After sterilization, the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at 20 ° C.
5.2.2.4 Malt extract agar (MEA)
For maintenance of fungal strains, sporulation and performance of viable counts.
Malt extract (food grade), e.g. Cristomalt powder from 30,0 g
Difal
Agar 15,0 g
Water (see 5.2.2.2) 1 000,0 ml

Cristomalt powder from Difal is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
The malt extract should be food grade (e.g. Cristomalt powder from Difal) or equivalent that is not highly
purified and not only based on maltose (e.g. Malt extract from OXOID™) . However, if there are problems
producing at least 75 % spiny spores see 5.4.1.3.
Sterilize in the autoclave [5.3.2.1 a)]. After sterilization, the pH of the medium shall be equivalent to
5,6 ± 0,2 when measured at (20 ± 1) °C.
In case of encountering problems with neutralization (5.5.2.3 and 5.5.2.4), it can be necessary to add
neutralizer to the MEA. Annex B gives guidance on the neutralizers that may be used.
5.2.2.5 Diluent
Tryptone sodium chloride solution:
Tryptone, pancreatic digest of casein 1,0 g
NaCl 8,5 g
Water (see 5.2.2.2) 1 000,0 ml
Sterilize in the autoclave (see 5.3.2.1). After sterilization the pH shall be equivalent to 7,0 ± 0,2 when
measured at 20 °C.
5.2.2.6 Neutralizer
The neutralizer shall be validated for the product under test in accordance with 5.5.2.3 and 5.5.2.4. 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 B.
5.2.2.7 Hard water for dilution of the products
Hard water for dilution of products shall be prepared as follows:
— solution A: Dissolve 19,84 g anhydrous MgCl and 46,24 g anhydrous CaCl in water (see 5.2.2.2)
2 2
and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.19) or in autoclave (5.3.2.1 a).
Autoclaving – if used – can 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 (5.3.2.15) for no longer than one
month;
— solution B: Dissolve 35,02 g NaHCO in water (see 5.2.2.2) and dilute to 1 000 ml. Sterilize by
membrane filtration (5.3.2.19). Store the solution in the refrigerator (5.3.2.15) for no longer than one
week.
Add at least 600 ml water (see 5.2.2.2) to 6,0 ml of solution A in a 1 000 ml volumetric flask, then add
8,0 ml solution B. Mix and dilute to 1 000 ml with water (see 5.2.2.2).
Sterilize by passing through a filter with a maximum effective pore size of 0,45 µm.
The pH of the hard water shall be 7,0 ± 0,2, when measured at (20 ± 1) °C (5.3.2.6). 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).

Malt extract from OXOID™ is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the test product solutions (5.4.2), the addition of the product to the hard water
produces a different final water hardness expressed as calcium carbonate (CaCO ) in each test tube. In any case, the
final hardness is lower than 375 mg/l of calcium carbonate.
5.2.2.8 Interfering substances
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 2 times its final concentration in the test.
For the additional interfering substances, the ionic composition (e.g. pH, calcium and/or magnesium
hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids, detergents)
shall be fully specified.
NOTE The term “interfering substance” is used even if it contains more than one substance.
The method of preparation and sterilization together with the composition shall be noted in the test
report (see 5.7).
5.2.2.8.2 Bovine albumin solutions
Bovine albumin solutions for the test conditions shall be prepared as follows:
— Preparation for clean conditions:
— dissolve 0,06 g of bovine albumin (Cohn fraction V for Dubos medium) in 100 ml of water (see
5.2.2.2);
— sterilize by 0,45 µm membrane filtration (see 5.3.2.19), keep in the refrigerator and use within
one month.
The final concentration of bovine albumin in the test procedure (see 5.5.2) is 0,3 g/l.
— Preparation for dirty conditions:
— dissolve 0,6 g of bovine albumin (Cohn fraction V for Dubos medium) in 100 ml of water (see
5.2.2.2);
— sterilize by 0,45 µm membrane filtration (see 5.3.2.19), keep in the refrigerator and use within
one month.
The final concentration of bovine albumin in the test procedure (see 5.5.2) is 3,0 g/l.
5.2.2.8.3 Milk (dairies)
Skimmed milk, guaranteed free of antibiotics or additives, shall be reconstituted at a rate of 100 g powder
per litre of water (see 5.2.2.2). The working solution shall be prepared as follows:
— prepare a solution of 2,0 % (v/v) in water (see 5.2.2.2) by adding 2 parts of reconstituted milk to 98
parts of water;
— sterilize for 30 min at 105 °C ± 3 °C (or 5 min at 121 °C ± 3 °C).
The final concentration of the reconstituted milk should be 1,0 % (v/v) in the test (see 5.5.2) or 1 g/l of
milk powder during the test.
5.2.3 Test surface
Stainless steel discs (2 cm diameter discs) 304 with grade 2b finish on both sides (EN 10088-1,
EN 10088-2). The surfaces should be flat.
The surfaces should be used only once.
Prior to use the surfaces should be placed in a beaker (minimum size: 50 ml) containing a commercial
alkaline cleaner in a concentration as recommended by the manufacturer for 60 min. Immediately rinse
the discs with running freshly distilled water for 10 s.
The surface shall not be allowed to dry to any extent. The discs shall only be handled with forceps. Rinse
the discs with water (see 5.2.2.2) for a further 10 s to ensure complete removal of the surfactant. To
supply a satisfactory flow of water, a sterilized fluid dispensing pressure vessel with suitable hose and
connectors or other suitable method can be used and regulated to supply approximately 2 000 ml per
min. To disinfect, place the clean disc in a bath containing 70 % (v/v) iso-propanol for 15 min. Remove
the disc and dry by evaporation under laminar air flow.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of 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:
+3
a) in the autoclave (see 5.3.2.1) by maintaining it at 121 °C for a minimum holding time of 15 min;
b) in the dry heat sterilizer (see 5.3.2.1) by maintaining it 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 for a minimum holding time of 2 h.
5.3.2 Usual microbiological laboratory equipment and in particular, the following:
5.3.2.1 Apparatus for sterilization
+3
a) For moist heat sterilization, an autoclave capable of being maintained at 121 °C for 15 min;
b) for dry heat treatment, 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 for a minimum holding time
of 2 h.
5.3.2.2 Temperature controlled cabinet capable of being controlled at test temperatures,
(θ ± 1) °C.
5.3.2.3 Water baths capable of being controlled at 20 °C ± 1 °C, at 45 °C ± 1 °C and at additional test
temperatures θ ± 1 °C (see 5.5.1).

Decon is an example of a suitable product available commercially. This information is given for the convenience of users
of this document and does not constitute an endorsement by CEN of this product.
Disposable equipment is an acceptable alternative to reusable glassware.
5.3.2.4 Incubator (for bactericidal activity), capable of being controlled at either 36 °C ± 1 °C or
37 °C ± 1 °C. An incubator at 37 °C ± 1 °C may be used if an incubator at 36 °C ± 1 °C is not available.
5.3.2.5 Incubator (for fungicidal or yeasticidal activity), capable of being controlled at 30 °C ± 1 °C.
5.3.2.6 pH meter, having an accuracy of calibration of 0,1 pH units at 20 °C ± 1 °C.
5.3.2.7 Stopwatch.
® 6
5.3.2.8 Vortex mixer (mechanical shaker or electromechanical agitator, i.e. Vortex mixer ).
5.3.2.9 Containers: Test tubes, culture bottles or flasks of suitable capacity.
5.3.2.10 Graduated pipettes of nominal capacities 10 ml, 1 ml, 0,1 ml and 0,05 ml, or calibrated
automatic pipettes.
5.3.2.11 Petri dishes of size 90 mm to 100 mm.
5.3.2.12 Glass beads (Diameter: ≤ 5 mm).
5.3.2.13 Volumetric flasks.
5.3.2.14 Mechanical shaker.
5.3.2.15 Refrigerator capable of being controlled at 2 °C to 8 °C.
5.3.2.16 Forceps.
5.3.2.17 Microbiological filtered laminar air flow cabinet.
5.3.2.18 Fritted filter: Porosity of 40 µm to 100 µm according to ISO 4793.
5.3.2.19 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).
5.3.2.20 Vacuum desiccator, with an active desiccant. Vacuum source may be a pump or central
supply and should achieve a vacuum of 20 mmHg to 25 mmHg (508-635 torr; 677-847 mbar; 68000-
85000 Pascal; conversion tables are readily available on the Internet for other units).
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions
5.4.1.1 Stock cultures of test organisms
Stocks cultures shall be kept in accordance with the requirements of EN 12353.

Vortex® in an example of a suitable product available commercially. This information is given for the convenience of users
of this document and does not constitute an endorsement by CEN of this product.
5.4.1.2 Working culture of test organisms
a) Bacteria:
In order to prepare the working culture of the test organism, subculture from the stock culture (see
5.4.1.1) by streaking on TSA slopes or plates (see 5.2.2.3) and incubate (see 5.3.2.4). 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.
NOTE 1 The second and/or third subculture are the working culture(s).
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 that the subculture has been kept in the incubator during
the 48 h period. In these circumstances, prepare a further 24 h subculture after proceeding. Do not
take a fourth subculture.
For additional strains (see 5.2.1), any departure from this method of culturing the bacteria or
preparing the suspensions shall be noted, giving the reasons in the test report.
b) Fungi:
In order to prepare the working culture of Candida albicans, subculture from the stock culture (see
5.4.1.1) by streaking onto MEA slopes or plates (see 5.2.2.4) and incubate (see 5.3.2.5). After 42 h to
48 h, prepare a second subculture from the first subculture in the same way and incubate for 42 h to
48 h. From this second subculture, a third subculture may be produced in the same way.
NOTE 2 The second and/or third subculture are the working culture(s).
For Aspergillus brasiliensis (5.2.1), use only the first subculture grown on MEA (5.2.2.4) in Petri dishes
or flasks with ventilated caps (5.3.2.17) and incubate at 30 °C ± 1 °C for 7 d to 9 d. No further
subculturing is needed. Do not stack the Petri dishes during the incubation to improve the
temperature homogenization.
At the end of incubation, all the cultures shall show a dark brown or black surface. Cultures with
appearance of rare and small white or grey areas might be kept.
5.4.1.3 Test suspensions
a) Bacterial test suspension:
Take 10 ml of diluent (see 5.2.2.5) and place in a 100 ml flask with 5 g of glass beads (see 5.3.2.12).
Take the working culture (see 5.4.1.2) and transfer loopfuls of the cells into the diluent. The cells
should be suspended in the diluent by immersing the loop in the diluent and rubbing it against the
side of the flask to dislodge the cells. Shake the flask for 3 min using a mechanical shaker (see
5.3.2.14). Aspirate the suspension from the glass beads and transfer to another flask. Adjust the
8 8
number of cells in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml.
The numbers of units shall be estimated by means of spectrophotometer or any other suitable means.
Maintain this suspension in the water bath at 20 °C ± 1 °C and use within 2 h.
b) Fungal test suspension:
1) Candida albicans:
Take 10 ml of diluent (see 5.2.2.5) and place in a 100 ml flask with 5 g of glass beads. Take the
working culture (see 5.4.1.2) and transfer loopfuls of the cells into the diluent. The cells should
be suspended in the diluent by immersing the loop in the diluent and rubbing it against the side
of the flask to dislodge the cells. Shake the flask for 3 min using a mechanical shaker (5.3.2.14).
Aspirate the suspension from the glass beads and transfer to another flask. Adjust the number
7 7
of cells in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml.
The numbers of units shall be estimated by means of spectrophotometer or any other suitable
means. Maintain this suspension in the water bath at 20 °C ± 1 °C and use within 2 h.
2) Aspergillus brasiliensis:
Take the working culture (see 5.4.1.2) and suspend the cells in 10 ml of sterile 0,05 % w/v
polysorbate 80 solution in water (see 5.2.2.2). Using a sterile glass rod or spatula detach the
conidiospores from the culture surface. Transfer the suspension into a flask and gently shake
by hand for one minute together with glass beads (see 5.3.2.12). The suspension is filtered
through a fritted filter (see 5.3.2.18).
Carry out a microscopic examination under × 400 magnification immediately after the preparation to
show:
— the presence of a high concentration (at least 75 % of spiny spores) of characteristic mature spores,
i.e. spiny spores (versus smooth spores);
— the absence of spore germination (check at least 10 fields of view);
— the absence of mycelia fragments (check at least 10 fields of view).
If germinated spores are present, discard the suspension.
nd
If mycelia are present, proceed to a 2 fritted filtration.
If mycelia are still present, discard the suspension.
If 75 % spiny spores are not achieved it can be due to the Aspergillus brasiliensis culture or the media
used to produce these spores. In this situation, it will be necessary to obtain the culture from another
culture collection and/or use a MEA from a different supplier.
7 7
Adjust the number of spores in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml using the diluent
(5.2.2.5), estimating the number of cfu by any suitable means. Use the suspension within 4 h in a water
bath controlled at 20 °C ± 1 °C (5.3.2.3). In any case, adjust the temperature according to 5.5.1 only
immediately before the start of the test (5.5.2).
The use of a cell counting device for adjusting the number of cells is highly recommended. When using a
suitable counting chamber, follow the instructions explicitly.
This test suspension shall not be stored more than 2 d at 2 °C to 8 °C.
The test suspension shall be mixed (see 5.3.2.8) immediately before use to re-suspend the spores.

Analytical quality, non hydrolysed in accordance with European Pharmacopoeia, Volume 1. TWEEN 80 is an example of a
suitable product available commercially. This information is given for the convenience of users of this document and does not
constitute an endorsement by CEN of this product.
5.4.1.4 Quantifying of bacterial and fungal test suspensions
−6 −7
Dilute the adjusted bacterial suspensions (see 5.4.1.3 a)) by 10 (serial dilutions) and 10 and dilute
−5 −6
the adjusted yeast and mould spore suspension (see 5.4.1.3 b)) by 10 and 10 using diluent (see
5.2.2.5). Mix the suspension (see 5.3.2.8).
Take a sample of 1,0 ml of each dilution in duplicate and inoculate pour plates or spread plates.
When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes (see 5.3.2.11)
and add 15 ml to 20 ml melted TSA (see 5.2.2.3) for the bacteria and 15 ml to 20 ml melted MEA (see
5.2.2.4) for the fungi, cooled to 45 °C ± 1 °C.
When using the spread plate technique, spread each 1,0 ml sample – divided into portions of
approximately equal size – on an appropriate number (at least two) of surface dried plates containing
TSA or MEA.
a) Counting of bacterial test suspensions
For the bacterial strains, incubate the plates at (36 ± 1) °C or at (37 ± 1) °C for 18 h to 24 h. Discard
any plates which are not countable for any reason. Count the plates and determine the number of
colony forming units. Incubate the plates for a further 18 h to 24 h. Do not recount plates which no
longer show countable quantities of colonies. Recount the remaining plates.
b) Counting of fungal test suspensions
For the fungal strains, incubate the plates at (30 ± 1) °C for 18 h to 24 h (Candida albicans), or for 42
h to 48 h (Aspergillus brasiliensis). Discard any pl
...