EN 13624:2021

SLOVENSKI STANDARD
01-januar-2022
Nadomešča:
SIST EN 13624:2013
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje fungicidnega delovanja ali delovanja na kvasovke v humani medicini -
Preskusna metoda in zahteve (faza 2, stopnja 1)
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)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich -
Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour
l’évaluation de l’activité fongicide ou levuricide en médecine - Méthode d'essai et
prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 13624:2021
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 13624
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 11.080.20 Supersedes EN 13624:2013
English Version
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)
Désinfectants chimiques et antiseptiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der
fongicide ou levuricide en médecine - Méthode d'essai fungiziden oder levuroziden Wirkung im
et exigences (phase 2, étape 1) humanmedizinischen Bereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 1)
This European Standard was approved by CEN on 3 October 2021.

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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13624:2021 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Requirements . 6
5 Test method . 7
5.1 Principle . 7
5.2 Materials and reagents . 8
5.3 Apparatus and glassware . 11
5.4 Preparation of test organism suspensions and product test solutions . 13
5.5 Procedure for assessing the fungicidal and yeasticidal activity of the product . 18
5.6 Experimental data and calculation . 26
5.7 Verification of methodology . 32
5.8 Expression of results and precision . 33
5.9 Interpretation of results – conclusion . 34
5.10 Test report . 36
Annex A (informative) Referenced strains in national collections . 38
Annex B (informative) Neutralizers and rinsing liquids. 39
Annex C (informative) Graphical representation of test procedures. 41
C.1 Dilution-neutralization method . 41
C.2 Membrane filtration method . 43
C.3 Dilution-neutralization method (modified method for ready-to-use products) . 45
C.4 Membrane filtration method (modified method for ready-to-use products) . 47
Annex D (informative) Example of a typical test report . 49
Annex E (informative) Precision of the test result . 54
Bibliography . 57

European foreword
This document (EN 13624:2021) 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 May 2022, and conflicting national standards shall be
withdrawn at the latest by May 2022.
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 13624:2013.
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:
— De-harmonization of the standard (Annex ZA and references to MDR/MDD deleted);
— Textile disinfection was added (see Table 1, 5.5.1.3);
— The amounts for the dirty conditions for the modified method for ready-to-use products were
reduced (5.2.2.8.4 b));
— Clarification that a neutralization time of 10 s shall be used for all products with contact times of
10 min or shorter (5.5.2.2. c) and 5.5.2.5 b));
— Explanation for alternative controls for chemo-thermal disinfection (5.5.2.3);
— The reference to CIP numbers for the fungi strains were deleted and the UMIP numbers were added
(Annex A);
— Harmonization of the text with EN 13727;
— Correction of editorial mistakes.
The changes of this revision have no impact on the test results obtained with reference to the version
EN 13624:2013. Those results are still valid.
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 suspension test for establishing whether a chemical disinfectant or an
antiseptic has a fungicidal or yeasticidal activity in the area and fields described in the scope.
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 can
influence its action in practical situations. Each utilization concentration of the chemical disinfectant or
antiseptic found by this test corresponds to the chosen experimental conditions.
1 Scope
This document specifies a test method and the minimum requirements for fungicidal or yeasticidal
activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable
preparation when diluted with hard water, or – in the case of ready-to-use products – with water.
Products can only be tested at a concentration of 80 % or less (97 % with a modified method for special
cases) as some dilution is always produced by adding the test organisms and interfering substance.
This document applies to products that are used in the medical area in the fields of hygienic handrub,
hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and
surface disinfection by wiping, spraying, flooding or other means.
This document applies to areas and situations where disinfection or antisepsis 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 1 The method described is intended to determine the activity of commercial formulations or active
substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test.
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 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical
disinfectants and antiseptics
ISO 4793:1980, Laboratory sintered (fritted) filters — Porosity grading, classification and designation
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 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
4 Requirements
The product shall demonstrate at least a 4 decimal logarithms (lg) reduction (for hygienic handwash at
least a 2 lg reduction), when tested in accordance with Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Hygienic
Textile
Test Surgical handrub Instrument Surface
handrub and
d
conditions and handwash disinfection disinfection disinfection
handwash
Minimum Candida albicans Candida albicans a) fungicidal a) fungicidal a) fungicidal
spectrum of (vegetative cells) (vegetative cells) activity: activity: activity:
test Aspergillus Aspergillus Aspergillus
organisms brasiliensis brasiliensis brasiliensis
(conidiospores) (conidiospores) (conidiospores)
Candida albicans Candida albicans Candida albicans
(veg. cells) (veg. cells) (veg. cells)
b) yeasticidal b) yeasticidal b) yeasticidal
activity: activity: activity:
Candida albicans Candida albicans Candida albicans
(veg. cells) (veg. cells) (veg. cells)
additional Any relevant test organism
Test according to the manufacturer's recommendation, but
temperature
generally between generally between
20°C and 50°C for 20°C and 50°C for
Candida spec. between 4 °C and Candida spec.
at 20 °C at 20 °C
and between and between
30 °C
20°C and 60°C for 20°C and 60°C for
e e
Aspergillus spec. Aspergillus spec.
Contact time according to the manufacturer's recommendation,
but between but no longer than
a
30 s and 60 s 1 min and 5 min 60 min
5 min or 60 min
Interfering 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine
substance albumin solution albumin solution albumin solution albumin solution
clean (hygienic (surgical
b b
conditions
handrub) handrub)
and/or and/or
dirty 3,0 g/l bovine 3,0 g/l bovine
conditions albumin solution albumin solution
3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine
plus 3,0 ml/l plus 3,0 ml/l
albumin solution albumin solution albumin solution
erythrocytes erythrocytes plus 3,0 ml/l plus 3,0 ml/l plus 3,0 ml/l
(hygienic (surgical erythrocytes erythrocytes erythrocytes
c c
handwash) handwash)
any relevant any relevant
additional — — —
substance substance
NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained
under the minimum test conditions.
Hygienic
Textile
Test Surgical handrub Instrument Surface
handrub and
d
conditions and handwash disinfection disinfection
disinfection
handwash
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.
b
Hygienic and surgical handrub shall be tested as a minimum under clean conditions.
c
Hygienic and surgical handwash shall be tested as a minimum under dirty conditions.
d
For further information see EN 16616:2015, Clause 4.
e
For further information regarding validity at elevated temperatures see EN 14885.
5 Test method
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use
products) is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an
interfering substance. The mixture is maintained at the temperature and the contact time specified in
Clause 4 and 5.5.1.1. At the end of this contact time, an aliquot is taken; the fungicidal and/or the
fungistatic action in this portion is immediately neutralized or suppressed by a validated method. The
method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane
filtration is used. The numbers of surviving fungi in each sample are determined and the reduction is
calculated.
NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded
as a ready-to-use product (5.4.2).
5.1.2 The test is performed using the vegetative cells of Candida albicans and the conidiospores of
Aspergillus brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal
activity) as test-organisms (Clause 4, Table 1).
5.1.3 Additional contact times and temperatures are specified (Clause 4, Table 1). Additional
interfering substances and test organisms may be used.
5.2 Materials and reagents
5.2.1 Test organisms
The fungicidal activity shall be evaluated using the following strains as test organisms selected
according to Clause 4 (Table 1) :
— Candida albicans ATCC 10231;
— Aspergillus brasiliensis (former “A. niger”) ATCC 16404.
The yeasticidal activity shall be evaluated using only Candida albicans.
Refer to Annex A for strain reference in some other culture collections.
The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3).
If additional test organisms are used, they shall be incubated under optimum growth conditions
(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms
selected do not correspond to the specified strains, their suitability for supplying the required inocula
shall be verified. If these additional test organisms are not classified at a reference centre, their
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or
national culture collection under a reference for five years.
5.2.2 Culture media and reagents
5.2.2.1 General
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 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 time limitation for use should be fixed.
All specified pH values are measured at (20 ± 1) °C.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water. If distilled water of
adequate quality is not available, water for injections (see bibliographic reference [2]) can be used.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used, e.g. for
preparation of culture media and subsequently sterilized.
See 5.2.2.7 for the procedure to prepare hard water.

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 the
product named.
5.2.2.3 Malt extract agar (MEA)
Malt extract agar, consisting of:
Malt extract [food grade (e.g. Christomalt powder 30,0 g
from Difal) or an 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 encountering problems with neutralization (5.5.1.2 and 5.5.1.3), 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 5.4.1.4.2.
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
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 (5.3.2.4) of the diluent shall be
equivalent to 7,0 ± 0,2.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and
5.5.2. It shall be sterile.
Information on neutralizers that have been found to be suitable for some categories of products is given
in Annex B.
5.2.2.6 Rinsing liquid (for membrane filtration)
The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and
5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter
membrane under the test conditions described in 5.5.3.
Information on rinsing liquids that have been found to be suitable for some categories of products is
given in Annex B.
This Malt extract from OXOID 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.
5.2.2.7 Hard water for dilution of products
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 the refrigerator
(5.3.2.8) 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)
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 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. If necessary, adjust the pH by using a solution of
approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l
(about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water
produces 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.
5.2.2.8 Interfering substance
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 (50
times in the case of the modified method, see 5.2.2.8.4).
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids and 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 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent
(5.2.2.4).
Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month.
The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l.
5.2.2.8.3 Dirty conditions (mixture of bovine albumin solutions – high concentration with sheep
erythrocytes)
Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent
(5.2.2.4).
Sterilize by membrane filtration (5.3.2.7).
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g
N
for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4).
Repeat this procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see
above). To avoid later contamination, this mixture should be split in portions probably needed per day
and kept in separate containers for a maximum of 7 d in a refrigerator (5.3.2.8).
The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be
3 g/l and 3 ml/l respectively.
5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4)
Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering
substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems
with the filtration.
a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent;
b) Dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml of diluent
(instead of 48,5 ml). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml
(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilized bovine albumin solution
to obtain 50 ml.
5.2.2.9 Defibrinated sheep blood
The defibrinated sheep blood should be sterile (aseptic blood-letting and preparation), pooled from
more than one sheep and 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 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 ( 180 ) °C for a minimum
+5 +5
holding time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at ( ) °C for a
0 0
minimum holding time of 2 h.
Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted
MEA in case of pour plate technique) and at additional test temperatures ±1 °C (5.5.1).
5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C.
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 (5.2.2.3).
5.3.2.5 Stopwatch.
5.3.2.6 Shakers.
® 4
mixer ;
a) Electromechanical agitator, e.g. Vortex
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 with a maximum pore size of 0,45 µm for sterilization of hard water (5.2.2.7),
bovine albumin (5.2.2.8.2, 5.2.2.8.3 and 5.2.2.8.4), and if the membrane filtration method is used (5.5.3).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the microorganisms 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, 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.
5.3.2.13 Centrifuge (800 g ).
N
5.3.2.14 Flasks with ventilated caps, Roux bottles or similar flasks.
5.3.2.15 Fritted filters: Porosity of 40 µm to 100 µm according to ISO 4793:1980, Clause 2.

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.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
5.4.1.1 General
For each test organism, two different suspensions have to 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 organisms and their 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 Candida albicans (yeast)
In order to prepare the working culture of Candida albicans (5.2.1), subculture from the stock culture
(5.4.1.2) by streaking onto MEA (5.2.2.3) slopes or plates and incubate (5.3.2.3). 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 subculture are the working cultures.
If it is not possible to prepare the second subculture on a particular day, a 72 h subculture may be used
for subsequent sub-culturing, provided that the subculture has been kept in the incubator (5.3.2.3)
during the 72 h period.
Never produce and use a fourth subculture.
5.4.1.3.2 Aspergillus brasiliensis (mould)
For Aspergillus brasiliensis (5.2.1) use only the first subculture grown on MEA (5.2.2.3) in Petri dishes
(5.3.2.10) or flasks with ventilated caps (5.3.2.14) and incubate for 7 d to 9 d. No further subculturing is
needed. Stacking the Petri dishes during the incubation could result in inhomogeneous temperature.
At the end of incubation, all the cultures have to show a dark brown or black surface with only a few
small white or grey spots. Examples of appropriate and inappropriate cultures are given in Figures 1
and 2.
Figure 1 — Photos N°1: Examples of appropriate cultures of A.brasiliensis ATCC 16404 after 7 d
of incubation at 30 °C
Figure 2 — Photo N°2: Example of inappropriate (not usable) culture of A.brasiliensis
ATCC 16404 after 7 d of incubation at 30 °C
5.4.1.3.3 Other test organisms (yeasts or moulds)
For additional test organisms, any departure from this method of culturing the yeast or the mould or of
preparing the suspensions shall be noted, giving the reasons in the test report.
5.4.1.4 Test suspension (N)
5.4.1.4.1 Candida albicans
a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take the
working culture (5.4.1.3.1) and transfer loopfuls of the cells into the diluent (5.2.2.4).The cells
should be suspended in the diluent by rubbing the loop against the wet wall of the flask to dislodge
the cells before immersing in the diluent. Shake the flask for 3 min using a mechanical shaker
[5.3.2.6 b)]. Aspirate the suspension from the glass beads and transfer to a tube.
7 5 7
b) Adjust the number of cells in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml using diluent
8 8
(5.2.2.4) (1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml in the case of the modified method, 5.5.4), estimating
the number of cfu by any suitable means. Maintain this test suspension in the water bath at 20 °C
and use within 2 h. Adjust the temperature according to 5.5.1.1 a) and 5.5.1.4 only immediately
before the start of the test.
The use of a spectrophotometer for adjusting the number of cells is highly recommended (about
620 nm wavelength – cuvette 10 mm path length). Each laboratory should therefore produce
calibration data knowing that suitable values of optical density are generally found between 0,200
and 0,350. To achieve reproducible results of this measurement it may be necessary to dilute the
test suspension, e.g. 1+9.
NOTE A colourimeter is a suitable alternative.
−5 −6 −6 −7
c) For counting, prepare 10 and 10 dilutions (10 and 10 dilutions in the case of the modified
method – 5.5.4) of the test suspension using diluent (5.2.2.4). Mix [(5.3.2.6a)].
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 (5.2.2.3), 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 (5.2.2.3).
cfu/ml = colony forming unit(s) per millilitre.
The technique used for counting of the test suspension has to be used for all other countings, 5.4.1.5 d),
5.5.2.2 c) and d), 5.5.2.3 b), 5.5.2.4 b) and 5.5.2.5 b).
For incubation and counting, see 5.4.1.6.
5.4.1.4.2 Aspergillus brasiliensis
The procedure for preparing the Aspergillus brasiliensis test suspension is as follows:
a) Take the working culture (5.4.1.3.2) and suspend the conidiospores in 10 ml of sterile 0,05 % (w/v)
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 beads (5.3.2.11). Filter the suspension through a fritted filter
(5.3.2.15);
b) Carry out a microscopic examination under × 400 magnification immediately after the preparation
to show:
1) the presence of a high concentration of characteristic mature conidiospores, i.e. spiny
conidiospores (versus smooth spores). See Figure 3 a) and b).

a) Photo N°3: Presence of smooth (a) and spiny b) Photo N°4: High concentration of
(b) spores [inappropriate (not usable) characteristic mature spores with spiny aspect
suspension] (appropriate suspension)
Figure 3 — Photos N°3 and N°4 Observation of conidiospores under light microscope
If there are less than 75 % spiny conidiospores it may be due to the Aspergillus strain used or to the
media. Therefore, use another strain, preferably from another supplier, and/or try out another
media;
2) the absence of conidiospore germination (check at least ten fields of view);
3) if germinated conidiospores are present, discard the suspension;
4) the absence of mycelia fragments (check at least ten fields of view).
If mycelia are present, proceed to a second fritted filtration. If mycelia are still present, discard the
suspension.
7 7
c) Adjust the number of conidiospores in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml
8 8
(1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml in the case of the modified method – 5.5.4) using the diluent
(5.2.2.4), estimating the number of cfu by any suitable means. Use the suspension within 4 h in a
water bath controlled at (20 ± 1) °C (5.3.2.2). In any case, adjust the temperature according to
5.5.1.4 only immediately before the start of the test (5.5.2, 5.5.3 or 5.5.4).
The use of a cell counting device for adjusting the number of cells is highly recommended. When
using a suitable counting chamber, follow the instructions explicitly.
Each laboratory should, therefore, produce calibration data to establish the relationship between
the counts obtained using the counting device and the counts (5.4.1.6) obtained by the pour plate
or the spread plate technique [d)]. Experienced laboratories found a better fit to the required
number of conidiospores when the conidiospore suspension count in the device was 10 % to 50 %
higher than the number aimed at.
−5 −6 −6 −7
d) For counting, prepare 10 and 10 dilutions (10 and 10 dilutions in the case of the modified
method 5.5.4) of the test suspension using diluent (5.2.2.4). 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 about half of each 1,0 ml sample into separate
Petri dishes (i.e. in duplicate = four plates) and add 15 ml to 20 ml of melted MEA (5.2.2.3),
cooled to (45 ± 1) °C;
2) when using the spread plate technique, spread about one quarter of each 1,0 ml sample on an
appropriate number (at least four) of surface dried plates containing MEA (5.2.2.3) (i.e. in
duplicate – at least eight plates).
The technique used for counting of the test suspension has to be used for all other countings, 5.4.1.5 d),
5.5.2.2 c) and d), 5.5.2.3 b), 5.5.2.4 b) and 5.5.2.5 b).
For incubation and counting, see 5.4.1.6.
5.4.1.5 Validation suspension (N and N )
V VB
a) To prepare the validation suspension (N ), dilute the test suspension N (see 5.4.1.4.1 and 5.4.1.4.2)
V
2 3
with the diluent (5.2.2.4) to obtain 3,0 × 10 cfu/ml to 1,6 × 10 cfu/ml (about one fourth (1 + 3) of
−4
the 10 dilution).
−4
NOTE In the case of the modified method (5.5.4), the procedure is the same, but one fourth (1+3) of the 10
3 4
dilution resulting in 3,0 × 10 cfu/ml to 1,6 × 10 cfu/ml.
b) For the neutralizer control N (5.5.2.4) dilute the test suspension (5.4.1.4) with the diluent to
VB
4 5 −2
obtain 3,0 × 10 cfu/ml to 1,6 × 10 cfu/ml [about one fourth (1+3) of the 10 dilution].
c) Maintain and use these validation suspensions (N and N ) the same way as the test suspension
V VB
[5.4.1.4.1 b) and 5.4.1.4.2 c)].
−1
d) For counting, prepare with diluent (5.2.2.4) a 10 dilution, in the case of the modified method
−2 −3
(5.5.4) a 10 dilution and in the case of the neutralizer control N [see b)] a 10 dilution. Mix
VB
[5.3.2.6 a)]. Take a sample of 1,0 ml in duplicate and inoculate using the pour plate or the spread
plate technique [with Candida albicans, see 5.4.1.4.1 c); with Aspergillus brasiliensis, see
5.4.1.4.2 d)].
For incubation and counting, see 5.4.1.6.
5.4.1.6 Incubation and counting of the test and the validation suspensions
a) Incubate (5.3.2.3) the plates for 42 h to 48 h. Discard any plates which are not countable (for any
reason). Count the plates and determine the number of cfu. Only for Aspergillus brasiliensis:
incubate the plates for a further 20 h to 24 h and – if the number of colonies has increased – for a
third additional period of 20 h to 24 h. Do not recount plates which no longer show well separated
colonies. Recount the remaining plates. If the number has increased, use only the higher number for
further evaluation.
b) Note for each plate the exact number of colonies but record > 165 (for moulds) or > 330 (for yeasts)
for any counts higher than 165 and 330 respectively and determine the V -values according to
C
5.6.2.2.
c) Calculate the numbers of cfu/ml in the test suspension N and in the validation suspensions N and
V
N (neutralizer control 5.5.2.4) using the method given in 5.6.2.3 and 5.6.2.5. Verify according to
VB
5.7.
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration (= real
test concentration) because it is diluted to 80 % during the test and the method validation (5.5.2 or
5.5.3).
Product test solutions shall be prepared in hard water (5.2.2.7) at minimum three different
concentrations to include one concentration in the active range and one concentration in the non-active
range (5.8.2). The product, as received, may be used as one of the product test solutions; in this case, the
highest tested concentration is 80 %. Ready to use products may be tested at 97 % [modified method
(5.5.4.)] In this case, the “real test concentration” is 97 %.
Dilutions of ready-to-use products shall be prepared in water (5.2.2.2) instead of hard water. Handwash
products are always prediluted with hard water (5.2.2.7) to achieve a 62,5 % solution. The solution
simulates the addition of tap water in practice (1:1). Such a product is nevertheless regarded as a
“ready-to-use product”. The modified method (5.5.4) cannot be used since 62,5 % represents the
highest accepted concentration (50 %), multiplied by 1,25.
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in
a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (= lower
concentrations) shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard
water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water in volumetric flasks
(5.3.2.12) on a volume/volume basis.
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogenous preparation, stable during the whole procedure. If during the procedure
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