SIST EN 17126:2019

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

This document specifies a test method and the minimum requirements for sporicidal activity of chemical disinfectant 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 European Standard applies to products that are used in the medical area in the fields of instrument disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means.
This European Standard applies to areas and situations where disinfection 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 may occur in the workplace and in the home. It may 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 sporiziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die sporizide Wirkung von chemischen Desinfektionsmitteln fest, die bei Verdünnung mit Wasser standardisierter Härte als homogene, physikalisch stabile Zubereitung vorliegen, bzw. bei gebrauchsfertigen Produkten bei der Verdünnung mit Wasser. Die Produkte können nur bei einer Konzentration bis zu 80 % (97 % bei einem modifizierten Verfahren für Sonderfälle) geprüft werden, da durch Zugabe der Prüforganismen und der Belastungssubstanz immer eine gewisse Verdünnung bewirkt wird.
Diese Europäische Norm gilt für Produkte, die im medizinischen Bereich bei der Instrumentendesinfektion durch Eintauchen und bei der Oberflächendesinfektion durch Abwischen, Besprühen, Einschwemmen oder auf sonstige Weise verwendet werden.
Diese Europäische Norm gilt für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen z. B. bei der Patientenbetreuung in
   Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich und
   medizinischen Einrichtungen in Schulen, Kindergärten und Heimen
vor und können auch am Arbeitsplatz und im häuslichen Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung von Patienten dienen.
ANMERKUNG 1   Das beschriebene Verfahren dient zur Bestimmung der Wirkung handelsüblicher Zubereitungen oder Wirkstoffe unter den Bedingungen, unter denen sie angewendet werden.
ANMERKUNG 2   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwendungsempfehlungen“ fest.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l’évaluation de l’activité sporicide des désinfectants chimiques utilisés dans le domaine médical - Méthodes d’essai et exigences (phase 2, étape 1)

Le présent document décrit une méthode d’essai et les exigences minimales relatives à l’activité sporicide des désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l’eau dure ou, dans le cas de produits prêts à l’emploi, dans l’eau. Les produits ne peuvent être soumis à l’essai qu’à la concentration de 80 % (97 % avec une méthode modifiée pour les cas particuliers) ou à des concentrations inférieures, car l’ajout des microorganismes d’essai et de la substance interférente s’accompagne forcément d’une dilution.
La présente Norme européenne s’applique aux produits utilisés en médecine dans la désinfection des instruments par immersion, ainsi que la désinfection des surfaces par essuyage, pulvérisation, rinçage ou d’autres moyens.
La présente Norme européenne s’applique dans les domaines et situations où la désinfection est indiquée en médecine humaine. 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 des 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 les conditions d’utilisation.
NOTE 2   Cette méthode correspond à un essai de phase 2, étape 1.
L’EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje sporocidnega delovanja kemičnih razkužil v humani medicini - Preskusna metoda in zahteve (faza 2, stopnja 1)

Ta evropski standard določa preskusno metodo in minimalne zahteve za sporocidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma z vodo pri proizvodih, ki so pripravljeni za uporabo. Proizvode je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji (s prilagojeno metodo v posebnih primerih 97-odstotni), ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje.  Ta evropski standard se uporablja za proizvode, ki se uporabljajo na področju zdravstva pri razkuževanju instrumentov s potapljanjem, površinskem razkuževanju z brisanjem, pršenjem, zalivanjem ali na druge načine. Ta evropski standard se uporablja za področja in primere, ko obstajajo zdravniške indikacije za razkuževanje ali antisepso. Te indikacije se pojavljajo pri negi bolnikov, na primer: – v bolnišnicah, javnih zdravstvenih in zobozdravstvenih ustanovah;
– v ambulantah šol, vrtcev in domov za starejše;
ter se lahko pojavijo na delovnem mestu ali doma. Vključujejo lahko tudi storitve, kot so pralnice in kuhinje, ki proizvode dostavljajo neposredno bolnikom. OPOMBA 1: Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo. OPOMBA 2: Ta metoda ustreza preskusu stopnje 1 faze 2. EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo.

General Information

• Status: Published
• Public Enquiry End Date: 31-Jul-2017
• Publication Date: 08-Jan-2019
• ICS: 11.080.20 - Disinfectants and antiseptics
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: KDS - Cosmetics, chemical disinfectants and surface active agents
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 05-Dec-2018
• Due Date: 09-Feb-2019
• Completion Date: 09-Jan-2019
• Directive: 93/38/EEC - Procurement procedures of entities operating in the water, energy, transport and telecommunications sectors
• Ref Project: EN 17126:2018 - Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants in the medical area - Test method and requirements (phase 2, step 1)

Overview

EN 17126:2018 (CEN) specifies a quantitative suspension test for evaluating the sporicidal activity of chemical disinfectants and antiseptics in the medical area (phase 2, step 1). The standard defines test method(s), minimum performance requirements and practical test conditions for products that form a homogeneous, physically stable preparation when diluted with hard water (or with water for ready‑to‑use products). Products may be tested at concentrations ≤ 80 % (97 % with a modified method for special cases). The method is intended to determine activity of commercial formulations under use‑relevant laboratory conditions.

Key Topics and Technical Requirements

• Test objective: Quantify sporicidal effect against defined spore‑forming organisms under controlled interference and temperature conditions.
• Minimum efficacy: Product shall demonstrate at least 4 decimal log (log10) reduction of spores under the specified conditions.
• Core test organisms: Clostridium difficile, Bacillus subtilis, Bacillus cereus (additional organisms may be used per manufacturer recommendation).
• Interfering substances: Simulates clean and dirty conditions:
  • Clean: 0.3 g/L bovine albumin
  • Dirty: 3.0 g/L bovine albumin + 3.0 mL/L erythrocytes
• Test temperatures and contact times: Set to reflect practical use; surface disinfectants intended for patient‑contact or frequently touched surfaces are tested with a maximum 15‑minute contact time, other applications up to 60 minutes. Exact temperature/contact time are chosen per application and manufacturer instructions.
• Methods described: dilution‑neutralization, membrane filtration, and modified methods for ready‑to‑use products. Neutralization and suppression must be validated.
• Product type constraints: Applies to immersion instrument disinfectants and surface/textile disinfectants used by wiping, spraying, flooding, etc. Products must be stable when diluted.

Applications and Who Uses This Standard

• Intended for manufacturers (formulation development and claims substantiation), independent test laboratories, and regulators assessing sporicidal claims for medical/healthcare products.
• Useful for infection prevention and control professionals, procurement officers, and clinical engineers to verify that disinfectants meet defined sporicidal performance for hospitals, dental clinics, nursing homes, school clinics, laundries and catering services supplying patient‑use items.
• Supports labeling and “use recommendations” when combined with other CEN standards.

Related Standards

• EN 14885 - Application of European Standards for chemical disinfectants and antiseptics (relationship between tests and use recommendations).
• EN 12353 - Preservation of test organisms used for activity determination.

Keywords: EN 17126:2018, sporicidal activity, chemical disinfectants, quantitative suspension test, phase 2 step 1, medical area, Clostridium difficile, dilution‑neutralization, membrane filtration.

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2019
.HPLþQDUD]NXåLODLQDQWLVHSWLNL.YDQWLWDWLYQLVXVSHQ]LMVNLSUHVNXV]D
YUHGQRWHQMHVSRURFLGQHJDGHORYDQMDNHPLþQLKUD]NXåLOYKXPDQLPHGLFLQL
3UHVNXVQDPHWRGDLQ]DKWHYHID]DVWRSQMD
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of sporicidal activity of chemical disinfectants in the medical area - Test method and
requirements (phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der sporiziden Wirkung im humanmedizinischen Bereich - Prüfverfahren
und Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l’évaluation de l’activité sporicide des désinfectants chimiques utilisés dans le domaine
médical - Méthodes d’essai et exigences (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 17126:2018
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17126
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2018
EUROPÄISCHE NORM
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of sporicidal activity of
chemical disinfectants in the medical area - Test method
and requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der
sporicide des désinfectants chimiques utilisés dans le sporiziden Wirkung im humanmedizinischen Bereich -
domaine médical - Méthodes d'essai et exigences Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
(phase 2, étape 1)
This European Standard was approved by CEN on 28 August 2018.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and 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
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17126:2018 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Requirements . 6
5 Test method . 7
5.1 Principle . 7
5.2 Materials and reagents . 8
5.2.1 Test organisms . 8
5.2.2 Culture media and reagents . 8
5.3 Apparatus and glassware . 14
5.3.1 General . 14
5.3.2 Usual microbiological laboratory equipment . 14
5.4 Preparation of test organism suspensions and product test solutions . 16
5.4.1 Test organism suspensions (test and validation suspension) . 16
5.4.2 Product test solutions . 21
5.5 Procedure for assessing the sporicidal activity of the product . 21
5.5.1 General . 21
5.5.2 Dilution-neutralization method . 23
5.5.3 Membrane filtration method . 25
5.5.4 Modified method for ready-to-use products . 27
5.6 Experimental data and calculation . 29
5.6.1 Explanation of terms and abbreviations . 29
5.6.2 Calculation . 30
5.7 Verification of methodology . 34
5.7.1 General . 34
5.7.2 Control of weighted mean counts . 34
5.7.3 Basic limits . 34
5.8 Expression of results and precision . 35
5.8.1 Reduction . 35
5.8.2 Control of active and non-active product test solution (5.4.2) . 35
5.8.3 Limiting test organism and sporicidal concentration . 35
5.8.4 Precision, repetitions . 36
5.9 Interpretation of results - conclusion . 36
5.9.1 General . 36
5.9.2 Sporicidal activity for surface disinfection products . 36
5.9.3 Sporicidal activity for instrument disinfection products . 36
5.9.4 Sporicidal activity for textile disinfection products . 36
5.9.5 Qualification for certain fields of application . 36
5.10 Test report . 36
Annex A (informative) Referenced strains in national collections . 39
Annex B (informative) Neutralizers and Rinsing Liquids . 40
Annex C (informative) Graphical representation of test procedures . 42
C.1 Dilution-neutralization method . 42
C.2 Membrane filtration method . 44
C.3 Dilution-neutralization method (modified method for ready-to-use products) . 46
C.4 Membrane filtration method (modified method for ready-to-use products) . 48
Annex D (informative) Example of a typical test report . 50
Annex E (informative) Precision of the test result. 54
Annex F (informative)  Graphical representation of spore preparation . 55
Annex G (informative) Guide for preparation of reference test solution . 56
Annex H (informative) Example for a titration Peracetic acids and hydrogen peroxide . 57
H.1 General . 57
H.2 Equipment . 57
H.3 Procedure . 57
Bibliography . 59

European foreword
This document (EN 17126:2018) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by June 2019, and conflicting national standards shall be
withdrawn at the latest by June 2019.
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.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.

Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant has
a sporicidal 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 may
influence its action in practical situations.
Each utilization concentration of the chemical disinfectant found by this test corresponds to the chosen
experimental conditions.
1 Scope
This document specifies a test method and the minimum requirements for sporicidal activity of
chemical disinfectant 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 European Standard applies to products that are used in the medical area in the fields of instrument
disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means.
This European Standard applies to areas and situations where disinfection 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 may occur in the workplace and in the home. It may 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
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
4 Requirements
The product shall demonstrate at least 4 decimal log (lg) reduction, when tested in accordance with
Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Instrument
Surface disinfection Textile disinfection
Test Conditions disinfection
Minimum spectrum of
test organisms
sporicidal activity
against Clostridium Clostridium difficile Clostridium difficile Clostridium difficile

difficile
Bacillus subtilis and Bacillus subtilis and Bacillus subtilis and
sporicidal activity
Bacillus cereus Bacillus cereus Bacillus cereus
Additional Any relevant test organism
according to the manufacturer´s recommendation, but between
Test temperature
4 °C and 30 °C 20 °C and 70 °C 20 °C and 80 °C
Contact time according to the manufacturer’s recommendation, but no longer than
15 min or 60 min 60 min
a
60 min
Interfering substance
clean conditions 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine
albumin solution albumin solution albumin solution
and/or and/or and/or
dirty conditions 3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine
albumin solution plus albumin solution plus albumin solution plus
3,0 ml/l erythrocytes 3,0 ml/l erythrocytes 3,0 ml/l erythrocytes
Additional any relevant substance any relevant substance any relevant substance
a
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 15 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.
NOTE For the additional conditions, the concentration defined as a result can be lower than the one
obtained under the obligatory 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 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 sporicidal action in this portion is immediately neutralized or
suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable
neutralizer cannot be found, membrane filtration is used. The numbers of surviving spores in each
sample are determined and the reduction is calculated.
5.1.2 The test is performed using spores of Clostridium difficile for a sporicidal activity
against Clostridium difficile and/or Bacillus subtilis and Bacillus cereus for sporicidal activity (Clause 4,
Table 1).
5.1.3 Additional and optional contact times and temperatures are specified (Clause 4, Table 1).
Additional interfering substances and test organisms may be used.
5.2 Materials and reagents
5.2.1 Test organisms
The sporicidal activity shall be evaluated using the following strains as test organisms selected
1)
according to Clause 4 (Table 1) .
a) Clostridium difficile R027 NCTC 13366
b) Bacillus subtilis ATCC 6633
c) Bacillus cereus CIP 105151
NOTE See Annex A for strain reference in some other culture collections.
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3).
The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test
and its control and validation.
If additional test organisms are used, they shall be incubated under optimum growth conditions
(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms
selected do not correspond to the specified strains, their suitability for supplying the required inocula
shall be verified. If these additional test organisms are not classified at a reference centre, their
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or
national culture collection under a reference for five years.
5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this European Standard refer to the anhydrous salts.
Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for
consequent molecular weight differences.
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.

1) The NCTC, CIP and ATCC numbers are the collection numbers of strains supplied by these culture collections.
This information is given for the convenience of users of this European Standard and does not constitute an
endorsement by CEN of the product named.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at 20 °C ± 1 °C.
5.2.2.2 Water
The water shall be free from substances that are toxic or inhibiting to the bacterial spores or to the
bacteria. It shall be freshly glass distilled water or deionized water.
Sterilize in the autoclave [5.3.2.1a)]. Sterilization is not necessary if the water is used e. g. for
preparation of culture media and subsequently sterilized.
NOTE See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Culture media for spore forming bacteria
a) BHIYT-L Agar for Clostridium difficile
BHIYT-L agar, consisting of:
Brain heart infusion 37,0 g
Yeast extract 5,0 g
L-Cysteine 1,0 g
Sodium taurocholate 1,0 g
Agar 15,0 g
Water (5.2.2.2.) to 1000,0 ml
Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH (5.3.2.4) of the medium shall be
equivalent to 7,0 ± 0,2. Let the medium cool down to 48 °C ± 2 °C. Dissolve 200 000 units of lysozyme in
10 ml water (5.2.2.2). Sterilize the enzymatic solution by membrane filtration (5.3.2.7).
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add
neutralizer to BHIYT-L. Annex B gives guidance on the neutralizers that may be used. It is
recommended not to use a neutralizer that causes opalescence in the agar.
b) Tryptone Soya Agar (TSA) for Bacillus species
Tryptone soya agar, consisting of:
Tryptone, pancreatic digest of casein 15,0 g
Soya peptone, papaic digest of Soybean meal 5,0 g
Sodium chloride (NaCl) 5,0 g
Agar 15,0 g
Water (5.2.2.2) to 1000,0 ml
Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH (5.3.2.4) of the medium shall be
equivalent to 7,2 ± 0,2. This agar should be used for counting of viable Bacillus spores.
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add
neutralizer to TSA. Annex B gives guidance on the neutralizers that may be used. It is recommended not
to use a neutralizer that causes opalescence in the agar.
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 1000,0 ml
Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH (5.3.2.4) of the medium 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.
NOTE 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.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is
given in Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 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 1000 ml. Sterilize by membrane filtration (5.3.2.7) or in the
autoclave [5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to 1000
ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (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
1000 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 1000 ml volumetric flask (5.3.2.12) and add 6,0 ml
(5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1000 ml with water (5.2.2.2). The
pH of the hard water shall be 7,0 ± 0,2, when measured at 20 °C ± 1 °C (5.3.2.4). If necessary, adjust
the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or
approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water
produces a different final water hardness in each test tube. In any case the final hardness expressed as calcium
carbonate (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 case of the modified method, 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).
Prepare at least 8,0 ml fresh sterile 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
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(5.2.2.4). Repeat this procedure at least 3 times, until the supernatant is colourless.
Resuspend 3,0 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 days in a refrigerator (5.3.2.8).
The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be
3,0 g/l and 3,0 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 in general 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
(5.2.2.4);
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.
5.2.2.10 Sporulation media
5.2.2.10.1 Brain Heart Infusion
Brain heart infusion, consisting of:
Brain infusion solids 12,5 g
Beef heart infusion solids 5,0 g
Proteose peptone 10,0 g
Glucose (C H O ) 2,0 g
6 12 6
Sodium chloride (NaCl) 5,0 g
Disodium hydrogen phosphate (Na HPO ) 2,5 g
2 4
Water (5.2.2.2) to 1000,0 ml
Sterilize in the autoclave [5.3.2.1. a)]. After sterilization the pH (5.3.2.4) of the medium shall be
equivalent to 7,4 ± 0,2.
5.2.2.10.2 Columbia Broth
Columbia broth, consisting of:
Pancreatic digest of casein 10,0 g
Yeast extract 5,0 g
Proteose peptone No. 3 5,0 g
Tryptic digest of beef heart 3,0 g
L-cysteine HCl 0,1 g
Dextrose (D-glucose) 2,5 g
Sodium chloride (NaCl) 5,0 g
Magnesium Sulfate (MgSO ) (anhydrous) 0,1 g
Ferrous sulfate (FeSO ) 0,02 g
Sodium carbonate (Na CO ) 0,6 g
2 3
Tris (hydroxmethyl) aminomethane (C H NO ) 0,83 g
4 11 3
Tris (hydroxmethyl) aminomethane HCL 2,86 g
Water (5.2.2.2) to 1000,0 ml
Sterilize in the autoclave [5.3.2.1. a)]. After sterilization the pH (5.3.2.4) of the medium shall be
equivalent to 7,5 ± 0,2.
5.2.2.10.3 Liquid Sporulation Medium
Liquid sporulation medium for preparation of Clostridium difficile spores, consisting of:
Prepare 1 L of the medium in a 2 L Erlenmeyer flask by adding the following in order given:
Water (5.2.2.2.) 700,0 ml
2) 10,0 g
Special peptone
2) Special peptone is a commercial available medium with a specially designed mixture of peptones, consisting
of: Total nitrogen 11,7 g, Amino nitrogen 3,8 g, Sodium chloride (NaCl) 3,5 g.
Potassium dihydrogenphosphate (KH PO ) 2,60 g
2 4
Ammonium sulfate [(NH ) SO ] 0,60 g
4 2 4
Calcium chloride monohydrate (CaCl ×H O) 0,08 g
2 2
Yeast extract powder 10,0 g
Potassium carbonate (K CO ) 3,48 g
2 3
Magnesium sulfate (MgSO ) 0,12 g
Water (5.2.2.2.) to 1000,0 ml
The pH (5.3.2.4) of the medium shall be 7,9 ± 0,2 before sterilization. If needed the adjustment should
be performed with KOH or HCl. Sterilize in the autoclave [5.3.2.1. a)] for 15 min at 121 °C.
5.2.2.10.4 Sodium Phosphate Buffer (0,1 M)
Sodium phosphate buffer (1 M), consisting of:
Disodium hydrogen phosphate (Na HPO ) 8,19 g
2 4
Sodium dihydrogen phosphate monohydrate (NaH PO ) 5,84 g
2 4
Water (5.2.2.2.) to 1000,0 ml
Sterilize in the autoclave [5.3.2.1. a)]. After sterilization the pH (5.3.2.4) of the medium shall to
equivalent to 7,0 ± 0,2.
5.2.2.10.5 Enzymatic Buffer
Enzymatic buffer, consisting of:
800 units of lysozyme and 250 units of trypsin per mg wet weight to 25 ml of 0,1 M Sodium phosphate
buffer (5.2.2.10.4.).
Sterilize by membrane filtration (5.3.2.7).
5.2.2.10.6 Tryptone Glucose Broth (TGB)
Tryptone glucose broth for preparation of the inoculum of Bacillus species, consisting of:
Yeast extract 2,5 g
Tryptone 5,0 g
Glucose (C H O ) 1,0 g
6 12 6
Water (5.2.2.2) to 1000,0 ml
Distribute in test tubes at a rate of 10 ml per tube. Sterilize in the autoclave [5.3.2.1. a)]. After
sterilization the pH (5.3.2.4) of the medium shall be equivalent to 7,2 ± 0,2 .
5.2.2.10.7 Meat Yeast extract Agar (MYA)
Meat yeast extract agar for preparation of Bacillus species spores consisting of:
Meat extract 10,0 g
Yeast extract 2,0 g
Manganese sulfate tetrahydrate (MnSO ×4H O) 0,053 g
4 2
Agar 15,0 g
Water (5.2.2.2) to 1000,0 ml
Distribute in sterile cell culture flasks with vented cap. Sterilize in the autoclave [5.3.2.1 a)] and allow to
cool to 55 °C before introducing into the flasks. After sterilization the pH of the medium shall be
equivalent to 7,0 ± 0,2.
5.2.2.11 Reference glutardialdehyde (Glutaral, 1,5-Pentanedial) CAS Number 111-30-8
Required chemical and physical parameters for use as reference standard for testing disinfectant
preparations are defined in Table 2.
Table 2 — Required chemical and physical parameters
Parameters Specifications
Solution 50 % Solution 25 %
a
clear liquid clear liquid
Appearance
pH-value 3,1 to 4,5 3,1 to 4,5
Concentration of glutardialdehyde (by
50,0 % to 52,0 % 25,0 % to 26,0 %
titration)
Stability (ambient conditions) 12 months 12 months
a
Yellowish colour indicates a beginning polymerization.
The specification above (see Table 2) should be checked on the certificate of analyses. The pH values
shall be tested and confirmed regularly by the laboratory. In case pH values and/or appearance are out
of specification the glutardialdehyde cannot be used anymore.
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)].
3)
5.3.2 Usual microbiological laboratory equipment
and, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)
+3
a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C
for a minimum holding time of 15 min;
+5
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 (160 ) °C for a
0 0
minimum holding time of 2 h.
3) Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C, at 45 °C ± 1 °C, at 70 °C ± 1 °C and
75 °C ± 1 °C (to maintain melted agar in case of pour plate technique, to perform the enzymatic
digestion during the spore preparation and for heat inactivation of harvest spores) and at additional
test temperatures ± 1 °C (5.5.1)
5.3.2.3 Incubator, capable of being controlled either at 36 °C ± 1 °C or 37 °C ± 1 °C (5.2.1). The
same temperature shall be used for incubations performed during a test and its control and validation.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 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 a) or b)) and sporulation media (5.2.2.10).
5.3.2.5 Stopwatch.
5.3.2.6 Shakers
® 4)
a) Electromechanical agitator, e. g. Vortex mixer
b) Mechanical shaker.
5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter
47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7), bovine albumin
(5.2.2.8.2, 5.2.2.8.3 and 5.2.2.8.4), and if the membrane filtration method is used (5.5.3).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml, 2 ml and 1 ml and 0,1 ml, or calibrated
automatic pipettes.
5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads (Diameter 3 mm to 4 mm).
5.3.2.12 Volumetric flasks.
5.3.2.13 Centrifuge, capable to being controlled at 2 °C to 8 °C (4000g ).
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5.3.2.14 Anaerobic jar with a gas generating kit or other anaerobic systems
5.3.2.15 Microscope, obligatory a phase-contrast type with magnification of at least 400x
5.3.2.16 Ice producing machine or commercially available ice
®
4) Vortex is an example of a suitable product available commercially. This information is given for the
convenience of users of this European Standard and does not constitute an endorsement by CEN of this product.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
5.4.1.1 General
For each test organism, two different suspensions have to be prepared: the “test suspension” to perform
the test and the “validation suspension” to perform the controls and method validation
5.4.1.2 Preservation and stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and maintained according to EN 12353.
The pellet of the freeze dried sample of Clostridium species should be rehydrated in BHI (5.2.2.10.1).
The inoculation should be done on BHIYT-L without Taurocholic acid sodium salt hydrate and
Lysozyme [5.2.2.3 a)] and incubate in an anaerobic jar (5.3.2.14) for 72 to 78 h.
The pellet of the freeze dried sample of Bacillus species should be rehydrated in TGB (5.2.2.10.6). The
inoculation should be done on TSA [5.2.2.3 b)].
5.4.1.3 Preparation of spore stock suspension
5.4.1.3.1 Preparation of Clostridium spore stock suspension (see Annex F)
Day 1: In order to prepare the spore stock suspension of the test organisms (5.2.1), prepare a
subculture from the stock culture (5.4.1.2) by streaking onto BHIYT-L agar [5.2.2.3 a)] slopes or plates
and incubate (5.3.2.3). Incubate for 48 h in an anaerobic jar (5.3.2.14) until the colonies are approx. 4
mm in diameter (see Annex F). Never produce and use a second subculture.
Day 2: Prepare a solution of columbia broth (5.2.2.10.2) following the manufacturer´s instructions and
sterilize by autoclaving. Keep one 15 ml open screw cap plastic tube containing 5 ml of columbia broth
(5.2.2.10.2) in an anaerobic jar (5.3.2.14) overnight (minimum 12 h) at room temperature to pre-reduce
oxygen level.
Day 3: Pick an isolated colony from the inoculated BHIYT-L plate [5.2.2.3 a)] and suspend it in the tube
containing 5 ml pre-reduced columbia broth (5.2.2.10.2). Incubate the tube anaerobically at 36 °C ± 1 °C
or 37 °C ± 1 °C for 24 h.
Place 50 ml open screw cap plastic tubes containing 20 ml of columbia broth (5.2.2.10.2) in an
anaerobic jar (5.3.2.14). Maintain at room temperature for minimum 12 h (overnight) to pre-reduce
oxygen level. Prepare one tube for each flask to be inoculated at day 4.
Day 4: Inoculate 50 µl of the 24 h culture into each 50 ml plastic tube containing 20 ml of pre-reduced
columbia broth (5.2.2.10.2). Incubate anaerobically at 36 °C ± 1 °C or 37 °C ± 1 °C for 20 h (open screw-
cap).
Prepare liquid sporulation medium by adding the ingredients in the order given [5.2.2.10.3.]. Add 500
ml of the medium into each 1 L flask and autoclave for 15 min at 121 °C. Wait for the temperature to
drop down 50 °C to 60 °C, then place the flaks in anaerobic jars (5.3.2.14) and incubate for 24 h at 36 °C
± 1 °C or 37 °C ± 1 °C to pre-reduce the oxygen level of the medium. Measure the pH at the end of this
period, it should be 8,2 ± 0,3.
Day 5: Pour the entire inoculum from a 20 ml plastic tube into a 500 ml culture flask with the liquid
sporulation medium (5.2.2.10.3). Incubate the flasks anaerobically for 10 days at 36 °C ± 1 °C or
37 °C ± 1 °C.
Day 15: Divide the sporulated culture from each flask equally into two 250 ml centrifuge tubes and
centrifuge the suspensions at 4000g for 10 min. Discard the supernatant and resuspend the sediment
N
from each 250 ml tube with 50 ml of deionized water [5.2.2.2.] and pool the resuspend material into
one 250 ml centrifuge tube. Adjust the volume to 250 ml with water (5.2.2.2.) and centrifuge for 10 min
at 4000g . Wash the pellet by centrifugation two more times with 250 ml water (5.2.2.2.) in each wash.
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After the third centrifugation step resuspend the pellet in 15 ml water (5.2.2.2) and transfer it into a
pre-weighed 50 ml centrifuge tube (to determine the wet weight of the pellet). Wash the original
centrifuge tube twice with 10 ml water (5.2.2.2) and collect the flushing liquid in the pre-weighed
centrifuge tube (end volume of 35 ml). After a further centrifugation step at 4000g for 10 min the
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pellet shall be weighed to determine the wet weight. Store the pellet overnight in the refrigerator
(5.3.2.8).
NOTE 1 The acceleration of centrifugation (e.g. 4000g ) and the volume of the centrifugation tube (< 250 ml)
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can be changed; if a better quality of spore purification can be achieved.
Day 16: For enzymatic digestion of remaining vegetative cells and cell debris resuspend the pellet in 10
ml of 0,1 M sodium phosphate buffer (5.2.2.10.4). If the wet weight of the pellet is higher than 700 mg,
divide the suspension equally into two separate tubes and adjust the volume in each to 10 ml with 0,1 M
sodium phosphate buffer (5.2.2.10.4). Mix well using shaker [5.3.2.6 a)]. Add 25 ml of a freshly prepared
mixture of enzymes as described in 5.2.2.10.5 and mix gently, but do not shake. Sonicate the suspension
in a ultrasonic water bath for 5 min and then incubate at 45 °C ± 1 °C for 6 h (5.3.2.2), repeating the
sonication step after 2 ± 0,2 h, 4 ± 0,2 h, 6 ± 0,2 h of incubation to break up the clumps. Store the
suspension refrigerated overnight (5.3.2.8.).
Day 17: Purification of Clostridium spore stock suspension
Centrifuge the suspension at 4000g for 10 min and wash the pellet three times with 10 ml of water
N
[5.2.2.2] in each wash. Resuspend the pellet in 30 ml of water [5.2.2.2] and heat it for 10 min in a water
bath at 70 °C ± 1 °C.
NOTE 2 Insert a thermometer in an identical tube with
...