SIST EN 13623:2021

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity against Legionella of chemical disinfectants for aqueous systems - Test method and requirements (phase 2, step 1)

This document specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant products intended to be used for treatment in aqueous systems against Legionella pneumophila that form a homogeneous, physically stable preparation when diluted with buffered ferrous hard water or hard water. Whenever Legionella pneumophila poses a risk to human health, this method is suitable for water used in cooling towers and water for general purposes, like spas, pools, showers and other uses. The method is not suitable for electro-chemical disinfection.
The document applies to products used as a single application shock treatment in order to kill Legionella pneumophila. It is not suitable for the evaluation of those products that are dosed continuously into water systems to control the growth of Legionella pneumophila.
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.
NOTE 3   This method does not take into account the fact that Legionella pneumophila is often found in cells of amoebae and/or biofilms and that thereby a product’s activity against the bacteria may be reduced.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendation".

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung gegen Legionella von chemischen Desinfektionsmitteln für wasserführende Systeme - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Dieses Dokument legt ein Prüfverfahren und die Mindestanforderungen an die bakterizide Wirkung von chemischen Desinfektionsmitteln zur Verwendung bei der Aufbereitung in wasserführenden Systemen gegen Legionella pneumophila fest, die bei Verdünnung mit gepuffertem eisenhaltigem Wasser standardisierter Härte oder Wasser standardisierter Härte als homogenes, physikalisch stabiles Präparat vorliegen. Immer wenn Legionella pneumophila ein Risiko für die menschliche Gesundheit darstellt, ist dieses Verfahren für in Kühltürmen genutztes Wasser und Wasser für allgemeine Zwecke, wie z. B. Bäder, Schwimm  und Bade¬becken, Duschen und andere Zwecke, geeignet. Das Verfahren ist nicht für die elektrochemische Desinfektion geeignet.
Das Dokument gilt für Produkte, die als einmalige Schockbehandlung zur Abtötung von Legionella pneumophila verwendet werden. Es ist nicht geeignet für die Bewertung derjenigen Produkte, die kontinuierlich in Wassersysteme dosiert werden, um das Wachstum von Legionella pneumophila zu kontrollieren.
ANMERKUNG 1   Das beschriebene Verfahren dient zur Bestimmung der Wirksamkeit von handelsüblichen Zube-reitungen oder Wirkstoffen unter den Bedingungen, unter denen sie angewendet werden.
ANMERKUNG 2   Das Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.
ANMERKUNG 3   Das vorliegende Verfahren berücksichtigt nicht die Tatsache, dass Legionella pneumophila oft in Zellen von Amöben und/oder Biofilmen zu finden ist und dass dadurch die Wirksamkeit des Produkts gegen die Bakterien möglicherweise verringert ist.
EN 14885 legt im Einzelnen die Beziehungen der verschiedenen Prüfungen untereinander und zu den „Anwendungsempfehlungen“ fest.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide contre des légionelles des désinfectants chimiques pour les systèmes aqueux - Méthode d'essai et prescriptions (phase 2, étape 1)

Le présent document décrit une méthode d’essai et les prescriptions minimales relatives à l’activité bactéricide des produits désinfectants chimiques destinés au traitement des systèmes aqueux vis-à-vis de Legionella pneumophila et donnant une préparation homogène, physiquement stable quand ils sont dilués dans de l’eau dure ferreuse tamponnée ou dans de l’eau dure. Lorsque Legionella pneumophila présente un risque pour la santé humaine, la présente méthode est adaptée à l’eau utilisée dans les tours de refroidissement et l’eau à usage général telle que l’eau utilisée dans les jacuzzis, les piscines, les douches et autres. La méthode n’est pas adaptée à la désinfection électrochimique.
Le document s’applique aux produits utilisés en traitement de choc à application unique destinés à tuer Legionella pneumophila. Il n’est pas adapté à l’évaluation des produits dosés en continu dans les réseaux d’approvisionnement en eau afin de contrôler la croissance de Legionella pneumophila.
NOTE 1   La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives dans les conditions dans lesquelles elles sont utilisées.
NOTE 2   La présente méthode correspond à un essai de phase 2, étape 1.
NOTE 3   La présente méthode ne tient pas compte du fait que Legionella pneumophila se trouve souvent dans des cellules amibiennes et/ou dans des biofilms, ce qui peut réduire l’activité du produit vis-à-vis des bactéries.
L’EN 14885 spécifie de manière détaillée la relation entre les différents essais et les « recommandations d’emploi ».

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje baktericidnega delovanja kemičnih razkužil za razkuževanje vode na bakterijo Legionella - Preskusna metoda in zahteve (faza 2, stopnja 1)

General Information

• Status: Published
• Public Enquiry End Date: 04-Jun-2018
• Publication Date: 06-Dec-2020
• 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: 16-Sep-2019
• Due Date: 21-Nov-2019
• Completion Date: 07-Dec-2020
• Directive: 93/42/EEC - Council Directive 93/42/EEC of 14 June 1993 concerning medical devices
• Mandate: M/023 - Standardization mandate to CEN/CENELEC concerning the development of European standards relating to medical devices
• Ref Project: EN 13623:2020 - Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity against Legionella of chemical disinfectants for aqueous systems - Test method and requirements (phase 2, step 1)

Relations

• Replaces: SIST EN 13623:2010 - Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity against Legionella of chemical disinfectants for aqueous systems - Test method and requirements (phase 2, step 1)
• Effective Date: 01-Jan-2021

Overview

EN 13623:2020 (CEN) specifies a laboratory quantitative suspension test for evaluating the bactericidal activity of chemical disinfectants against Legionella pneumophila in aqueous systems. It is a Phase 2, Step 1 test method designed for products intended as single shock treatments (not continuous dosing) in systems such as cooling towers, spas, pools, showers and other waters where Legionella poses a health risk. The method applies to homogeneous, physically stable formulations diluted in hard water or buffered ferrous hard water and excludes electro‑chemical disinfection technologies.

Key topics and technical requirements

• Test organism: Legionella pneumophila (obligatory strain ATCC 33152; referenced strains listed in Annex A).
• Efficacy threshold: product shall achieve at least a 4 decimal logarithm (≥ 4 log) reduction of viable Legionella under specified conditions.
• Test temperatures: (20 ± 1) °C and (30 ± 1) °C (obligatory conditions).
• Contact times: 60 min (rapid acting), and additional longer times (2 h, 6 h, 15 h, 40 h, 48 h) depending on product type; 15 h for slower‑acting products.
• Interfering substance: 0.005% yeast extract used in test suspension to simulate organic challenge.
• Diluent: hard water or buffered ferrous hard water as specified.
• Neutralization and recovery: validated dilution‑neutralization is the preferred method; membrane filtration is used if neutralizers are unsuitable.
• Limitations noted: the method does not account for Legionella protected within amoebae or biofilms, and results are linked to defined laboratory conditions (see EN 14885 for use recommendations).
• Documentation: detailed procedures, calculations, verification, and reporting requirements are included; Annexes cover strains, neutralizers and graphical procedures.

Applications

• Supporting product development and label claims for bactericidal activity against Legionella.
• Comparative laboratory evaluation of shock‑treatment disinfectants for cooling water, water for general purposes (pools, spas, showers), and similar aqueous systems.
• Evidence for regulatory submissions, conformity assessment, and quality control in disinfectant manufacturing.

Who uses this standard

• Chemical disinfectant manufacturers and formulation scientists
• Accredited microbiology and testing laboratories
• Water treatment professionals, facility managers (cooling towers, pools, spas)
• Regulators and procurement specialists assessing disinfectant efficacy

Related standards

• EN 14885 - application of European standards for disinfectants and antiseptics (relationship of tests to use recommendations).
• EN 12353 and other referenced methods for organism preservation and cultural methods are used in conjunction with EN 13623.

Keywords: EN 13623:2020, Legionella pneumophila, bactericidal activity, quantitative suspension test, chemical disinfectants, aqueous systems, cooling towers, shock treatment.

Standards Content (Sample)

SLOVENSKI STANDARD
01-januar-2021
Nadomešča:
SIST EN 13623:2010
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje baktericidnega delovanja kemičnih razkužil za razkuževanje vode na
bakterijo Legionella - Preskusna metoda in zahteve (faza 2, stopnja 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of bactericidal activity against Legionella of chemical disinfectants for aqueous systems -
Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der bakteriziden Wirkung gegen Legionella von chemischen
Desinfektionsmitteln für wasserführende Systeme - Prüfverfahren und Anforderungen
(Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l'évaluation de l'activité bactéricide contre des légionelles des désinfectants chimiques
pour les systèmes aqueux - Méthode d'essai et prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 13623:2020
ICS:
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 13623
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2020
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 13623:2010
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of bactericidal activity
against Legionella of chemical disinfectants for aqueous
systems - 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
bactéricide contre des légionelles des désinfectants bakteriziden Wirkung gegen Legionella von
chimiques pour les systèmes aqueux - Méthode d'essai chemischen Desinfektionsmitteln für wasserführende
et prescriptions (phase 2, étape 1) Systeme - Prüfverfahren und Anforderungen (Phase 2,
Stufe 1)
This European Standard was approved by CEN on 19 August 2019.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13623:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
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 . 11
5.3.1 General . 11
5.3.2 Usual microbiological laboratory equipment . 11
5.4 Preparation of test organism suspensions and test solutions . 12
5.4.1 Test organism suspension (test suspension N and validation suspension N ) . 12
V
5.4.2 Product test solution . 14
5.5 Procedure for assessing the bactericidal activity of the product . 14
5.5.1 General . 14
5.5.2 Dilution-neutralization method . 16
5.5.3 Membrane filtration method . 18
5.6 Experimental data and calculations . 19
5.6.1 Explanation of terms and abbreviations . 19
5.6.2 Calculation . 20
5.7 Verification of methodology . 25
5.7.1 General . 25
5.7.2 Control of weighted mean counts . 25
5.7.3 Basic limits . 25
5.8 Expression of results and precision . 25
5.8.1 Reduction . 25
5.8.2 Control of active and non-active product test solution (5.4.2) . 26
5.8.3 Bactericidal concentration . 26
5.8.4 Precision, repetition . 26
5.9 Interpretation of results – conclusion . 26
5.9.1 General . 26
5.9.2 Bactericidal activity for general purposes . 26
5.9.3 Bactericidal activity for specific purposes . 26
5.10 Test report . 26
Annex A (informative) Referenced strains in national collections . 28
Annex B (informative) Determination of the bactericidal activity against Legionella
pneumophila . 29
Annex C (informative) Neutralizer . 32
Annex D (informative) Graphical representation of test procedures . 34
Bibliography . 38
European foreword
This document (EN 13623:2020) 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 April 2021, and conflicting national standards shall be
withdrawn at the latest by April 2021.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 13623:2010.
This document was revised to adapt it to the latest state of CEN/TC 216, to correct errors and
ambiguities. The following is a list of significant changes since the last edition:
— the temperature range for incubation of plates from (36 ± 1) °C or (37 ± 1) °C was changed to the
range (36 ± 2) °C as given in the new ISO 11731 standard for Legionella culture;
— a new paragraph was added to the scope to state that the method is not suitable for continuously
dosed products;
— new Annex A " Referenced strains in national collections" was added;
— the calculation errors in Table A.2 (now Table B.2) were corrected.
The changes mentioned above have no impact on the test results obtained with reference to the
previous version. Those results are still valid.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant has
a bactericidal activity against Legionella pneumophila in the fields described in the scope. This standard
is specifically prepared for water treatment products, but it may also be possible to use it for other
products.
Proliferation of Legionella only occurs in waters under certain conditions, and predominantly poses a
risk when aerosolised. Many systems containing water do not require treatment. A decision to add
chemical disinfectants to any water should be based on an appropriate assessment according to
national regulations.
If the product complies with the requirements of this standard, it can be considered bactericidal against
Legionella pneumophila, but it should not necessarily be inferred that the product is acceptable for a
specific site of application without consideration of other relevant factors such as the pH, water,
chemistry, temperature and degree of biological fouling at that site of application. It does not take into
account the protective effect conveyed by biofilm on the organisms.
The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each concentration of the chemical disinfectant found by this test corresponds to defined
experimental conditions. However, for some applications the recommendations of use of a product may
differ and therefore additional test conditions need to be used.
1 Scope
This document specifies a test method and the minimum requirements for bactericidal activity of
chemical disinfectant products intended to be used for treatment in aqueous systems against Legionella
pneumophila that form a homogeneous, physically stable preparation when diluted with buffered
ferrous hard water or hard water. Whenever Legionella pneumophila poses a risk to human health, this
method is suitable for water used in cooling towers and water for general purposes, like spas, pools,
showers and other uses. The method is not suitable for electro-chemical disinfection.
The document applies to products used as a single application shock treatment in order to kill
Legionella pneumophila. It is not suitable for the evaluation of those products that are dosed
continuously into water systems to control the growth of Legionella pneumophila.
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.
NOTE 3 This method does not take into account the fact that Legionella pneumophila is often found in cells of
amoebae and/or biofilms and that thereby a product’s activity against the bacteria may be reduced.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use
recommendation".
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 and the following 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 https://www.iso.org/obp
3.1
cooling water
water used to remove heat from a process or environment
3.2
water for general purposes
water used in premises other than water used as cooling water
4 Requirements
The product shall demonstrate at least a four decimal logarithm (lg) reduction, when diluted with
buffered ferrous hard water (5.2.2.10) or hard water (5.2.2.7), and tested in accordance with the
requirements of Table 1 and Clause 5. Where indicated, additional specific bactericidal activity shall be
determined applying other contact times and test organisms (in accordance with 5.2.1 and 5.5.1.1) in
order to take into account intended specific use conditions.
Table 1 — Minimum and additional test conditions
Obligatory test conditions
Additional test
Bactericidal activity
Bactericidal activity
conditions
in water for general
in cooling water
purposes
Other strains and
Legionella
Test organisms Legionella pneumophila species of Legionella
pneumophila
can be used
No other
temperatures are
Test temperature 20 °C  30 °C
considered relevant
to this test
0,000 5 % yeast
Interfering substances 0,000 5 % yeast extract None
extract
60 min ( for rapid 60 min (for rapid
acting products) acting products)
2 h, 6 h, 15 h, 40 h,
Contact time or or
48 h
15 h (for slower acting 15 h (for slower acting
products) products)
Buffered ferrous hard
Diluent Hard water None
water
NOTE For these additional conditions, the concentration defined as a result can be lower than the one
obtained under the obligatory test conditions.
5 Test methods
5.1 Principle
5.1.1 A sample of the product diluted with hard water (5.2.2.7 or 5.2.2.10) is added to a test
suspension of bacteria in a solution of an interfering substance. The mixture is maintained at either
(20 ± 1) °C or (30 ± 1) °C for 60 min ± 10 s or (15 ± 1) h (obligatory test conditions). At the end of the
chosen contact time, an aliquot is taken, and the bactericidal and/or the bacteriostatic activity 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 bacteria in each sample are determined and the reduction is calculated.
5.1.2 The test is performed using Legionella pneumophila as test organism (obligatory test
conditions).
5.1.3 Additional and optional contact times are specified. Additional test organisms may be used.
5.2 Materials and reagents
5.2.1 Test organism
1)
The bactericidal activity shall be evaluated using the following strain as test organism :
Legionella pneumophila subsp. pneumophila: ATCC 33152.
NOTE See Annex A for strain reference in some other culture collections.
If required for specific applications, additional test organisms may be used, e.g. Legionella pneumophila
serogroup 1 Benidorm (NCTC 12006, ATCC 43108).
The required incubation temperature for this test organism is (36 ± 2) °C (5.3.2.3). The same
temperature 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
Unless specifically stated, all weights of chemical substances given in this 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.
For each culture medium and reagent, a limitation for use should be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water. If distilled water of
adequate quality is not available, water for injections (bibliographic reference [1]) can be used.
Sterilize in the autoclave (5.3.2.1, a)). Sterilization is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilized.
NOTE See 5.2.2.10 for the procedure to prepare buffered ferrous hard water.

1)
The NCTC and ATCC numbers are the collection numbers of strains supplied by the National Type Culture
Collection (NCTC) and American Type Culture Collection (ATCC). This information is given for the convenience of
users of this standard and does not constitute an endorsement by CEN of the product named.
5.2.2.3 Buffered Charcoal Yeast Extract (BCYE) Agar
BCYE agar, consisting of
—  yeast extract (bacteriological grade) 10,0 g
—  agar 12,0 g
—  activated charcoal 2,0 g
—  alpha-ketoglutarate, monopotassium salt 1,0 g
—  ACES buffer (N-2-acetamido-2-aminoethanesulfonic acid) 10,0 g
—  potassium hydroxide (KOH) (pellets) 2,8 g
—  L-cysteine hydrochloride monohydrate 0,4 g
—  iron(III) pyrophosphate [Fe (P 0 ) ] 0,25 g
4 2 7 3
—  distilled water to 1 000,0 ml
Preparation
a) Cysteine and iron solutions
Prepare fresh solutions of L-cysteine hydrochloride and iron(III) pyrophosphate by adding 0,4 g and
0,25 g respectively to 10-ml-volumes of water (5.2.2.2). Sterilize each solution by membrane filtration
(5.3.2.7). Store in clean sterile containers at (20 ± 3) °C for not more than three months.
b) ACES buffer
Add the ACES granules to 500 ml of water (5.2.2.2) and dissolve by standing in a water bath at 45 °C to
50 °C. To a separate 480 ml of water (5.2.2.2), add all the potassium hydroxide pellets and dissolve with
gentle shaking. To prepare the ACES buffer, mix the two solutions.
NOTE ACES buffer can cause denaturation of the yeast extract if the following sequence is not followed.
c) Final medium
Add sequentially to the 980 ml of ACES buffer, the charcoal, yeast extract and α-ketoglutarate. Prepare a
0,1 mol/l solution of potassium hydroxide (KOH) by dissolving 5,6 g in 1 l of water (5.2.2.2). Prepare a
0,1 mol/l solution of sulphuric acid (H SO ) by carefully adding 5,3 ml of H SO to 1 l of water
2 4 2 4
(5.2.2.2). Use the solutions of 0,1 mol/l potassium hydroxide or 0,1 mol/l sulphuric acid as appropriate
to adjust the pH to 6,9 ± 0,2. Add the agar, mix and autoclave (5.3.2.1, a)). After autoclaving, allow to
cool to (47 ± 2) °C in a water bath (5.3.2.2).
Add the L-cysteine and the iron(III) pyrophosphate solutions aseptically, mixing well between
additions.
Dispense in 20 ml volumes into Petri dishes of 90 mm to 100 mm diameter. The pH of the final medium
is 6,9 ± 0,2 at 25 °C. Allow excess moisture on the plates to dry and store at (4 ± 2) °C in airtight
containers in the dark for up to four weeks.
Prolonged heating during sterilisation or heating at too high a temperature shall be avoided, as it can
affect the nutritional qualities of BCYE medium. Batch-to-batch variation of the ingredients of the
medium (particularly α-ketoglutarate) can also affect its performance. Therefore it is essential to check
the quality of each newly prepared batch of media for its ability to support the growth of the test
organism within three days of incubation using the validation suspension N (5.4.1.5).
V
The ability of the media to support the growth of Legionella should be assessed quantitatively using
either known quantities of the obligatory Legionella strain or by direct comparison to previous batches.
Commercially supplied media may be used without testing if it has been performance tested in a
laboratory accredited to EN ISO/IEC 17025:2017 for that purpose.
5.2.2.4 BCYE Broth
Prepared by the same method as BCYE agar (5.2.2.3), but omitting the addition of the agar.
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 C.
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 C.
5.2.2.7 Hard water for general purposes (HWGP)
For the preparation of 1 l of hard water, the procedure is as follows:
a) prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the
autoclave (5.3.2.1, a)). Autoclaving – if used – may cause a loss of liquid. In this case make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator
(5.3.2.8) for no longer than one month;
b) prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8)
for no longer than one week;
c) place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml
(5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The
pH of the hard water shall be 7,0 ± 0,2, when measured at (20 ± 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 1 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 is lower than 300 mg/l
of calcium carbonate (CaCO ) in the test tube.
NOTE 2 This hard water represents typical conditions of non-cooling water.
5.2.2.8 Interfering substance (yeast extract)
—  yeast extract 0,5 g
—  water to 1 000,0 ml
Sterilize in the autoclave (5.3.2.1, a)).
Final concentration of the yeast extract in the test is 0,000 5 %.
5.2.2.9 Page’s Saline
Saline solution, consisting of:
—  sodium chloride (NaCl) 0,120 g
—  magnesium sulphate (MgSO · 7H 0) 0,004 g
4 2
—  calcium chloride (CaCl · 2H 0) 0,004 g
2 2
—  disodium hydrogen phosphate (Na HPO ) 0,142 g
2 4
—  potassium dihydrogenphosphate 0,136 g
(KH PO )
2 4
—  water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave (5.3.2.1, a)).
To aid accurate preparation, it is recommended that a 10 l volume of Page’s Saline is prepared and
dispensed in smaller volumes as required for autoclaving. Alternatively the salt solutions may be made
up individually in concentrated form for dilution when the product is required.
5.2.2.10 Buffered ferrous hard water for treatment of cooling water (BFHW)
For the preparation of 1 l of BFHW, the procedure is as follows:
a) prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the
autoclave (5.3.2.1, a)). Autoclaving – if used – may cause a loss of liquid. In this case make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator
(5.3.2.8) for no longer than one month;
b) prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8)
for no longer than one week;
c) place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 70 ml of
boric acid (12,37 g of Boric acid made up to 1 000 ml with distilled water), 30 ml of 0,05 M Borax
(19,07 g of Borax made up to 1 000 ml with distilled water) and 6,0 ml (5.3.2.9) of solution A, then
-3
8,0 ml of solution B. Finally add 1,0 ml ferric sulphate solution (3,0 x 10 mol/l). Mix and dilute to
1 000 ml with water (5.2.2.2). The pH of the hard water shall be 8,0 ± 0,2, when measured at (20
± 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). Sterilize by membrane filtration (5.3.2.7).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE 1 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 is lower than 300 mg/l
of calcium carbonate (CaCO ) in the test tube.
NOTE 2 This buffered ferrous hard water represents typical conditions of cooling water.
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 that 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)].
2)
5.3.2 Usual microbiological laboratory equipment
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 +5
holding time of 30 min, at ( 170 ) °C for a minimum holding time of 1 h or at ( 160 ) °C for a
0 0
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, (30 ± 1) °C and at (47 ± 2) °C. An
oven or incubator capable of being controlled at either of these temperatures may be the alternative.
5.3.2.3 Incubator, capable of being controlled at (36 ± 2) °C. Plates will need to be incubated in
sealed bags or containers to prevent drying out of the agar.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
NOTE It is advised to use a puncture electrode or a flat membrane electrode for measuring the pH of the agar
media (5.2.2.3).
5.3.2.5 Stopwatch
)
Disposable equipment is an acceptable alternative to reusable glassware.
5.3.2.6 Shaker
3)
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.
The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters
of diameter 47 mm to 50 mm and with a maximum pore size of 0,45 µm for sterilization of solutions and
suspensions and if the membrane filtration 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, 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 Centrifuge (2 000 g ).
N
5.3.2.12 Volumetric flasks.
5.4 Preparation of test organism suspensions and test solutions
5.4.1 Test organism suspension (test suspension N and validation suspension N )
V
5.4.1.1 General
Two different suspensions shall be prepared: the "test suspension N" to perform the test and the
"validation suspension N " to perform the controls and method validation.
V
5.4.1.2 Preservation and stock cultures of test organism
The test organisms and their stock cultures shall be reconstituted in accordance with the supplier's
instructions and kept in accordance with EN 12353.
Subculture onto BCYE agar (5.2.2.3). After incubation (5.3.2.3), make a suspension from the resulting
growth just visible to the naked eye and dispense in Page’s Saline (5.2.2.9) or water (5.2.2.2) for storage
at a temperature equal or lower than − 70 °C. In case of no growth on BCYE agar a BCYE broth may be
inoculated (5.2.2.4), and a subculture from this broth may be incubated (5.3.2.3) and prepared as
described above.
NOTE Commercially available cryo-preservation systems can be used providing they produce satisfactory
recovery of the organism.
3)
Vortex® is an example of a suitable product available commercially. This information is given for the
convenience of users of this standard and does not constitute an endorsement by CEN of this product.
5.4.1.3 Working cultures of test organism
In order to prepare the working culture of the test organism (5.2.1), prepare a subculture from the
frozen suspension (5.4.1.2) by streaking onto BCYE plates (5.2.2.3) and incubate (5.3.2.3). This
subculture is the working culture.
5.4.1.4 Test suspensions (N)
a) Take the working culture (5.4.1.3) and remove growth from the plate by suspending with Page’s
saline (5.2.2.9). Aspirate the suspension and transfer into a tube. Mix (5.3.2.6) for 10 s, then
centrifuge (5.3.2.11) for 15 min. Re-suspend the pellet in Page’s saline (5.2.2.9).
8 4) 8
b) Adjust the number of cells in the suspension to 1,5 x 10 cfu/ml to 5 x 10 cfu/ml using Page’s
saline, (5.2.2.9), estimating the number of cfu by any suitable means. Maintain this test suspension
in the water bath at either 20 °C for products intended for non-cooling water applications or 30 °C
for cooling water products (5.5.1.1, a)) and use within 2 h.
The use of spectrophotometer for adjusting the number of cells is highly recommended (about 620
nm wavelength - cuvette 10 mm path length). Each laboratory should therefore produce calibration
data for each test organism knowing that suitable values of optical density are found between 0,150
and 0,400. A colorimeter is a suitable alternative.
-6 -7
c) For counting, prepare 10 and 10 dilutions of the test suspension using Page’s saline (5.2.2.9).
Mix (5.3.2.6). Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the spread
plate technique. Spread each 1,0 ml sample – divided into portions of approximately equal size
(max. 0,2 ml) – on an appropriate number of plates containing BCYE (5.2.2.3).
For incubation and counting see 5.4.1.6.
5.4.1.5 Validation suspension (N , N )
V VB
a) To prepare the validation suspension, dilute the test suspension (5.4.1.4) with Page’s saline
2 3 -5
(5.2.2.9) to obtain 3,0 x 10 cfu/ml to 1,6 x 10 cfu/ml (about one fourth (1+3) of the 10
dilution).
-1
b) For counting prepare a 10 dilution with Page’s saline (5.2.2.9). Mix (5.3.2.6). Take a sample of
1,0 ml in duplicate and inoculate using the spread plate technique (5.4.1.4, c)).
For incubation and counting see 5.4.1.6.
5.4.1.6 Incubation and counting of the test and validation suspensions
a) Incubate (5.3.2.3) the plates for seven days. Discard any plates that are not countable for any
reason. Count the plates and determine the number of cfu.
b) Note for each plate the exact number of colonies but record "> 330" for any counts higher than 330
and determine the V -values according to 5.6.2.2.
C
c) Calculate the numbers of cfu/ml in the test suspension N and in the validation suspension N using
V
the methods given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7.

)
cfu/ml: colony forming unit(s) per millilitre.
5.4.2 Product test solution
The concentration of a product test solution shall be ten times the desired test concentration because it
is diluted to 10 % during the test and the method validation (5.5.2 or 5.5.3). Product test solutions shall
be prepared in hard water at minimum three different concentrations to include one concentration in
the active range and one concentration in the non-active range (5.8.2). The type of hard water is
selected according to the intended use: BFHW (5.2.2.10) for products intended to be used for treatment
of cooling water and HWGP (5.2.2.7) for products intended to be used for treatment of water for general
purposes. The product, as received, may be used as one of the product test solutions; in this case the
highest tested concentration is 10 %.
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in
a volumetric flask and filling up with hard water (5.2.2.7 or 5.2.2.10). Subsequent dilutions (lower
concentrations) shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard
water (5.2.2.7 or 5.2.2.10).
For liquid products, dilutions of the product shall be prepared with hard water on a volume/volume
basis using volumetric flasks (5.3.2.12).
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous preparation that is stable during the whole procedure. If during the procedure
a visible inhomogeneity appears due to the formation of a precipitate or flocculant (for example,
through the addition of the interfering substance), it shall be recorded in the test report.
NOTE Counting micro-organisms embedded in a precipitate or flocculant is difficult and unreliable.
The concentration of the product stated in the test report shall be the desired test concentration.
Record the test concentration in terms of mass per volume or volume per volume and details of the
product sample as received.
5.5 Procedure for assessing the bactericidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions (obligatory and additional)
Besides the obligatory, contact time and test organisms, additional experimental conditions (including
test organisms) may be selected according to the practical use considered for the product (Clause 4), as
follows:
a) temperature θ [in degrees Celsius (°C)]:
1) the obligatory temperature to be tested for cooling water (3.1) is θ = 30 °C;
2) the obligatory temperature to be tested for water for general purposes (3.2) is θ = 20 °C;
3) the allowed deviation for the chosen temperature is ± 1 °C;
NOTE No additional temperatures are considered relevant to this test.
b) contact time t [in minutes (min)]:
1) for fast acting products such as oxidising substances the obligatory contact time to be tested is
t = 60 min;
2) for slow acting products the obligatory contact time is 15 h;
3) the additional contact times may be chosen from 2 h, 6 h, 15 h, 40 h, 48 h;
4) the allowed deviation for the chosen contact time of 60 min or less is ± 10 s, for contact times of
6 h or less it is ± 5 min and ± 1 h for all other contact times;
c) interfering substance: the obligatory interfering substance to be tested is at the final concentration
of 0,000 5 % yeast extract in the test mixture;
d) test organism:
1) the obligatory test organism is Legionella pneumophila (5.2.1);
2) additional test organisms (other strains of Legionella pneumophila or species of Legionella)
may be used.
5.5.1.2 Choice of test method
The method of choice is the dilution-neutralization method. To determine a suitable neutralizer carry
out the validation of the dilution neutralization method (5.5.2.3, 5.5.2.4 and 5.5.2.5 in connection with
5.5.2.6) using a neutralizer, chosen according to laboratory experience and published data.
If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into
account the information given in Annex C.
If both neutralizers are found to be invalid, the membrane filtration method (5.5.3) may be used.
5.5.1.3 General instructions for validation and control procedures
The neutralization and/or removal of the bactericidal or bacteriostatic activity of the product shall be
controlled and validated – only for the highest product test concentration – for each of the used test
organisms and for each experimental condition (contact time). These procedures (experimental
condition control, neutralizer or filtration control and method validation) shall be performed at the
same time with the test and with the same neutralizer – or rinsing liquid – used in the test.
The same hard water (5.2.2.7 or 5.2.2.10) used in the test (5.5.2.2) shall be used in the validation and
controls.
The same agar (5.2.2.3) used in the test (5.5.2.2) shall be used in the validation and controls.
5.5.1.4 Equilibration of temperature
Prior to testing, equilibrate all reagents [product test solutions (5.4.2), test suspension (5.4.1.4),
validation suspension (5.4.1.5) hard water (5.2.2.7 or 5.2.2.10) and interfering substance (5.2.2.8)] to
the test temperature of either 20 °C for products intended for non-cooling water applications or 30 °C
for cooling water products (5.5.1.1, a)) using the water bath (5.3.2.2) controlled at (20 ± 1) °C or
(30 ± 1) °C.
Check that the temperature of the reagents is stabilized at the chosen temperature.
The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at a
temperature of (20 ± 1) °C.
5.5.1.5 Precautions for manipulation of test organisms
Do not touch the upper part of the test tube sides when adding the test or the validation suspensions
(5.4.1).
5)
5.5.2 Dilution-neutralization method
5.5.2.1 General
The test and the control and validation procedures (5.5.2.2 to 5.5.2.5) shall be carried out at the same
time.
5.5.2.2 Test N – determination of bactericidal conce
...