kSIST FprEN 16615:2026

SLOVENSKI STANDARD
oSIST prEN 16615:2022
01-december-2022
Kemična razkužila in antiseptiki - Kvantitativna preskusna metoda za vrednotenje
baktericidnega delovanja in delovanja na kvasovke in/ali fungicidnega in/ali
tuberkulocidnega in/ali mikobaktericidnega delovanja na neporoznih površinah z
mehanskim delovanjem z odvzemom brisa v medicini (4-področni preskus) -
Preskusna metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Quantitative test method for the evaluation of
bactericidal and yeasticidal and/or fungicidal and/or tuberculocidal and/or
mycobactericidal activity on non-porous surfaces with mechanical action employing
wipes or mops in the medical area (4- field test) - Test method and requirements (phase
2, step 2)
Chemische Desinfektion und Antiseptika - Quantitativer Prüfversuch zur Bestimmung der
bakteriziden und levuriziden und/oder fungiziden und/oder tuberkuloziden und/oder
mykobakteriziden Wirkung auf nicht-porösen Oberflächen mit mechanischer Einwirkung
mit Hilfe von Tüchern oder Mops im humanmedizinischen Bereich (4-Felder-Test) -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Méthode d’essai quantitative pour l’évaluation
de l’activité bactéricide et levuricide et/ou fongicide et/ou tuberculocide et/ou
mycobactéricide des surfaces non poreuses, avec action mécanique à l’aide de lingettes
ou de serpillières dans le domaine médical (essai à 4 zones) - Méthode d'essai et
prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: prEN 16615
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
oSIST prEN 16615:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 16615:2022
oSIST prEN 16615:2022
DRAFT
EUROPEAN STANDARD
prEN 16615
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2022
ICS 11.080.20 Will supersede EN 16615:2015
English Version
Chemical disinfectants and antiseptics - Quantitative test method
for the evaluation of bactericidal and yeasticidal and/or
fungicidal and/or tuberculocidal and/or mycobactericidal
activity on non-porous surfaces with mechanical action
employing wipes or mops in the medical area (4- field test) - Test
method and requirements (phase 2, step 2)
Antiseptiques et désinfectants chimiques - Méthode d'essai Chemische Desinfektionsmittel und Antiseptika - Quantitatives
quantitative pour l'évaluation de l'activité bactéricide et Prüfverfahren zur Bestimmung der bakteriziden und
levuricide et/ou fongicide et/ou tuberculocide et/ou levuroziden und/oder fungiziden und/oder tuberkuloziden
mycobactéricide sur des surfaces non poreuses, avec action und/oder mykobakteriziden Wirkung auf nicht-porösen
mécanique à l'aide de lingettes ou de serpillières dans le Oberflächen mit mechanischer Einwirkung mit Hilfe von
domaine médical (essai à 4 zones) - Méthode d'essai et Tüchern oder Mops im humanmedizinischen Bereich (4-
exigences (phase 2, étape 2) Felder-Test) - Prüfverfahren 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, Türkiye 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 NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
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. prEN 16615:2022 E
worldwide for CEN national Members.

oSIST prEN 16615:2022
prEN 16615:2022 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 6
3 Terms and definitions . 6
4 Requirements . 6
5 Test methods . 7
5.1 Principle . 7
5.2 Materials and reagents . 8
5.2.1 Test organism . 8
5.2.2 Culture media and reagents . 9
5.3 Apparatus and glassware . 13
5.4 Preparation of test organism suspensions and product test solutions . 17
5.4.1 Test organism suspensions . 17
5.4.2 Product test solution . 22
5.5 Procedure for assessing the bactericidal and yeasticidal and/or fungicidal and/or
tuberculocidal and/or mycobactericidal activity of the product . 23
5.5.1 General . 23
5.5.2 Method . 25
5.6 Experimental data and calculation . 28
5.6.1 Explanation of terms and abbreviations . 28
5.6.2 Calculation . 29
5.7 Verification of methodology . 34
5.7.1 General . 34
5.7.2 Control of weighted mean counts . 34
5.7.3 Basic limits . 35
5.8 Expression of results and precision . 35
5.8.1 Overview of the different suspensions/test mixtures . 35
5.8.2 V -values . 36
c
5.8.3 Limiting test organism and bactericidal and yeasticidal concentration. 36
5.8.4 Precision, repetitions . 37
5.9 Interpretation of results – conclusion . 38
5.10 Test report . 39
Annex A (informative) Referenced strains in national collections . 41
Annex B (informative) Neutralizers . 42
Annex C (informative) Graphical representations of the test method . 43
Annex D (informative) Example of a typical test report . 46
Annex E (informative) Alternative drying endpoint . 50
Annex F (informative) Additional test temperature . 52
Bibliography . 53

oSIST prEN 16615:2022
prEN 16615:2022 (E)
European foreword
This document (prEN 16615:2022) 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 16615:2015.
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 standard and thereby make it more understandable.
The following is a list of significant technical changes since the last edition:
— Annex ZA was deleted, the document was de-harmonized;
— N was deleted;
— Harmonization of the text with EN 13727;
— Implementation of different applications forms;
— Implementation different swab material;
— Implementation of the definition of room temperature;
— Water control always be carried out with hard water (= process and method validation);
— Polysorbate 80 should be weighed 1 g instead 1 ml;
— Implementation of Apparatus Pre-cleaning of surfaces with 2-Propanol instead of n-Propanol;
— Correction of calculation and editorial mistakes;
— Implementation of additional test surfaces;
— Implementation of standard wiping cloth (wipe) and specified wiping cloth;
— Implementation of fungicidal, tuberculocidal and mycobactericidal activity;
— Water control: Test field 1 to be taken into account for calculation of the water control;
— Implementation of Annex E and Annex F.
The changes of this revision have no impact on the test results obtained with reference to the version
EN 16615:2015. Those results are still valid.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
Introduction
This document specifies a carrier test for establishing whether a chemical disinfectant for use on surfaces
administered with wipes has a bactericidal and yeasticidal activity in the fields described in the scope.
The laboratory test closely simulates practical conditions of application such as contact time,
temperature and interfering substances, including pre-drying specified test organisms on a test-surface
as carrier and wiping the product on the test-surface with a wipe. The conditions are intended to cover
general purposes. However, if for some applications the recommendations of use of a product differ
additional test conditions may be used or may need to be used.
Each utilization concentration of the product found by this test corresponds to defined experimental
conditions.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
1 Scope
This document specifies a test method and the minimum requirements for bactericidal and yeasticidal,
fungicidal, tuberculocidal and/or mycobactericidal activity of chemical disinfectant products that form a
homogeneous, physically stable preparation when diluted with hard water – or in the case of ready-to-
use products – with water.
This document applies to products that are used in the medical area for disinfecting non-porous surfaces
including surfaces of medical devices by wiping or mopping – regardless if they are covered by the
93/42/EEC Directive on Medical Devices or not.
Due to the new methods of application of surface disinfectants like pre-impregnated wipes this standard
was established to cover the different application method.
prEN 16615 is applicable for four methods of application of products for wiping and/or mopping:
a) soaking any non-specified wipe or mop with product;
b) spraying the product on any non-specified wipe and / or mop or a specified wipe or mop;
c) impregnation of specified wipes or mops by the user with the product according to the
manufacturer’s recommendation;
d) pre-impregnation of specified wipes or mop by the manufacturer as ready-to-use wipes or mops.
In all types of application, the water control is done with the standard wipe [5.3.2.17.1], because it is a
process or method control.
This document does not apply to products that are sprayed on or flooding surfaces, without wiping in the
contact time. In this case, the methods of phase 2/ stage 2 without mechanical action apply.
The test surface (5.3.2.16) was selected as standard surface to cover all non-porous surfaces. This
document does not apply to the testing of the influence of different surfaces.
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 can occur in the workplace and in the home. It can also include services such as laundries and
kitchens supplying products directly for the patients.
NOTE This method corresponds to a phase 2, step 2 test.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 12353, Chemical disinfectants and antiseptics - Preservation of test organisms used for the
determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal
(including bacteriophages) activity
EN 14885, Chemical disinfectants and antiseptics - Application of European Standards for chemical
disinfectants and antiseptics
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
ready-to-use wipe
pre-impregnated wipe
wipe containing disinfectant added by the wipe manufacturer at the manufacturing site
3.2
impregnated wipe
wipe containing disinfectant added by the user
Note 1 to entry: Examples include a wipe soaked in disinfectant, a dry wipe sprayed with disinfectant.
4 Requirements
The product, when diluted with hard water or – in the case of ready-to-use products – with water, shall
demonstrate at least a 5 decimal log (lg) reduction for bacteria and at least a 4 decimal log (lg) reduction
for fungi and mycobacteria on test field 1, when tested in accordance with Table 1 and Clause 5.
The mean of the number of cfu per 25 cm on the test fields 2 to 4 of the product under test shall be equal
or less than 50, the mean of the number of cfu per 25 cm on the test fields 1 to 4 of the water control
shall be equal or more than 10. Details on the precision and repetition are given in 5.8.4 and EN 14885.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
Table 1 — Experimental conditions
Test Bactericidal Yeasticidal Fungicidal Tuberculocidal Mycobactericidal
conditions activity activity activity activity activity
Staphylococcus Aspergillus Mycobacterium
Minimum
aureus brasiliensis avium
Enterococcus Candida Mycobacterium
spectrum of
Candida Mycobacterium
hirae albicans terrae
albicans terrae
test Pseudomonas
organisms aeruginosa
additional any relevant test organism
according to the manufacturer’s recommendation, but between
Test
(4 ± 1) °C to (30 ± 1) °C
temperature
For tests performed at room temperature, the range shall be (21,5 ± 3,5)°C
according to the manufacturer’s recommendation, but at minimum 1 min
Contact time
a
and no longer than 5 min or 60 min (from 1 min to 5 min at intervals of 1 min and
from 5 min to 60 min at intervals of 5 min)
Interfering substance
clean
0,3 g/l bovine albumin
conditions
dirty
3,0 g/l bovine albumin plus 3,0 ml/l erythrocytes
conditions
additional any relevant substance
NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained
under the minimum test conditions.
a
The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions
of the product. The recommended contact time for the use of the product is within the responsibility of the
manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient and/or
the medical staff and surfaces, which are frequently touched by different people, leading to the transmission of
microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same applies where
the contact time of the product shall be limited for practical reasons. Products for other surfaces than stated
above may be tested with a contact time of maximum 60 min.
5 Test methods
5.1 Principle
5.1.1 A test-surface is marked with 4 squares of 5 × 5 cm, the “test fields”, in a row. Test field 1 on the
test-surface is inoculated with a test suspension of bacteria, yeasts, fungal spores or mycobacteria in a
solution of interfering substances. The inoculum is dried. A wipe is soaked with a sample of the product
as delivered and/or diluted with hard water (5.2.2.7) (for ready to use products: water). The test-surface
is wiped with the soaked wipe across the four marked test fields, starting in front of test field 1, turning
immediately after test field 4 and wiped back to the starting point. In parallel a water control (5.5.2.2 h)
is performed: a wipe is soaked with hard water (5.2.2.7) instead of the product in the same way to ensure
that the test organisms are spread on the 4 fields and their number reaches a certain level.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
NOTE For the purposes of this document, references to wiping, wipe and wiped can be equated to mopping,
mop and mopped when the standard method is used to test a mopping application.
Temperature, soiling and contact time are employed as recommended by the manufacturer. At the end of
the contact time, the test organisms are recovered from each test field with moistened FLOQSwabs. The
swabs are brought into a tube containing broth and neutralizer and the test organisms are to be severed
from the swab by shaking. The numbers of surviving test organisms in each sample are determined, and
the reduction is calculated by comparing the results of the drying control D and the results obtained
ct
with the product. The test is performed using P. aeruginosa, S. aureus, E. hirae and C. albicans (minimum
test condition) and/or A. brasiliensis and/or M. terrae and/or M. avium ( optional test condition) as test
organisms.
5.1.2 Additional test organisms (only bacterial or fungal strains), contact times and interfering
substances can be used.
5.2 Materials and reagents
5.2.1 Test organism
The bactericidal activity shall be evaluated using the following strains as test organisms :
— Staphylococcus aureus ATCC 6538;
— Pseudomonas aeruginosa, ATCC 15442;
— Enterococcus hirae ATCC 10541.
The yeasticidal activity shall be evaluated using the following strain as test organism :
— Candida albicans ATCC 10231.
The fungicidal activity shall be evaluated using the following strains as test organisms :
— Aspergillus brasiliensis ATCC 16404;
— Candida albicans ATCC 10231.
The tuberculocidal activity shall be evaluated using the following strain as test organism :
— Mycobacterium terrae ATCC 15755.
The mycobactericidal activity shall be evaluated using the following strains as test organisms :
— Mycobacterium terrae ATCC 15755;
— Mycobacterium avium ATCC 15769.
See Annex A for strain references in some other culture collections.
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, their suitability for supplying the required inocula
shall be verified. If these additional test organisms are not classified at a reference centre, their
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or
national culture collection under a reference for five years.

The ATCC numbers are the collection numbers of strains supplied by these culture collections. This information is given
for the convenience of users of this document and does not constitute an endorsement by CEN of this product.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
The required incubation temperature for these test bacteria is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3). The
same temperature (36 °C or 37 °C) shall be used for all incubations performed during its control and
validation. The required incubation temperature for Candida albicans and Aspergillus brasiliensis is
(30 ± 1) °C (5.3.2.3).
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 test organism.
To improve reproducibility, it is recommended that commercially available dehydrated material is used
for the preparation of culture media if it complies with the formulae given below. 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.
All specified pH values are measured at (21,5 ± 3,5)°C (5.3.2.4).
5.2.2.2 Water
The water shall be freshly glass-distilled or deionized and demineralized water. If distilled water or
deionized and demineralized water of adequate quality is not available, water for injections (see [1]) may
be used.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used, e.g. for preparation
of culture media and subsequently sterilized.
See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Medium
a) Soya Agar (TSA)
— Tryptone, pancreatic digest of casein 15,0 g
— Soya peptone, papaic digest of soybean meal 5,0 g
— Sodium chloride (NaCl) 5,0 g
— Agar 15,0 g
— Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave (5.3.1). After sterilization the pH (5.3.2.4) of the medium shall be equivalent to
7,2 ± 0,2.
In case of encountering problems with neutralization (5.5.1.2) it may be necessary to add neutralizer to
TSA. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use a
neutralizer that causes opalescence in the agar.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
b) Malt Extract Agar (MEA)
Malt extract agar, consisting of:
— Malt extract [food grade (e.g. Christomalt powder from Difal) or an 30,0 g
equivalent extract that is not highly purified and not only based on
maltose (e.g. Malt extract from OXOID)]
— Agar 15,0 g
— Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave (5.3.1). After sterilization, the pH (5.3.2.4) of the medium shall be equivalent to
5,6 ± 0,2.
In case of an encountering problems with neutralization (5.5.1.2) it may be necessary to add neutralizer
to MEA. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use
neutralizer that causes opalescence in the agar.
If there are problems with producing at least 75 % spiny conidiospores, see 5.4.1.4 b 2).
c) Middlebrook and Cohn 7H10 medium with 10% OADC enrichment (MCO)
— Middlebrook 7H10 agar-powder 19,0 g
— Glycerol [bibliographic reference 1] 5,0 ml
— Water (5.2.2.2) to 900,0 ml
Heat to boiling to dissolve completely. Sterilize in the autoclave (5.3.1) and cool to 50 °C to 55 °C. Add 100
ml Middlebrook OAOC enrichment under aseptic conditions. Fill 18 ml to 20 ml per plate (5.3.2.10). The
pH of the medium shall be equivalent to 6,6 ± 0,2 (5.3.2.4).
In case of an encountering problems with neutralization (5.5.1.2) it may be necessary to add neutralizer
to MCO. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use
neutralizer that causes opalescence in the agar.
d) Middlebrook 7H10 broth with 10% ADC enrichment (MADC-broth)
— Middlebrook 7H10 broth-powder 4,7 g
— Glycerol [bibliographic reference 1] 100,0 ml
— Water (5.2.2.2) 750,0 ml
Sterilize in the autoclave (5.3.1) and cool to 45 °C. Add 100 ml Middlebrook ADC enrichment under
aseptic conditions and sterilized water (5.2.2.2) to 1 000,0 ml. The pH of the medium shall be equivalent
to 6,6 ± 0,2 (5.3.2.4).
This Malt extracts from Difal and OXOID are examples 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.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
5.2.2.4 Diluent
a) General Diluent
Tryptone Sodium Chloride Solution:
— 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 (5.3.1). After sterilization the pH (5.3.2.4) of the general diluent shall be
equivalent to 7,0 ± 0,2.
b) Glycerol Diluent (for Pseudomonas aeruginosa only)
Tryptone Sodium Chloride Glycerol Solution:
— Tryptone, pancreatic digest of casein 1,0 g
— Sodium chloride (NaCl) 8,5 g
— Glycerol [bibliographic reference 1] 2,0 g
— Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave (5.3.1). After sterilization the pH (5.3.2.4) of the diluent shall be equivalent to
7,0 ± 0,2.
This modified diluent [5.2.2.4 b)] should be only used for the preparation of the test suspension of
Pseudomonas aeruginosa (5.4.1.4). All further dilutions should be done with the general diluent [5.2.2.4
a)].
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2 and 5.5.2. It
shall be sterile.
Information on neutralizer that has been found to be suitable for some categories of products is given in
Annex B.
5.2.2.6 Sterile defibrinated sheep blood
The sterile defibrinated sheep blood can be acquired from a commercial supplier.
5.2.2.7 Hard water for dilution of products
a) Hard water general
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 [5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in a refrigerator (5.3.2.8)
for no longer than one month.
— Prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water and dilute to 1 000 ml.
Sterilize by membrane filtration (5.3.2.7). Store the solution in a refrigerator (5.3.2.8) for no longer
than one week.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
— Place 600 ml to 700 ml water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml
(5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The
pH (5.3.2.4) of the hard water shall be 7,0 ± 0,2 (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.
When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces
a different final water hardness in each test tube. In any case the final hardness expressed as calcium
carbonate (CaCO ) is in the test tube lower than 375 mg/l.
b) Hard water with the addition of polysorbate 80
Use the procedure described in 5.2.2.7 a). At the end add 1 g of polysorbate 80 per litre. Sterilized by
membrane filtration. The hard water with the addition of polysorbate 80 shall be freshly prepared under
aseptic conditions and used within 12 h.
5.2.2.8 Interfering substances
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids, detergents) shall be defined.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of general
diluent [5.2.2.4 a)]
Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within 1 month.
The final concentration of the bovine albumin in the test procedure (5.5) is 0,3 g/l.
5.2.2.8.3 Dirty conditions (mixture of bovine albumin solutions – high concentration with sheep
erythrocytes)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of general
diluent [5.2.2.4 a)].
Sterilize by membrane filtration (5.3.2.7).
Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (5.2.2.6). Centrifuge the sheep blood at 800
g for 10 min. After discarding the supernatant, resuspend erythrocytes in general diluent [5.2.2.4 a)].
N
Repeat this procedure at least 3 times, until the supernatant is colourless. Resuspend 3,0 ml of the packed
sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see above). To avoid contamination
this mixture should be split in portions probably needed per day and kept in separate containers for a
maximum of 7 days in a refrigerator at 2 °C to 8 °C.
The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be
3,0 g/l and 3,0 ml/l respectively.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
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 moist and dry heat sterilization:
+3
a) For moist heat sterilization, an autoclave capable of being maintained at 121 °C for a minimum
( )
holding time of 15 min;
+ 5
b) for dry heat sterilization, a hot air oven capable of being maintained at ( ) °C for a minimum
+ 5 + 5
holding time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a
0 0
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at (21,5 ± 3,5)°C and at (45 ± 1) °C [to maintain
melted TSA in case of pour plate technique] or (55 ± 1) °C [to prepare MCO and at additional test
temperatures ± 1 °C (5.5.1)].
5.3.2.3 Incubator, capable of being controlled at either (36 ± 1) °C or at (37 ± 1) °C (bacteria and
mycobacteria) or (30 ± 1) °C (yeasts and fungi). The same temperature shall be used for all incubations
of the bacteria 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
(21,5 ± 3,5)°C. A puncture electrode or a flat membrane electrode should be used for measuring the pH
of the agar-media (5.2.2.3).
5.3.2.5 Stopwatch
5.3.2.6 Shakers
® 4
a) Electromechanical agitator, e.g. Vortex mixer .
b) Mechanical shaker.
5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm
to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin (5.2.2.8.2
and 5.2.2.8.3).
Disposable sterile equipment is an acceptable alternative to reusable glassware.
4 ®
Vortex in 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.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic
pipettes may be used.
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.
5.3.2.13 Centrifuge (800 g ).
N
5.3.2.14 Rectangular glass spatula (4 cm edge length).
5.3.2.15 Loop (metal or plastic).
5.3.2.16 Test-surface, PVC with or without PUR (Polyurethan) surface coating, thickness 2 mm;
measuring 20 cm x 50 cm. Clean the test-surfaces before using with 2-propanol 70 %. To assure uniform
precleaning a standard wiping cloth is impregnated with 16 ml 70 % 2-propanol (v/v) and, using the
unitary weight, is wiped once back and forth over the test surface (in case of FOREX material see 5.3.2.16
b) clean for the first use the foil free surface and in the second test the surface after removing the foil).
After drying mark with a pencil or a permanent marker four squares as test fields 1 to 4, each measuring
5 cm x 5 cm, figuring a row at a distance of 5 cm from one another. The row should be approximately in
the middle of the test-surface (see Figure 1 and Figure 2). The drying controls D and D are performed
c0 ct
on a smaller test-surface measuring minimum 7 cm x 13 cm - marked with two squares of 5 cm x 5 cm.
Each plate (for FOREX plate side) can be used only once, no reprocessing is allowed.
Example for the test-surface:
a) Mipolam Troplan PUR Evercare , (art. nr. 85931006, Gerflor Mipolam GmbH, Mülheimer Straße 27,
TM
53840 Troisdorf, Germany) .
b) PVC plate free foam (20 x 50 cm, thickness 2 mm) FOREX classic, white matt finished, one side with
foil, (art. nr. SFSFOXC020RW1F, thyssenkrupp Plastics, Widdersdorfer Str. 158, 50825 Cologne,
Germany) .
This test-surface 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.
This test-surface 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.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
Figure 1 —PVC plate free foam (20 x 50 cm, thickness 2 mm) FOREX classic, white matt finished,
one side with foil, art. nr. SFSFOXC020RW1F marked with 4 fields

Key
Schematic representation of the test-surface
a = 50 cm b = 20 cm
with four test areas T1 to T4 (5 cm x 5 cm) and a given range of wiper wipe.
c = 5 cm d = 10 cm e = 5 cm
Size of the unitary weight:
f = 8,6 cm g = 12,1 cm
Test field 1 is inoculated with 0,05 ml of the test organisms / interfering substance mixture (bacteria and
9 7
mycobacteria 1,5 to 5,0 × 10 cfu/ml, corresponding to a final number on test field 1 of 6,75 × 10 to
8 8
2,25 × 10 cfu; for Candida albicans and Aspergillus brasiliensis 1,5 to 5,0 × 10 cfu/ml, corresponding to
6 7
a final number on test field 1 of 6,75 × 10 to 2,25 × 10 cfu). The arrow shows the cleaning sweep with
the wipe (see Annex C). The starting point is in front of test field 1 and the turn is immediately after test
field 4. The end point of the wiping process is the starting point after passing test field 1 for the second
time.
Figure 2 — Scheme of the markings and the wipe sweep over four test fields on the test-surface
oSIST prEN 16615:2022
prEN 16615:2022 (E)
5.3.2.17 Wiping cloth
5.3.2.17.1 Standard wiping cloth (wipe): For testing according to 5.5.2.2 a) and for the water control
[5.5.2.2 h)] a standard wiping cloth with the dimension of 16,5 cm x 30 cm and composition of 55 % pulp,
45 % polyethylene terephthalate (PET) is used; (example: Tork Low-Lint Cleaning”, art. nr. 190491,
supplier: “SCA Tork) . Wipes are used only once.
5.3.2.17.2 Specified wiping cloth (wipe): For ready-to-use systems or impregnated wipe systems the
specified wiping cloth is used in combination with the disinfectant. The manufacturer describes precisely
their composition and how they are to be used (e.g. the number of layers). The wipes are used only once.
5.3.2.18 Unitary weight, granite block or comparable innert material, 12,1 cm long, 8,6 cm wide and
8,6 cm high (the height may differ with other materials), weighing (2,3 to 2,5) kg. The use of the unitary
weight standardizes the wiping procedure and simulates the average pressure when wiping is performed
in practice.
5.3.2.19 Swabs (sterile for single use only), the soakable part shall be made from nylon (FLOQSwabs),
or pure cotton free from substances which might inhibit or promote the action of the product test
solution and from substances inhibiting the test organisms. (Suggestion for nylon swabs: FLOQSwabs
Copan Diagnostics Inc., art.no. 502CS01, suggestion for cotton swabs: Greiner BioOne GmbH, art. no.
421084)
5.3.2.20 Parafilm (for single use only), to protect the lower horizontal surface and the vertical
surfaces of the unitary weight from any contamination during the wiping procedure a parafilm covering
these parts is used. This parafilm shall be replaced by a new one after each wiping procedure (example:
Parafilm® M (100 mm) Art.Nr. 7016 05, BRAND GMBH + CO KG, Postfach 11 55, 97861 Wertheim,
Germany) .
5.3.2.21 Rubber, to fix the wipe at the unitary weight (wide 1 cm, diameter 8,5 cm)
5.3.2.22 Flasks with ventilated caps: Roux bottles or similar flasks
5.3.2.23 Spectrophotometer or other suitable technique
5.3.2.24 Microscope
5.3.2.25 Fritted filter: Porosity of 40 µm to 100 µm (ISO 4793, [3])
5.3.2.26 Coned bottom screw cap tubes, contents of 50 ml (diameter about 28 mm)
5.3.2.27 Counting chamber
This wipe 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.
These swabs are examples of a suitable products 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.
Parafilm 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.
oSIST prEN 16615:2022
prEN 16615:2022 (E)
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions
5.4.1.1 General
For each test organism, two different suspensions shall be prepared: the “test suspension” to perform the
test and the “validation suspension” to perform the controls and method validation.
5.4.1.2 Preservation and stock cultures of test organisms
The test organism and its stock cultures shall be prepared and kept in accordance with EN 12353.
5.4.1.3 Working culture of test organisms
5.4.1.3.1 Bacteria
In order to prepare the working culture of test organisms (5.2.1), prepare a subculture from the stock
culture (5.4.1.2) by streaking onto TSA [5.2.2.3 a)] slopes or plates and incubate (5.3
...