SIST EN 1657:2025

SLOVENSKI STANDARD
01-marec-2025
Nadomešča:
SIST EN 1657:2016
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje fungicidnega delovanja ali delovanja kemičnih razkužil in antiseptikov
na kvasovke v veterini - Preskusna metoda in zahteve (faza 2, stopnja 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the
veterinary area - Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel
und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2,
Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants
chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (ph
Ta slovenski standard je istoveten z: EN 1657:2024
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 1657
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2024
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 1657:2016
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of fungicidal or
yeasticidal activity of chemical disinfectants and
antiseptics used in the veterinary area - Test method and
requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der
fongicide ou levuricide des antiseptiques et des fungiziden oder levuroziden Wirkung chemischer
désinfectants chimiques utilisés dans le domaine Desinfektionsmittel und Antiseptika für den
vétérinaire - Méthode d'essai et exigences (phase 2, Veterinärbereich - Prüfverfahren und Anforderungen
étape 1) (Phase 2, Stufe 1)
This European Standard was approved by CEN on 18 November 2024.

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, Türkiye 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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 1657:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms, definitions and abbreviated terms . 6
3.1 Terms and definitions . 6
3.2 Symbols and abbreviated terms . 7
4 Requirements . 7
5 Test method . 8
5.1 Principle . 8
5.2 Materials and reagents . 9
5.2.1 Test organisms . 9
5.2.2 Culture media and reagents . 9
5.3 Apparatus and glassware . 12
5.3.1 General . 12
5.3.2 Usual microbiological laboratory equipment and, in particular, the following . 12
5.4 Preparation of test organism suspensions and product test solutions . 13
5.4.1 Test organism suspensions (test and validation suspension) . 13
5.4.2 Product test solutions . 18
5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product . 18
5.5.1 General . 18
5.5.2 Dilution-neutralization method . 19
5.5.3 Membrane filtration method . 22
5.6 Experimental data and calculation . 24
5.6.1 Explanation of terms and abbreviations . 24
5.6.2 Calculation . 24
5.7 Verification of methodology . 27
5.7.1 General . 27
5.7.2 Control of weighted mean counts . 27
5.7.3 Basic limits . 28
5.7.4 Additional limits for Aspergillus brasiliensis . 28
5.8 Expression of results and precision . 28
5.8.1 Reduction . 28
5.8.2 Control of active and non-active product test solution (5.4.2) . 28
5.8.3 Limiting test organism and fungicidal/yeasticidal concentration . 28
5.8.4 Precision, replicates . 29
5.9 Interpretation of results – conclusion . 29
5.9.1 General . 29
5.9.2 Fungicidal activity for general purposes . 29
5.9.3 Fungicidal activity for specific purposes . 29
5.9.4 Yeasticidal activity for general purposes . 29
5.9.5 Yeasticidal activity for specific purposes . 29
5.9.6 Yeasticidal activity for teat disinfectants . 30
5.10 Test report . 30
Annex A (informative) Referenced strains in national collections . 32
Annex B (informative) Neutralizers and rinsing liquids . 33
Annex C (informative) Graphical representation of test procedures . 35
C.1 Dilution-neutralization method . 35
C.2 Membrane filtration method . 37
Annex D (informative) Example of a typical test report . 40
Annex E (informative) Precision of the test result. 45
Bibliography . 48

European foreword
This document (EN 1657:2024) 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 June 2025, and conflicting national standards shall be
withdrawn at the latest by June 2025.
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 1657:2016.
This document was revised to correct obvious 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 obligatory conditions have been removed and the preparation of the milk interfering substance has
been amended.
The data obtained using the 2016 version of EN 1657 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, Türkiye and the United
Kingdom.
Introduction
This document specifies a suspension test method for establishing whether a chemical disinfectant or
antiseptic has a fungicidal or yeasticidal activity in the areas 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 may influence
its action in practical situations.
The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test
corresponds to defined experimental conditions. However, for some applications the recommendations
of use of a product may differ and therefore additional test conditions need to be used.
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, as some dilution is always produced by
adding the test organisms and interfering substance.
This document applies to products that are used in the veterinary area – i.e. in the breeding, husbandry,
production, veterinary care facilities, transport and disposal of all animals except when in the food chain
following death and entry into processing industry. This document also applies to products used for teat
disinfection.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
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.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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, Laboratory sintered (fritted) filters — Porosity grading, classification and designation
3 Terms, definitions and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 apply.
ISO and IEC maintain terminology 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/
3.2 Symbols and abbreviated terms
For the purposes of this document, the following symbols and abbreviated terms apply.
c is the sum of V values taken into account
C
cfu Colony forming units
n is the number of V values taken into account
C
N Test Suspension
Nv Validation suspension
Na Test
A Control A (Experimental conditions control)
B Control B (Neutralization control)
C Control C (Dilution neutralization validation)
N Test at time 0
Nv Validation suspension at time 0
R Reduction
Vc is the number of cfu counted per sample of 1,0ml
4 Requirements
The product shall demonstrate at least a 4 decimal log (lg) reduction when diluted with hard water
(5.2.2.7) or – in the case of ready-to-use products – with water (5.2.2.2) and tested in accordance with
Table 1 and Clause 5 under simulated low-level soiling (3,0 g/l bovine albumin) or high level soiling
(10,0 g/l yeast extract and 10,0 g/l bovine albumin) or 10,0 g/l milk for teat disinfectants or 3,0 g/l
bovine albumin for pre-milking teat disinfectants in additional test conditions.
Table 1 —Test conditions
Test conditions Fungicidal activity Yeasticidal activity Yeasticidal activity for
teat disinfectants
Test organisms Aspergillus brasiliensis Candida albicans Candida albicans
Candida albicans
additional any relevant test any relevant test any relevant test organism
organism organism
Test temperature At intervals of 5°C
Minimum
5°C ± 1°C 5°C ± 1°C 20°C ± 1°C
Maximum
40°C ± 1°C 40°C ± 1°C 30°C ± 1°C
Contact time At intervals of 30 s from 30 s to 5 min and at intervals
Minimum of 5 min from 5 min to 120 min
Maximum 1 min ± 5 s 1 min ± 5 s 1 min ± 5 s for post-milking
teat disinfectants
30 s ± 5 s for pre-milking
teat disinfectants
120 min ± 10 s 120 min ± 10 s 5 min ± 10 s for post-
milking teat disinfectants
3 min ± 10 s for pre-
milking teat disinfectants
Interfering substance
low level soiling 3,0 g/l bovine albumin 3,0 g/l bovine albumin Post milking: 10,0 g/l of
reconstituted milk
high level soiling 10 g/l yeast extract plus 10 g/l yeast extract plus
10 g/l bovine albumin 10 g/l bovine albumin Pre-milking: 3,0 g/l bovine
albumin
additional any relevant substance any relevant substance any relevant substance
NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the
standard test conditions.
The recommended test conditions for the use of the product are within the responsibility of the manufacturer.
Any additional specific fungicidal or yeasticidal activity shall be determined in accordance with 5.2.1 and
5.5.1.1 in order to take into account intended specific use conditions.
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 temperature θ for the test contact time t in accordance with
Table 1. At the end of this contact time, an aliquot is taken, and the fungicidal/yeasticidal and/or the
fungistatic/yeastistatic activity in this portion is immediately neutralized or suppressed by a validated
method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot be found,
membrane filtration is used. The number of surviving fungi in each sample are determined and the
reduction is calculated.
5.2 Materials and reagents
5.2.1 Test organisms
The fungicidal activity shall be evaluated using the following strains as test organisms:
— Candida albicans;
— Aspergillus brasiliensis.
(formerly Aspergillus niger)
The yeasticidal activity shall be evaluated using only Candida albicans.
NOTE See Annex A for strain references in some culture collections.
The required incubation temperature for these test organisms is 30 °C± 1 °C (see 5.3.2.3). The same
temperature shall be used for all incubations performed during a test and its control and validation.
If additional test organisms are used, they shall be kept and used under optimum growth conditions
(temperature, time, atmosphere, media) and 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 Standard refer to the anhydrous salts. Hydrated forms
may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
If additional strains do not grow on the media (5.2.2.3) or cannot be used with diluent (5.2.2.4) alternative
media shall be used and shall be reported as well as alternative incubation conditions.
To improve reproducibility, it is recommended that commercially available dehydrated material (if
appropriate) is used for the preparation of culture media. The manufacturer's instructions relating to the
preparation of these products should be rigorously followed.
Ready to use media may be used if it complies with the required specification.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at 20 °C± 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 [1]) can be used.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used e.g. for preparation
of culture media and subsequently sterilized.
NOTE 1 See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Malt extract agar (MEA)
Malt extract agar, consisting of:
a
Malt extract 30,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml
a
The malt extract should be of food grade (Cristomalt poudre from Difal is recommended) or equivalent that is
not highly purified and not only based on maltose (Malt extract from OXOID is recommended) .
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 °C ± 1 °C.
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add
neutralizer to the MEA. Annex B gives guidance on the neutralizers that may be used.
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 of the diluent shall be equivalent to
7,0 ± 0,2 when measured at 20 °C ± 1 °C.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and
5.5.2. It shall be sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given
in Annex 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.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is
given in Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:

This information is given for the convenience of users of this European Standard and does not constitute an endorsement by
CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.
— 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 of the hard water shall be 7,0 ± 0,2, when measured at 20 °C ± 1 °C (5.3.2.4). If necessary, adjust
the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or
approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water
produces a different final water hardness in each test tube. In any case the final hardness is lower than 375 mg/l of
calcium carbonate (CaCO ) in the test tube.
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.
For any additional interfering substance 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 Low-level and pre-milking soiling (bovine albumin solution)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 90 ml of water
(5.2.2.2) in a 100ml volumetric flask (5.3.2.12). Make up to the mark with water.
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l.
5.2.2.8.3 High-level soiling (mixture of bovine albumin solution with yeast extract)
Dissolve 50,0 g yeast extract powder in 150 ml of water (5.2.2.2) in a 250 ml volumetric flask (5.3.2.12)
and allow foam to collapse. Make up to the mark with water (5.2.2.2). Transfer to a clean dry bottle and
sterilize in an autoclave [5.3.2.1 a)]. Allow to cool to 20 °C ± 1 °C.
Pipette 25 ml of this solution into a 50 ml volumetric flask (5.3.2.12) and add 10 ml of water (5.2.2.2).
Dissolve 5,0 g of bovine albumin fraction V (suitable for microbiological purposes) in the solution with
shaking and allow foam to collapse. Make up to the mark with water (5.2.2.2), sterilize by membrane
filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration in the test procedure (5.5) is 10,0 g/l yeast extract and 10,0 g/l bovine albumin.
5.2.2.8.4 Milk for teat disinfectants
Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder
per litre of water (5.2.2.2), shall be prepared as follows:
— prepare a solution of 10,0 % (v/v) in water (5.2.2.2) by adding 10 parts of reconstituted milk to 90
parts of water. Heat for 30 min at (105 ± 3) °C [or 5 min at (121 ± 3) °C].
The final concentration of reconstituted milk in the test procedure (5.5) is 1,0 % (v/v) of reconstituted
milk.
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
+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 ( ) °C for a minimum holding time of 1 h or at ( ) °C for a
170 160
0 0
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at the specified temperatures and at 45 °C ± 1 °C (to
maintain melted MEA in case of pour plate technique).
5.3.2.3 Incubator, capable of being controlled at the specified temperatures ± 1 °C.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 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
Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.2.6 Shakers
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.
The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with 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), 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, 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, 3 mm to 4 mm in diameter.
5.3.2.12 Volumetric flasks.
5.3.2.13 Fritted filter, with porosity of 40 µm to 100 µm according to ISO 4793.
5.3.2.14 Flasks with ventilated caps.
5.3.2.15 Microscope capable of x 400 magnification
5.3.2.16 Containers; test tubes, culture bottles or flasks of suitable capacity.
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 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 working stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.

Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of
this European Standard and does not constitute an endorsement by CEN of this product.
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), prepare a subculture from the stock
culture (5.4.1.2) by streaking onto MEA (5.2.2.3) slopes or plates (5.3.2.10) 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 subcultures 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 subculturing, 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 or
flasks with ventilated caps (5.3.2.14) and incubate for 7 days to 9 days at 30 °C ± 1°C. No further
subculturing is needed. Do not stack the Petri dishes during the incubation to improve the temperature
homogenization. At the end of incubation, all the cultures have to show a dark brown or black surface.
Cultures with rare and small white or grey areas may be used (see Figure 1).

Figure 1 — Photo No 1: A. brasiliensis ATCC 16404 after 7 d of incubation at 30°C
Figure 2 — Photo No.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
The procedure for preparing the Candida albicans test suspension is as follows:
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 [5.3.2.6b)].
Aspirate the suspension from the glass beads and transfer to a tube;
7 4 7
b) adjust the number of cells in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml using diluent
(5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the
water bath at 20 °C ± 1 °C [5.3.2.2] and use within 2 h.
The use of a spectrophotometer for adjusting the number of cells is highly recommended (approximately
620 nm wavelength – cuvette 10 mm path length). Each laboratory should therefore produce calibration
data for each test organism knowing that suitable values of optical density are generally found between
0,200 and 0,350. A colourimeter is a suitable alternative.
−5 −6
c) For counting, prepare 10 and 10 dilutions 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 °C ± 1 °C.

cfu/ml = colony-forming unit(s) per millilitre
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).
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 spores in 10 ml of sterile 0,05 % (w/v)
polysorbate 80 solution in water (5.2.2.2). Using a sterile 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
(5.3.2.13).
b) Carry out a microscopic examination under x 400 magnification (5.3.2.15) immediately after the
preparation to show:
1) the presence of a high concentration (at least 75 %) of characteristic mature spores i.e. spiny
spores (versus smooth spores) [see Figures 3 and 4];
If there are less than 75 % spiny conidiospores it may be due to the Aspergillus brasiliensis
culture or the media used to produce the spores. In this situation, it will be necessary to obtain
the culture from another culture collection and/or use a MEA from a different supplier.
2) the absence of spore germination (check at least 10 fields of view);
3) If germinated spores are present, discard the suspension;
4) the absence of mycelia fragments (check at least 10 fields of view).
If mycelia are present, proceed to a 2nd fritted filtration.
If mycelia are still present, discard the suspension.

Figure 3 — Photo No 3: Observation of conidiospores under light microscope: presence of
smooth (a) and spiny (b) spores (insufficient spiny spores)
Figure 4 — Photo No 4: Observation of conidiospores under light microscope: High
concentration of characteristic mature spores with spiny aspect (sufficient spiny spores)
7 7
c) Adjust the number of spores in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml using the
diluent (5.2.2.4), estimating the number of cfu by any suitable means. Use the suspension within 4 h,
maintain in a water bath controlled at 20°C ± 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 or 5.5.3).
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. Experienced laboratories
found a better fit to the required number of spores when the spore suspension count in the device was
10 % to 50 % higher than the number aimed at.
−5 −6
d) For counting, prepare 10 and 10 dilutions 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 °C ± 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).
For incubation and counting, see 5.4.1.6.
5.4.1.5 Validation suspension (“Nv”)
a) To prepare the validation suspension, dilute the test suspension (5.4.1.4.1 and 5.4.1.4.2) with the
2 3
diluent (5.2.2.4) to obtain the fungal count of 3,0 × 10 cfu/ml to 1,6 × 10 cfu/ml [about one-quarter
−4
(1 + 3) of the 10 dilution].
−1
b) For counting, prepare a 10 dilution with diluent (5.2.2.4). Mix [5.3.2.6a)]. Take a sample of 1,0 ml
in duplicate and inoculate using the pour plate or the spread plate technique [with Candida albicans,
5.4.1.4.1 c); with Aspergillus brasiliensis, 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
For incubation and counting of the test and the validation suspensions, the procedure is as follows:
a) incubate (5.3.2.3) the plates for 42 h to 48 h. Discard any plates that 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 that no longer
show well-separated colonies. Recount the remaining plates. If the number has increased, use only the
higher number for further evaluation;
b) count 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 Vc values according
to 5.6.2.2;
c) calculate the numbers of cfu/ml in the test suspension “N” and in the validation suspension “Nv” using
the methods given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7.
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired 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 %.
Dilutions of ready-to-use products, i.e. products that are not diluted when applied, shall be prepared in
water (5.2.2.2).
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 (5.2.2.7) on a
volume/volume basis using volumetric flasks (5.3.2.12).
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous preparation that is stable during the whole procedure. If during the procedure
a visible inhomogeneity appears due to the formation of a precipitate or flocculant (for example, through
the addition of the interfering substance), it shall be recor
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