Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test method and requirements (phase 2, step 2)

This document specifies a test method and the minimum requirements for the microbicidal activity of a defined disinfection process for the treatment of contaminated textile. This procedure is carried out by using a washing machine as defined in 5.3.2.18 and refers to the disinfection step without prewash. This procedure is not limited to certain types of textile. The suppliers instructions shall be sufficient to allow the method in the standard to be carried out fully (e.g. dosing disinfectant in whatever washing phase e.g. main wash, rinsing, disinfecting at 40 °C). This document applies to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example: — in hospitals, in community medical facilities, and in dental institutions; — in clinics of schools, of kindergartens, and of nursing homes; and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients. The method described is intended to determine the activity of a product or product combination under the conditions in which they are used. This is a phase 2, step 2 laboratory test that simulates the conditions of application of the product. NOTE This method corresponds to a phase 2, step 2 test (see EN 14885). EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”.

Chemisches Desinfektionsmittel und Antiseptika - Chemothermische Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die mikrobizide Wirkung eines festgelegten Desinfektionsprozesses zur Behandlung von kontaminierten Textilien fest. Dieses Ver-fahren wird unter Einsatz einer in 5.3.2.18 festgelegten Waschmaschine durchgeführt und bezieht sich auf den Desinfektionsschritt ohne Vorwaschen. Dieses Verfahren ist nicht auf bestimmte Arten von Textilien beschränkt. Die Anweisungen des Lieferanten werden als ausreichend angenommen, wenn sie die im Prüfverfahren angegebenen Prozessparameter (z. B. Dosierung des Desinfektionsmittels in einer beliebigen Waschphase, z. B. Haupt¬waschgang, Spülen, Desinfektion bei 40 °C) enthalten.
Dieses Dokument ist anzuwenden für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen bei der Patientenbetreuung vor, z. B.:
- in Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
- in medizinischen Einrichtungen von Schulen, Kindergärten und Heimen;
und könnten auch am Arbeitsplatz und im häuslichen Bereich gegeben sein.
Eingeschlossen sein könnten auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung der Patienten dienen.
Das beschriebene Verfahren dient der Bestimmung der Wirksamkeit eines Produkts oder einer Kombination von Produkten unter den Bedingungen, unter denen es/sie angewendet wird/werden. Es ist eine Labor¬prüfung der Phase 2, Stufe 2, die die Bedingungen der Produktanwendung simuliert.
EN 14885 legt im Einzelnen die Beziehungen der verschiedenen Prüfungen untereinander und zu den „Anwendungsempfehlungen“ fest.

Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile - Méthode d'essai et prescriptions (phase 2, étape 2)

Le présent document spécifie une méthode d’essai et les exigences minimales relatives à l’activité microbicide d’un processus spécifié de désinfection destiné au traitement de textile contaminé. Cette procédure consiste à utiliser une machine à laver comme spécifié en 5.3.2.18 et se rapporte à l’étape de désinfection sans prélavage. Cette procédure ne se limite pas à certains types de textile. Il est escompté que les instructions du fournisseur seront suffisantes si elles comprennent les paramètres de processus identifiés pour l’essai (par exemple, le dosage du désinfectant quelle que soit l’étape de lavage, par exemple, le lavage, le rinçage, la désinfection à 40 °C).
Le présent document s’applique aux secteurs et situations pour lesquels la désinfection est médicalement préconisée. Ces préconisations touchent les soins délivrés aux patients, par exemple :
— dans les hôpitaux, les cabinets médicaux et dentaires ;
— dans les infirmeries des écoles, des écoles maternelles et des maisons de repos ;
et pourraient concerner les lieux de travail et le domicile des particuliers.
Le présent document pourrait également s’appliquer aux textiles entretenus par des blanchisseries et aux articles textiles de cuisine en contact direct avec les patients.
La méthode décrite a pour objet de déterminer l’activité d’un produit ou d’une combinaison de produits dans leurs conditions d’emploi. Il s’agit d’un essai en laboratoire de phase 2, étape 2 qui simule les conditions d’application du produit.
L’EN 14885 spécifie en détail la relation entre les divers essais et la façon « d’appliquer les recommandations ».

Kemična razkužila in antiseptiki - Termokemično razkuževanje tekstila - Preskusna metoda in zahteve (faza 2, stopnja 2)

Ta dokument določa preskusno metodo in minimalne zahteve za mikrobicidno delovanje definiranega postopka razkuževanja pri obdelavi kontaminiranega tekstila. Ta postopek se izvaja z uporabo pralnega stroja, kot je določeno v točki 5.3.2.18, in se navezuje na korak razkuževanja brez predpranja. Ta postopek ni omejen na določene vrste tekstila. Navodila dobavitelja zadoščajo za popolno izvedbo metode iz standarda (npr. odmerjanje razkužila v kateri koli fazi pranja, npr. glavno pranje, izpiranje, razkuževanje pri 40 °C). Ta dokument se uporablja za področja in primere, ko obstajajo zdravniške indikacije za razkuževanje. Te indikacije se pojavljajo pri negi bolnikov, na primer: – v bolnišnicah, javnih zdravstvenih in zobozdravstvenih ustanovah; – v ambulantah šol, vrtcev in domov za starejše; in lahko nastanejo na delovnem mestu ali doma. Vključujejo lahko tudi storitve, kot so pralnice in kuhinje, ki proizvode dostavljajo neposredno bolnikom. Opisana metoda je namenjena določevanju dejavnosti izdelka ali kombinacije izdelkov pod pogoji, v katerih se uporabljajo. To je 2. stopnja laboratorijskega preskusa faze 2, ki simulira pogoje uporabe izdelka. OPOMBA: Ta metoda ustreza 2. stopnji preskusa faze 2 (glej standard EN 14885). EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo.

General Information

Status
Published
Public Enquiry End Date
01-Feb-2021
Publication Date
30-Aug-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
11-Aug-2022
Due Date
16-Oct-2022
Completion Date
31-Aug-2022

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 16616:2022
01-november-2022
Nadomešča:
SIST EN 16616:2015
Kemična razkužila in antiseptiki - Termokemično razkuževanje tekstila - Preskusna
metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test
method and requirements (phase 2, step 2)
Chemisches Desinfektionsmittel und Antiseptika - Chemothermische
Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile -
Méthode d'essai et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 16616:2022
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 16616:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 16616:2022

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SIST EN 16616:2022


EN 16616
EUROPEAN STANDARD

NORME EUROPÉENNE

August 2022
EUROPÄISCHE NORM
ICS 11.080.20 Supersedes EN 16616:2015
English Version

Chemical disinfectants and antiseptics - Chemical-thermal
textile disinfection - Test method and requirements (phase
2, step 2)
Désinfectants chimiques et antiseptiques - Désinfection Chemische Desinfektionsmittel und Antiseptika -
thermochimique du textile - Méthode d'essai et Chemothermische Wäschedesinfektion - Prüfverfahren
prescriptions (phase 2, étape 2) und Anforderungen (Phase 2, Stufe 2)
This European Standard was approved by CEN on 27 June 2022.

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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16616:2022 E
worldwide for CEN national Members.

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SIST EN 16616:2022
EN 16616:2022 (E)
Contents Page
European foreword . 4
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Requirements . 8
5 Test methods . 9
5.1 Principle . 9
5.2 Materials and reagents . 9
5.2.1 Test organisms . 9
5.2.2 Culture media and reagents . 10
5.3 Apparatus and glassware . 12
5.3.1 General . 12
5.3.2 Usual microbiological laboratory equipment . 13
5.4 Preparation of test organism suspensions (test suspension) . 15
5.4.1 General . 15
5.4.2 Preservation and stock cultures of test organisms. 15
5.4.3 Working culture and test organisms . 16
5.4.4 Test suspension (N) . 16
5.4.5 Inoculation of the carriers . 20
5.5 Procedure for assessing the microbicidal activity of the product . 21
5.5.1 General . 21
5.5.2 Test procedure . 22
5.6 Experimental data and calculation . 24
5.6.1 Explanation of terms and abbreviations . 24
5.6.2 Calculation . 25
5.7 Verification of methodology . 27
5.7.1 General . 27
5.7.2 Control of weighted mean counts . 28
5.7.3 Basic limits . 28
5.8 Expression of results and precision . 28
5.8.1 Reduction . 28
5.8.2 Repetitions . 29
5.9 Interpretation of results – conclusion . 29
5.9.1 General . 29
5.9.2 Microbicidal activity . 29
5.10 Test report . 30
Annex A (informative) Referenced strains in national collections . 32
Annex B (informative) Suitable neutralizers . 34
B.1 General . 34
B.2 Neutralizers . 34
B.3 Neutralizer added to the agar for counting . 35
Annex C (informative) Graphical representations of the test method . 36
2

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SIST EN 16616:2022
EN 16616:2022 (E)
Annex D (informative) Example of washing machine specification . 37
Annex E (informative) Preparation of hard water for using in the test and reference
procedures . 38
Annex F (informative) Test report (example) . 39
Annex G (informative) Example for loading the washing machine . 46
Bibliography . 48

3

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SIST EN 16616:2022
EN 16616:2022 (E)
European foreword
This document (EN 16616:2022) 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 February 2023, and conflicting national standards shall
be withdrawn at the latest by February 2023.
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 16616:2015.
The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to
harmonize the structure and wording with other tests of CEN/TC 216 existing or in preparation and to
improve the readability of the document and thereby make it more understandable. The following is a
list of significant technical changes since the last edition, EN 16616:2015:
— the scope is adapted to the scope of the WG 1;
— in Clause 4 the requirements for phase 2, step 1 tests are deleted;
— addition of water for the test and reference control (5.2.2.4);
— the example for washing machines in 5.3.2.18 of the previous version is switched to Annex D;
— addition of Potter S 1 apparatus (5.3.2.22);
— adaption of 5.4.3.1 to 5.4.3.3 to other current standards;
— review of 5.4.4 (editorial changes and better description);
— re-wording of the description of the neutralization (5.5.1.2) and addition of a reference to phase 2,
step 1 tests;
— addition of an information on using a spectrophotometer for counting cell numbers of mycobacteria
in 5.4.4.3 (NOTE);
— addition of the documentation and justification of the choice of the neutralizer in the test report
(5.5.1.2);
— addition of a new NOTE in 5.5.2.1;
— addition of a NOTE and the reference to Annex G in 5.5.2.2;
— addition of the reference control (5.5.2.3);
— RII is deleted;
— a reference to phase 2, step 1 tests was added;
— correction and adaption to the current tests of Table 2 (5.6.1.1);
4

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SIST EN 16616:2022
EN 16616:2022 (E)
— addition of two paragraphs in 5.6.2.1 (former 5.6.2.3);
— in 5.6.2.2 the V -values will be expressed per carrier (former per ml);
C
— addition of N and N in the calculation (5.6.2.3);
0
— in 5.6.2.4 the calculation is changed to values per carrier, the formula is corrected and the weighted
mean is added in all calculations;
— in 5.6.2.5 calculation is changed: only N will be calculated, RI is not counted and RII, B and C are no
w
longer used in the standard;
— correction of the example in 5.7.2;
— adaption of 5.7.3 to the tests in the current version;
— addition of the requirements of the test report in 5.10;
— adaption of Annex A to EN 12353;
— correction of Annex C;
— addition of a new Annex D “Example of washing machine specification”;
— addition of a new Annex E “Preparation of hard water for using in the test and reference procedures”;
— addition of a new Annex F “Test report (example)”;
— addition of a new Annex G “Example for loading the washing machine”;
— document editorially revised, clauses not applied (from the old version) deleted;
— de-harmonization of the standard, Annex ZA deleted.
The changes of this revision have no impact on the test results obtained with reference to the version
EN 16616:2015. Those results are still valid except the reduction of the reference control N was higher
w
than 3 lg and/or the calculation of the results followed the wrong way of version 2015.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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.
5

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SIST EN 16616:2022
EN 16616:2022 (E)
Introduction
This document specifies a carrier test for establishing whether a single-wash disinfecting product or
combination of products for the treatment of contaminated textile has or does not have necessary
microbicidal activity. This document only intends to validate the disinfection part of the laundry process.
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 could influence its
action in practice.
The conditions are intended to cover general purposes and to allow reference between microbiological
laboratories and types of detergents and disinfectants. Each effective dosage of the chemical disinfectant
found by this test corresponds only to the chosen experimental conditions. Where actual conditions vary
additional testing in microbiological laboratories is needed to determine the effective dosage.
Instructions for use are the responsibility of manufactures of detergents or disinfectants.
6

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SIST EN 16616:2022
EN 16616:2022 (E)
1 Scope
This document specifies a test method and the minimum requirements for the microbicidal activity of a
specified disinfection process for the treatment of contaminated textile. This procedure is carried out by
using a washing machine as specified in 5.3.2.18 and refers to the disinfection step without prewash. This
procedure is not limited to certain types of textile. The suppliers' instructions are expected to be sufficient
if they content the process parameters identified in the test (e.g. dosing disinfectant in whatever washing
phase e.g. main wash, rinsing, disinfecting at 40 °C).
This document applies to areas and situations where disinfection is medically indicated. Such indications
occur in patient care, for example:
— in hospitals, in community medical facilities, and in dental institutions;
— in clinics of schools, of kindergartens, and of nursing homes;
and could occur in the workplace and in the home.
It could also include services such as laundries and kitchens supplying products directly for the patients.
The method described is intended to determine the activity of a product or product combination under
the conditions in which they are used. This is a phase 2, step 2 laboratory test that simulates the
conditions of application of the product.
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 13624, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of
fungicidal or yeasticidal activity in the medical area - Test method and requirements (phase 2, step 1)
EN 13727, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of
bactericidal activity in the medical area - Test method and requirements (phase 2, step 1)
EN 14348, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of
mycobactericidal activity of chemical disinfectants in the medical area including instrument disinfectants -
Test methods and requirements (phase 2, step 1)
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 https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
7

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SIST EN 16616:2022
EN 16616:2022 (E)
3.1
liquor ratio
ratio of the weight of dry textile in kilograms and volume of wash liquor in litres (m/V)
3.2
disinfection process
process taking into account practical conditions of application of the product including contact time(s),
temperature(s), product(s), concentration(s), liquor ratio, test organisms and interfering substances to
disinfect the textile
3.3
treatment of contaminated textile
handling the textile according the disinfection process to obtain disinfected textile
4 Requirements
The test results shall fulfil the basic limits (see 5.7.3). The product shall demonstrate at least the decimal
logarithms (lg) reduction specified in 5.9.2, when tested in accordance with Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Textile disinfection Textile disinfection
Test conditions
temperature < 60 °C temperature ≥ 60 °C
b
Test organism E. coli K12
E. faecium
E. hirae
P. aeruginosa
S. aureus
C. albicans
Additional test organism
Fungicidal activity A. brasiliensis
Tuberculocidal activity M. terrae
Mycobactericidal activity M. terrae and M. avium
Temperature As recommended by the As recommended by the
a a
manufacturer and < 60 °C manufacturer and ≥ 60 °C
Contact time As recommended by the As recommended by the
manufacturer manufacturer
Interfering substance Sheep blood Sheep blood
NOTE The implementation of bacterial spores and viruses was discussed. Further development is necessary
to make it technically feasible.
a
The temperature and the contact time shall be chosen on the basis of the practical conditions of the product application
and within the responsibility of the manufacturer.
b
This includes bactericidal, yeasticidal, fungicidal, tuberculocidal and mycobactericidal activity.
8

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SIST EN 16616:2022
EN 16616:2022 (E)
5 Test methods
5.1 Principle
Carriers made of cotton fabric (5.3.2.16) are contaminated with a test suspension of microorganisms in
defibrinated sheep blood (5.2.2.12). After drying the carriers are transferred into cotton towels and then
the disinfection process in the washing machine is performed at test temperatures either t < 60 °C or
t ≥ 60 °C. The process refers to the disinfection step without prewash. At the end of the disinfection step
of the procedure, the towels with the carriers have to be taken out. Each carrier is transferred into a
separate tube containing neutralizer (5.2.2.11) and glass beads (5.3.2.11). The microorganisms should be
recovered from the carriers by shaking. The number of surviving microorganisms in each sample is
determined and the reduction rate is calculated.
5.2 Materials and reagents
5.2.1 Test organisms
1
The bactericidal activity shall be evaluated using the following strains as test organisms :
— Pseudomonas aeruginosa ATCC® 15442™
— Escherichia coli (K12) NCTC 10538
— Staphylococcus aureus ATCC® 6538™
— Enterococcus hirae ATCC® 10541™
— Enterococcus faecium ATCC® 6057™
The yeasticidal/fungicidal activity shall be evaluated using the following test organisms:
— Candida albicans ATCC® 10231™
2

Aspergillus brasiliensis
ATCC® 16404™

The tuberculocidal/mycobactericidal activity shall be evaluated using the following test organisms:
— Mycobacterium terrae ATCC® 15755™
— Mycobacterium avium ATCC® 15769™
NOTE See Annex A for strain reference in some other culture collections.
The required incubation temperatures for these test organisms are (36 ± 1) °C (5.3.2.3) [C. albicans and
A. brasiliensis: (30 ± 1) °C]. The same temperature shall be used for all incubations performed during a
test and its controls and validation.
If additional test organisms are used, they shall be incubated under optimum growth conditions
(temperature, time, atmosphere and media) noted in the test report. If the additional test organisms
selected do not correspond to the specified strains/species, their suitability for supplying the required
inocula shall be verified. If these additional test organisms are not classified at a reference centre, their

1
The ATCC® numbers are the collection numbers of strains supplied by the American Type Culture Collection® (ATCC®).
This information is given for the convenience of users of this document and does not constitute an endorsement by CEN
of these products.
2
Formerly: Aspergillus niger ATCC® 16404™.
9

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SIST EN 16616:2022
EN 16616:2022 (E)
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or
national culture collection stored 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 document refer to the anhydrous salts. Hydrated forms
may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the growth of 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 used for preparation of media
The water shall be fresh distilled water and not just demineralized water. Sterilize in the autoclave
[5.3.2.1 a)].
NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently
sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections (see [1]) can be used.
5.2.2.3 Hard water for dilution of products for validation tests
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
2
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the
2
autoclave [5.3.2.1a)]. Autoclaving, if used, could 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) at (2 to
8) °C 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
3
1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) at
(2 to 8) °C for no longer than one week.
— 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 with the
use of a pipette (5.3.2.9) 6,0 ml 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.
5.2.2.4 Water for the test and reference control
Water with potable water quality is necessary (the water should contain less than 100 cfu/ml of bacteria
at 36 °C and 22 °C). Water hardness shall be logged and mentioned in the laboratory protocol and shall
be documented in the test report. The final hardness shall be equal or higher than 4 mmol/l alkaline earth
2+ 2+
ions (Mg and Ca ). The temperature of the water influx should be between (12 and 20) °C.
10

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NOTE 1 Annex E indicates how to adjust the water hardness for the test and reference procedure using solutions
A and B from 5.2.2.3. The water hardness can be measured using commercially available test kits.
NOTE 2 Water supplier data can be used to document water hardness.
5.2.2.5 Tryptone Soy Agar (TSA)
Tryptone, pancreatic digest of casein 15,0 g
Soy 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 1 000,0 ml

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at (20 ± 1) °C.
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it could be necessary to add
neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used.
5.2.2.6 Tryptone Soy Broth (TSB)
EXAMPLE
Tryptone, pancreatic digest of casein 15,0 g
Soy peptone, papaic digest of soybean meal 5,0 g
Sodium chloride (NaCl) 5,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.7 Brain Heart Infusion Agar (BHI)
EXAMPLE
Brain heart infusion 12,5 g
Beef heart infusion 5,0 g
Proteose-Peptone 10,0 g
Glucose 2,0 g
Sodium chloride (NaCl) 5,0 g
Disodiumhydrogen phosphate 2,5 g
Agar 10,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at (20 ± 1) °C.
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5.2.2.8 Malt Extract Agar (MEA)
Malt extract 30,0 g
Soy peptone, papaic digest of soybean meal 3,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to
5,6 ± 0,2 when measured at (20 ± 1) °C.
5.
...

SLOVENSKI STANDARD
oSIST prEN 16616:2021
01-januar-2021
Kemična razkužila in antiseptiki - Termokemično razkuževanje tekstila - Preskusna
metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test
method and requirements (phase 2, step 2)
Chemisches Desinfektionsmittel und Antiseptika - Chemothermische
Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile -
Méthode d'essai et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: prEN 16616
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
oSIST prEN 16616:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 16616:2021


DRAFT
EUROPEAN STANDARD
prEN 16616
NORME EUROPÉENNE

EUROPÄISCHE NORM

December 2020
ICS 11.080.20 Will supersede EN 16616:2015
English Version

Chemical disinfectants and antiseptics - Chemical-thermal
textile disinfection - Test method and requirements (phase
2, step 2)
Désinfectants chimiques et antiseptiques - Désinfection Chemisches Desinfektionsmittel und Antiseptika -
thermochimique du textile - Méthode d'essai et Chemothermische Wäschedesinfektion - Prüfverfahren
prescriptions (phase 2, étape 2) und Anforderungen (Phase 2, Stufe 2)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 216.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

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

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE 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. prEN 16616:2020 E
worldwide for CEN national Members.

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Contents Page
European foreword . 4
Introduction . 7
1 Scope . 8
2 Normative references . 8
3 Terms and definitions . 9
4 Requirements . 9
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents . 10
5.2.1 Test organisms . 10
5.2.2 Culture media and reagents . 11
5.3 Apparatus and glassware . 13
5.3.1 General . 13
5.3.2 Usual microbiological laboratory equipment . 13
5.4 Preparation of test organism suspensions (test suspension) . 16
5.4.1 General . 16
5.4.2 Preservation and stock cultures of test organisms. 16
5.4.3 Working culture and test organisms . 16
5.4.4 Test suspension (N) . 17
5.4.5 Inoculation of the carriers . 21
5.5 Procedure for assessing the microbicidal activity of the product . 21
5.5.1 General . 21
5.5.2 Method . 23
5.6 Experimental data and calculation . 25
5.6.1 Explanation of terms and abbreviations . 25
5.6.2 Calculation . 25
5.7 Verification of methodology . 28
5.7.1 General . 28
5.7.2 Control of weighted mean counts . 28
5.7.3 Basic limits . 28
5.8 Expression of results and precision . 29
5.8.1 Reduction . 29
5.8.2 Repetitions . 29
5.9 Interpretation of results – conclusion . 29
5.9.1 General . 29
5.9.2 Microbicidal activity . 29
5.10 Test report . 30
Annex A (informative) Referenced strains in national collections . 32
Annex B (informative) Suitable neutralizers and rinsing liquids . 34
B.1 B.1 General . 34
B.2 B.2 Neutralizers . 34
B.3 B.3 Neutralizer added to the agar for counting . 35
Annex C (informative) Graphical representations of the test method . 36
2

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Annex D (informative) Example of washing machine specification . 37
Annex E (informative) Preparation of hard water for using in the test and reference
procedures . 38
Annex F (informative) Test report (example) . 39
Annex G (informative) Example for loading the washing machine . 46
Annex ZA (informative) Relationship between this European Standard and the and the
General Safety and Performance Requirements of Regulation (EU) 2017/745 aimed
to be covered . 48
Bibliography . 50

3

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European foreword
This document (prEN 16616:2020) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 16616:2015.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of
EU Directive.
For relationship with EU Directive, see informative Annex ZA, which is an integral part of this
document.
The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to
harmonize the structure and wording with other tests of CEN/TC 216 existing or in preparation and to
improve the readability of the standard and thereby make it more understandable. The following is a
list of significant technical changes since the last edition:
— the scope is adapted to the scope of the WG 1;
— in Clause 4 the requirements for phase 2, step 1 tests were deleted;
— in 5.1. some editorial changes were done;
— in 5.2.2.4 water for the test and reference control were added;
— old 5.2.2.6 was deleted, because it was not used in the standard;
— the example for washing machines in 5.3.2.18 of the previous version was switched to Annex D;
— Potter S1 apparatus was added (5.3.2.21);
— 5.4.3.1 to 5.4.3.3 were adapted to other current standards;
— 5.4.4 was reviewed (editorial changes and better description);
— the description of the neutralization (5.5.1.2) was re-worded, a reference to phase 2, step 1 tests
was added;
— in 5.4.3.1 and 5.4.3.2 an information on using a spectrophotometer for counting cell numbers of
mycobacteria was added (NOTE);
— in 5.4.5 some editorial changes were done;
— 5.4.2 (old version) was deleted;
— the choice of the neutralizer shall be documented and justified in the test report (5.5.1.2);
— 5.5.1.4 was deleted, no longer necessary;
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— in 5.5.2.1 a new NOTE was added;
— in 5.5.2.2 a NOTE and the reference to Annex G was added, c) is deleted, no longer necessary;
— the reference control was added (5.5.2.3);
— RII was deleted (5.5.2.5);
— 5.5.2.6 to 5.5.2.8 were deleted, a reference to phase 2, step 1 tests was added;
— in 5.5.2.9 some editorial changes were done;
— in 5.6.1.1 the table was corrected and adapted to the current tests;
— in 5.6.2.1 two paragraphs are added (former 5.6.2.3);
— in 5.6.2.2 the V -values will be expressed per carrier (former per ml);
c
— In 5.6.2.3 the calculation of N and N was added, the formula was corrected;
0
— in 5.6.2.4 the calculation was changed to values per carrier, the formula was corrected and the
weighted mean was added in all calculations;
— in 5.6.2.4 the requirements for N were corrected;
w
— 5.6.2.5 was deleted, a reference to phase 2, step 1 test was added;
— 5.6.2.6 calculation was changed: only N will be calculated, RI is not counted and RII, B and C are no
w
longer used in the standard;
— the example in 5.7.2 was corrected;
— 5.7.3 was adapted to the tests in the current version;
— in 5.8.1 some editorial changes were done;
— in 5.10 a new test report was added;
— Annex A was adapted to EN 12353;
— Annex C was corrected;
— addition of a new Annex D “Example of a washing machine”;
— addition of a new Annex E “Preparation of hard water for using in the test and reference
procedure”;
— addition of a new Annex F “Example of a test report”;
— addition of a new Annex G “Example for loading the washing machine”.
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The changes of this revision have no impact on the test results obtained with reference to the version
EN 16616:2015. Those results are still valid except the reduction of the reference control N was
w
higher than 3 lg and/or the calculation of the results followed the wrong way of version 2015.
6

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Introduction
This document specifies a carrier test for establishing whether a single-wash disinfecting product or
combination of products for the treatment of contaminated textile has or does not have necessary
microbicidal activity. This document only intends to validate the disinfection part of the laundry
process.
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 could
influence its action in practice.
The conditions are intended to cover general purposes and to allow reference between microbiological
laboratories and types of detergents and disinfectants. Each effective dosage of the chemical
disinfectant found by this test corresponds only to the chosen experimental conditions. Where actual
conditions vary additional testing in microbiological laboratories is needed to determine the effective
dosage. Instructions for use are the responsibility of manufactures of detergents or disinfectants.
7

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1 Scope
This document specifies a test method and the minimum requirements for the microbicidal activity of a
defined disinfection process for the treatment of contaminated textile. This procedure is carried out by
using a washing machine as defined in 5.3.2.18 and refers to the disinfection step without prewash. This
procedure is not limited to certain types of textile. The suppliers' instructions are expected to be
sufficient to allow the method in this document to be carried out fully (e.g. dosing disinfectant in
whatever washing phase e.g. main wash, rinsing, disinfecting at 40 °C).
This document applies to areas and situations where disinfection is medically indicated. Such
indications occur in patient care, for example:
— in hospitals, in community medical facilities, and in dental institutions;
— in clinics of schools, of kindergartens, and of nursing homes;
and could occur in the workplace and in the home.
It could also include services such as laundries and kitchens supplying products directly for the
patients.
The method described is intended to determine the activity of a product or product combination under
the conditions in which they are used. This is a phase 2, step 2 laboratory test that simulates the
conditions of application of the product.
NOTE This method corresponds to a phase 2, step 2 test (see EN 14885).
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 13624, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of
fungicidal or yeasticidal activity in the medical area — Test method and requirements (phase 2, step 1)
EN 13727, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of
bactericidal activity in the medical area — Test method and requirements (phase 2, step 1)
EN 14348, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of
mycobactericidal activity of chemical disinfectants in the medical area including instrument disinfectants -
Test methods and requirements (phase 2, step 1)
EN 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical
disinfectants and antiseptics
8

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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
liquor ratio
ratio of the weight of dry textile in kilograms and volume of wash liquor in litres (w/v)
3.2
disinfection process
process taking into account practical conditions of application of the product including contact time,
temperature, test organisms and interfering substances to disinfect the textile
3.3
treatment of contaminated textile
handling the textile according the disinfection process to obtain disinfected textile
4 Requirements
The test results shall fulfil the basic limits (see 5.7.3).
Table 1 — Minimum and additional test conditions
Test conditions Textile disinfection Textile disinfection
temperature < 60°C temperature ≥ 60°C
Test organism E. coli K12 b
E. faecium
E. hirae
P. aeruginosa
S. aureus
C. albicans
Additional test organism A. brasiliensis
Fungicidal activity M. terrae
Tuberculocidal activity M. terrae and M. avium
Mycobactericidal activity
Temperature As recommended by the As recommended by the
a a
manufacturer and < 60°C manufacturer and ≥ 60°C
Contact time As recommended by the As recommended by the
manufacturer manufacturer
Interfering substance Sheep blood Sheep blood
NOTE The implementation of bacterial spores and viruses was discussed. Further development is necessary to
make it technically feasible.
a
The temperature and the contact time shall be chosen on the basis of the practical conditions of the product application
and within the responsibility of the manufacturer.
b
This includes bactericidal, yeasticidal, fungicidal, tuberculocidal and mycobactericidal activity.
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5 Test methods
5.1 Principle
Carriers made of cotton fabric (5.3.2.16) are contaminated with a test suspension of microorganisms in
defibrinated sheep blood (5.2.2.12). After drying the carriers are transferred into cotton towels and
then the disinfection process in the washing machine is performed at test temperatures either t < 60 °C
or t ≥ 60 °C. The process refers to the disinfection step without prewash. At the end of the disinfection
step of the procedure, the towels with the carriers have to be taken out. Each carrier is transferred into
a separate tube containing neutralizer (5.2.2.11) and glass-beads (5.3.2.11). The microorganisms should
be recovered from the carriers by shaking. The number of surviving microorganisms in each sample is
determined and the reduction rate is calculated.
5.2 Materials and reagents
5.2.1 Test organisms
1
The bactericidal activity shall be evaluated using the following strains as test organisms
— Pseudomonas aeruginosa ATCC 15442
— Escherichia coli (K12) NCTC 10538
— Staphylococcus aureus ATCC 6538
— Enterococcus hirae ATCC 10541
— Enterococcus faecium ATCC 6057
The yeasticidal/fungicidal activity shall be evaluated using the following test organisms:
— Candida albicans ATCC 10231
— Aspergillus brasiliensis
ATCC 16404
(formerly Aspergillus niger ATCC 16404)
The tuberculocidal/mycobactericidal activity shall be evaluated using the following test organisms:
— Mycobacterium terrae ATCC 15755
— Mycobacterium avium ATCC 15769
NOTE See Annex A for strain reference in some other culture collections.
The required incubation temperatures for these test organisms are (36 ± 1) °C (5.3.2.3) [C. albicans and
A. brasiliensis: (30 ± 1) °C]. The same temperature shall be used for all incubations performed during a
test and its controls and validation.
If additional test organisms are used, they shall be incubated under optimum growth conditions
(temperature, time, atmosphere and media) noted in the test report. If the additional test organisms
selected do not correspond to the specified strains/species, 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 stored under a reference for five years.

1
The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC).
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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5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this document refer to the anhydrous salts. Hydrated forms
may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the growth of 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 used for preparation of media
The water shall be fresh distilled water and not just demineralized water. Sterilize in the autoclave
[5.3.2.1a)].
NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently
sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference
[1]) can be used.
5.2.2.3 Hard water for dilution of products for validation tests
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
2
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the
2
autoclave [5.3.2.1a)]. Autoclaving, if used, could 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) at (2 to 8) °C 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
3
1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) at
(2 to 8) °C for no longer than one week.
— 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 with the
use of a pipette (5.3.2.9) 6,0 ml 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.
5.2.2.4 Water for the test and reference control
Water with potable water quality is necessary (the water should contain less than 100 cfu/ml of
bacteria at 36 °C and 22 °C). Water hardness shall be logged and mentioned in the laboratory protocol
and shall be documented in the test report. The final hardness shall be equal or higher than 4 mmol/l
2+ 2+
alkaline earth ions (Mg and Ca ). The temperature of the water influx should be between (12 and
20) °C.
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NOTE 1 Annex E indicates how to adjust the water hardness for the test and reference procedure using
solutions A and B from 5.2.2.3. The water hardness could be measured using commercial available test kits.
NOTE 2 Water supplier data can be used to document water hardness.
5.2.2.5 Tryptone Soy Agar (TSA)
Tryptone, pancreatic digest of casein 15,0 g
Soy 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 1 000,0 ml
Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at (20 ± 1) °C.
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it could be necessary to add
neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used.
5.2.2.6 Tryptone Soy Broth (TSB)
Tryptone, pancreatic digest of casein 15,0 g
Soy peptone, papaic digest of soybean meal 5,0 g
Sodium chloride (NaCl) 5,0 g
Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.7 Brain Heart Infusion Agar (BHI)
EXAMPLE
Brain heart infusion 12,5 g
Beef heart infusion 5,0 g
Proteose-Peptone 10,0 g
Glucose 2,0 g
Sodium chloride (NaCl) 5,0 g
Disodiumhydrogen phosphate 2,5 g
Agar 10,0 g
Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.8 Malt Extract Agar (MEA)
Malt extract 30,0 g
Soy peptone, papaic digest of soybean meal 3,0 g
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...

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