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 European Standard specifies a test method and the minimum requirements for sporicidal 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 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 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 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)

Diese Europäische Norm legt ein Prüfverfahren für und die Mindestanforderungen an die sporizide Wirkung von chemischen Desinfektionsmitteln und Antiseptika 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 sind 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 Produkte für die direkte Patientenversorgung liefern.
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)

La présente Norme européenne 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, inondation 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
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
05-Dec-2018
Due Date
09-Feb-2019
Completion Date
09-Jan-2019

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SLOVENSKI STANDARD
SIST EN 17126:2019
01-februar-2019
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YUHGQRWHQMHVSRURFLGQHJDGHORYDQMDNHPLþQLKUD]NXåLOYKXPDQLPHGLFLQL
3UHVNXVQDPHWRGDLQ]DKWHYH ID]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
SIST EN 17126:2019 en,fr,de

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

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SIST EN 17126:2019
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SIST EN 17126:2019
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.
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EN 17126:2018 (E)
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

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EN 17126:2018 (E)

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

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SIST EN 17126:2019
EN 17126:2018 (E)
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.
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EN 17126:2018 (E)
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.
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EN 17126:2018 (E)
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.
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EN 17126:2018 (E)
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
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

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

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EN 17126:2018 (E)

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

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.
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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:
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EN 17126:2018 (E)
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.

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SIST EN 17126:2019
EN 17126:2018 (E)

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

(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 accord
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