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 European Standard 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 European Standard applies to products used to treat water in order to kill 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
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
16-Sep-2019
Due Date
21-Nov-2019
Completion Date
07-Dec-2020

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SLOVENSKI STANDARD
SIST EN 13623:2021
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
SIST EN 13623:2021 en,fr,de

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

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SIST EN 13623:2021
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SIST EN 13623:2021
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.
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SIST EN 13623:2021
EN 13623:2020 (E)
Contents Page

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

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

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

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

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

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

5 Test 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

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

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SIST EN 13623:2021
EN 13623:2020 (E)
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.
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SIST EN 13623:2021
EN 13623:2020 (E)
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.
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SIST EN 13623:2021
EN 13623:2020 (E)
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
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SIST EN 13623:2021
EN 13623:2020 (E)
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.

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SIST EN 13623:2021
EN 13623:2020 (E)
5.2 Materials and reagents
5.2.1 Test organism

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.

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.

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SIST EN 13623:2021
EN 13623:2020 (E)
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).

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SIST EN 13623:2021
EN 13623:2020 (E)

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.
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SIST EN 13623:2021
EN 13623:2020 (E)
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;
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