oSIST prEN 14349:2023
(Main)Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-porous surfaces without mechanical action - Test method and requirements (phase 2, step 2)
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area on non-porous surfaces without mechanical action - Test method and requirements (phase 2, step 2)
This European Standard specifies a test method and the minimum requirements for bactericidal 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.
This European Standard applies to products that are used in the veterinary area on non-porous surfaces without mechanical action 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 2 test.
NOTE 3 This method cannot be used to evaluate the activity of products against mycobacteria.
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf nicht-porösen Oberflächen ohne mechanische Wirkung - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Dieses Dokument legt ein Prüfverfahren und die Mindestanforderungen an die bakterizide Wirkung von che
mischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte oder— im Fall gebrauchsfertiger Produkte— mit Wasser eine homogene, physikalisch stabile Zubereitung bilden.
Das beschriebene Verfahren ist für die Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen unter den Bedingungen, unter denen sie verwendet werden, bestimmt. Dieses Dokument gilt für Produkte, die im Veterinärbereich zur Desinfektion nicht-poröser Oberflächen ohne mechanische Wirkung angewendet werden, 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 eintreten.
EN14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwen
dungsempfehlungen“ fest.
ANMERKUNG Dieses Verfahren entspricht einer Prüfung der Phase2, Stufe2.
Dieses Verfahren schließt die Bewertung der Wirkung gegen Hefen, Pilzsporen, Mykobakterien und bakterielle Sporen aus.
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire sur des surfaces non poreuses sans action mécanique -
Le présent document spécifie une méthode d’essai et les exigences minimales relatives à l’activité bactéricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l’eau dure ou, dans le cas de produits prêts à l’emploi, dans l’eau.
La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives dans les conditions d’utilisation. Le présent document s’applique aux produits utilisés dans le domaine vétérinaire pour désinfecter des surfaces non poreuses sans action mécanique, à savoir dans la reproduction, l’élevage, la production, le transport et l’abattage de tous les animaux, à l’exception de la chaîne alimentaire qui suit l’abattage et l’entrée dans l’industrie de transformation.
L’EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».
NOTE Cette méthode correspond à un essai de phase 2, étape 2.
Cette méthode exclut l’évaluation de l’activité des produits vis-à-vis des levures, des spores fongiques, des mycobactéries et des spores bactériennes.
Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah brez mehanskega delovanja za vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 14349:2023
01-januar-2023
Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah
brez mehanskega delovanja za vrednotenje baktericidnega delovanja kemičnih
razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of
bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area
on non-porous surfaces without mechanical action - Test method and requirements
(phase 2, step 2)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur
Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika
für den Veterinärbereich auf nicht-porösen Oberflächen ohne mechanische Wirkung -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation
de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le
domaine vétérinaire sur des surfaces non poreuses sans action mécanique -
Ta slovenski standard je istoveten z: prEN 14349
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
oSIST prEN 14349:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 14349:2023
DRAFT
EUROPEAN STANDARD
prEN 14349
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2023
ICS 71.100.35 Will supersede EN 14349:2012
English Version
Chemical disinfectants and antiseptics - Quantitative
surface test for the evaluation of bactericidal activity of
chemical disinfectants and antiseptics used in the
veterinary area on non-porous surfaces without
mechanical action - Test method and requirements (phase
2, step 2)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de surface pour l'évaluation de l'activité Quantitativer Oberflächenversuch zur Bestimmung der
bactéricide des antiseptiques et des désinfectants bakteriziden Wirkung chemischer Desinfektionsmittel
chimiques utilisés dans le domaine vétérinaire sur des und Antiseptika für den Veterinärbereich auf nicht-
surfaces non poreuses sans action mécanique - porösen Oberflächen ohne mechanische Wirkung -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 216.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE 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 14349:2023 E
worldwide for CEN national Members.
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prEN 14349:2023 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms, definitions and abbreviations . 5
4 Requirements . 6
5 Test method . 7
Annex A (informative) Referenced strains in national collections . 27
Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of
chemical disinfectants and antiseptics . 28
Annex C (informative) Graphical representation of the method . 30
Annex D (informative) Example of a typical test report . 34
Bibliography . 37
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European foreword
This document (prEN 14349:2023) 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 14349:2012.
Data obtained using the former version of EN 14349 may still be used.
It 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.
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Introduction
This document specifies a surface test for establishing whether a chemical disinfectant or antiseptic has
bactericidal 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 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 the 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 bactericidal 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.
The method described is intended to determine the activity of commercial formulations or active
substances under the conditions in which they are used. This document applies to products that are used
in the veterinary area for disinfecting non-porous surfaces without mechanical action 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 This method corresponds to a Phase 2 Step 2 test.
This method excludes the evaluation of the activity of products against yeasts, fungal spores,
mycobacteria and bacterial spores.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 12353, Chemical disinfectants and antiseptics - Preservation of test organisms used for the
determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal
(including bacteriophages) activity
EN 14885, Chemical disinfectants and antiseptics - Application of European Standards for chemical
disinfectants and antiseptics
3 Terms, definitions and abbreviations
3.1 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 apply.
3.2 Symbols and abbreviations
c is the sum of V -values taken into account
C
cfu colony forming units
d is the dilution taken into account, lower dilution factor
n is the number of V -values taken into account
C
N number of cells per ml in the test suspension
N number of cfu recovered from the test surface in the water control
w
B counting of the cfu in the neutralizer control
Na number of cfu recovered from the test surface in the test
C counting of the cfu in the method validation
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N number of colony forming units remaining on the test surface
ts
R reduction
V is the volume of the inoculated into the plate expressed in ml
Nv is the validation suspension
4 Requirements
The product shall demonstrate at least a 4 decimal log (lg) reduction when diluted with hard water
(5.2.2.6) 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 g/l bovine albumin) or high level soiling (10 g/l
yeast extract and 10 g/l bovine albumin) on a surface.
Table 1 - Test conditions
Test conditions Bactericidal activity on non-porous surfaces
without mechanical action in the veterinary
area
Minimum spectrum of test
Enterococcus hirae
organisms
Pseudomonas aeruginosa
Staphylococcus aureus
additional any relevant test organism
Test temperature According to the manufacturer’s recommendation
but between
Minimum 4 °C ± 1 °C
Maximum 40 °C ± 1 °C
At intervals of 5°C
Contact time
Minimum 1 min ± 5 s
Maximum 120 min ± 10 s
At intervals of 30 s from 30 s to 5 min and at
intervals of 5 min from 5 min to 120 min
Interfering substance
low level soiling 3,0 g/l bovine albumin
high level soiling 10 g/l yeast extract plus 10 g/l bovine albumin
additional any relevant substance
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.
NOTE For the additional conditions, the concentration defined as a result can be lower
than the one obtained under the minimum test conditions.
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Any additional specific bactericidal 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
A test suspension of bacteria in a solution of interfering substances (5.2.2.7) is inoculated onto a test
stainless steel surface and dried. A sample of the product under test is applied in a manner that
completely covers the dried test organisms. The surface is maintained at the test temperature and contact
time exemplified in Clause 4 and (5.5.2). At the end of the contact time the surface is transferred to a
previously validated neutralization medium to suppress any product activity immediately. The number
of surviving test organisms which can be recovered from the surface is determined quantitatively.
The number of bacteria on a test surface treated with hard water (or water in the case of ready to use
products) in place of the product under test is also determined and the reduction in viable counts
attributed to the product is calculated as the difference between the two test surfaces’ results.
5.2 Materials and reagents
5.2.1 Test organisms
The bactericidal activity shall be evaluated using the following strains:
— Enterococcus hirae
— Pseudomonas aeruginosa
— Staphylococcus aureus
NOTE See Annex A for strain references in some culture collections.
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3). The
same temperature (either 36 °C or 37 °C) 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.
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) additional
media shall be used and shall be reported as well as additional incubation conditions.
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To improve the reproducibility, it is recommended that commercially available dehydrated material is
used for the preparation of culture media. The manufacturers’ 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.
5.2.2.2 Water
The water shall be freshly glass-distilled and not demineralized water. If distilled water of adequate
quality is not available, water for injections (see bibliographic reference [1]) may be used.
Sterilize in the autoclave [5.3.2.1a)]. Sterilization is not necessary if the water is used e.g. for preparation
of culture media and subsequently sterilized.
NOTE The procedure to prepare hard water is described in 5.2.2.6.
5.2.2.3 Tryptone Soya Agar (TSA)
Tryptone soya agar, consisting of:
— Tryptone, pancreatic digest of casein 15,0 g;
— Soya peptone, papaic digest of soybean meal 5,0 g;
— Sodium chloride (NaCl) 5,0 g;
— Agar 15,0 g;
— Water (5.2.2.2) to1 000 ml.
Sterilize in the autoclave [5.3.2.1 a)].
After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C.
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add
neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used. It is recommended
not to use a neutralizer that causes opalescence in the agar.
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 ml.
Sterilize in the autoclave [5.3.2.1 a)].
After sterilization the pH of the diluent shall be equivalent to 7,0 ± 0,2, when measured at (20 ± 1) °C.
5.2.2.5 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. The neutralizer shall be sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given
in Annex B.
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5.2.2.6 Hard water for dilution of products
For the preparation of 1 000 ml of hard water, the procedure is as follows:
— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride
2
(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 [5.3.2.1a)]. 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
3
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 of
solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The pH of the hard
water shall be 7,0 ± 0,2, when measured at (20 ± 1) °C (5.3.2.4). If necessary, adjust the pH by using a
solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l
(about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
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 (CaCO3) is in the test tube lower than 375 mg/l.
5.2.2.7 Interfering substances
5.2.2.7.1 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 2 times its final concentration in the test.
For any additional interfering substances, 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.
5.2.2.7.2 Low level soiling (bovine albumin solution)
Dissolve 0,6 g of bovine albumin V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2).
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) is 3 g/l.
5.2.2.7.3 High level soiling (mixture of bovine albumin solution with yeast extract)
Dissolve 10 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 the autoclave [5.3.2.1 a)]. Allow to cool to 20 °C ± 5 °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 1 g of bovine albumin fraction V (suitable for microbiological purposes) in the solution in the
flask 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) and keep in 10 ml portions in a refrigerator (5.3.2.8) and use within one
month.
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The final concentration in the test procedure (5.5) is 10 g/l yeast extract and 10 g/l bovine albumin.
5.2.3 Test surface
Stainless steel discs (2 cm diameter discs) 304 (bibliography references [2] [3]) with grade 2b finish on
both sides. The surfaces should be flat.
The surfaces should be used only once and only one side of the disc shall be used. The discs shall only be
handled with forceps.
Prior to use the surfaces should be placed in a beaker (minimum size 50 ml) containing not less than
1)
20 ml of 5 % (V/V) Decon® for 60 min. Immediately rinse the discs with water (5.2.2.2) for 10 s.
The surface shall not be allowed to dry to any extent. Rinse the discs with water for a further 10 s to
ensure complete removal of the surfactant. To supply a satisfactory flow of water, a sterile fluid
dispensing pressure vessel with suitable hose and connectors or other suitable method can be used and
regulated to supply approximately 2 000 ml per min. Place the clean discs in a bath containing 70 % (V/V)
iso-propanol for 15 min. Remove the discs and dry by evaporation under laminar air flow (5.3.2.15)
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 an autoclave [5.3.2.1 a)]
b) by dry heat, in a hot air oven [5.3.2.1 b)]
2)
5.3.2 Usual microbiological laboratory equipment
and, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)
+3
a) for moist heat sterilization, an autoclave capable of being maintained at ( ) °C for a minimum
121
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 ( ) °C for a
160
0
0
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at 4 and 40°C ± 1 °C as required by the test
conditions, and 45 ± 1 °C (to maintain melted TSA,in case of pour plate technique).
5.3.2.3 Incubator, capable of being controlled either at (36 ± 1) °C or (37 ± 1) °C (5.2.1).
5.3.2.4 pH meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
1)
Decon® is an example of a suitable product available commercially. This information is given for the convenience of users
of this standard and does not constitute endorsement by CEN of this product.
2) Disposable sterile equipment is an acceptable alternative to reusable glassware.
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5.3.2.5 A puncture electrode or a flat membrane electrode should be used for measuring the pH
of the agar media (5.2.2.3).
5.3.5.6 Stopwatch.
5.3.5.7 Shakers
3
a) Electromechanical agitator, e.g. Vortex® mixer ;
b) Mechanical shaker.
5.3.5.8 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm
to 50 mm and 0,45 μm pore size for sterilization of hard water (5.2.2.6) and bovine albumin (5.2.2.7.2
and 5.2.2.7.3).
5.3.5.9 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.5.10 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 0,1 ml and 0,05 ml, or calibrated
automatic pipettes.
5.3.5.11 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.5.12 Glass beads (diameter 3 mm to 4 mm).
5.3.5.13 Volumetric flasks.
5.3.5.14 Temperature controlled cabinet, capable of being controlled at test temperatures.
5.3.5.15 Containers: Test tubes, culture bottles or flasks of suitable capacity
5.3.5.16 Laminar air flow: Microbiological filtered laminar air flow cabinet
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, one suspension has to be prepared: this is used as the bacterial “test suspension”
to perform the test and the “validation suspension” to perform the controls and method validation.
5.4.1.2 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
In order to prepare the working culture of the test organisms (5.2.1), prepare a subculture from the stock
culture (5.4.1.2) by streaking onto TSA (5.2.2.3) slopes or plates and incubate (5.3.2.3). After 18 h to 24 h
prepare a second subculture from the first subculture in the same way and incubate for 18 h to 24 h. From
3) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this d
ocument
and does not constitute an endorsement by CEN of this product.
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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 48 h subculture may be used
for subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during
the 48 h period.
Never produce and use a fourth subculture.
For additional test organisms, any departure from this method of culturing the bacteria or preparing the
suspensions shall be noted, giving the reasons in the test report.
5.4.1.4 Test suspension (“N”)
a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask (5.3.2.12) with 5 g of glass beads (5.3.2.11).
Take the working culture (5.4.1.3) 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.
9 9 4)
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/ml by spectrophotometer or any other suitable means.
Maintain this suspension in the water bath at (20 ± 1) °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.
−7 −8
c) For counting of the bacterial test suspension 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 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 TSA (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
TSA (5.2.2.3).
For incubation and counting see 5.4.1.6.
5.4.1.5 Incubation and counting of the test and the validation suspensions
a) Incubate (5.3.2.3) the plates for 20 h - 24 h. Discard any plates that are not countable for any reason.
Count the cfu on the plates and determine the total number of cfu. Incubate the plates for a further
20 h - 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) Note for each plate the exact number of colonies but record > 330 for any counts higher than 330 and
determine the V values according to 5.6.2.2.
C
4)
cfu/ml = colony forming unit(s) per millilitre
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c) Calculate the numbers of cfu per 0,025 ml in the test suspension N using the methods given in 5.6.2.3.
Verify according to 5.7.
NOTE 0,05 ml of an equal parts mixture of the test suspension and interfering substance is added to the surface
therefore 0,025 ml of the suspension is added.
5.4.2 Product test solutions
Product test solutions shall be prepared in hard water (5.2.2.6) at a minimum thr
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