Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity in the medical area - Test method and requirements (phase 2, step 1)

This document specifies a test method and the minimum requirements for fungicidal or yeasticidal activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water, or - in the case of ready-to-use products - with water. Products can only be tested at a concentration of 80 % or less (97 % with a modified method for special cases) as some dilution is always produced by adding the test organisms and interfering substance.
This document applies to products that are used in the medical area in the fields of hygienic handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means.
This document applies to areas and situations where disinfection or antisepsis is medically indicated. Such indications occur in patient care, for example:
-   in hospitals, in community medical facilities and in dental institutions;
-   in clinics of schools, of kindergartens and of nursing homes;
and can occur in the workplace and in the home. It can also include services such as laundries and kitchens supplying products directly for the patients.
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.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendations".

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Dieses Dokument legt ein Prüfverfahren und Mindestanforderungen für die fungizide oder levurozide (Hefen abtötende) Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte bzw. – im Fall von gebrauchsfertigen Produkten – mit Wasser als homogenes physikalisch stabiles Präparat vorliegen. Die Produkte können nur in einer Konzentration bis zu 80 % (97 % bei einem modifizierten Verfahren in Sonderfällen) geprüft werden, da durch Zugabe der Prüforganismen und der Belastungssubstanz stets eine gewisse Verdünnung bewirkt wird.
Dieses Dokument ist anzuwenden für Produkte, die im humanmedizinischen Bereich für hygienische Händedesinfektion, hygienische Händewaschung, chirurgische Händedesinfektion, chirurgische Händewaschung, für die Instrumentendesinfektion durch Eintauchen sowie für die Oberflächendesinfektion mittels Wischen, Sprühen, Spülen o. Ä. verwendet werden.
Dieses Dokument ist anzuwenden für Bereiche und Bedingungen, wo eine Desinfektion oder Antisepsis aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen z. B. bei der Patientenbetreuung in
—   Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
—   medizinischen Einrichtungen in Schulen, Kindergärten und Heimen;
vor und können auch am Arbeitsplatz oder im häuslichen Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung der Patienten dienen.
ANMERKUNG 1   Das beschriebene Verfahren dient zur Bestimmung der Wirksamkeit kommerziell erhältlicher Zubereitungen oder Wirkstoffe unter den jeweiligen Anwendungsbedingungen.
ANMERKUNG 2   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.
EN 14885 legt detailliert die Beziehungen der unterschiedlichen Prüfungen untereinander und zu „Anwendungsempfehlungen“ fest.

Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour l’évaluation de l’activité fongicide ou levuricide en médecine - Méthode d'essai et prescriptions (phase 2, étape 1)

Le présent document spécifie une méthode d’essai et les exigences minimales relatives à l’activité fongicide et levuricide des désinfectants chimiques et des produits antiseptiques 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 de l’eau. Les produits ne peuvent être soumis à l’essai qu’à une concentration inférieure ou égale à 80 % (97 % avec une méthode modifiée dans certains cas particuliers), car l’ajout des souches d’essai et de la substance interférente entraîne toujours une dilution.
Le présent document s’applique aux produits employés en médecine pour la friction et le lavage hygiéniques des mains, pour la friction et le lavage chirurgicaux des mains, pour la désinfection des instruments par immersion et pour la désinfection de surface par essuyage, pulvérisation, rinçage ou autre.
Le présent document s’applique dans les zones et situations où la désinfection ou l’antisepsie est médicalement préconisée. De telles indications se rencontrent dans le cadre des soins apportés aux patients, par exemple :
-   dans des hôpitaux, dans des centres de soins médicaux et des cabinets dentaires ;
-   dans des infirmeries d’écoles, de jardins d’enfants et de maisons de retraite ;
et peuvent aussi se rencontrer sur les lieux de travail ou à domicile. Elles peuvent également concerner des services tels que des blanchisseries et cuisines qui fournissent des produits directement aux patients.
NOTE 1   La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives dans leurs conditions d’utilisation.
NOTE 2   Cette méthode correspond à un essai de type phase 2, étape 1.
L’EN 14885 précise de manière détaillée la relation entre les différents essais et les « recommandations d’utilisation ».

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje fungicidnega delovanja ali delovanja na kvasovke v humani medicini - Preskusna metoda in zahteve (faza 2, stopnja 1)

General Information

Status
Published
Public Enquiry End Date
19-Oct-2019
Publication Date
08-Dec-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Nov-2021
Due Date
24-Jan-2022
Completion Date
09-Dec-2021

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SLOVENSKI STANDARD
SIST EN 13624:2022
01-januar-2022
Nadomešča:
SIST EN 13624:2013
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje fungicidnega delovanja ali delovanja na kvasovke v humani medicini -
Preskusna metoda in zahteve (faza 2, stopnja 1)

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation

of fungicidal or yeasticidal activity in the medical area - Test method and requirements

(phase 2, step 1)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur

Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich -

Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour

l’évaluation de l’activité fongicide ou levuricide en médecine - Méthode d'essai et

prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 13624:2021
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
SIST EN 13624:2022 en,fr,de

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

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SIST EN 13624:2022
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SIST EN 13624:2022
EN 13624
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 11.080.20 Supersedes EN 13624:2013
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of fungicidal or
yeasticidal activity in the medical area - Test method and
requirements (phase 2, step 1)

Désinfectants chimiques et antiseptiques - Essai Chemische Desinfektionsmittel und Antiseptika -

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

fongicide ou levuricide en médecine - Méthode d'essai fungiziden oder levuroziden Wirkung im

et exigences (phase 2, étape 1) humanmedizinischen Bereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 1)
This European Standard was approved by CEN on 3 October 2021.

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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13624:2021 E

worldwide for CEN national Members.
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SIST EN 13624:2022
EN 13624:2021 (E)
Contents Page

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

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

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

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Requirements ................................................................................................................................................... 6

5 Test method ...................................................................................................................................................... 7

5.1 Principle ............................................................................................................................................................. 7

5.2 Materials and reagents .................................................................................................................................. 8

5.3 Apparatus and glassware .......................................................................................................................... 11

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

5.5 Procedure for assessing the fungicidal and yeasticidal activity of the product .................... 18

5.6 Experimental data and calculation ........................................................................................................ 26

5.7 Verification of methodology ..................................................................................................................... 32

5.8 Expression of results and precision ...................................................................................................... 33

5.9 Interpretation of results – conclusion .................................................................................................. 34

5.10 Test report ...................................................................................................................................................... 36

Annex A (informative) Referenced strains in national collections ......................................................... 38

Annex B (informative) Neutralizers and rinsing liquids............................................................................. 39

Annex C (informative) Graphical representation of test procedures..................................................... 41

C.1 Dilution-neutralization method ............................................................................................................. 41

C.2 Membrane filtration method ................................................................................................................... 43

C.3 Dilution-neutralization method (modified method for ready-to-use products) .................. 45

C.4 Membrane filtration method (modified method for ready-to-use products) ........................ 47

Annex D (informative) Example of a typical test report .............................................................................. 49

Annex E (informative) Precision of the test result ........................................................................................ 54

Bibliography ................................................................................................................................................................. 57

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SIST EN 13624:2022
EN 13624:2021 (E)
European foreword

This document (EN 13624:2021) 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 2022, and conflicting national standards shall be

withdrawn at the latest by May 2022.

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 13624:2013.

The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to

harmonize the structure and wording with other tests of CEN/TC 216 existing or in preparation and to

improve the readability of the standard and thereby make it more understandable. The following is a

list of significant technical changes since the last edition:
— De-harmonization of the standard (Annex ZA and references to MDR/MDD deleted);
— Textile disinfection was added (see Table 1, 5.5.1.3);

— The amounts for the dirty conditions for the modified method for ready-to-use products were

reduced (5.2.2.8.4 b));

— Clarification that a neutralization time of 10 s shall be used for all products with contact times of

10 min or shorter (5.5.2.2. c) and 5.5.2.5 b));
— Explanation for alternative controls for chemo-thermal disinfection (5.5.2.3);

— The reference to CIP numbers for the fungi strains were deleted and the UMIP numbers were added

(Annex A);
— Harmonization of the text with EN 13727;
— Correction of editorial mistakes.

The changes of this revision have no impact on the test results obtained with reference to the version

EN 13624:2013. Those results are still valid.

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, Turkey and the

United Kingdom.
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SIST EN 13624:2022
EN 13624:2021 (E)
Introduction

This document specifies a suspension test for establishing whether a chemical disinfectant or an

antiseptic has a fungicidal or yeasticidal activity in the area and 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 substances, i.e. conditions which can

influence its action in practical situations. Each utilization concentration of the chemical disinfectant or

antiseptic found by this test corresponds to the chosen experimental conditions.
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SIST EN 13624:2022
EN 13624:2021 (E)
1 Scope

This document specifies a test method and the minimum requirements for fungicidal or yeasticidal

activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable

preparation when diluted with hard water, or – in the case of ready-to-use products – with water.

Products can only be tested at a concentration of 80 % or less (97 % with a modified method for special

cases) as some dilution is always produced by adding the test organisms and interfering substance.

This document applies to products that are used in the medical area in the fields of hygienic handrub,

hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and

surface disinfection by wiping, spraying, flooding or other means.

This document applies to areas and situations where disinfection or antisepsis is medically indicated.

Such indications occur in patient care, for example:
— in hospitals, in community medical facilities and in dental institutions;
— in clinics of schools, of kindergartens and of nursing homes;

and can occur in the workplace and in the home. It can also include services such as laundries and

kitchens supplying products directly for the patients.

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.

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, Chemical disinfectants and antiseptics — Application of European Standards for chemical

disinfectants and antiseptics

ISO 4793:1980, 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 terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
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SIST EN 13624:2022
EN 13624:2021 (E)
4 Requirements

The product shall demonstrate at least a 4 decimal logarithms (lg) reduction (for hygienic handwash at

least a 2 lg reduction), when tested in accordance with Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Hygienic
Textile
Test Surgical handrub Instrument Surface
handrub and
conditions and handwash disinfection disinfection disinfection
handwash

Minimum Candida albicans Candida albicans a) fungicidal a) fungicidal a) fungicidal

spectrum of (vegetative cells) (vegetative cells) activity: activity: activity:
test Aspergillus Aspergillus Aspergillus
organisms brasiliensis brasiliensis brasiliensis
(conidiospores) (conidiospores) (conidiospores)
Candida albicans Candida albicans Candida albicans
(veg. cells) (veg. cells) (veg. cells)
b) yeasticidal b) yeasticidal b) yeasticidal
activity: activity: activity:
Candida albicans Candida albicans Candida albicans
(veg. cells) (veg. cells) (veg. cells)
additional Any relevant test organism
Test according to the manufacturer's recommendation, but
temperature
generally between generally between
20°C and 50°C for 20°C and 50°C for
Candida spec. between 4 °C and Candida spec.
at 20 °C at 20 °C
and between and between
30 °C
20°C and 60°C for 20°C and 60°C for
e e
Aspergillus spec. Aspergillus spec.
Contact time according to the manufacturer's recommendation,
but between but no longer than
30 s and 60 s 1 min and 5 min 60 min
5 min or 60 min
Interfering 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine
substance albumin solution albumin solution albumin solution albumin solution
clean (hygienic (surgical
b b
conditions
handrub) handrub)
and/or and/or
dirty 3,0 g/l bovine 3,0 g/l bovine
conditions albumin solution albumin solution
3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine
plus 3,0 ml/l plus 3,0 ml/l
albumin solution albumin solution albumin solution
erythrocytes erythrocytes plus 3,0 ml/l plus 3,0 ml/l plus 3,0 ml/l
(hygienic (surgical erythrocytes erythrocytes erythrocytes
c c
handwash) handwash)
any relevant any relevant
additional — — —
substance substance

NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained

under the minimum test conditions.
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SIST EN 13624:2022
EN 13624:2021 (E)
Hygienic
Textile
Test Surgical handrub Instrument Surface
handrub and
conditions and handwash disinfection disinfection
disinfection
handwash

The contact times for surface disinfectants stated in this table are chosen on the basis of the practical

conditions of the product. The recommended contact time for the use of the product is within the

responsibility of the manufacturer. Products intended to disinfect surfaces that are likely to come into

contact with the patient and/or the medical staff and surfaces, which are frequently touched by different

people, leading to the transmission of microorganisms to the patient, shall be tested with a contact time of

maximum 5 min. The same applies where the contact time of the product shall be limited for practical

reasons. Products for other surfaces than stated above may be tested with a contact time of maximum

60 min.

Hygienic and surgical handrub shall be tested as a minimum under clean conditions.

Hygienic and surgical handwash shall be tested as a minimum under dirty conditions.

For further information see EN 16616:2015, Clause 4.

For further information regarding validity at elevated temperatures see EN 14885.

5 Test method
5.1 Principle

5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use

products) is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an

interfering substance. The mixture is maintained at the temperature and the contact time specified in

Clause 4 and 5.5.1.1. At the end of this contact time, an aliquot is taken; the fungicidal and/or the

fungistatic action in this portion is immediately neutralized or suppressed by a validated method. The

method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane

filtration is used. The numbers of surviving fungi in each sample are determined and the reduction is

calculated.

NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded

as a ready-to-use product (5.4.2).

5.1.2 The test is performed using the vegetative cells of Candida albicans and the conidiospores of

Aspergillus brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal

activity) as test-organisms (Clause 4, Table 1).

5.1.3 Additional contact times and temperatures are specified (Clause 4, Table 1). Additional

interfering substances and test organisms may be used.
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SIST EN 13624:2022
EN 13624:2021 (E)
5.2 Materials and reagents
5.2.1 Test organisms

The fungicidal activity shall be evaluated using the following strains as test organisms selected

according to Clause 4 (Table 1) :
— Candida albicans ATCC 10231;
— Aspergillus brasiliensis (former “A. niger”) ATCC 16404.
The yeasticidal activity shall be evaluated using only Candida albicans.
Refer to Annex A for strain reference in some other culture collections.

The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3).

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 document refer to the anhydrous salts. Hydrated forms

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

molecular weight differences.

The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be

free from substances that are toxic or inhibitory to the test organisms.

To improve reproducibility, it is recommended that commercially available dehydrated material is used

for the preparation of culture media. The manufacturer's instructions relating to the preparation of

these products should be rigorously followed.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at (20 ± 1) °C.
5.2.2.2 Water

The water shall be freshly glass-distilled water and not demineralized water. If distilled water of

adequate quality is not available, water for injections (see bibliographic reference [2]) can be used.

Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used, e.g. for

preparation of culture media and subsequently sterilized.
See 5.2.2.7 for the procedure to prepare hard water.

The ATCC numbers are the collection numbers of strains supplied by these 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.
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SIST EN 13624:2022
EN 13624:2021 (E)
5.2.2.3 Malt extract agar (MEA)
Malt extract agar, consisting of:
Malt extract [food grade (e.g. Christomalt powder 30,0 g
from Difal) or an equivalent extract that is not highly purified and not
only based on maltose (e.g. Malt extract from OXOID)]
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave (5.3.1). After sterilization, the pH (5.3.2.4) of the medium shall be equivalent to

5,6 ± 0,2.

In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add

neutralizer to MEA. Annex B gives guidance on the neutralizers that may be used. It is recommended

not to use neutralizer that causes opalescence in the agar.

If there are problems with producing at least 75 % spiny conidiospores, see 5.4.1.4.2.

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 (5.3.2.4) of the diluent shall be

equivalent to 7,0 ± 0,2.
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.

Information on neutralizers that have been found to be suitable for some categories of products is given

in Annex B.
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.

Information on rinsing liquids that have been found to be suitable for some categories of products is

given in Annex B.

This 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.
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SIST EN 13624:2022
EN 13624:2021 (E)
5.2.2.7 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:

— prepare solution A: dissolve 19,84 g magnesium chloride (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 (5.3.2.4) of the hard water shall be 7,0 ± 0,2. If necessary, adjust the pH by using a solution of

approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l

(about 1 mol/l) of hydrochloric acid (HCl).

The hard water shall be freshly prepared under aseptic conditions and used within 12 h.

NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water

produces different final water hardness in each test tube. In any case, the final hardness expressed as calcium

carbonate (CaCO ) is in the test tube lower than 375 mg/l.
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 (50

times in the case of the modified method, see 5.2.2.8.4).

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 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent

(5.2.2.4).

Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month.

The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l.

5.2.2.8.3 Dirty conditions (mixture of bovine albumin solutions – high concentration with sheep

erythrocytes)

Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent

(5.2.2.4).
Sterilize by membrane filtration (5.3.2.7).
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Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g

for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4).

Repeat this procedure at least 3 times, until the supernatant is colourless.

Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see

above). To avoid later contamination, this mixture should be split in portions probably needed per day

and kept in separate containers for a maximum of 7 d in a refrigerator (5.3.2.8).

The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be

3 g/l and 3 ml/l respectively.

5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4)

Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering

substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems

with the filtration.

a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent;

b) Dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml of diluent

(instead of 48,5 ml). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml

(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilized bovine albumin solution

to obtain 50 ml.
5.2.2.9 Defibrinated sheep blood

The defibrinated sheep blood should be sterile (aseptic blood-letting and preparation), pooled from

more than one sheep and can be acquired from a commercial supplier.
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 moist and dry heat sterilization:

a) for moist heat sterilization, an autoclave capable of being maintained at ( 121 ) °C for a minimum

holding time of 15 min;

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

+5 +5

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

160
0 0
minimum holding time of 2 h.

Disposable sterile equipment is an acceptable alternative to reusable glassware.

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5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted

MEA in case of pour plate technique) and at additional test temperatures ±1 °C (5.5.1).

5.3.2.3 Incubator, capable of being controlle
...

SLOVENSKI STANDARD
oSIST prEN 13624:2019
01-oktober-2019
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje fungicidnega delovanja ali delovanja na kvasovke v humani medicini -
Preskusna metoda in zahteve (faza 2, stopnja 1)

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation

of fungicidal or yeasticidal activity in the medical area - Test method and requirements

(phase 2, step 1)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur

Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich -

Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour

l'évaluation de l'activité fongicide ou levuricide en médecine - Méthode d'essai et

prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: prEN 13624
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
oSIST prEN 13624:2019 en,fr,de

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

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oSIST prEN 13624:2019
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oSIST prEN 13624:2019
DRAFT
EUROPEAN STANDARD
prEN 13624
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2019
ICS 11.080.20 Will supersede EN 13624:2013
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of fungicidal or
yeasticidal activity in the medical area - Test method and
requirements (phase 2, step 1)

Désinfectants chimiques et antiseptiques - Essai Chemische Desinfektionsmittel und Antiseptika -

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

fongicide ou levuricide en médecine - Méthode d'essai fungiziden oder levuroziden Wirkung im

et prescriptions (phase 2, étape 1) humanmedizinischen Bereich - Prüfverfahren und

Anforderungen (Phase 2, Stufe 1)

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 216.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
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. prEN 13624:2019 E

worldwide for CEN national Members.
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oSIST prEN 13624:2019
prEN 13624:2019 (E)
Contents Page

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

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

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

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Requirements ................................................................................................................................................... 6

5 Test method ...................................................................................................................................................... 7

5.1 Principle ............................................................................................................................................................. 7

5.2 Materials and reagents .................................................................................................................................. 8

5.3 Apparatus and glassware .......................................................................................................................... 11

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

5.5 Procedure for assessing the fungicidal and yeasticidal activity of the product .................... 18

5.6 Experimental data and calculation ........................................................................................................ 26

5.7 Verification of methodology ..................................................................................................................... 32

5.8 Expression of results and precision ...................................................................................................... 33

5.9 Interpretation of results – conclusion .................................................................................................. 34

5.10 Test report ...................................................................................................................................................... 35

Annex A (informative) Referenced strains in national collections ........................................................... 37

Annex B (informative) Neutralizers and rinsing liquids ............................................................................... 38

Annex C (informative) Graphical representation of test procedures ....................................................... 40

C.1 Dilution-neutralization method ............................................................................................................. 40

C.2 Membrane filtration method ................................................................................................................... 42

C.3 Dilution-neutralization method (modified method for ready-to-use products) .................. 44

C.4 Membrane filtration method (modified method for ready-to-use products) ........................ 46

Annex D (informative) Example of a typical test report................................................................................ 48

Annex E (informative) Precision of the test result .......................................................................................... 53

Annex ZA (informative) Relationship between this European Standard and the General

Safety and Performance Requirements of Regulation (EU) 2017/745 aimed to be

covered............................................................................................................................................................. 56

Bibliography ................................................................................................................................................................. 58

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oSIST prEN 13624:2019
prEN 13624:2019 (E)
European foreword

This document (prEN 13624:2019) has been prepared by Technical Committee CEN/TC 216 “Chemical

disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 13624:2013.

This document has been prepared under a standardization request given to CEN by the European

Commission and the European Free Trade Association, and supports essential requirements of

EU Directive(s).

For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this

document.

The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to

harmonize the structure and wording with other tests of CEN/TC 216 existing or in preparation and to

improve the readability of the standard and thereby make it more understandable. The following is a

list of significant technical changes since the last edition:

— new Annex ZA was added and the reference was changed to the Medical Device Regulation (instead

of the Medical Device Directive) (European foreword and Annex ZA);
— Textile disinfection was added (see Table 1, 5.5.1.3);

— The amounts for the dirty conditions for the modified method for ready-to-use products were

reduced (5.2.2.8.4 b));

— Clarification that a neutralization time of 10 s shall be used for all products with contact times of 10

min or shorter (5.5.2.2. c) and 5.5.2.5 b));
— Explanation for alternative controls for chemo-thermal disinfection (5.5.2.3);
— The reference to CIP numbers for the fungi strains were deleted (Annex A);
— Harmonization of the text with EN 13727;
— Correction of editorial mistakes.

The changes of this revision have no impact on the test results obtained with reference to the version

EN 13624:2013. Those results are still valid.
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Introduction

This document specifies a suspension test for establishing whether a chemical disinfectant or an

antiseptic has a fungicidal or yeasticidal activity in the area and 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 substances, i.e. conditions which can

influence its action in practical situations. Each utilization concentration of the chemical disinfectant or

antiseptic found by this test corresponds to the chosen experimental conditions.
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1 Scope

This document specifies a test method and the minimum requirements for fungicidal or yeasticidal

activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable

preparation when diluted with hard water, or – in the case of ready-to-use products – with water.

Products can only be tested at a concentration of 80 % or less (97 % with a modified method for special

cases) as some dilution is always produced by adding the test organisms and interfering substance.

This document applies to products that are used in the medical area in the fields of hygienic handrub,

hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and

surface disinfection by wiping, spraying, flooding or other means.

This document applies to areas and situations where disinfection or antisepsis is medically indicated.

Such indications occur in patient care, for example:
— in hospitals, in community medical facilities and in dental institutions;
— in clinics of schools, of kindergartens and of nursing homes;

and can occur in the workplace and in the home. It can also include services such as laundries and

kitchens supplying products directly for the patients.

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.

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, Chemical disinfectants and antiseptics — Application of European Standards for chemical

disinfectants and antiseptics

ISO 4793:1980, 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 terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
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4 Requirements

The product shall demonstrate at least a 4 decimal logarithms (lg) reduction (for hygienic handwash at

least a 2 lg reduction), when tested in accordance with Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Hygienic
Textile
Test Surgical handrub Instrument Surface
handrub and
conditions and handwash disinfection disinfection disinfection
handwash

Minimum Candida albicans Candida albicans a) fungicidal a) fungicidal a) fungicidal

spectrum of (vegetative cells) (vegetative cells) activity: activity: activity:
test Aspergillus Aspergillus Aspergillus
organisms brasiliensis brasiliensis brasiliensis
(conidiospores) (conidiospores) (conidiospores)
Candida albicans Candida albicans Candida albicans
(veg. cells) (veg. cells) (veg. cells)
b) yeasticidal b) yeasticidal b) yeasticidal
activity: activity: activity:
Candida albicans Candida albicans Candida albicans
(veg. cells) (veg. cells) (veg. cells)
additional Any relevant test organism
Test according to the manufacturer's recommendation, but
temperature
between 4 °C and
at 20 °C at 20 °C at 20 °C or above at 20 °C or above
30 °C
Contact time according to the manufacturer's recommendation,
but between but no longer than
contact time
according to
30 s and 60 s 1 min and 5 min 60 min
5 min or 60 min
manufacturers
recommendations
Interfering 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine
substance albumin solution albumin solution albumin solution albumin solution
clean (hygienic (surgical
b b
conditions
handrub) handrub)
and/or and/or
dirty 3,0 g/l bovine 3,0 g/l bovine
conditions albumin solution albumin solution
3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine
plus 3,0 ml/l plus 3,0 ml/l
albumin solution albumin solution albumin solution
erythrocytes erythrocytes plus 3,0 ml/l plus 3,0 ml/l plus 3,0 ml/l
(hygienic (surgical and erythrocytes erythrocytes erythrocytes
c c
handwash) handwash)
any relevant any relevant
additional — — —
substance substance
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oSIST prEN 13624:2019
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Hygienic
Textile
Test Surgical handrub Instrument Surface
handrub and
conditions and handwash disinfection disinfection
disinfection
handwash

The contact times for surface disinfectants stated in this table are chosen on the basis of the practical

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

of the manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient

and/or the medical staff and surfaces, which are frequently touched by different people, leading to the

transmission of microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same

applies where the contact time of the product shall be limited for practical reasons. Products for other surfaces

than stated above may be tested with a contact time of maximum 60 min.

Hygienic and surgical handrub shall be tested as a minimum under clean conditions.

Hygienic and surgical handwash shall be tested as a minimum under dirty conditions.

For further information see EN 16616:2015, Clause 4

NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained

under the minimum test conditions.
5 Test method
5.1 Principle

5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use

products) is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an

interfering substance. The mixture is maintained at the temperature and the contact time specified in

Clause 4 and 5.5.1.1. At the end of this contact time, an aliquot is taken; the fungicidal and/or the

fungistatic action in this portion is immediately neutralized or suppressed by a validated method. The

method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane

filtration is used. The numbers of surviving fungi in each sample are determined and the reduction is

calculated.

NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded

as a ready-to-use product (5.4.2).

5.1.2 The test is performed using the vegetative cells of Candida albicans and the conidiospores of

Aspergillus brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal

activity) as test-organisms (Clause 4, Table 1).

5.1.3 Additional contact times and temperatures are specified (Clause 4, Table 1). Additional

interfering substances and test organisms may be used.
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5.2 Materials and reagents
5.2.1 Test organisms

The fungicidal activity shall be evaluated using the following strains as test organisms selected

according to Clause 4 (Table 1) :
— Candida albicans ATCC 10231;
— Aspergillus brasiliensis (former “A. niger”) ATCC 16404.
The yeasticidal activity shall be evaluated using only Candida albicans.
Refer to Annex A for strain reference in some other culture collections.

The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3).

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 document refer to the anhydrous salts. Hydrated forms

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

molecular weight differences.

The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be

free from substances that are toxic or inhibitory to the test organisms.

To improve reproducibility, it is recommended that commercially available dehydrated material is used

for the preparation of culture media. The manufacturer's instructions relating to the preparation of

these products should be rigorously followed.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at (20 ± 1) °C.
5.2.2.2 Water

The water shall be freshly glass-distilled water and not demineralized water. If distilled water of

adequate quality is not available, water for injections (see bibliographic reference [2]) can be used.

Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used, e.g. for

preparation of culture media and subsequently sterilized.
See 5.2.2.7 for the procedure to prepare hard water.

The ATCC numbers are the collection numbers of strains supplied by these 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.
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5.2.2.3 Malt extract agar (MEA)
Malt extract agar, consisting of:
Malt extract [food grade (e.g. Christomalt powder 30,0 g
from Difal) or an equivalent extract that is not highly purified and not
only based on maltose (e.g. Malt extract from OXOID)]
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave (5.3.1). After sterilization, the pH (5.3.2.4) of the medium shall be equivalent to

5,6 ± 0,2.

In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add

neutralizer to MEA. Annex B gives guidance on the neutralizers that may be used. It is recommended

not to use neutralizer that causes opalescence in the agar.

If there are problems with producing at least 75 % spiny conidiospores, see 5.4.1.4.2.

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 (5.3.2.4) of the diluent shall be

equivalent to 7,0 ± 0,2.
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.

Information on neutralizers that have been found to be suitable for some categories of products is given

in Annex B.
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.

Information on rinsing liquids that have been found to be suitable for some categories of products is

given in Annex B.

This 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.
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5.2.2.7 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:

— prepare solution A: dissolve 19,84 g magnesium chloride (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 (5.3.2.4) of the hard water shall be 7,0 ± 0,2. If necessary, adjust the pH by using a solution of

approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l

(about 1 mol/l) of hydrochloric acid (HCl).

The hard water shall be freshly prepared under aseptic conditions and used within 12 h.

NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water

produces different final water hardness in each test tube. In any case, the final hardness expressed as calcium

carbonate (CaCO ) is in the test tube lower than 375 mg/l.
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 (50

times in the case of the modified method, see 5.2.2.8.4).

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 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent

(5.2.2.4).

Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month.

The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l.

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5.2.2.8.3 Dirty conditions (mixture of bovine albumin solutions – high concentration with sheep

erythrocytes)

Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent

(5.2.2.4).
Sterilize by membrane filtration (5.3.2.7).

Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g

for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4).

Repeat this procedure at least 3 times, until the supernatant is colourless.

Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see

above). To avoid later contamination, this mixture should be split in portions probably needed per day

and kept in separate containers for a maximum of 7 d in a refrigerator (5.3.2.8).

The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be

3 g/l and 3 ml/l respectively.

5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4)

Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering

substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems

with the filtration.

a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent;

b) Dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml of diluent

(instead of 48,5 ml). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml

(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilized bovine albumin solution

to obtain 50 ml.
5.2.2.9 Defibrinated sheep blood

The defibrinated sheep blood should be sterile (aseptic blood-letting and preparation), pooled from

more than one sheep and can be acquired from a commercial supplier.
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)].
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oSIST prEN 13624:2019
prEN 13624:2019 (E)

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

5.3.2.1 Apparatus for moist and dry heat sterilization:

a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum

holding time of 15 min;
180 ) °C for a minimum
b) for dry heat sterilization, a hot air oven capable of being maintained at (
+5 +5

holding time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a

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

MEA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1).

5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C.

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

A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar

media (5.2.2.3).
5.3.2.5 Stopwatch.
5.3.2.6 Shakers.
® 4
a) Electromechanical agitator, e.g. Vortex mixer ;
...

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