prEN 17846
(Main)Chemical disinfectants and antiseptics - Quantitative test method for the evaluation of sporicidal activity against Clostridioides difficile on non-porous surfaces with mechanical action employing wipes in the medical area (4- field test) - Test method and requirements (phase 2, step 2)
Chemical disinfectants and antiseptics - Quantitative test method for the evaluation of sporicidal activity against Clostridioides difficile on non-porous surfaces with mechanical action employing wipes in the medical area (4- field test) - Test method and requirements (phase 2, step 2)
This document specifies a test method and the minimum requirements for sporicidal activity against spores of Clostridioides difficile of chemical disinfectant products that form a homogeneous, physically stable preparation when diluted with hard water - or in the case of ready-to-use products - with water.
This document applies to products that are used in the medical area for disinfecting non-porous surfaces including surfaces of medical devices by wiping - regardless if they are covered by the 93/42/EEC Directive on Medical Devices or not.
Due to the new methods of application of surface disinfectants like pre-impregnated wipes this document was established to cover the different application method.
The document is applicable for four method of application of products for wiping and/or mopping:
a) soaking any non-specified wipe or mop with product;
b) spraying the product on any non-specified wipe and / or mop or a specified wipe or mop;
c) impregnation of specified wipes or mops by the user with the product according to the manufacturer’s recommendation;
d) preimpregnation of specified wipes or mops by the manufacturer as ready-to-use wipes or mops.
In all types of application the water control has to be done with the standard wipe [5.3.2.17 a)], because it is a process or method control.
This document does not apply to products that are sprayed on or flooding surfaces, then left until the contact application phase 2, step 2 standards without mechanical action should be used and their methods performed.
The test surface (5.3.2.16) was selected as standard surface and should cover all non-porous surfaces. It was not intended to cover the influence of each different surface.
This document 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 This method corresponds to a phase 2, step 2 test.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendations".
Chemische Desinfektionsmittel und Antiseptika - Quantitatives Prüfverfahren zur Bestimmung der sporiziden Wirkung gegen Clostridioides difficile auf nicht-porösen Oberflächen mit mechanischer Einwirkung mit Hilfe von Tüchern im humanmedizinischen Bereich (4‑Felder-Test) - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die sporizide Wirkung gegen Sporen von Clostridioides difficile von chemischen Desinfektionsmitteln fest, die bei Verdünnung mit Wasser standardisierter Härte - bzw. bei gebrauchsfertigen Produkten bei der Verdünnung mit Wasser - als homogene, physikalisch stabile Zubereitung vorliegen.
Dieses Dokument gilt für Produkte, die im medizinischen Bereich zur Desinfektion von nicht-porösen Oberflächen, einschließlich Oberflächen auf Medizinprodukten, durch Abwischen verwendet werden - unabhängig davon, ob sie in der Richtlinie 93/42/EWG über Medizinprodukte erfasst sind oder nicht.
Aufgrund der neuen Verfahren zur Anwendung von Oberflächendesinfektionsmitteln wie vorgetränkten Tüchern wurde dieses Dokument erstellt, um die verschiedenen Anwendungsmethoden zu behandeln.
Das Dokument gilt für vier Verfahren zur Anwendung von Produkten zum Wischen und/oder Abputzen:
a) Tränken eines nicht-spezifizierten Tuchs oder Putzlappens mit dem Produkt;
b) Sprühen des Produkts auf ein nicht-spezifiziertes Tuch und/oder einen Putzlappen oder ein spezifiziertes Tuch oder Putzlappen;
c) Tränken spezifizierter Tücher oder Putzlappen mit dem Produkt entsprechend den Herstellerempfehlungen durch den Anwender;
d) Vortränken spezifizierter Tücher oder Putzlappen als gebrauchsfertige Tücher oder Putzlappen durch den Hersteller.
Bei allen Anwendungsarten muss mit dem Standardtuch [5.3.2.17 a)] der Kontrollversuch mit Wasser durchgeführt, da es sich um eine Prozess- oder Verfahrenskontrolle handelt.
Dieses Dokument gilt nicht für Produkte, die auf Oberflächen gesprüht oder geschwemmt und bis zur Einwirkungsanwendung darauf belassen werden; dann sollten Normen der Phase 2, Stufe 2 ohne mechanische Einwirkung und die zugehörigen Verfahren durchgeführt werden.
Die Prüffläche (5.3.2.16) wurde als Standardoberfläche gewählt und sollte alle nicht-porösen Oberflächen abdecken. Es war nicht beabsichtigt, den Einfluss der einzelnen unterschiedlichen Oberflächen zu behandeln.
Dieses Dokument 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,
- 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 der Patienten dienen.
ANMERKUNG Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den "Anwendungsempfehlungen" fest.
Antiseptiques et désinfectants chimiques - Méthode d’essai quantitative pour l’évaluation de l’activité sporicide contre Clostrididioides difficile sur des surfaces non poreuses, avec action mécanique à l’aide de lingettes dans le domaine médical (essai à 4 zones) - Méthode d'essai et prescriptions (phase 2, étape 2)
Le présent document spécifie une méthode d’essai et les exigences minimales relatives à l’activité sporicide contre les spores de Clostridioides difficile des désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans l'eau dure ou - dans le cas de produits prêts à l’emploi - dans l’eau.
Ce document s’applique aux produits utilisés dans le secteur médical pour désinfecter les surfaces non poreuses, y compris les surfaces de dispositifs médicaux par essuyage - qu’elles soient couvertes ou non par la directive 93/42/CEE relative aux dispositifs médicaux.
En raison des nouvelles méthodes d’application de désinfectants de surface, par exemple les lingettes pré-imprégnées, ce document a été rédigé pour couvrir les différentes méthodes d’application.
Le document est applicable pour quatre méthodes d’application de produits pour l’essuyage et/ou le lavage :
a) imprégnation d’une lingette ou lavette non spécifiée avec le produit ;
b) pulvérisation du produit sur une lingette et/ou lavette non spécifiée ou sur une lingette ou lavette spécifiée ;
c) imprégnation des lingettes ou lavettes spécifiées par l’utilisateur avec le produit, conformément aux recommandations du fabricant ;
d) pré-imprégnation des lingettes ou lavettes spécifiées par le fabricant sous forme de lingettes ou lavettes prêtes à l’emploi.
Dans tous les types d’application, le contrôle de l’eau doit être effectué avec la lingette standard [5.3.2.17 a)], car il s’agit d’un contrôle du processus ou de la méthode.
Ce document ne s’applique pas aux produits pulvérisés sur des surfaces inondables puis laissés jusqu’à ce que les normes de phase 2, étape 2 sur l’application de contact sans action mécanique soient utilisées et leurs méthodes effectuées.
La surface d’essai (5.3.2.16) a été choisie comme surface standard et il convient qu’elle englobe toutes les surfaces non poreuses. Le présent document n’est pas destiné à traiter de l’influence de chaque surface différente.
Le présent document s’applique aux secteurs et situations où la désinfection est médicalement préconisée. Ces indications relèvent des soins aux patients, par exemple :
- dans les hôpitaux, les établissements médicaux, les centres de soins dentaires ;
- dans des infirmeries d’écoles, de jardins d’enfants et de crèches ;
et peuvent également concerner le lieu de travail et le domicile. Il peut également s’agir de services, comme des blanchisseries ou des cuisines, fournissant directement des produits pour le patient.
NOTE Cette méthode correspond à un essai de type phase 2, étape 2.
L’EN 14885 précise de manière détaillée la relation entre les différents essais et les « recommandations d’utilisation ».
Kemična razkužila in antiseptiki - Kvantitativna preskusna metoda za vrednotenje sporocidnega delovanja na Clostridioides difficile na neporoznih površinah z mehanskim delovanjem z odvzemom brisa v medicini (4-področni preskus) - Preskusna metoda in zahteve (faza 2, stopnja 2)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17846:2022
01-junij-2022
[Not translated]
Chemical disinfectants and antiseptics - Quantitative test method for the evaluation of
sporicidal activity against Clostridioides difficile on non-porous surfaces with mechanical
action employing wipes in the medical area (4- field test) - Test method andrequirements (phase 2, step 2)
Chemische Desinfektion und Antiseptika - Quantitativer Prüfversuch zur Bestimmung der
sporiziden Wirkung gegen Clostridioides difficile auf nicht-porösen Oberflächen mit
mechanischer Einwirkung mit Hilfe von Tüchern im humanmedizinischen Bereich (4-Felder-Test) - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Méthode d’essai quantitative pour l’évaluation
de l’activité sporicide contre Clostrididioides difficile sur des surfaces non poreuses, avec
action mécanique à l’aide de lingettes dans le domaine médical (essai à 4 zones) -
Méthode d'essai et prescriptions (phase 2, étape 2)Ta slovenski standard je istoveten z: prEN 17846
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
oSIST prEN 17846:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17846:2022
DRAFT
EUROPEAN STANDARD
prEN 17846
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2022
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative test
method for the evaluation of sporicidal activity against
Clostridioides difficile on non-porous surfaces with
mechanical action employing wipes in the medical area (4-
field test) - Test method and requirements (phase 2, step
Antiseptiques et désinfectants chimiques - Méthode Chemische Desinfektion und Antiseptika -
d'essai quantitative pour l'évaluation de l'activité Quantitativer Prüfversuch zur Bestimmung der
sporicide contre Clostrididioides difficile sur des sporiziden Wirkung gegen Clostridioides difficile auf
surfaces non poreuses, avec action mécanique à l'aide nicht-porösen Oberflächen mit mechanischer
de lingettes dans le domaine médical (essai à 4 zones) - Einwirkung mit Hilfe von Tüchern im
Méthode d'essai et prescriptions (phase 2, étape 2) humanmedizinischen Bereich (4-Felder-Test) -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)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 N O R M A L I S A T IO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17846:2022 E
worldwide for CEN national Members.---------------------- Page: 3 ----------------------
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Contents Page
European foreword ....................................................................................................................................................... 3
Introduction .................................................................................................................................................................... 4
1 Scope .................................................................................................................................................................... 5
2 Normative references .................................................................................................................................... 5
3 Terms and definitions ................................................................................................................................... 6
4 Requirements ................................................................................................................................................... 6
5 Test methods .................................................................................................................................................... 6
5.1 Principle ............................................................................................................................................................. 6
5.2 Materials and reagents .................................................................................................................................. 7
5.2.1 Test organism ................................................................................................................................................... 7
5.2.2 Culture media and reagents ........................................................................................................................ 7
5.3 Apparatus and glassware .......................................................................................................................... 10
5.3.1 General ............................................................................................................................................................. 10
5.3.2 Usual microbiological laboratory equipment ................................................................................... 10
5.4 Preparation of test organism suspensions and product test solutions .................................... 13
5.4.1 Test organism suspensions ...................................................................................................................... 13
5.4.2 Product test solution ................................................................................................................................... 15
5.5 Procedure for assessing the sporicidal activity against C. difficile of the product ............... 15
5.5.1 General ............................................................................................................................................................. 15
5.5.2 Method ............................................................................................................................................................. 17
5.6 Experimental data and calculation ........................................................................................................ 21
5.6.1 Explanation of terms and abbreviations ............................................................................................. 21
5.6.2 Calculation ...................................................................................................................................................... 21
5.7 Verification of methodology ..................................................................................................................... 26
5.7.1 General ............................................................................................................................................................. 26
5.7.2 Control of weighted mean counts ........................................................................................................... 27
5.7.3 Basic limits ..................................................................................................................................................... 27
5.8 Expression of results and precision ...................................................................................................... 27
5.8.1 Overview of the different suspensions / test mixtures .................................................................. 27
5.8.2 V -values ......................................................................................................................................................... 27
5.8.3 Limiting test organism and sporicidal concentration .................................................................... 28
5.8.4 Precision, repetitions ................................................................................................................................. 29
5.9 Interpretation of results – conclusion .................................................................................................. 29
5.10 Test report ...................................................................................................................................................... 29
Annex A (informative) Referenced strains in national collections ........................................................... 32
Annex B (informative) Neutralizers ..................................................................................................................... 33
Annex C (informative) Graphical representations of the test method ..................................................... 35
Annex D (informative) Example of a typical test report................................................................................ 37
Bibliography ................................................................................................................................................................. 42
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European foreword
This document (prEN 17846:2022) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.This document is a working document.
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Introduction
This document specifies a carrier test for establishing whether a chemical disinfectant for use on surfaces
administered with wipes has a sporicidal activity against Clostridioides difficile in the fields described in
the scope.The laboratory test closely simulates practical conditions of application such as contact time,
temperature and interfering substances, including pre-drying specified test organisms on a test-surface
as carrier and wiping the product on the test-surface with a wipe. The conditions are intended to cover
general purposes. However, if for some applications the recommendations of use of a product differ
additional test conditions may be or need to be used.Each utilization concentration of the product found by this test corresponds to defined experimental
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1 Scope
This document specifies a test method and the minimum requirements for sporicidal activity against
spores of Clostridioides difficile of chemical disinfectant products that form a homogeneous, physically
stable preparation when diluted with hard water – or in the case of ready-to-use products – with water.
This document applies to products that are used in the medical area for disinfecting non-porous surfaces
including surfaces of medical devices by wiping – regardless if they are covered by the 93/42/EEC
Directive on Medical Devices or not.Due to the new methods of application of surface disinfectants like pre-impregnated wipes this document
was established to cover the different application method.The document is applicable for four method of application of products for wiping and/or mopping:
a) soaking any non-specified wipe or mop with product;b) spraying the product on any non-specified wipe and / or mop or a specified wipe or mop;
c) impregnation of specified wipes or mops by the user with the product according to the
manufacturer’s recommendation;d) preimpregnation of specified wipes or mops by the manufacturer as ready-to-use wipes or mops.
In all types of application the water control has to be done with the standard wipe [5.3.2.17 a)], because
it is a process or method control.This document does not apply to products that are sprayed on or flooding surfaces, then left until the
contact application phase 2, step 2 standards without mechanical action should be used and their
methods performed.The test surface (5.3.2.16) was selected as standard surface and should cover all non-porous surfaces. It
was not intended to cover the influence of each different surface.This document 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 This method corresponds to a phase 2, step 2 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
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EN 17126, 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)EN 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical
disinfectants and antiseptics3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 apply.
3.1pre-impregnated wipe
ready-to-use wipe
wipe containing disinfectant added by the wipe manufacturer at the manufacturing site
3.2impregnated wipe
wipe containing disinfectant added by the user
Note 1 to entry: Examples include a wipe soaked in disinfectant, a wipe sprayed with disinfectant.
4 RequirementsThe product, when diluted with hard water or – in the case of ready-to-use products – with water, and
tested in accordance with Clause 5 under simulated clean conditions (0,3 g/l bovine albumin) or
simulated dirty conditions (3,0 g/l bovine albumin + 3,0 ml/l sheep erythrocytes) according to its
practical applications and under the following test conditions: temperature between 4 °C and 30 °C,
contact time min. 1 min and max. either 30 min or 60 min shall demonstrate at least a decimal log (lg)
reduction in counts of 4 on test field 1. The mean of the number of cfu per 25 cm on the test fields 2 to 4
shall be equal or less than 50, the mean of the number of cfu on test fields 1 to 4 of the water control shall
be equal or more than 10. Details on the precision and repetition are given in 5.8.4 and EN 14885.
The sporicidal activity against Clostridioides difficile spores shall be evaluated using the following test
organisms: Clostridioide difficile R027.Where indicated, additional specific sporicidal activity shall be determined applying other contact times
and test organisms in order to take into account intended specific use conditions.
NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained
under the minimum test conditions.5 Test methods
5.1 Principle
5.1.1 A test-surface is marked with 4 squares of 5 × 5 cm, the “test fields”, in a row. Test field 1 on the
test-surface is inoculated with a test suspension of Clostridioides difficile (C. difficile) spores in a solution
of interfering substances. The inoculum is dried. A wipe is soaked with a sample of the product as
delivered and/or diluted with hard water (for ready to use products: water). The test-surface is wiped
with the soaked wipe across the four marked test fields, starting in front of test field 1, turning
) See 5.5.1.1 b).---------------------- Page: 8 ----------------------
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immediately after test field 4 and wiped back to the starting point. In parallel a water control is
performed: a wipe is soaked with hard water [5.5.2.2 e)] instead of the product.NOTE For the purposes of this document references to wiping, wipe and wiped can be equated to mopping,
mop and mopped when the standard method is used to test a mopping application. Temperature, soiling and contact
time are employed as recommended by the manufacturer. At the end of the contact time, the test organisms are
recovered from each test field with moistened swabs. The swabs are brought into a tube containing broth and
neutralizer and the test organisms are to be severed from the swab by shaking. The numbers of surviving test
organisms in each sample are determined, and the reduction is calculated by comparing the results of the drying
control D and the results obtained with the product. In parallel to the test with the product water is applied in the
same way to ensure that the test organisms are spread on the 4 fields and their number reaches a certain level. The
test is performed using C. difficile as test organism (minimum test conditions).5.1.2 Additional test organisms (only sporicidal strains), contact times and interfering substances can
be used.5.2 Materials and reagents
5.2.1 Test organism
The sporicidal activity against C. difficile shall be evaluated using the following strain as test organism :
— Clostridioides difficile R027 NCTC 13366See Annex A for strain references in some other culture collections.
If additional test organisms are used, they shall be incubated under optimum growth conditions
(temperature, time, atmosphere and 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.The required incubation temperature for these test bacteria is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3) under
anaerobic conditions. The same temperature (36 °C or 37 °C) and anaerobic conditions shall be used for
all incubations performed during its control and validation.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 organism.To improve reproducibility, it is recommended that commercially available dehydrated material is used
for the preparation of culture media if it complies with the formulas given below. The manufacturer's
instructions relating to the preparation of these products should be rigorously followed.
For each culture medium and reagent a limitation for use should be fixed.) The NCTC numbers are the collection numbers of strains supplied by the National Collection of Type Cultures
(NCTC). This information is given for the convenience of users of this document and does not constitute an
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All specified pH values are measured at 20 °C ± 1 °C (5.3.2.4).
5.2.2.2 Water
The water shall be freshly glass-distilled or deionized and demineralized water. If distilled water or
deionized and demineralized water of adequate quality is not available, water for injections (see [1]) may
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.
5.2.2.3 Medium
a) BHIYT-L Agar
— 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 1 000,0 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.5.2.2.4 Diluent
a) General Diluent
Tryptone Sodium Chloride Solution:
— 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 (5.3.1). After sterilization the pH (5.3.2.4) of the general 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 and 5.5.2. It
shall be sterile.Information on neutralizer that has been found to be suitable for some categories of products is given in
Annex B.5.2.2.6 Sterile defibrinated sheep blood
The sterile defibrinated sheep blood can be acquired from a commercial supplier.
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5.2.2.7 Hard water for dilution of products
a) Hard water general
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 a refrigerator (5.3.2.8)
for no longer than one month.Prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water and dilute to 1 000 ml.
Sterilize by membrane filtration (5.3.2.7). Store the solution in a refrigerator (5.3.2.8) for no longer
than one week.Place 600 ml to 700 ml 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.
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 substances
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids, 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 general
diluent [5.2.2.4 a)].Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within 1 month.
The final concentration of the bovine albumin in the test procedure (5.5) is 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 general
diluent [5.2.2.4 a)].Sterilize by membrane filtration (5.3.2.7).
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oSIST prEN 17846:2022
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Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (5.2.2.6). Centrifuge the sheep blood at 800
g for 10 min. After discarding the supernatant, resuspend erythrocytes in general diluent [5.2.2.4 a)].
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 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 at 2 °C to 8 °C.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.5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.2.1 a)];b) by dry heat, in the hot air oven [5.3.2.1 b)].
5.3.2 Usual microbiological laboratory equipment
and in particular, the following:
5.3.2.1 Apparatus for sterilization:
+ 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 + 5holding time of 30 min, at ( 170 ) °C for a minimum holding time of 1 h or at ( 160 ) °C for a
0 0minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at 20° C ± 1 °C and at 45 °C ± 1 °C [to maintain
melted agar 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 either 36 °C ± 1 °C or at 37 °C ± 1 °C (5.2.1). The
same temperature shall be used for all incubations of the C. difficile spores performed during a test and
its controls 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).5.3.2.5 Stopwatch.
5.3.2.6 Shakers.
Error! Bookmark not defined.
) Disposable sterile equipment is an acceptable alternative to reusable
glassware.
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oSIST prEN 17846:2022
prEN 17846:2022 (E)
® 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) and bovine albumin (5.2.2.8.2
and 5.2.2.8.3).The vacuum source used shall give an even filtration flow rate.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic
pipettes may be used.5.3.2.10 Petri dishes (plates) of size 90 mm to 100 mm. (The Petri dishes are needed for pour plate
technique and for pre-moistening the standard wipes cloth with 16 ml product test solution or hard
water)5.3.2.11 Glass beads (diameter: 3 mm to 4 mm).
5.3.2.12 Volumetric flasks.
5.3.2.13 Centrifug
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