SIST EN 16616:2022
(Main)Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test method and requirements (phase 2, step 2)
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 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”.
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
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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.
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
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
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);
— 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);
— 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.
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.
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
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.
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
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™
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
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.
Formerly: Aspergillus niger ATCC® 16404™.
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
(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 [5.3.2.1a)]. 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
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.
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.
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.
5.2.2.9 Middlebrook and Cohn 7H10 medium incl. 10 % OADC (7H10)
Middlebrook 7H10 agar 19,0 g
Glycerol 5,0 ml
Water (5.2.2.2) to 900,0 ml
Heat to boiling to dissolve completely. Sterilize for 10 min in the autoclave [5.3.2.1 a)] and cool to 50 °C
to 55 °C. Add 100 ml Middlebrook OADC enrichment under aseptic conditions. The final pH of the medium
shall be equivalent 6,6 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.10 Diluent
Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride (NaCl) 8,5 g
Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave [5.3.2.1 a)]. After sterilization, the pH of the diluent shall be equivalent to
7,0 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.11 Neutralizer
Information on neutralizers that have been found to be suitable for some categories of products is given
in Annex B.
5.2.2.12 Sterile defibrinated sheep blood
The sterile defibrinated sheep blood (aseptic blood-letting and preparation) pooled from more than one
sheep can be acquired from a commercial supplier.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
5.3.2 Usual microbiological laboratory equipment
and, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)
+3
a) for moist heat sterilization, an autoclave capable of being maintained at ( ) °C for a minimum
contact time of 15 min [2];
+5
b) for dry heat sterilization, a hot air oven capable of being maintained at ( ) °C for a minimum
+5
+5
contact time of 30 min, at ( 170 )°C for a minimum contact time of 1 h or at ( 160 ) °C for a
0 0
minimum contact time of 2 h.
5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (36 ± 1) °C or (37 ± 1) °C, at
(45 ± 1) °C (to maintain melted medium in case of pour plate technique) and at additional test
temperatures ± 1 °C, temperatures till (70 ± 1) °C shall be adjustable.
5.3.2.3 Incubator, capable of being controlled either at (30 ± 1) °C or (36 ± 1) °C. The same
temperature shall be used for incubations performed during a test and its controls and validation.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media
(5.2.2.5 to 5.2.2.9).
5.3.2.5 Stopwatch, a digital stopwatch is recommended.
5.3.2.6 Shakers
® 4
a) Electromechanical agitator, e.g. Vortex mixer ;
-1
b) Orbital shaker (at 400 min ).
5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm
to 50 mm and 0,22 µm pore size for sterilization of hard water (5.2.2.3).
5.3.2.8 Refrigerator, capable of being controlled at (2 to 8) °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 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 Glass beads (diameter 3 mm to 4 mm).
5.3.2.12 Volumetric flasks
Disposable sterile equipment is an acceptable alternative to reusable glassware.
4 ®
Vortex is an example of a suitable product available commercially. This information is given for the convenience of
users of this document and does not constitute an endorsement by CEN of this product.
5.3.2.13 Centrifuge (3 000 g ).
N
5.3.2.14 Coned bottom screw cap tubes (contents of 50 ml, diameter: about 28 mm).
5.3.2.15 Cylindrical screw vial (contents about 15 ml to 50 ml) for recovery of the test organisms
from the carriers.
5.3.2.16 Cotton carriers, 1 cm : The carriers are prepared by using standard cotton fabric cuted into
1 cm pieces, cooked in double-distilled water three times and sterilized in the autoclave [see 5.3.2.1a)].
carriers are put in the pockets of a bigger cotton towel during the test
For practical reasons the 1 cm
procedure in the washing machine.
NOTE 1 The incorporation of the test suspension can be improved if the carriers are not dried after autoclaving.
2 2
Mass per unit area: (170 ± 10) g/m (real 160 g/m )
Fibrous material (warp and weft): cotton, double corded
Fibre length (warp and weft): at least 27 mm
29,5 ± 1 mg/m [(29,5 ± 1) mg/m corresponds to (295
Yarn linear density (warp and weft):
± 10 dtex)]
Yarn twist (warp and weft): Z-twist (700 ± 25) t/m
Weave: plain weave 1
Threads per unit length: 270 threads/dm each
The cotton control cloth shall be bleached, unfinished and not brightened.
After three washes the maximum tensile strength, wet, in the warp should be (63 ± 5) daN.
NOTE 2 Cotton proofed in accordance with ISO 2267 [3] fulfils the requirements.
5.3.2.17 Machine ballast load and preparation of ballast load
Textile of poly cotton (65 % polyester / 35 % cotton) shall be used as ballast load. For special purposes
textile of cotton (100 %) can be used.
NOTE 1 The bound liquor is higher in pure cotton than in poly cotton.
Amount of ballast load: 70 % ± 10 % of the max. capacity (in kg).
The ballast load should be washed for 30 min at (80 to 90) °C without additives followed by at least 3
rinsing steps and sterilized in the autoclave prior to first use.
The ballast load should be used for not more than 100 washing cycles (incl. preparation and disinfection
cycles).
After disinfection testing the ballast load should be washed for 30 min at (80 to 90) °C followed by at least
5 rinsing steps (at least 5 min each) prior to use.
NOTE 2 The ballast load can be sterilized additionally in the autoclave.
NOTE 3 If foam is present in last rinsing step, additional rinsing might be necessary.
5.3.2.18 Washing machine and preparation of the machine
The washing machine and additional equipment shall guarantee the following specifications:
a) heating time from 40 °C to 60 °C is ≤ 15 min;
b) thermostatic temperature control + 0,5 °C in the range from (30 to 90) °C temperature;
c) switch on temperature ≤ 4 °C below switch-off temperature, accuracy at switch off ± 1 °C;
d) timer needs to be programmable;
e) equipment for calibration of the water supply, e.g. a transparent tube for the control of the water
level in the washing machine;
f) separate addition of washing agent and disinfectant shall be possible. Dosage possibility for powder
surfactants and liquid surfactants is necessary;
g) program sequence shall be interrupted at any time and possibility for taking samples shall be given;
h) water supply: Water supply should be controlled with a margin of ± 0,5 l or ± 0,5 kg (this can be
achieved by for example a water flow meter or a weighbridge).
NOTE An example of washing machine specification is given in Annex D.
It is recommended to keep the machine in an air conditioned room and control environmental conditions
in temperature range of (20 to 25) °C and between 40 % to 60 % relative humidity.
Before each test run the machine should be run one time for 30 min at (80 to 90) °C without additives.
No ballast load is needed for machine preparation.
It is recommended to document adequate machine parameters during the test runs to ensure a proper
function of the machine and the process. This includes temperature in the wash liquor, amount of water
influx, reversing profile and if possible on/off-status of heating element.
5.3.2.19 Fritted filter, porosity of 40 µm to 100 µm (see ISO 4793)
5.3.2.20 Roux bottles or similar flasks
5.3.2.21 Glass wool for filtration
5.3.2.22 Potter S 1 apparatus
5.4 Preparation of test organism suspensions (test suspension)
5.4.1 General
For each test organism a test suspension to perform the test shall be prepared.
5.4.2 Preservation and stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.
5.4.3 Working culture and test organisms
5.4.3.1 Bacteria
In order to prepare the working culture of the bacteria, subculture from the stock culture by streaking
onto at least three plates containing TSA (5.2.2.5) [E. faecium: also BHI (5.2.2.7) can be used] and incubate
at (36 ± 1) °C (5.3.2.3).
After 18 h to24 h incubation at (36 ± 1) °C or (37 ± 1) °C prepare a second subculture from the first
subculture in the same way and incubate for 18 h to24 h. From this second subculture, a third subculture
may be produced in the same way. The second and (if produced) third subculture are the working
cultures.
Do not take a fourth subculture.
5.4.3.2 Yeasts and moulds
Candida albicans (yeast)
In order to prepare the working culture of Candida albicans, subculture from the stock culture by
streaking onto MEA (5.2.2.8) and incubate (5.3.2.3) at (30 ± 1) °C. After 42 h to 48 h prepare a second
subculture from the first subculture in the same way and incubate for 42 h to 48 h.
From this second subculture, a third subculture may be produced in the same way. The second and (if
produced) third subcultures are the working culture(s).
Do not take a fourth subculture.
Aspergillus brasiliensis (mould)
In order to prepare the working culture of Aspergillus brasiliensis, subculture from the stock culture by
streaking onto MEA (5.2.2.8) and incubate at (30 ± 1) °C (5.3.2.3). For Aspergillus brasiliensis use only the
first subculture grown on MEA (5.2.2.8) in Roux bottles (5.3.2.20) and incubate for 7 d to 9 d. No further
subculturing is needed.
At the end of incubation, all the cultures shall show a dark brown or black surface with only a few small
white or grey spots, see EN 13624.
5.4.3.3 Mycobacteria
In order to prepare the working culture of the mycobacteria (M. avium and M. terrae), subculture from
the stock culture by streaking onto at least three plates for each test containing 7H10 (5.2.2.9) and
incubate at (36 ± 1) °C (5.3.2.3) for 21 d.
The first culture will be the working culture. Do not take any subculture.
In the case of M. terrae, it is possible to prepare carrier with a shelf life of 12 weeks (storage conditions
see 5.4.5). To prepare 100 carriers with M. terrae, use 10 Petri dishes.
5.4.4 Test suspension (N)
5.4.4.1 Bacterial test suspension (N)
For harvesting the bacteria, 5 ml or 10 ml of 9 g/l NaCl solution are pipetted into the plates or the Roux
bottles respectively and the bacteria are washed off and resuspended. If necessary the suspension is
purified by filtration through glass wool (5.3.2.21). The filtrate is aliquoted in screw cap tubes and
centrifuged 15 min at 3 000 g . The liquid is removed and the pellet is mixed with suitable volume of
N
9 5
9 g/l NaCl solution. The number of cells in the suspension shall be at least 1,5 x 10 cfu/ml . The numbers
of cfu shall be estimated by means of spectrophotometer or any other suitable technique.
NOTE For preparation of 10 carriers, two Petri dishes or one Roux bottle are needed.
For P. aeruginosa centrifugation at 4 700 g is recommended.
N
To get the test suspension at the appropriate cfu/ml, centrifuge it again and resuspend it with
defibrinated sheep blood (5.2.2.12) in the same volume.
Spread a loop of this test suspension on a suitable agar plate and incubate for purity control.
−7 −8
For counting, prepare 10 and 10 dilutions of the test suspension using the diluent (5.2.2.10 and the
primary used culture medium respectively). Mix [5.3.2.6 a)]. Take a sample of 1,0 ml of each dilution in
duplicate and inoculate using the pour plate or the spread plate technique.
When using the pour plate technique, transfer each 1 ml sample into separate Petri dishes and add 15 ml
to 20 ml melted TSA (5.2.2.5) [E. faecium: also BHI (5.2.2.7) can be used], cooled to (45 ± 1) °C.
When using the spread plate technique, spread each 1,0 ml sample – divided into portions of
approximately equal size – on an appropriate number (at least two) of surface dried plates containing
TSA (5.2.2.5) [E. faecium: also BHI (5.2.2.7) can be used].
For incubation and counting see 5.5.2.5.
5.4.4.2 Fungal test suspension (N)
5.4.4.2.1 Candida albicans
For harvesting the cells, 5 ml or 10 ml 9 g/l NaCl solution are pipetted into the plates or the Roux bottles
respectively and the cells are washed off and resuspended. The suspension is aliquoted in screw cap tubes
and centrifuged for 15 min at 3 000 g . The liquid is removed and the pellet is mixed with suitable volume
N
of 9 g/l NaCl solution. The number of cells in the suspension shall be at least 1,5 x 10 cfu/ml. The
numbers of cfu shall be estimated by means of spectrophotometer or any other suitable technique.
To get the test suspension at the appropriate cfu/ml, centrifuge it again and resuspend it with
defibrinated sheep blood (5.2.2.12) in the same volume.
Spread a loop of this test suspension on a suitable agar plate and incubate for purity control.
−6 −7
For counting, prepare 10 and 10 dilutions of the test suspension using diluent (5.2.2.10). Mix
[5.3.2.6 a)].
Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread plate
technique.
1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add
15 ml to 20 ml melted MEA (5.2.2.8), cooled to (45 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of
approximately equal size - on an appropriate number (at least two) of surface dried plates containing
MEA (5.2.2.8).
For incubation and counting, see 5.5.2.5.
cfu/ml = colony forming unit(s) per millilitre.
5.4.4.2.2 Aspergillus brasiliensis
The procedure for preparing the Aspergillus brasiliensis test suspension is as follows:
a) Take the working culture (5.4.3.2) and suspend the conidiospores in 10 ml of sterile 0,5 g/l
polysorbate 80 solution in water (5.2.2.2). Using a glass rod or spatula, detach the conidiospores from
the culture surface. Transfer the suspension into a flask and gently shake by hand for one minute
together with 5 g of glass bea
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