SIST EN 14204:2026

SLOVENSKI STANDARD
oSIST prEN 14204:2024
01-maj-2024
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje mikobaktericidnega delovanja kemičnih razkužil in antiseptikov v
veterini - Preskusna metoda in zahteve (faza 2, stopnja 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of mycobactericidal 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 mykobakteriziden 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 mycobactéricide des antiseptiques et des désinfectants
chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase
2, é
Ta slovenski standard je istoveten z: prEN 14204
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
oSIST prEN 14204:2024 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 14204:2024
oSIST prEN 14204:2024
DRAFT
EUROPEAN STANDARD
prEN 14204
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2024
ICS 71.100.35 Will supersede EN 14204:2012
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of mycobactericidal
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
mycobactéricide des antiseptiques et des désinfectants mykobakteriziden Wirkung chemischer
chimiques utilisés dans le domaine vétérinaire - Desinfektionsmittel und Antiseptika für den
Méthode d'essai et prescriptions (phase 2, é 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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 14204:2024 E
worldwide for CEN national Members.

oSIST prEN 14204:2024
prEN 14204:2024 (E)
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 . 6
5.1 Principle . 6
5.2 Materials and reagents . 6
5.2.1 Test organisms . 6
5.2.2 Culture media and reagents . 7
5.3 Apparatus and glassware . 9
5.3.1 General. 9
5.3.2 Usual microbiological laboratory equipment and, in particular, the following . 9
5.4 Preparation of mycobacterial test suspension and product test solutions . 11
5.4.1 Test organism suspensions (test and validation suspension) . 11
5.4.2 Product test solutions . 13
5.5 Procedure for assessing the mycobactericidal activity of the product . 13
5.5.1 General. 13
5.5.2 Test procedure - Dilution-neutralization method . 14
5.5.3 Test procedure - Membrane filtration method . 17
5.6 Experimental data and calculation . 19
5.6.1 Explanation of terms and abbreviations . 19
5.6.2 Calculation . 19
5.7 Verification of methodology . 23
5.7.1 General. 23
5.7.2 Control of weighted mean counts . 24
5.7.3 Basic limits . 24
5.8 Expression of results . 24
5.8.1 Reduction . 24
5.8.2 Control of active and non-active product test solution (5.4.2) . 25
5.8.3 Mycobactericidal concentration . 25
5.9 Interpretation of results - conclusion. 25
5.9.1 General. 25
5.9.2 Mycobactericidal activity for general purposes . 25
5.9.3 Qualification for certain fields of application . 25
5.10 Test report . 25
Annex A (informative) Referenced strain in national collections . 27
Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of
chemical disinfectants and antiseptics and rinsing liquids . 28
Annex C (informative) Graphical representations of dilution-neutralization method . 30
Annex D (informative) Example of a typical test report . 32
Bibliography . 35

oSIST prEN 14204:2024
prEN 14204:2024 (E)
European foreword
This document (prEN 14204:2024) 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 supersedes EN 14204:2012 and was revised to harmonize the structure and wording
with other quantitative suspension tests of CEN/TC 216 (existing or in preparation).
Results obtained using the previous version of this standard are still valid.
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Introduction
This document specifies a suspension test for establishing whether a chemical disinfectant or antiseptic
has mycobactericidal activity in the area 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 chosen experimental conditions. However, for some applications the
recommendations of use of a product may differ and therefore additional test conditions need to be
used.
oSIST prEN 14204:2024
prEN 14204:2024 (E)
1 Scope
This document specifies a test method and the minimum requirements for mycobactericidal 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.
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 for equipment disinfection by immersion, surface
disinfection by wiping, spraying or flooding or other means and teat disinfection in the veterinary area
– i.e. in the breeding, husbandry, production, veterinary care facilities, transport and disposal of all
animals except when in the food chain following death and entry 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 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, Chemical disinfectants and antiseptics — Application of European Standards for chemical
disinfectants and antiseptics
3 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/
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 (3,0 g/l bovine albumin) or high-level soiling
(10 g/l yeast extract and 10 g/l bovine albumin) or in additional test conditions.
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Table 1 — Test conditions
Test conditions Mycobactericidal activity
Minimum Spectrum of target organism Mycobacterium avium
additional any relevant test organism
Test temperature
minimum 5 °C ± 1 °C
maximum 40 °C ± 1 °C
Contact time
minimum 1 min ± 5 s
maximum 120 min ± 10 s
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
Any additional specific mycobactericidal activity shall be determined in accordance with 5.2.1
and 5.5.1.1 in order to take into account intended specific use conditions.
5 Test method
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted in hard water (or water for ready to use
products) is added to a test suspension of mycobacteria in a solution of an interfering substance.
The mixture is maintained at the test temperature Ɵ for the test contact time t. At the end of this contact
time, an aliquot is taken and the mycobactericidal action in this portion is immediately neutralized or
suppressed by a validated method.
The method of choice is the dilution-neutralization. If a suitable neutralizer cannot be found, membrane
filtration is used. The numbers of surviving mycobacteria in each sample are determined and the
reduction in viable counts calculated.
5.1.2 For general disinfectant product, the test is performed using Mycobacterium avium as test
organism.
5.1.3 Additional and optional contact times and temperatures are specified (Clause 4, Table 1).
Additional interfering substances and test organisms may be used.
5.2 Materials and reagents
5.2.1 Test organisms
The mycobactericidal activity shall be evaluated using the following strain as test organisms:
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Mycobacterium avium   ATCC® 15769™
NOTE See Annex A for corresponding strain numbers in some other culture collections.
The required incubation temperature for this test organism is 36 ± 1 °C or 37 ± 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 document refer to the anhydrous salts. Hydrated forms
may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
To improve the 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 shelf life should be fixed.
5.2.2.2 Water
The water shall be freshly glass distilled and not demineralized water
Sterilize in the autoclave [5.3.2.1 a)].
Refer to 5.2.2.7 for the procedure to prepare hard water.
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] Pharmacopoeia) can be used.
5.2.2.3 Middlebrook and Cohn 7H10 medium + 10 % oleic acid albumin dextrose
complex (OADC) (reported as 7H10 in the text)
7H10, consisting of:
middlebrook 7H10 agar 19 g;
glycerol 5 ml;
water (5.2.2.2) to 895 ml.
The ATCC numbers are the collection number of strains supplied by the American Type Culture Collections
(ATCC). This information is given for the convenience of users of this document and does not constitute an
endorsement by CEN of the product named.
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Heat to boiling to dissolve completely. Sterilize for 10 min in the autoclave [5.3.2.1 a)] and cool to 50 °C
to 55 °C. Add 100 ml Middlebrook OADC enrichment under aseptic conditions. Final pH = 6,6 ± 0,2
at 25 °C.
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it can be necessary to add
neutralizer to the 7H10. Annex B gives guidance on the neutralizers that may be used.
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
— tryptone, pancreatic digest of casein  1,0 g;
— sodium chloride (NaCl)  8,5 g;
— water (see 5.2.2.2) to  1 000 ml.
— sterilize in the autoclave [see 5.3.2.1 a)].
— after sterilization the pH of the medium 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. It shall be sterile.
Information on neutralizers that have been found to be suitable for some categories of products is given
in Annex B.
5.2.2.6 Rinsing liquid (for membrane filtration)
The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3
and 5.5.3.
It shall be sterile, compatible with the filter membrane and capable of filtration through the filter
membrane under the test conditions described in 5.5.3.
Information on rinsing liquids that have been found to be suitable for some categories of products is
given in Annex B.
5.2.2.7 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 [5.3.2.1 a)]. Autoclaving- if used- can 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 a 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. If necessary, adjust the pH by using a
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prEN 14204:2024 (E)
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.
5.2.2.8 Interfering substances
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
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 90 ml of
water (5.2.2.2) in a 100 ml volumetric flask (5.3.2.12). 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 of the bovine albumin in the test procedure (5.5.2) 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 ± 1) °C.
Pipette 25 ml of this solution into a 50 ml volumetric flask 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) and keep 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.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
5.3.2 Usual microbiological laboratory equipment and, in particular, the following
5.3.2.1 Apparatus for sterilization

Disposable Sterile Equipment is an acceptable alternative to reusable glassware.
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prEN 14204:2024 (E)
+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 between 5 and 40 °C ± 1 °C as required by the test
conditions.
5.3.2.3 CO Incubator, capable of being controlled at either (36 ± 1) °C or (37 ± 1) °C. An incubator
at (36 ± 1) °C or (37 ± 1) °C may be used if a CO incubator is not available.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar
media (5.2.2.3).
5.3.2.5 Stopwatch
5.3.2.6 Shaker
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 filtration of hard water (5.2.2.6), bovine
albumin (5.2.2.7.2 and 5.2.2.7.3) and if the membrane filtration method (5.5.3) is used.
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the microorganisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic
pipettes may be used.
5.3.2.10 Petri dishes (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads 3 mm to 4 mm in diameter.
5.3.2.12 Volumetric flasks.
5.3.2.13 High Speed homogenizer, e.g. Potter ® apparatus .

Vortex  is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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5.3.2.14 Containers, test tubes, flasks or bottles of suitable capacity.
5.3.2.15 Coned bottom screw cap tube.
5.4 Preparation of mycobacterial test suspension 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 of test organism
The test organism and stock cultures shall be prepared and kept in accordance with EN 12353.
5.4.1.3 Working culture of test organism
In order to prepare a working culture of the test organism (5.2.1) subculture from the stock
culture (5.4.1.2) by streaking on to at least two plates of 7H10 medium (5.2.2.3) and incubate (5.3.2.3).
If a CO incubator is not used, plates shall be placed into polyethylene bags or sealed with insulating
tape to prevent drying of the medium.
After 21 days prepare a second subculture from the first subculture in the same way and incubate
for 21 days.
The first and/or the second subculture is the working cultures. Never produce and use a third
subculture.
For additional strains, any departure from this method of culturing the mycobacteria or preparing the
suspensions shall be noted, giving the reasons in the test report.
5.4.1.4 Test suspension (N)
a) Prepare a suitable homogeneous suspension from the working culture (5.4.1.3) using
— either homogenization by glass beads: with the aid of a plastic disposable loop transfer the test
organisms from at least two plates of the working culture (5.4.1.3) into a coned bottom screw
cap tube (5.3.2.15) containing 6-7 g of dry glass beads (5.3.2.11). The test organisms together
with 2 ml of water (5.2.2.2) are homogenized by mixing [5.3.2.6 a)] for at least 5 min to
distribute them homogeneously on the beads and on most of the parts of the screw cap tube’s
internal surface. Add 10 ml water (5.2.2.2) drop by drop and re-suspend them by
mixing [5.3.2.6 a)]. After 20 min sedimentation time the supernatant is transferred to a tube;
— or homogenization by Potter S 1 apparatus: pipette 5,0 ml water (5.2.2.2) on each of the plates
(at least two) of the working culture (5.4.1.3) and recover with a glass spatula the test
organisms. Pipette all of the liquid from the plates into a 25 ml centrifugation tube. Add up
to 18 ml water (5.2.2.2). Make three consecutive washings with water (5.2.2.2) (centrifuging
each time at approximately 2 000 gN for 15 min). After each centrifuging, discard the
supernatant, re-suspend by mixing [5.3.2.6 a)] and fill up to the original volume with
water (5.2.2.2).
Potter  Apparatus is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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After the last centrifuging discard the supernatant and transfer the sediment into a 15 ml glass
vessel of the Potter S 1 apparatus. Fill up to 15 ml with water (5.2.2.2). Mix, cool with ice and
homogenize for 15 min.
After 20 min sedimentation time, during which enough ice for cooling should be present, the
supernatant is transferred to a tube.
NOTE 1 Other methods of homogenization are allowed provided that the number of colony forming units
per millilitre obtained is appropriate and stable during the time of the test and microscopic examination
shows that the suspension is as homogeneous as after the two procedures described above.
Do not use tensio-active substances (surfactants).
8 8
b) Adjust the number of cells in the (supernatant) suspension to 3,0 × 10 cfu/ml to 8,0 × 10 cfu/ml
using water (5.2.2.2). Maintain this test suspension in the water bath at (20 ± 1) °C and use at the
day of preparation. Adjust the temperature according to 5.5.1.1 a) and 5.5.1.4 only immediately
before the start of the test.
The use of a spectrophotometer for adjusting the number of cells is highly recommended
(about 620 nm wavelength - cuvette 10 mm path length). Each laboratory should therefore produce
a calibration curve for each test organism knowing that suitable values of optical density are
generally found between 0,200 and 0,400. To achieve reproducible results of this measurement it
can be necessary to dilute the test suspension, e.g. 1+9.
NOTE 2 A colourimeter is a suitable alternative.
−6 −7
c) For counting prepare 10 and 10 dilutions of the test suspension using diluent (5.2.2.4). Mix
[(5.3.2.6 a)].
d) Take a sample of 1,0 ml of each dilution in duplicate. Divide each sample in two nearly equal
amounts and spread onto separate surface dried plates (5.3.2.10) containing 18 ml to 20 ml
of 7H10 medium (5.2.2.3), i.e. four plates per duplicate 1,0 ml samples.
For incubation and counting see 5.4.1.6.
5.4.1.5 Validation suspension (N )
V
a) To prepare the validation suspension dilute the test suspension (5.4.1.4) with the diluent (5.2.2.4)
2 3
to obtain 3 × 10 cfu/ml to 1,6 × 10 cfu/ml.
−1
b) For counting prepare a 10 dilution with diluent (5.2.2.4). Mix [5.3.2.6 a)]. Take a sample of 1,0 ml
in duplicate. Divide each sample in two nearly equal amounts and spread onto separate surface
dried plates (5.3.2.10) containing 18 ml to 20 ml of 7H10 medium (5.2.2.3), i.e. four plates per
duplicate 1,0 ml samples.
For incubating and counting see 5.4.1.6.
5.4.1.6 Incubation and counting of the test and validation suspensions
a) Incubate (5.3.2.3) the plates for 21 days at (36 ± 1) °C or (37 ± 1) °C. Discard any which are not
countable. Count the plates to determine the total number of colony forming units (cfu). Incubate
the plates for a further 7 days. Do not recount plates which no longer show well separated colonies.
Recount the remaining plates. If the number has increased, use only the higher number for further
evaluation.
cfu/ml = colony forming unit(s) per millilitre.
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prEN 14204:2024 (E)
b) Note for each plate the exact number of colonies but record > 330 for any counts higher
than 330 and determine V values according to 5.6.2.2.
C
c) Calculate the number of cfu/ml in the test suspension N and in the validation suspension N using
V
the method given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7.
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration because
it is diluted to 80 % during the test and the method validation (5.5.2).
Product test solutions shall be prepared in hard water (5.2.2.7) 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, in this case the
highest tested concentration is 80 %.
Dilutions of ready-to-use products, i.e. products that are not diluted when applied, shall be prepared in
water (5.2.2.2).
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in
a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (lower concentrations)
shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.6).
For liquid products, dilutions of the product shall be prepared with hard water (5.2.2.7) or
water (5.2.2.2) for ready to use products on a volume/volume basis using volumetric flasks (5.3.2.12).
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous preparation that is stable during the whole procedure. If during the procedure
a visible inhomogeneity appears due to the formation of a precipitate or flocculent (for example,
through the addition of the interfering substance), it shall be recorded in the test report.
NOTE Counting microorganisms embedded in a precipitate or flocculent is difficult and unreliable.
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 mycobactericidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions
Experimental conditions may be selected according to the practical use considered for the
product (Clause 4):
a) temperature: θ (in °C);
The temperatures to be tested are specified in Clause 4, Table 1; the allowed deviation for each chosen
temperature is ± 1 °C;
b) contact time: t (in min and s);
The contact times to be tested are specified in Clause 4, Table 1; the allowed deviation for each chosen
contact time is ± 10 s, (except for 1 min ± 5 s);
c) interfering substance;
oSIST prEN 14204:2024
prEN 14204:2024 (E)
The interfering substance to be tested are specified in Clause 4, Table 1. Additional interfering
substances may be tested according to specific fields of application.
d) test organism;
The test organisms are as specified in Clause 4, Table 1 and 5.2.1. Additional test organisms may be
tested.
5.5.1.2 Choice of test method (dilution-neutralization or membrane filtration)
The method of choice is the dilution-neutralization method (5.5.2). To determine a suitable neutralizer,
carry out the validation of the dilution neutralization method (5.5.2.3, 5.5.2.4 and 5.5.2.5 in connection
with 5.5.2.6) using a 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.
In special circumstances it can be necessary to add neutralizer to 7H10 medium (5.2.2.3). If neutralizer
is added to 7H10 medium, the same amount shall be added to 7H10 medium used in the test procedure.
5.5.1.3 General instructions for validation and control procedures
The neutralization and/or removal of the mycobactericidal and/or mycobacteriostatic 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 (experimental condition control, neutralizer or filtration control and method
validation) shall be performed at the same time with the test and with the same neutralizer – or rinsing
liquid – used in the test.
If because of problems with neutralization, a neutralizer has been added to 7H10 medium (5.2.2.3) used
for the validation and control procedures, the 7H10 medium used for the test shall contain the same
amount of this neutralizer as well.
5.5.1.4 Equilibration of temperature
Prior to testing, equilibrate all reagents (product test solutions (5.4.2), test suspension (5.4.1.4),
validation suspension (5.4.1.5), diluent (5.2.2.4), hard water (5.2.2.6) and interfering
substance (5.2.2.7) to the test temperature [5.5.1.1 a)] using the water bath (5.3.2.2) controlled at θ.
Check that the temperature of the reagents is stabilized at θ.
The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.8) and water (5.2.2.2) shall be equilibrated at a
temperature of (20 ± 1) °C.
In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to θ.
5.5.1.5 Precautions for manipulation of test organisms
Do not touch the upper part of the test tube sides when adding the test or the validation
suspensions (5.4.1).
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 5.5.2.6) shall be carried out in
parallel and separately for each experimental condition.
NOTE For a graphical representation of this method, see Annex C.
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prEN 14204:2024 (E)
5.5.2.2 Dilution-neutralization method Test Na – Determination of mycobactericidal
concentration
The procedure for determining mycobactericidal concentration is as follows:
a) Pipette 1,0 ml of the interfering substance (5.2.2.8) into a tube. Add 1,0 ml of the test
suspension (5.4.1.4). Start the stopwatch (5.3.2.5) immediately, mix [5.3.2.6 a)], and place the tube
in a water bath controlled at the chosen test temperature θ [5.5.1.1 a)] for 2 min ± 10 s.
At the end of this time, add 8,0 ml of one of the product test solutions (5.4.2). Restart the stopwatch
at the beginning of the addition. Mix [5.3.2.6 a)] and place the tube in a water bath controlled at θ
for the chosen contact time t [5.5.1.1 b)]. Just before the end of t, mix [5.3.2.6 a)] again.
b) At the end of t, take a 1,0 ml of the test mixture Na and transfer into a tube containing 8,0 ml
neutralizer (5.2.2.5) and 1,0 ml water (5.2.2.2). Mix [5.3.2.6 a)] and place in a water bath controlled
at (20 ± 1) °C. After a neutralization time of 5 min ± 10 s (in case of contact times of 10 min or
shorter, after a neutralization time of 5 min ± 1 s), immediately take a sample of 1,0 ml of the
neutralized mixture “Na” (containing neutralizer, product test solution, interfering substance and
test suspension) and transfer to a tube containing 9,0 ml diluent (5.2.2.4). Take a sample of 1,0 ml
of the neutralized and ten-fold diluted test mixture Na (containing neutralizer, product test
solution, interfering substance and test suspension) in duplicate. Divide each sample in two
portions of approximately equal size and inoculate 7H10 plates (5.2.2.3) using the spread plate
technique.
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 7H10
For incubation and counting see 5.5.2.6.
c) Perform the procedures a) and b) using the other product test solutions at the same time.
d) Perform the procedures a) to c) applying the other obligatory and – if appropriate – other
additional experimental conditions (5.5.1.1).
5.5.2.3 Experimental conditions control “A” – Validation of the selected experimental conditions
and/or verification of the absence of any lethal effect in the test conditions
To validate the selected experimental conditions and/or verify the absence of any lethal effect in the
test conditions, the procedure is as follows.
a) Pipette 1,0 ml of the interfering substance used in the test (5.5.2.2) into a tube. Add 1,0 ml of the
validation suspension (5.4.1.5). Start the stopwatch immediately, mix [5.3.2.6 a)] and place the tube
in a water bath controlled at θ for 2 min ± 10 s.
At the end of this time, add 8,0 ml of hard water (5.2.2.7). [In the case of ready-to-use products:
water (5.2.2.2) instead of hard water]. Restart the stopwatch at the beginning of the addition.
Mix [5.3.2.6 a)] and place the tube in a water bath controlled at θ for t. Just before the end of t,
mix [5.3.2.6 a)] again.
b) At the end of t, take a sample of 1,0 ml of this mixture A in duplicate divide each sample in two
portions of approximately equal size and inoculate 7H10 plates (5.2.2.3) using the spread plate
technique.
For incubation and counting, see 5.5.2.6.
oSIST prEN 14204:2024
prEN 14204:2024 (E)
5.5.2.4 Neutralizer control B – Verification of the absence of toxicity of the neutralizer
To verify the absence of toxicity of the neutralizer, the procedure is as follows.
a) Pipette 8,0 ml of the neutralizer – used in the test (5.5.2.2) — and 1,0 ml of water (5.2.2.2) into a
tube. Add 1,0 ml of the validation suspension (5.4.1.5). Start the stopwatch at the beginning of the
addition, mix [5.3.2.6 a)], and place the tube in a water bath controlled at (20 ± 1) °C
for 5 min ± 10 s. Just before the end of this time, mix [5.3.2.6 a)].
b) At the end of this time, take a sample of 1,0 ml of this mixture B in duplicate divide each sample in
two portions of approximately equal size and inoculate 7H10 plates (5.2.2.3) using th
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