SIST EN 1276:2019

SLOVENSKI STANDARD
01-november-2019
Nadomešča:
SIST EN 1276:2010
SIST EN 1276:2010/AC:2010
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v živilski in
drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in
zahteve (faza 2, stopnja 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial,
domestic and institutional areas - Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in
den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen -
Prüfverfahren und Anforderungen (Phase 2, Stufe 1); Deutsche und Englische Fassung
prEN 1276:2016
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
lévaluation de lactivité bactéricide des antiseptiques et des désinfectants chimiques
utilisés dans le domaine de lagro-alimentaire, dans lindustrie, dans les domaines
domestiques et en collectivité - Méthode dessai et prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 1276:2019
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 1276
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2019
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 1276:2009
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of bactericidal activity of
chemical disinfectants and antiseptics used in food,
industrial, domestic and institutional areas - 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
bactéricide des antiseptiques et des désinfectants bakteriziden Wirkung chemischer Desinfektionsmittel
chimiques utilisés dans le domaine de l'agro- und Antiseptika in den Bereichen Lebensmittel,
alimentaire, dans l'industrie, dans les domaines Industrie, Haushalt und öffentliche Einrichtungen -
domestiques et en collectivité - Méthode d'essai et Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
prescriptions (phase 2, étape 1)
This European Standard was approved by CEN on 17 June 2019.

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, Turkey 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 1276:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 6
3 Terms and definitions . 6
4 Requirements . 7
5 Test method . 8
5.1 Principle . 8
5.2 Materials and reagents . 9
5.3 Apparatus and glassware . 12
5.4 Preparation of test organism suspensions and product test solutions . 13
5.5 Procedure for assessing the bactericidal activity of the product . 15
5.6 Experimental data and calculation . 21
5.7 Verification of methodology . 24
5.8 Expression of results and precision . 25
5.9 Interpretation of results - conclusion . 25
5.10 Test report . 26
Annex A (informative) Referenced strains in national collections . 28
Annex B (informative) Neutralizers and rinsing liquids. 29
Annex C (informative) Graphical representations of dilution neutralization method and
membrane filtration method . 31
Annex D (informative) Example of a typical test report . 35
Annex E (informative) Precision of the test result . 39

European foreword
This document (EN 1276:2019) 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 February 2020 and conflicting national standards shall
be withdrawn at the latest by February 2020.
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 1276:2009.
Data obtained by using the latest version of EN 1276 are still valid.
The main changes in relation to EN 1276:2009 are:
— handrub and handwash test conditions and test requirements have been harmonized with
EN 13727;
— interfering substance for breweries, soft drinks, cosmetics and cleaning in place have been deleted.
A sentence to allow additional interfering substance for specific applications has been added;
— the obligatory conditions (temperature and contact time) have been deleted. The text has been
harmonized with EN 13727 keeping specified time intervals and temperature steps;
— test conditions for temperatures ≥ 40 °C have been added.
According to the CEN-CENELEC Internal Regulations, the national standards organizations 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, Turkey and the
United Kingdom.
Introduction
This document describes a suspension test for establishing whether a chemical disinfectant or
antiseptic has or does not have bactericidal activity in the fields described in the scope.
This laboratory test takes into account practical conditions of application of the product, including
contact time, temperature, test organisms and interfering substance, i.e. conditions which may influence
its action in practical situations.
The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test
corresponds to defined test conditions. However, for some applications, the recommendations of use of
a product can differ and therefore additional test conditions need to 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. Products can only
be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test
organisms and interfering substance.
This document applies to products that are used in food, industrial, domestic and institutional areas
excluding areas and situations where disinfection is medically indicated and excluding products used
on living tissues except those for hand hygiene in the above considered areas. The following areas are at
least included:
a) processing, distribution and retailing of:
1) food of animal origin:
— milk and milk products;
— meat and meat products;
— fish, seafood, and related products;
— eggs and egg products;
— animal feeds;
— etc.
2) food of vegetable origin:
— beverages;
— fruits, vegetables and derivatives (including sugar, distillery, etc.);
— flour, milling and baking;
— animal feeds;
— etc.
b) institutional and domestic areas:
— catering establishments;
— public areas;
— public transports;
— schools;
— nurseries;
— shops;
— sports rooms;
— waste containers (bins, etc.);
— hotels;
— dwellings;
— clinically non sensitive areas of hospitals;
— offices;
— etc.
c) other industrial areas:
— packaging material;
— biotechnology (yeast, proteins, enzymes, etc.);
— pharmaceutical;
— cosmetics and toiletries;
— textiles;
— space industry, computer industry;
— etc.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
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 14885:2018, 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 14885:2018 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Requirements
The product shall demonstrate at least a 5 decimal logarithm (lg) reduction (3 lg for handwashes) 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 Clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution-
5.2.2.8.2) or simulated dirty conditions (3 g/l bovine albumin solution - 5.2.2.8.3) according to its
practical applications and under the suitable test conditions as described in 5.5.1.1, Tables 1 and 2 here
below.
Table 1 — Test conditions for general purpose disinfection
Test Conditions Bactericidal activity
Enterococcus hirae
Escherichia coli
Test organism
(see 5.2.1)
Pseudomonas aeruginosa
obligatory
Staphylococcus aureus
E. faecium (for temperatures ≥ 40 °C)
Example of Salmonella Typhimurium
additional test Lactobacillus brevis
microorganisms Enterobacter cloacae
Test temperature
in a range from 4 °C to 60 °C

Contact time
in a range from 1 min to 60 min (from 1 min to 5 min at intervals of 1
min and from 5 min to 60 min at intervals of 5 min)

0,3 g/l Bovine Albumin for Staphylococcus aureus, Enterococcus hirae,
Escherichia coli and
Clean conditions
Pseudomonas aeruginosa
3,0 g/l Bovine Albumin for Staphylococcus aureus, Enterococcus hirae,
Dirty conditions
Pseudomonas aeruginosa and Escherichia coli
additional any relevant substance
Log reduction (decimal lg) ≥ 5 lg
The recommended contact time for the use of the product is within the responsibility of the
manufacturer.
Table 2 — Test conditions for hand hygiene
Test Conditions Bactericidal activity
Enterococcus hirae
Test organisms
Escherichia coli K12 (NCTC 10538)
(see 5.2.1)
Pseudomonas aeruginosa
obligatory
Staphylococcus aureus
Test temperature
20 °C
Contact time
30 s or 60 s
clean conditions
0,3 g/l Bovine Albumin
(for hygienic handrubs)
Dirty conditions
3,0 g/l Bovine Albumin
(for hygienic handwashes)
≥ 5 lg for handrubs
Log reduction (decimal lg)
≥ 3 lg for handwashes
Where indicated, additional specific bactericidal activity shall be determined applying other interfering
substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in order to take into
account intended specific use conditions.
5 Test method
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use
products with the exception of handwash products whose first dilution is done in hard water (5.4.2)) is
added to a test suspension of bacteria in a solution of an interfering substance. The mixture is
maintained at the chosen test temperature for the adopted contact time. At the end of this contact time,
an aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately
neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a
suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving bacteria in
each sample are determined and the reduction is calculated.
5.1.2 The test is performed using Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus
and Enterococcus hirae as test organisms. For temperatures ≥ 40 °C only Enterococcus faecium shall be
used.
For testing of hand hygiene products, Pseudomonas aeruginosa, Escherichia coli K12, Staphylococcus
aureus and Enterococcus hirae are used as test organisms.
5.1.3 Additional test organisms can be used.
5.2 Materials and reagents
5.2.1 Test organisms
The bactericidal activity shall be evaluated using the following strains as test organisms:
— Pseudomonas aeruginosa ATCC 15442;

— Escherichia coli ATCC 10536;

— Staphylococcus aureus ATCC 6538;

— Enterococcus hirae ATCC 10541;

— Escherichia coli K12 NCTC 10538;

— Enterococcus faecium ATCC 6057.
If required for specific applications, additional strains may be chosen, for example from:
— Salmonella Typhimurium ATCC 13311;

— Lactobacillus brevis DSM 6235;

— Enterobacter cloacae DSM 6234.
Refer to Annex A for strain references in some other culture collections.
The required temperature for growing these test organisms 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 for growing microorganisms
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 European Standard refer to the anhydrous salts.
Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for
consequent molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
To improve reproducibility, it is recommended that commercially available dehydrated material is used
for the preparation of culture media. The manufacturer's instructions relating to the preparation of
these products are to be rigorously followed.
NOTE For each culture medium and reagent, a limitation for use is to be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave (see 5.3.2.1 a).
NOTE 1 Sterilization is not necessary if the water is used, e.g. for preparation of culture media and
subsequently sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections can be used.
See 5.2.2.7 for the procedure to prepare hard water.
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.
NOTE In the case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it can be necessary to
add neutralizer to the TSA. Annex B gives guidance on the neutralizers that can be used.
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride (NaCl) 8,5 g
Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave (5.3.2.1 a). After sterilization, the pH of the diluent shall be equivalent to
7,0 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and
5.5.2. It shall be sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is
given in Annex B.
5.2.2.6 Rinsing liquid (for membrane filtration)
The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and
5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter
membrane under the test conditions described in 5.5.3.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is
given in Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 000 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 - 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.
When preparing the product test solutions (5.4.2), the addition of the product to the hard water
produces a different final water hardness in each test tube. In any case, the final hardness is lower than
375 mg/l of calcium carbonate (CaCO3) in the test tube.
5.2.2.8 Interfering substance
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids and detergents) shall be defined.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water
(5.2.2.2).
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) is 0,3 g/l.
5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water
(5.2.2.2).
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l.
5.2.2.8.4 Milk (dairies, etc.)
Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder
per litre of water (5.2.2.2), shall be prepared as follows:
— prepare a solution of 10,0 % (v/v) in water (5.2.2.2) by adding 10 parts of reconstituted milk to 90
parts of water. Heat for 30 min at (105 ± 3) °C [or 5 min at (121 ± 3) °C].
The final concentration of reconstituted milk in the test procedure (5.5) is 1,0 % (v/v) of reconstituted
milk.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave (5.3.2.1 a);
b) by dry heat, in the hot air oven (5.3.2.1 b).
1)
5.3.2 Usual microbiological laboratory equipment and, in particular, the following
5.3.2.1 Apparatus for sterilization:
+3
a) for moist heat sterilization, an autoclave capable of being maintained at for a minimum
()121 °C
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 for a
()160 °C
0 0
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted
TSA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1).
5.3.2.3 Incubator, capable of being controlled 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.
NOTE A puncture electrode or a flat membrane electrode are to be used for measuring the pH of the agar
media (5.2.2.3).
1) Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.2.5 Stopwatch
5.3.2.6 Shaker
® 2)
a) Electromechanical agitator, e.g. Vortex mixer .
b) Mechanical shaker
5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered.
The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters
of diameter 47 mm to 50 mm and 0,45 μm pore size for sterilization of hard water (5.2.2.7), bovine
albumin (5.2.2.8.2 and 5.2.2.8.3), and if the membrane filtration method is used (5.5.3).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the microorganisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml, or calibrated
automatic pipettes.
5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads, 3 mm to 4 mm in diameter.
5.3.2.12 Volumetric flasks
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
5.4.1.1 General
For each test organism, two different suspensions shall be prepared: the “test suspension” to perform
the test and the “validation suspension” to perform the controls and method validation.
5.4.1.2 Preservation and stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.
5.4.1.3 Working culture of test organisms
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 slopes (5.2.2.3) or plates (5.3.2.10) 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.
®
2) Vortex 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 an endorsement by CEN of this product.
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 test organisms 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 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 another tube.
8 3) 8
Adjust the number of cells in the suspension to (1,5 x 10 ) cfu/ml to (5 x 10 ) cfu/ml using diluent
(5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the
water bath at the test temperature θ ± 1 °C (5.5.1.1) 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.
−6 −7
b) For counting, prepare 10 and 10 dilutions of the test suspension using diluent (5.2.2.4). Mix
(5.3.2.6 a). Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate
or the spread plate technique.
1) When using the pour plate technique, transfer 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 Validation suspension (“Nv”)
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 the bacterial count of (3,0 x 10 ) cfu/ml to (1,6 × 10 ) cfu/ml [about one fourth (1 + 3) of
−5
the 10 dilution].
−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 and inoculate using the pour plate or the spread plate technique (5.4.1.4).
For incubation and counting, see 5.4.1.6.
5.4.1.6 Incubation and counting of the test and the validation suspensions
For incubation and counting of the test and validation suspension, the procedure is as follows:

3) cfu/ml = colony forming unit(s) per millilitre.
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 to 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) Note for each plate the exact number of colonies but record “> 330” for any counts higher than 330
and determine the Vc values according to 5.6.2.2.
c) Calculate the numbers of cfu/ml in the test suspension “N” and in the validation suspension “Nv”
using the methods given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7.
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration because
it is diluted to 80 % during the test and the method validation (5.5.2 or 5.5.3). Product test solutions
shall be prepared in hard water (5.2.2.7) at a minimum of three different concentrations to include one
concentration in the active range and one concentration in the non-active range (5.8.2). The product
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).
Handwash products shall be tested at 50 % concentration as highest concentration (1:1 dilution, in
order to simulate real use conditions) and therefore shall be pre-diluted in hard water (5.2.2.7) at
62,5 % concentration. Such a product is nevertheless regarded as a ready-to-use product and the
subsequent dilutions shall be performed with water (5.2.2.2).
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in
a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (lower concentrations)
shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water (5.2.2.7) on a
volume/volume basis using volumetric flasks (5.3.2.12).
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous preparation that is stable during the whole procedure. If during the procedure
a visible inhomogeneity appears due to the formation of a precipitate or 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 bactericidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions
For general purpose disinfection and hand hygiene, the experimental conditions in Table 1 and Table 2
apply:
The recommended contact time for the use of the product is within the responsibility of the
manufacturer.
For contact times equal or below 1 min, 5 s tolerance shall apply. For longer contact times, 10 s
tolerance shall apply.
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.
If both neutralizers are found to be invalid, the membrane filtration method (5.5.3) may be used.
NOTE In special circumstances, it can be necessary to add neutralizer to TSA (5.2.2.3).
5.5.1.3 General instructions 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 (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.
In the case of ready-to-use-products, use water (5.2.2.2) instead of hard water.
If because of problems with neutralization, a neutralizer has been added to TSA (5.5.1.2) used for the
validation and control procedures, the TSA 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 (5.2.2.7) and interfering substance
(5.2.2.8)) to the test temperature (θ ± 1) °C (5.5.1.1) using the water bath (5.3.2.2) controlled at
(θ ± 1)°C.
Check that the temperature of the reagents is stabilized at (θ ± 1) °C.
The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at
(20 ± 1) °C.
In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to (20± 1) °C.
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).
4)
5.5.2 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 in
parallel and separately for each experimental condition (5.5.1.1).

4) For a graphical representation of this method, see Annex C, C.1.
5.5.2.2 Test “Na” – determination of bactericidal concentrations
The procedure for determining bactericidal concentrations 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 ± 1°C (5.5.1.1) 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). Just before the end of t, mix (5.3.2.6 a) again.
b) At the end of t, take a 1,0 ml sample 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 (5.3.2.6a)) and place in a water bath
controlled at the chosen temperature. After a neutralization time of 5 min ± 10 s (in case of contact
times of 10 min or shorter only (10 ± 1)s), mix and immediately take a sample of 1,0 ml of the
neutralized test mixture “Na” (containing neutralizer, product test solution, interfering substance
and test suspension) in duplicate and inoculate using the 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.
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 chosen experimental conditions (5.5.1.1).
For handwashes, two additional decimal dilution shall be plated.
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 the chosen temperature (θ ± 1) °C 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 the chosen temperature (θ ± 1) °C 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 and inoculate using the pour
plate or the spread plate technique (5.5.2.2 b)).
For incubation and counting, see 5.5.2.6.
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 the chosen temperature
(θ ± 1) °C for 5 min ± 10 s (10 s ± 1 s for contact times equal to or shorter than 10 min). 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 and inoculate using
the pour plate or the spread plate technique (5.5.2.2 b)).
For incubation and counting, see 5.5.2.6.
5.5.2.5 Method validation “C” – dilution-neutralization validation
To validate the dilution neutralization method, 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
diluent (5.2.2.4) and then, starting a stopwatch, add 8,0 ml of the product test solution only of the
highest concentration used in the test (5.5.2.2). Mix (5.3.2.6 a)) and place the tube in a water bath
controlled at the chosen temperature (θ ± 1) °C  for t. Just before the end of t, mix (5.3.2.6 a))
again.
b) At the end of t transfer 1,0 ml of the mixture into a tube containing 8,0 ml of neutralizer (used in
5.5.2.2). Restart the stopwatch immediately at the beginning of the addition. Mix (5.3.2.6 a)) and
place the tube in a water bath controlled at the chosen temperature (θ ± 1) °C for 5 min ± 10 s
((10 ± 1) s for contact times equal or shorter than 10 min). Add 1,0 ml of the validation suspension
(5.4.1.5). Start a stopwatch at the beginning of the addition and mix (5.3.2.6 a)). Place the tube in a
water bath controlled at (20 ± 1) °C for (30 ± 1) min. Just before the end of this time, mix (5.3.2.6
a)) again. At the end of this time, take a sample of 1,0 ml of the mixture “C” in duplicate and
inoculate using the pour plate or the spread plate technique (5.5.2.2 b)).
For
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