oSIST prEN 1657:2023
(Main)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)
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)
This European Standard 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 European Standard applies to products that are used in the veterinary area - i.e. in the breeding, husbandry, production, transport and disposal of all animals except when in the food chain following death and entry to the processing industry.
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 (Annex F).
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)
Dieses Dokument legt ein Prüfverfahren und die Mindestanforderungen an die fungizide oder levurozide Wir
kung von chemischen Desinfektionsmitteln und Antiseptika fest, die in Wasser standardisierter Härte oder– im Fall gebrauchsfertiger Produkte– in Wasser eine homogene, physikalisch stabile Zubereitung bilden. Die
dukte können nur bis zu einer Konzentration von 80% geprüft werden, da immer eine bestimmte Verdünnung durch Zugabe der Prüforganismen oder der Belastungssubstanz erfolgt.
Dieses Dokument gilt für Produkte für die Anwendung im Veterinärbereich, d.h. bei der Aufzucht, Haltung, Produktion und beim Transport von Tieren sowie bei der Tierkörperbeseitigung, außer wenn die Tiere nach der Tötung durch Zuführung in die weiterverarbeitende Industrie in die Nahrungsmittelkette gelangen.
EN14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwen
dungsempfehlungen“ fest.
ANMERKUNG1 Das beschriebene Verfahren ist zur Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen unter deren Anwendungsbedingungen vorgesehen.
ANMERKUNG2 Dieses Verfahren entspricht einer Prüfung der Phase2, Stufe1.
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
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)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 1657:2023
01-april-2023
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: prEN 1657
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
oSIST prEN 1657:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 1657:2023
DRAFT
EUROPEAN STANDARD
prEN 1657
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2023
ICS Will supersede 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 prescriptions (ph Veterinärbereich - Prüfverfahren und Anforderungen
(Phase 2, Stufe 1)
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 NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 1657:2023 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Requirements . 5
5 Test method . 7
5.1 Principle . 7
5.2 Materials and reagents . 7
5.3 Apparatus and glassware . 10
5.4 Preparation of test organism suspensions and product test solutions . 12
5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product . 17
5.6 Experimental data and calculation . 22
5.7 Verification of methodology . 26
5.8 Expression of results and precision . 27
5.9 Interpretation of results – conclusion . 27
5.10 Test report . 28
Annex A (informative) Referenced strains in national collections . 30
Annex B (informative) Suitable neutralizers and rinsing liquids . 31
Annex C (informative) Graphical representation of test procedures . 33
Annex D (informative) Example of a typical test report . 37
Annex E (informative) Precision of the test result . 41
Bibliography . 44
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European foreword
This document (prEN 1657:2023) 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 1657:2016.
This document was revised to harmonize the preparation of the fungal spore suspension with other
fungicidal tests of CEN/TC 216 and to incorporate amendments applicable to all European Standards.
An additional requirement has been added for the Aspergillus spore suspension and therefore results
obtained using EN 1657:2005 and not fulfilling this additional requirement will need to be confirmed
by repeating the tests using EN 1657:2015.
The test conditions for teat disinfectants have been added.
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Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant or
antiseptic has a fungicidal or yeasticidal activity in the 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 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.
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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, transport and disposal of all animals except when in the food chain following death and
entry to the processing industry.
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 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 14885:2022, 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 and definitions
For the purposes of this document, the terms and definitions given in EN 14885:2022 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/
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 g/l yeast extract and 10 g/l bovine albumin) or 10 g/l skimmed milk for teat disinfectants or in
additional test conditions.
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Table 1 — Obligatory and additional test conditions
Yeasticidal activity for teat
Test conditions Fungicidal activity Yeasticidal activity
disinfectants
Test organisms Aspergillus brasiliensis
Candida albicans Candida albicans
obligatory Candida albicans
additional any relevant test organism any relevant test organism any relevant test organism
At intervals of 5°C
Test temperature
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
At intervals of 30 s from 30 s to 5 min and at intervals
of 5 min from 5 min to 120 min
1 min ± 5 s for post-milking
teat disinfectants
Contact time
1 min ± 5 s 1 min ± 5 s
30 s ± 5 s for pre-milking teat
Minimum
disinfectants
Maximum
30 min ± 10 s for post-milking
teat disinfectants
a a
120 min ± 10 s 120 min ± 10 s
3 min ± 10 s for pre-milking
teat disinfectants
Interfering
substance
Post milking: 10,0 g/l of milk
3,0 g/l bovine albumin 3,0 g/l bovine albumin
low level soiling powder
10 g/l yeast extract plus 10 g/l yeast extract plus
high level soiling Pre-milking: 3,0 g/l bovine
10 g/l bovine albumin 10 g/l bovine albumin
albumin
additional any relevant substance any relevant substance any relevant substance
The obligatory contact times for disinfectants stated in Table 1 were chosen to enable comparison of standard
conditions.
NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained
under the obligatory test conditions.
a
The recommended contact time for the use of the product is within the responsibility of the manufacturer.
Any additional specific fungicidal 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.
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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 temperatures 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 numbers of surviving fungi in each sample are determined and the
reduction is calculated.
5.1.2 The test is performed using the vegetative cells of Candida albicans and the spores of Aspergillus
brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as
test organisms (obligatory test conditions).
5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms
can be used.
5.2 Materials and reagents
5.2.1 Test organisms
1)
The fungicidal activity shall be evaluated using the following strains as test organisms:
— Candida albicans ATCC 10231;
— Aspergillus brasiliensis ATCC 16404.
(formerly A.niger)
The yeasticidal activity shall be evaluated using only Candida albicans.
NOTE See Annex A for strain references in some other culture collections.
The required incubation temperature for these test organisms is (30 ± 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 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.
1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC). This information is given for the convenience
of users of this European Standard and does not constitute an endorsement by CEN of the product named.
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5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this European 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.
To improve reproducibility, it is recommended that commercially available dehydrated material is used
for the preparation of culture media. The manufacturer's instructions relating to the preparation of
these products should be rigorously followed.
For each culture medium and reagent, a limitation for use should be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave [5.3.2.1 a)].
NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently
sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference
[1]) can be used.
NOTE 3 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
2)
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.
2) 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.
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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:
— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride
(CaCl2) 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 (NaHCO3) 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 300 mg/l
of calcium carbonate (CaCO3) in the test tube.
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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.
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 soiling (bovine albumin solution)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of 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 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 100 g milk powder in 1 000 ml water (5.2.2.2). Heat for 30 min at 105°C ± 3 °C
or 5 min at 121 °C ± 3 °C.
The final concentration of Skimmed milk powder in the test procedure (5.5) is 10,0 g/l.
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)].
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3)
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
0
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
0
+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 4°C ± 1 °C, 10 °C ± 1 °C, at 20 °C ± 1 °C, at
30°C ± 1 °C, 40°C ± 1 °C at 45 °C ± 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 °C ± 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
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.
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.
3)
Disposable sterile equipment is an acceptable alternative to reusable glassware.
4)
Vortex® is an example of a suitable product available commercially. This information is given for the
convenience of users of this European Standard and does not constitute an endorsement by CEN of this product.
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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
5.4 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), 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 (5.3.2.15) 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).
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oSIST prEN 1657:2023
prEN 1657:2023 (E)
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 O
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