SIST EN 14349:2025

SLOVENSKI STANDARD
01-september-2025
Nadomešča:
SIST EN 14349:2013
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 - Méthode
d’essai et exigences (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 14349:2025
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 14349
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2025
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes 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 -
Méthode d'essai et exigences (phase 2, étape 2) Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
This European Standard was approved by CEN on 7 April 2025.

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
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14349:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms, definitions and abbreviations . 6
3.1 Terms and definitions . 6
3.2 Symbols and abbreviations . 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
5.2.3 Test surface . 11
5.3 Apparatus and glassware . 12
5.3.1 General . 12
5.3.2 Usual microbiological laboratory equipment . 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 . 15
5.5 Procedure for assessing the bactericidal activity of the product . 15
5.5.1 General . 15
)
5.5.2 Test procedure (Dilution-neutralization method) . 16
5.5.3 Observation of test surface agar . 20
5.6 Experimental data and calculation . 21
5.6.1 Explanation of terms and abbreviations . 21
5.6.2 Calculation . 21
5.7 Verification of methodology . 24
5.7.1 General . 24
5.7.2 Control of weighted mean counts . 24
5.7.3 Basic limits . 25
5.8 Expression of results and precision . 25
5.8.1 Reduction . 25
5.8.2 Control of active and non-active product test solution (5.4.2) . 25
5.8.3 Limiting test organism and bactericidal concentration . 25
5.8.4 Precision, replicates . 25
5.9 Interpretation of results – conclusion . 26
5.9.1 General . 26
5.9.2 Bactericidal activity for general purposes . 26
5.9.3 Bactericidal activity for specific purposes . 26
5.10 Test report . 26
Annex A (informative) Referenced strains in national collections . 28
Annex B (informative) Neutralizers . 29
Annex C (informative) Graphical representation of the method . 31
Annex D (informative) Example of a typical test report . 35
Bibliography . 39

European foreword
This document (EN 14349:2025) 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 November 2025, and conflicting national standards shall
be withdrawn at the latest by November 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 14349:2012.
This European Standard 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.
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 surface test method for establishing whether a chemical disinfectant or
antiseptic has bactericidal 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 the specified experimental conditions. However, for some applications, the
recommendations of use of a product may differ and therefore additional test conditions need to be used.
Data obtained using the former version of EN 14349 may still be used.
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.
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, veterinary care facilities,
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 in the conditions in which they are used.
NOTE 2 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
1 1
EN 14885:2022 , 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.
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/

As impacted by EN 14885:2022/AC:2023.
3.2 Symbols and abbreviations
For the purposes of this document, the following symbols and abbreviations apply.
c sum of V values taken into account
C
cfu colony forming units
d dilution taken into account, lower dilution factor
n 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
N number of colony forming units remaining on the test surface
ts
R reduction
Vc number of cfu counted per sample of 1,0 ml
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 (3,0 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 5 °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 1min 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 recommended contact time for the use of the product is within the
responsibility of the manufacturer.
NOTE For the additional conditions, the concentration specified as a result can be lower
than the one obtained under the minimum test conditions.
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 substance (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 in accordance with Table 1. At the end of the contact time the surface is transferred to a previously
validated neutralization medium so that the action of the disinfectant is immediately neutralized. The
number of surviving test organisms which can be recovered from the surface is determined
quantitatively.
The number of bacteria on a 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 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 document refer to the anhydrous salts. Hydrated forms
may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
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 the reproducibility, it is recommended that commercially available dehydrated material (if
appropriate) 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)  to 1 000,0 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.
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.
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
(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 [5.3.2.1a)]. 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
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 ± 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 is lower than 375 mg/l of
calcium carbonate (CaCO ) in the test tube.
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 90 ml of water (5.2.2.2) in a
volumetric flask (5.3.2.13). Make up to the mark with water.
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 bovine albumin in the test procedure (5.5) is 3,0 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 ± 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 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.
The final concentration in the test procedure (5.5) is 10,0 g/l yeast extract and 10,0 g/l bovine albumin.
5.2.3 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
20 ml of a commercial alkaline cleaner in a concentration as recommended by the manufacturer for
60 min. Immediately rinse the discs with running freshly distilled 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)]
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 ( 121 ) °C for a minimum
holding time of 15 min;
+5
b) for dry heat sterilization, a hot air oven capable of being maintained at ( 180 ) °C for a minimum
+5 +5
holding time of 30 min, at ( 170 ) °C for a minimum holding time of 1 h or at ( 160 ) °C for a
0 0
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at the specified temperatures ± 1 °C, and (45 ± 1) °C
(to maintain melted TSA, 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 ± 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.
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.
3 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, 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).
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 0,1 ml and 0,05 ml, or calibrated
automatic pipettes.
5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads (diameter 3 mm to 4 mm).
5.3.2.12 Volumetric flasks.
5.3.2.13 Temperature controlled cabinet, capable of being controlled at test temperatures.
5.3.2.14 Containers: Test tubes, culture bottles or flasks of suitable capacity.
5.3.2.15 Laminar air flow: Microbiological filtered laminar air flow cabinet.
5.3.2.16 Vacuum desiccator, with an active desiccant. Vacuum source may be a pump or central
supply and should achieve a vacuum of 20 mmHg to 25 mmHg (508 torr to 635 torr; 677 mbar to
847 mbar; 68000 Pascal to 85000 Pascal; conversion tables are readily available on the Internet for other
units).
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 shall 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 Preservation and working 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
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 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”)
The procedure for preparing the bacterial test suspensions is as follows:
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 any suitable means. Maintain this suspension in the
water bath at the test temperature and use within 2 h.
The use of spectrophotometer for adjusting the number of cells is highly recommended
(approximately 620 nm wavelength - cuvette 10 mm path length). Therefore each laboratory
produces calibration data for each test organism knowing that suitable values of optical density are
generally found between 0,150 and 0,460. A colourimeter is a suitable alternative.
−7 −8
c) 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 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 5.4.1.5.
5.4.1.5 Incubation and counting of the test suspension
a) incubate (5.3.2.3) the plates for 20 h to 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 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 > 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
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 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. Dilutions of ready-
to-use products, i.e. products that are not diluted when applied, shall be prepared in water (5.2.2.2)
instead of hard water.
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 (5.2.2.6). Subsequent dilutions (lower concentrations)
shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.6).
For liquid products, dilutions of the product shall be prepared with hard water (5.2.2.6) in volumetric
flasks (5.3.2.12) on a volume/volume basis.
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous preparation, 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 recorded on the test report.
The concentration of the product stated in the test report shall be the desired test concentration. Record
the test concentration in terms of mass per volume or volume per volume and details of the product
sample as received.
5.5 Procedure for assessing the bactericidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions
The experimental conditions may be selected according to the practical use considered for the product
(Clause 4, Table 1):
a) Temperature θ (in °C);
b) contact time t (in min);
c) interfering substance;
d) test organisms;
5.5.1.2 Neutralization
To determine a suitable neutralizer carry out the validation of the dilution-neutralization method (5.5.2.4
and 5.5.2.5 in connection with 5.5.2.6) using a suitable neutralizer, chosen according to laboratory
experience and published data.
If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into
account the information given in Annex B.
5.5.1.3 General instruction for validation and control procedures
The neutralization and/or removal of the bactericidal and/or bacteriostatic activity of the product shall
be controlled and validated - only for the highest product test concentration - for each of the used test
organisms and for each experimental condition (interfering substance, temperature, contact time). These
procedures (water control, neutralizer control and method validation) shall be performed at the same
time with the test and with the same neutralizer used in the test.
5.5.1.4 Equilibration of temperature
Prior to testing, equilibrate the inoculated and dried stainless steel discs and all reagents; product test
solutions (5.4.2), test suspensions (5.4.1.4), hard water (5.2.2.6) [or in the case of ready-to-use products,
water (5.2.2.2)] and interfering substance to the test temperature of θ [5.5.1.1 a)] using the water bath
(5.3.2.2) and/or the temperature controlled cabinet (5.3.2.13) controlled at θ. Check that the temperature
of the reagents is stabilized at θ.
The neutralizer (5.2.2.5) and diluent (5.2.2.4) shall be equilibrated at a temperature of (20 ± 1) °C.
5)
5.5.2 Test procedure (Dilution-neutralization method)
5.5.2.1 General
The test and the control and validation procedures (5.5.2.2 through 5.5.2.5) shall be carried out at the in
parallel and separately for each experimental condition (5.5.1.1).
5.5.2.2 Test “N ” – determination of bactericidal concentrations
a
The procedure for determining bactericidal concentrations is as follows:
a) to prepare the microbial test suspension pipette 1,0 ml of the interfering substance (5.2.2.7) into a
tube. Add 1,0 ml of the test suspension (5.4.1.5). Start the stopwatch (5.3.2.5) immediately, mix
[5.3.2.6 a)] and place the tube in a water bath or temperature controlled cabinet at the chosen test
temperature θ [5.5.1.1 a)] for 2 min ± 10 s.
Immediately before addition, the test suspension should be well mixed to fully re-suspend the
organisms.
b) place the test surfaces (5.2.3) in a sterile Petri dish (5.3.2.10) and ensure that the dish is in a
horizontal position. Prepare the test surfaces by inoculating 0,05 ml of the microbial test suspension
[5.5.2.2 a)] on to each test surface (Figure 1). The inoculum shall not be spread over the surface. Dry
the surfaces at 37 °C until they are visibly dry (Figure 2).
It is understood that drying of the test surfaces will occur at different rates due to the ambient conditions
of the laboratory and the design of the incubator (e.g. with or without a fan). For this reason no time
duration is given and the minimum required time for the surfaces to become visibly dry should be
established for each laboratory. The drying time should not exceed 60 min and if it does, alternative
drying conditions shall be used.
Alternatively, place surfaces in an open Petri dish to dry, inoculated side uppermost at 18 °C to 25 °C in a
vacuum desiccator for not longer than 60 min. Ensure that the petri dish is partially open by placing its
lid off centre. Check that the desiccator is properly sealed. Attach the outlet of the desiccator to a vacuum
source and start evacuation of the air to achieve a vacuum of 20 mmHg to 25 mmHg (508 torr to 635 torr;

5)
For a graphical representation of this method see Annex C (C.1)
677 mbar to 847 mbar; 68000 Pascal to 85000 Pascal; conversion tables are readily available on the
Internet for other units).
Allow the test surfaces to equilibrate with the chosen test temperature θ.

Figure 1 — Inoculated carrier
Figure 2 — Visibly dry inoculum
Figure 3 — Product applied onto inoculated carrier
c) for the disinfectant test (N ) pipette 0,1 ml of each product test solution (5.4.2) to be tested on to
a
separate dried surfaces ensuring that the dried inoculum is totally covered by the test product
(Figure 3).
Place the surfaces in a temperature-controlled cabinet (5.3.2.13) at the chosen test temperature θ
and incubate for the contact time t.
d) at the end of t, transfer each of the surfaces (N ) to a separate container (5.3.2.14) containing 10 ml
a
of neutralizer (5.2.2.5) together with sufficient glass beads, for example 5 g (5.3.2.11) to support the
surface. The surfaces should be placed with the inoculated surface downwards in contact with the
beads. Shake the containers for a minimum of 1 min. The shaking should be sufficiently vigorous to
ensure that the test surface moves constantly over the beads. Ensure that the beads are able to move
freely.
−1 −2
After the neutralization time of 5 min ± 10 s prepare a series of ten-fold dilutions from 10 to 10
of the neutralized mixture in the diluent (5.2.2.4). Take a 1,0 ml sample of the neutralized mixture
and each of the dilutions in duplicate and inoculate using pour plate or spread plate technique.
1) When using the pour plate technique, pipette each 1,0 ml sample into separate Petri dishes and
add 15 ml to 20 ml of 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 5.5.2.6.
e) recover the test surface (“N ”), let the neutralizer drain off and rinse with 10 ml of water (5.2.2.2).
a
Transfer to a Petri dish (5.3.2.10) containing 10 ml of solidified TSA (5.2.2.3) and place on top of the
agar test side uppermost. Add 10 ml of TSA (5.2.2.3) melted and cooled to 45 °C.
f) perform the procedure a) to e) using the other product test solutions at the same time.
g) perform the proc
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