EN 13727-2012 Plus A2-2015

BS EN 13727:2012+A1:2013
13727:2012+A2:2015
BSI Standards Publication
Chemical disinfectants and

antiseptics — Quantitative
suspension test for the
evaluation of bactericidal
activity in the medical area
— Test method and
requirements (phase 2, step 1)
BS EN 13727:2012+A2:2015 BRITISH STANDARD
National foreword
This British Standard is the UK implementation of
EN 13727:2012+A2:2015. It supersedes BS EN 13727:2012+A1:2013 which
is withdrawn.
The start and finish of text introduced or altered by amendment is
indicated in the text by tags. Tags indicating changes to CEN text carry
the number of the CEN amendment. For example, text altered by CEN
amendment A1 is indicated by .
The UK participation in its preparation was entrusted to Technical
Committee CH/216, Chemical disinfectants and antiseptics.
A list of organizations represented on this committee can be obtained
on request to its secretary.
This publication does not purport to include all the necessary provisions
of a contract. Users are responsible for its correct application.
© The British Standards Institution 2015.
Published by BSI Standards Limited 2015
ISBN 978 0 580 89232 5
ICS 11.080.20
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the Standards
Policy and Strategy Committee on 31 October 2012.
Amendments/corrigenda issued since publication
Date Text affected
28 February 2014 Implementation of CEN amendment A1:2013
30 November 2015 Implementation of CEN amendment A2:2015
EUROPEAN STANDARD EN 13727:2012+A2
NORME EUROPÉENNE
EUROPÄISCHE NORM October 2015
ICS 11.080.20 Supersedes EN 13727:2012+A1:2013
English Version
Chemical disinfectants and antiseptics - Quantitative

suspension test for the evaluation of bactericidal activity in
the medical 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
bactéricide en médecine - Méthode d'essai et bakteriziden Wirkung im humanmedizinischen Bereich
prescriptions (Phase 2, Étape 1) - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
This European Standard was approved by CEN on 14 October 2013 and includes Amendment 2 approved by CEN on 3 August
2015.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13727:2012+A2:2015 E
worldwide for CEN national Members.
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Contents Page
European foreword....................................................................................................................................................... 4
Introduction .................................................................................................................................................................... 5
1 Scope .................................................................................................................................................................... 6
2 Normative references .................................................................................................................................... 6
3 Terms and definitions ................................................................................................................................... 6
4 Requirements ................................................................................................................................................... 6
5 Test method ...................................................................................................................................................... 8
5.1 Principle ............................................................................................................................................................. 8
5.2 Materials and reagents.................................................................................................................................. 8
5.2.1 Test organisms ................................................................................................................................................. 8
5.2.2 Culture media and reagents ........................................................................................................................ 9
5.3 Apparatus and glassware .......................................................................................................................... 11
5.3.1 General ............................................................................................................................................................. 11
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 Dilution-neutralization method.............................................................................................................. 17
5.5.3 Membrane filtration method ................................................................................................................... 19
5.5.4 Modified method for ready-to-use products ...................................................................................... 21
5.6 Experimental data and calculation ........................................................................................................ 23
5.6.1 Explanation of terms and abbreviations ............................................................................................. 23
5.6.2 Calculation ...................................................................................................................................................... 23
5.7 Verification of methodology ..................................................................................................................... 28
5.7.1 General ............................................................................................................................................................. 28
5.7.2 Control of weighted mean counts ........................................................................................................... 28
5.7.3 Basic limits ..................................................................................................................................................... 29
5.8 Expression of results and precision ...................................................................................................... 29
5.8.1 Reduction ........................................................................................................................................................ 29
5.8.2 Control of active and non-active product test solution (5.4.2) ..................................................... 29
5.8.3 Limiting test organism and bactericidal concentration................................................................. 30
5.8.4 Precision, repetitions ................................................................................................................................. 30
5.9 Interpretation of results - conclusion ................................................................................................... 30
5.9.1 General ............................................................................................................................................................. 30
5.9.2 Bactericidal activity for handrub and handwash products .......................................................... 30
5.9.3 Bactericidal activity for instrument disinfection products .......................................................... 30
5.9.4 Bactericidal activity for surface disinfection products .................................................................. 31
5.9.5 Qualification for certain fields of application .................................................................................... 31
5.10 Test report ...................................................................................................................................................... 31
Annex A (informative) Referenced strains in national collections ........................................................... 33
Annex B (informative) Neutralizers and rinsing liquids ............................................................................... 34
2
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Annex C (informative) Graphical representation of test procedures ....................................................... 36

Annex D (informative) Example of a typical test report ................................................................................ 44
Annex E (informative) Precision of the test result........................................................................................... 48
Annex ZA (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC ........................................................................................... 51
Bibliography ................................................................................................................................................................. 52
3
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
European foreword
This document (EN 13727:2012+A2:2015) 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 April 2016, and conflicting national standards shall be
withdrawn at the latest by April 2016.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document includes Amendment 1 approved by CEN on 2013-10-14 and Amendment 2 approved
by CEN on 2015-08-03.
This document supersedes #EN 13727:2012+A1:2013$.
The start and finish of text introduced or altered by amendment is indicated in the text by tags !"
and #$.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this
document.
#deleted text$
!Data obtained using the former version of EN 13727 may still be used, if a neutralization time of 10 s
for all products with contact times of 10 min or shorter has been demonstrated to be sufficient. Data
obtained by using the prEN 12054 should not be used as this project was abandoned in 2001."
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
4
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant or
an antiseptic has a bactericidal activity in the area and fields described in the scope.
This laboratory test takes into account practical conditions of application of the product including
contact time, temperature, test organisms and interfering substances, i.e. conditions which may
influence its action in practical situations. Each utilization concentration of the chemical disinfectant or
antiseptic found by this test corresponds to the chosen experimental conditions.
5
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
1 Scope
This European Standard specifies a test method and the minimum requirements for bactericidal activity
of chemical disinfectant and antiseptic products that form a homogeneous, physically stable
preparation when diluted with hard water, or - in the case of ready-to-use products - with water.
Products can only be tested at a concentration of 80 % or less (97 % with a modified method for special
cases) as some dilution is always produced by adding the test organisms and interfering substance.
This European Standard applies to products that are used in the medical area in the fields of hygienic
handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by
immersion, and surface disinfection by wiping, spraying, flooding or other means.
This European Standard applies to areas and situations where disinfection or antisepsis is medically
indicated. Such indications occur in patient care, for example:
— in hospitals, in community medical facilities and in dental institutions;
— in clinics of schools, of kindergartens and of nursing homes;
and may occur in the workplace and in the home. It may also include services such as laundries and
kitchens supplying products directly for the patients.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active
substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test.
NOTE 3 This method cannot be used to evaluate the activity of products against Legionella in watersystems
against mycobacteria and against bacterial spores.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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.
4 Requirements
The product shall demonstrate at least a 5 decimal log (lg) reduction (for hygienic hand wash at least a
3 lg reduction), when tested in accordance with Table 1 and Clause 5.
6
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Table 1 — Minimum and additional test conditions
Test Hygienic handrub Surgical handrub Instrument Surface

Conditions and handwash and handwash disinfection disinfection
Minimum P. aeruginosa, P. aeruginosa, P. aeruginosa, P. aeruginosa,
spectrum of
S. aureus, S. aureus, S. aureus, S. aureus,
test organisms
E. hirae, E. hirae, E. hirae, E. hirae
E. coli K12 E. coli K12 when temperature is
40 °C or higher: only
E. faecium
additional Any relevant test organism
Test according to the manufacturer’s recommendation, but between
temperature
20 °C and 20 °C 20 °C and 20 °C 20 °C and 70 °C 4°C and 30 °C
Contact time according to the manufacturer’s recommendation
but between but no longer than
30 s and 60 s 1 min and 5 min 60 min 5 min or
60 min a
Interfering substance
clean 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine
conditions albumin solution albumin solution albumin solution albumin solution
(hygienic handrub) b (surgical handrub) b
and/or and/or
dirty 3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine
conditions albumin solution albumin solution albumin solution albumin solution plus
plus 3,0 ml/l plus 3,0 ml/l plus 3,0 ml/l 3,0 ml/l erythrocytes
erythrocytes erythrocytes erythrocytes
(hygienic handwash) (surgical handwash)
c c
additional clean or dirty; clean or dirty; any relevant any relevant

any relevant any relevant substance substance
substance substance
NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained
under the minimum test conditions.
aThe contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of
the product. The recommended contact time for the use of the product is within the responsibility of the
manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient and / or
the medical staff and surfaces, which are frequently touched by different people, leading to the transmission of
microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same applies where the
contact time of the product shall be limited for practical reasons. Products for other surfaces than stated above may
be tested with a contact time of maximum 60 min.
b hygienic and surgical handrub shall be tested as a minimum under clean conditions.
c hygienic and surgical handwash shall be tested as a minimum under dirty conditions.
7
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5 Test method
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use
products) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture
is maintained at one of the temperatures and the contact times specified in Clause 4 and 5.5.1.1. At the
end of this contact time, an aliquot is taken; the bactericidal and/or the bacteriostatic action 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.
NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded
as a ready-to-use product (5.4.2).
5.1.2 The test is performed using Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae
and for certain product types Escherichia coli K12 as test-organisms (Clause 4, Table 1).
5.1.3 Other contact times and temperatures within the limits specified in Clause 4, Table 1 may be
used. Additional interfering substances and test organisms may 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 selected
according to Clause 4 (Table 1) 1) :
a) Escherichia coli K12, NCTC 10538
b) Pseudomonas aeruginosa, ATCC 15442
c) Staphylococcus aureus, ATCC 6538
d) Enterococcus hirae, ATCC 10541
e) Enterococcus faecium, ATCC 6057
NOTE See Annex A for strain reference in some other culture collections.
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3).

The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test
and its control and validation.
If additional test organisms are used, they shall be incubated under optimum growth conditions
(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms
selected do not correspond to the specified strains, their suitability for supplying the required inocula
shall be verified. If these additional test organisms are not classified at a reference centre, their
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or
national culture collection under a reference for five years.
1) The NCTC and ATCC numbers are the collection numbers of strains supplied by these culture collections. This information
is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product
named.
8
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
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.
NOTE 1 To improve reproducibility, it is recommended that commercially available dehydrated material is
used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these
products should be rigorously followed.
NOTE 2 For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at 20 °C ± 1 °C.

5.2.2.2 Water
The water shall be freshly glass-distilled water 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 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 (5.3.2.4) of the medium shall be
equivalent to 7,2 ± 0,2.
NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add
neutralizer to TSA. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use a
neutralizer that causes opalescence in the agar.
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:

Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride 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 (5.3.2.4) of the diluent shall be
equivalent to 7,0 ± 0,2.
9
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and
5.5.2. It shall be sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is
given in Annex B.
5.2.2.6 Rinsing liquid (for membrane filtration)
The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and
5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter
membrane under the test conditions described in 5.5.3.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is
given in Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:

— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride
(CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the
autoclave [5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator
(5.3.2.8) for no longer than one month;
— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to
1000 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 (5.3.2.4) of the hard water shall be 7,0 ± 0,2. (5.3.2.4). If necessary, adjust the pH by using a
solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately
36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water
produces different final water hardness in each test tube. In any case, the final hardness expressed as calcium
carbonate (CaCO3) is lower than 375 mg/l 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 (50
times in the case of the modified method, 5.2.2.8.4).
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.
10
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent
(5.2.2.4).
Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month.
The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l ;
5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solution – high concentration with sheep
erythrocytes)
Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent
(5.2.2.4).
Sterilize by membrane filtration (5.3.2.7).
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 gN
for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4).
Repeat this procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see
above). To avoid later contamination this mixture should be split in portions probably needed per day
and kept in separate containers for a maximum of 7 days in a refrigerator (5.3.2.8).
The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be
3 g/l and 3 ml/l respectively.
5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4)
Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering
substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems
with the filtration.
a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent;
b) Dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml of diluent
(instead of 48,5 ml). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml
(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilized bovine albumin solution
to obtain 50 ml.
5.2.2.9 Defibrinated sheep blood
The defibrinated sheep blood should be sterile (aseptic blood-letting and preparation), pooled from
more than one sheep and can be acquired from a commercial supplier.
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)].
11
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.3.2 Usual microbiological laboratory equipment 2)
and, in particular, the following:

5.3.2.1 Apparatus for sterilization (moist and dry heat)
a) For moist heat sterilization, an autoclave capable of being maintained at ( 121 0+3 ) °C for a minimum
holding time of 15 min;
b) for dry heat sterilization, a hot air oven capable of being maintained at ( 180 0+5 ) °C for a minimum
+5
holding time of 30 min, at (170 0 ) °C for a minimum holding time of 1 h or at ( 160 0+5 ) °C for a
minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C, at 45 °C ± 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 °C ± 1 °C or 37 °C ± 1 °C (5.2.1). The same

temperature shall be used for incubations performed during a test and its control and validation.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.
NOTE A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar
media (5.2.2.3).
5.3.2.5 Stopwatch.
5.3.2.6 Shakers
a) Electromechanical agitator, e.g. Vortex® mixer 3);
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 (5.2.2.7), bovine albumin (5.2.2.8.2,
5.2.2.8.3 and 5.2.2.8.4), 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 micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 100 µl, 1 µl or calibrated automatic
pipettes.
5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
2) Disposable sterile equipment is an acceptable alternative to reusable glassware.

3) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users
of this European Standard and does not constitute an endorsement by CEN of this product.
12
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.3.2.11 Glass beads (Diameter 3 mm to 4 mm).
5.3.2.12 Volumetric flasks.
5.3.2.13 Centrifuge (800 gN).
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
5.4.1.1 General
For each test organism, two different suspensions have to be prepared: the “test suspension” to perform
the test and the “validation suspension” to perform the controls and method validation.
5.4.1.2 Preservation and stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.
5.4.1.3 Working culture of test organisms
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 subculture are the working cultures.
If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used
for subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3)
during the 48 h period.
Never produce and use a fourth subculture.
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 a tube.
b) Adjust the number of cells in the suspension to 1,5 x 108 cfu/ml 4) to 5,0 x 108 cfu/ml using diluent
(5.2.2.4) (1,5 x 109 cfu/ml to 5,0 x 109 cfu/ml in the case of the modified method – 5.5.4), estimating
the number of cfu by any suitable means. Maintain this test suspension in the water bath at 20 °C
and use within 2 h. Adjust the temperature according to 5.5.1.1 a) and 5.5.1.4 only immediately
before the start of the test.
NOTE 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 calibration data for
each test organism knowing that suitable values of optical density are generally found between 0,150 and 0,460.
To achieve reproducible results of this measurement it may be necessary to dilute the test suspension, e.g. 1+9. A
colorimeter is a suitable alternative.
4) cfu/ml = colony forming unit(s) per millilitre.
13
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
c) For counting, prepare 10-6and 10-7 dilutions (10-7 and 10-8 dilutions in the case of the modified
method – 5.5.4) 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 °C ± 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).
The technique used for counting of the test suspension has to be used for all other countings, 5.4.1.5 d),
5.5.2.2.c) and d), 5.5.2.3 b), 5.5.2.4 b) and 5.5.2.5 b).
For incubation and counting see 5.4.1.6.
5.4.1.5 Validation suspension (NV, NVB)
a) To prepare the validation suspension (NV), dilute the test suspension (5.4.1.4) with the diluent
(5.2.2.4) to obtain 3,0 x 102 cfu/ml to 1,6 x 103 cfu/ml [about one fourth (1+3) of the 10-5 dilution].
NOTE In the case of the modified method (5.5.4) the procedure is the same but only one fourth (1+3) of the
10-4 dilution is used resulting in 3,0 x 103 cfu/ml to 1,6 x104 cfu/ml.
b) To prepare the validation suspension for the neutralizer control NVB (5.5.2.4) (NVB) dilute the test
suspension (5.4.1.4) with the diluent (5.2.2.4) to obtain 3,0 x 104 cfu/ml to 1,6 x 105 cfu/ml [about
one fourth (1+3) of the 10-3 dilution] (NVB).
c) Maintain and use these validation suspensions (NV and NVB) the same way as the test suspension
[5.4.1.4 b)].
d) For counting prepare with diluent (5.2.2.4) a 10-1dilution, in the case of the modified method 10-2
dilution,] and in case of the neutralizer control NVB [see b)] a 10-3 dilution.
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 c)].
For incubation and counting see 5.4.1.6.

5.4.1.6 Incubation and counting of the test and the validation suspensions
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 plates and determine the 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.
14
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
c) Calculate the numbers of cfu/ml in the test suspension N and in the validation suspensions NV and
NVB (neutralizer control 5.5.2.4) 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 (= real
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 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 %. !Ready to use products may be tested at 97 % (see 5.5.4.)." In
this case, the “real test concentration” is 97 %.
Dilutions of ready-to-use products shall be prepared in water (5.2.2.2) instead of hard water. Handwash
products are always prediluted with hard water (5.2.2.7) to achieve a 62,5 % solution. This solution
simulates the addition of tap water in practice (1:1). Such a product is nevertheless regarded as a
“ready-to-use product”. The modified method (5.5.4) cannot be used, since 62,5 % represents the
highest accepted concentration (50 %), multiplied by 1,25.
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 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 homogenous preparation, stable during the whole procedure. If during the procedure a
visible inhomogeneity appears due to the formation of a precipitate or flocculate (for example, through
the addition of the interfering substance), it shall be recorded in the test report.
NOTE Counting micro-organisms embedded in a precipitate or flocculate 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
The 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):
15
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
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 or less where it is ± 5 s.
c) interfering substance:
The interfering substance to be tested is either 0,30 g/l bovine albumin (5.2.2.8.2) representing clean
conditions or a mixture of 3 ml/l sheep erythrocytes and 3,0 g/l bovine albumin (5.2.2.8.3) representing
dirty conditions – according to Clause 4, Table 1 and practical applications. Additional interfering
substances may be tested according to specific fields of application.
d) test organisms:
Escherichia coli K12, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae, Enterococcus
faecium 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
The method of choice is the dilution-neutralization method. 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 may 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, but observe the
exception with handwash products (5.1.1, NOTE).
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 of θ [5.5.1.1 a)] using the water bath (5.3.2.2) controlled at θ. Observe
the provisions laid down in 5.4.1.4 b). Check that the temperature of the reagents is stabilized at θ.
The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at a
temperature of 20 °C ± 1 °C.
In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to θ.
16
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
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 Dilution-neutralization method 5)
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
same time.
5.5.2.2 Test Na – determination of bactericidal concentrations
The procedure for determining bactericidal concentrations is as follows:

a) The procedure for determining bactericidal concentrations is as follows: 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.
b) 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.
c) 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 [5.3.2.6 a)] and place in a water bath controlled
at 20 °C ± 1 °C. After a neutralization time of 5 min ± 10 s (in case of contact times of 10 min or
shorter only 10 s ± 1 s), mix [5.3.2.6 a)] 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 °C ± 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).
d) #Additionally transfer 0,5 ml of this mixture into a tube containing 4,5 ml of neutralizer to obtain
10-1 dilution of Na, with hygienic handwash products mix and dilute additionally with neutralizer to
obtain a 10-2 dilution of Na. Take samples of 1,0 ml from each dilution tube in duplicate and
inoculate using the pour plate or spread plate technique.
In the case of hygienic handwash products only the 10-2 dilution of Na (and not the 100 [see 5.5.2.2
c)] and the 10-1 dilution) may be inoculated and incubated.
For incubation and counting, see 5.5.2.6.$
e) Perform the procedure a) to d) using the other product test solutions at the same time.
5) For a graphical representation of this method see Annex C, Figures C.1 and C.2
17
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
f) Perform the procedure a) to e) applying the other minimum 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.
b) 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
(except handwash products (5.4.2)): 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.
c) 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 c)].
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 9,0 ml of the neutralizer used in the test (5.5.2.2) into a tube. Add 1,0 ml of the validation
suspension (NVB) [5.4.1.5 b)] containing 3,0 x 104 cfu/ml to 1,6 x 105 cfu/ml. Start the stopwatch at
the beginning of the addition, mix [5.3.2.6 a)]. Transfer 0,5 ml of this mixture into a tube containing
4,5 ml of neutralizer to obtain 10-1 dilution of NVB, repeat this procedure to obtain 10-2 dilution of
NVB. Place the tubes of the 10-2 dilution of NVB in a water bath controlled at 20 °C ± 1 °C for
5 min ± 10 s (in case of contact times of 10 min or shorter only 10 s ± 1 s). Just before the end of
this time, mix [5.3.2.6 a)].
#NOTE The high amount of neutralizer in relation to the test organisms reflects the additional dilutions with
neutralizer – in the case of Na [5.5.2.2 d)] 10-1 and for hygienic handwash products10-1 and 10-2.$
b) At the end of this time, take a sample of 1,0 ml of this mixture B (10-2 dilution of NVB) in duplicate
and inoculate using the pour plate or the spread plate technique [5.5.2.2 c)].

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 θ 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 at the beginning of the addition. Mix [5.3.2.6 a)] and place the tube
in a water bath controlled at 20 °C ± 1 °C for 5 min ± 10 s (in case of contact times of 10 min or
shorter only 10 s ± 1 s). Add 1,0 ml of the validation suspension (5.4.1.5). Start a stopwatch at the
18
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
beginning of the addition and mix [5.3.2.6 a)]. Place the tube in a water bath controlled at
20 °C ± 1 °C for 30 min ± 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 c)].

5.5.2.6 Incubation and counting of the test mixture and the control and validation mixtures
For incubation and counting of the test mixture and the control and validation mixtures, the procedure
is as follows:
a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any
reason). Count the plates and determine the number of cfu. Incubate the plates for a further 20 h to
24 h. 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.
c) Calculate the numbers of cfu/ml in the test mixture Na and in the validation mixtures A, B and C
using the method given in 5.6.2.4 and 5.6.2.6. Verify according to 5.7.
5.5.3 Membrane filtration method 6)
5.5.3.1 General
The test and the control and validation procedures (5.5.3.2 through 5.5.3.5) shall be carried out at the
same time.
Each membrane filtration apparatus (5.3.2.7) is filled with 50 ml of the rinsing liquid (5.2.2.6). The time
required for filtering — if longer than one minute in exceptional cases — shall be recorded in the test
report. When transferring the membranes to the surface of an agar plate, care should be taken to ensure
that the test organisms are on the upper side of the membrane when placed on the plate, and to avoid
trapping air between the membrane and agar surface.
5.5.3.2 Test Na – determination of the bactericidal concentrations
#The procedure for determining the bactericidal concentrations is as follows:

a) See 5.5.2.2 a) and b)
b) At the end of t, take a sample of 0,1 ml of the test mixture Na (for hygienic hand wash products 1 μl)
in duplicate and transfer each 0,1 ml (1 μl) sample into a separate membrane filtration apparatus
(5.5.3.1). Filter immediately. Filter through at least 150 ml but no more than 500 ml of rinsing
liquid (5.2.2.6). If the rinsing liquid is not water, complete the procedure by filtering 50 ml of water
(5.2.2.2). Then transfer each of the membranes to the surface of separate TSA plates.
NOTE The amount of 1 μl takes into account the 100 fold dilution of Na [10-2 dilution in 5.5.2.2 d)] which
enables the measurement of a 3 lg reduction. Since it is not recommended to pipette microbial suspensions in
6) For a graphical representation of this method, see Annex C, Figures C.3 and C.4
19
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
1 μl portions, for example 1 ml in 1 000 ml rinsing liquid (or 100 μl in 100 ml) may be beforehand diluted and
1 ml of this mixture may be poured into the membrane filtration apparatus.$
5.5.3.3 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) See 5.5.2.3 a) and b);
b) At the end of t, take a sample of 1,0 ml of this mixture A in duplicate and transfer each 1,0 ml
sample into a separate membrane filtration apparatus (5.5.3.1). Filter immediately and additionally
with 50 ml of water (5.2.2.2). Then transfer each of the membranes to the surface of separate TSA
plates (5.2.2.3).

5.5.3.4 Filtration control B – validation of the filtration procedure
To validate the filtration procedure proceed as follows:

Take 0,1 ml of the validation suspension NV [5.4.1.5 a)] (Attention: not NVB as described in 5.5.2.4.) in
duplicate (suspension for control B) and transfer each 0,1 ml sample into a separate membrane
filtration apparatus (5.5.3.1).
Filter immediately. Filter through the rinsing liquid (5.2.2.6) the same way as in the test [5.5.3.2 b)]. If
the rinsing liquid is not water, complete the procedure by filtering 50 ml of water (5.2.2.2). Then
transfer each of the membranes to the surface of separate TSA plates (5.2.2.3).
5.5.3.5 Method validation C – validation of the membrane filtration method or counting of the bacteria
on the membranes which have previously been in contact with the mixture of product and interfering
substance
For validation of the membrane filtration method or counting of the bacteria on the membranes which
have previously been in contact with the mixture of product and interfering substance, the procedure is
as follows:
a) See 5.5.2.5 a);
b) At the end of t, take 0,1 ml of the mixture in duplicate and transfer each 0,1 ml sample into a
separate membrane filtration apparatus (5.5.3.1). Filter immediately. Filter through the rinsing
liquid (5.2.2.6) the same way as in the test [5.5.3.2 b)], then cover the membranes with 50 ml of the
rinsing liquid (5.2.2.6) and add 0,1 ml of the validation suspension NV [5.4.1.5 a)]. Filter
immediately again and additionally with 50 ml of water (5.2.2.2), then transfer each of the
membranes to the surface of separate TSA plates (5.2.2.3).

5.5.3.6 Incubation and counting of test mixture and the validation mixtures
For incubation and counting of the test mixture and the control and validation mixtures, the procedure
is as follows:
a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any
reason). Count the plates and determine the number of colony forming units. Incubate the plates for
20
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
a further 20 h to 24 h. 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.
c) Calculate the numbers of cfu/ml in the test mixture Na and in the validation mixtures A, B and C
using the method given in 5.6.2.4 and 5.6.2.6. Verify according to 5.7.
5.5.4 Modified method for ready-to-use products 7)
5.5.4.1 General
!Ready to use products may be tested according to the following modified test procedure, if the
product does not pass the procedure in 5.5.2 or 5.5.3." The test suspension N and the validation
suspension NV have to be prepared in tenfold higher numbers, as described in 5.4.1.4 and 5.4.1.5.
The interfering substance has to be prepared in fivefold higher concentrations as described in 5.2.2.8.4.
NOTE The concentration of the product in the modified method is 97 %.
5.5.4.2 Modified dilution-neutralization method
In the following only the modifications to 5.5.2 are described. See also Figures C.5 and C.6.
5.5.4.2.1 Test Na
Pipette 0,2 ml of the 5 fold concentrated interfering substance (5.2.2.8.4) into a tube. Add 0,1 ml of the
10 fold concentrated 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 [5.5.1.1 a)] for
2 min ± 10 s.
At the end of this time, add 9,7 ml of the undiluted product test solution (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. Follow the instructions in c) d), and where applicable, f).
5.5.4.2.2 Experimental conditions control A – validation of the selected experimental conditions
Pipette 0,2 ml of the interfering substance used in the test (5.5.4.2.1) into a tube. Add 0,1 ml of the 10
fold concentrated validation suspension [5.4.1.5 a)] as described for this modified method. 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 9,7 ml of water (5.2.2.2). Restart the stopwatch at the beginning of the
addition. Follow the instructions in 5.5.2.3 b) (last two sentences) and c).
5.5.4.2.3 Neutralizer control B – verification of the absence of toxicity of the neutralizer
Follow the procedure as described in 5.5.2.4.
7) For a graphical representation of this method, see Annex C, Figures C.5 to C.8
21
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.5.4.2.4 Method validation C – dilution-neutralization validation
a) Pipette 0,2 ml of the interfering substance used in the test (5.5.4.2.1) into a tube. Add 0,1 ml of the
diluent (5.2.2.4) and then, starting a stopwatch, add 9,7 ml of the product test solution. 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 transfer 1,1 ml of the mixture into a tube containing 8,8 ml of neutralizer (used in
5.5.4.2.1). 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 20 °C ± 1 °C for 5 min ± 10 s (in case of contact times of 10 min or
shorter only 10 s ± 1 s). Add 0,1 ml of the 10 fold concentrated validation suspension [5.4.1.5 a)] as
described for this modified method. 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 °C ± 1 °C for 30 min ± 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 c)].

5.5.4.3 Modified membrane filtration method
In the following only the modifications to 5.5.3 are described. See also Figures C.7 und C.8.
5.5.4.3.1 Test Na
See 5.5.4.2.1, but follow after the mixing at the end of t 5.5.2.2 b), c) and e).
5.5.4.3.2 Experimental conditions control A – validation of the selected experimental conditions
See 5.5.4.2.2 but do not follow 5.5.2.3 c), but 5.5.3.3 b).
5.5.4.3.3 Filtration control B – validation of the filtration procedure
Take 0,01 ml of the validation suspension NV [5.4.1.5 a)] (Attention: not NVB as described in 5.5.2.4.) in
duplicate (suspension for control B) and transfer each 0,01 ml sample into a separate membrane
filtration apparatus (5.5.3.1).
Follow 5.5.3.4.
5.5.4.3.4 Method validation C – validation of the membrane filtration method or counting of the
bacteria on the membranes which have previously been in contact with the mixture of product
and interfering substance
a) Follow 5.5.4.2.4 a).
b) At the end of t, take 0,01 ml of the mixture in duplicate and transfer each 0,01 ml sample into a
separate membrane filtration apparatus (5.5.3.1). Filter immediately. Filter through the rinsing
liquid (5.2.2.6) the same way as in the [5.5.4.3.1, i. e. 5.5.3.2 b)], then cover the membranes with
50 ml of the rinsing liquid (5.2.2.6) and add 0,01 ml of the 10 fold concentrated validation
suspension [5.4.1.5 a)]. Filter immediately again and additionally with 50 ml of water (5.2.2.2), then
transfer each of the membranes to the surface of separate TSA plates (5.2.2.3).
22
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.6 Experimental data and calculation
5.6.1 Explanation of terms and abbreviations
5.6.1.1 Overview of the different suspensions and test mixtures
N, NV and NVB, represent the bacterial suspensions, Na represents the bactericidal test mixture, A
(experimental conditions control), B (neutralizer or filtration control), C (method validation) represent
the different control test mixtures.
N, NV, NVB, N0, NV0, Na and A, B and C represent the number of cells counted per ml in the different test
mixtures in accordance with Table 2.
Table 2 — Number of cells counted per ml in the different test mixtures
Number of cells per Number of cells per Number of survivors per

ml in the bacterial ml in the test ml in the test mixtures at
suspensions mixtures at the the end of the contact time
beginning of the t (A) or 5 min (B) or 30
contact time (time 0 ) min (C)
Test N N0a (=N/10) Na (before neutralization or
Test suspension filtration)
Controls NV NV0a (=NV/10 A, B, C

Validation
suspension = NVB/1000)
NVB
Validation
suspension for
control B
a In the case of the modified method – 5.5.4 N0 is N /100 and NV0 is NV /100
5.6.1.2 VC-values
All experimental data are reported as VC-values:

a) in the dilution-neutralization method (test and controls), a VC-value is the number of cfu counted
per 1,0 ml sample;
b) in the membrane filtration method, a VC-value is the number of cfu counted per 0,1 ml sample of
test mixture Na, (1 μl in the case of hygienic handwash), of filtration control (B) and of method
validation (C) and per 1,0 ml sample in the experimental condition control A (for the modified
method 5.5.4.3 it is 0,1 ml of Na, 1,0 ml of A and 0,01 ml of B and C).
5.6.2 Calculation
5.6.2.1 General
The first step in the calculation is the determination of the VC-values, the second the calculation of N, N0,
Na, NV, NV0, NVB, A, B and C. The third step is the calculation of the reduction R (5.8).
23
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.6.2.2 Determination of VC-values
The VC-values are determined as follows.

a) The usual limits for counting bacteria on agar plates are between 15 and 300 colonies. In this
European Standard, a deviation of 10 % is accepted, so the limits are 14 and 330 colonies. On
membranes, the usual upper limits are different: 150, i.e. with the 10 % deviation: 165.
NOTE 1 The lower limit (14) is based on the fact that the variability increases the smaller the number counted
in the sample (1 ml or 0,1 ml) is and therefore subsequent calculations may lead to wrong results. The lower limit
refers only to the sample (and not necessarily to the counting on one plate), e.g. three plates per 1 ml sample with
3 cfu, 8 cfu and 5 cfu give a VC-value of 16. The upper limits (330, 165) reflect the imprecision of counting
confluent colonies and growth inhibition due to nutriment depletion. They refer only to the counting on one plate
and not necessarily to the sample.
b) #For counting the test suspension N (5.4.1.6), the validation suspensions NV and NVB (5.4.1.6)
and for all countings of the dilution-neutralization method (5.5.2.6), determine and record the VC-
values according to the number of plates used per 1 ml (or other volumes for membrane filtration
and/or hygienic handwash products) sample (5.6.1.2).$
NOTE 2 If more than one plate per 1 ml sample has been used to determine the VC-value, the countings per
plate should be noted.
If the count on one plate is higher than 330, report the number as “>330”. If more than one plate per
1 ml sample has been used and at least one of them shows a number higher than 330, report this VC-
value as “more than sum of the counts,” e.g. for “>330, 310, 302”, report “ > 942”.
If a VC-value is lower than 14, report the number (but substitute by “<14” for further calculations in the
case of Na).
For the membrane-filtration method (5.5.3), the countings on the membranes are the VC-values
(5.6.1.2). Report the VC-values below the lower limit (14) or above the upper limit (165) as described
above.
c) Only VC-values within the counting limits are taken into account for further calculation, except in
the case of Na (5.6.2.4).
5.6.2.3 Calculation of N and N0
N is the number of cells per ml in the test suspension (5.4.1.4; 5.6.1.1).

Since two dilutions of the test suspension (5.4.1.4 in connection with 5.4.1.6) are evaluated, calculate
the number of cfu/ml as the weighted mean count using the following formula:
c
N= (1)
( n1 + 0,1 n2 ) 10−6
where
c is the sum of VC-values taken into account;
n1 is the number of VC-values taken into account in the lower dilution, i.e. 10-6;
n2 is the number of VC-values taken into account in the higher dilution, i.e. 10-7;
10-6 is the dilution factor corresponding to the lower dilution.
!NOTE 1 For the modified method (5.5.4), the lower dilution is 10-7 and the higher dilution is 10-8."
24
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Round off the results calculated to two significant figures. For this, if the last figure is below 5, the
preceding figure is not modified; if the last figure is 5 or more than 5, the preceding figure is increased
by one unit. Proceed stepwise until two significant figures are obtained. As a result, the number of
cfu/ml is expressed by a number between 1,0 and 9,9 multiplied by the appropriate power of 10.
EXAMPLE
168 + 213 + 20 + 25 426

N= = =1,9363 × 108 =1,9 × 108 (cfu / ml )
( 2 + 0,1 × 2 ) 10 −6
2, 2 × 10 −6
NOTE!2" For the modified method (5.5.4) N is tenfold higher and therefore the dilutions to be evaluated
are tenfold higher (10-7 instead of 10-6, and 10-8 instead of 10-7). The formula given above has to be changed
accordingly.
N0 is the number of cells per ml in the test mixture [5.5.2.2 a)] at the beginning of the contact time (time
“zero” = 0). It is one-tenth of the weighted mean of N due to the tenfold dilution by the addition of the
product and interfering substance. It is one-hundredth in the case of the modified method (5.5.4) as
only 0,1 ml of N are used in the test.
5.6.2.4 Calculation of Na
Na is the number of survivors per ml in the test mixture [5.5.2.2 a) or 5.5.3.2 a)] at the end of the contact
time and before neutralization or membrane filtration. It is tenfold higher than the VC-values due to the
addition of neutralizer and water [5.5.2.2 b)] or the sample volume of 0,1 ml [5.5.3.2 b)] in the
membrane filtration method.
a) #Calculate the mean for each dilution step Na 0, Na -1 and for hygienic handwash products
additionally or only Na -2 [see also 5.5.2.2 d)] using the following formula:$
N a 0 , N a −1 , N a −2 = 10 c / n (2)
where
n is the number of VC-values taken into account.
If one or both of the duplicate VC-values are either below the lower or above the upper limit, express the
results as “less than” or “more than”.
#EXAMPLES
a1 duplicate VC-values Na -1: 2, 16
( < 14 + 16) × 10 1
−1
Na = × 10 = < 150 × 101 = < 1500 = < 1,5 × 103
2
a2 duplicate VC-values Na -2 (membrane filtration): >165, >165
( > 165 + > 165) × 10 2

Na
−2
= × 10 = > 1650 × 102 = > 165 000 = > 1,65 × 105$
2
a3 duplicate VC-values (two spread plates per 1,0 ml sample): > 660, 600
25
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Na0 =
( > 660 + 600 ) × 10 = > 6,3 × 103
> 6300 =
2
b) #For calculation of Na use only Na 0, Na -1, Na -2 results, where one or both VC-values are within
the counting limits. Exceptions and rules for special cases:
NOTE Although 10-2 dilutions are only prepared when hygienic handwash products are tested, the following
examples include this dilution step.
b1 If all subsequent dilutions of Na show mean values of „more than”, take only the highest dilution (10-
1, with hygienic handwash products 10-2) as result for N .$
a
EXAMPLE 1
VC1 VC2 mean x 10 Na = > 6600 x 102 = > 6,6 x 105

Na 0 > 660 > 660 > 6600
Na -1 > 660 > 660 > 6600
Na -2 > 660 > 660 > 6600
#b2 If all subsequent dilutions of Na show mean values of „less than”, take only the lowest dilution
(100) as result for Na.
EXAMPLE 2
VC1 VC2 mean x 10 Na = < 160 x 100 = < 1,6 x 102

Na 0 < 14 18 < 160
Na -1 < 14 < 14 < 140
Na -2 < 14 < 14 < 140
$
b3 If one or both duplicate VC-values in only one dilution of Na are within the counting limits, use this
result as Na.
EXAMPLE 3
VC1 VC2 mean x 10 Na = 1015 x 101 = 1,0 x 104

Na 0 > 660 > 660 > 6600
Na -1 96 107 1015
Na -2 < 14 < 14 < 140
b4 If the higher dilution in two subsequent dilutions of Na shows a mean value of „less than” and the
lower dilution shows a mean value of „more than”, take only the lower dilution as Na value.
EXAMPLE 4
VC1 VC2 mean x 10 Na = > 6600 x 100 = > 6,6 x 103

Na 0 > 660 > 660 > 6600
Na -1 < 14 29 < 215
26
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Na -2 < 14 < 14 < 140
c) Use maximum 2 subsequent dilutions for calculating Na as a weighted mean. Exceptions and rules
for special cases:
c1 If one or both duplicate VC-values in three or more subsequent dilutions of Na (including Na 0) are
within the counting limits (e.g. Na -2: 17, 23; Na -1: 120, 135; Na 0: 308, > 330) the whole test is invalid
(5.7.1).
c2 If two subsequent dilutions of Na show duplicate VC-values within the counting limits calculate Na as
the weighted mean using the Formula (3):
c × 10
Na = (3)
2, 2 × 10 Z
where
Z is the dilution factor corresponding to the lower dilution, e.g. Na 0 is the lower dilution in
comparison with Na -1.
c3 If in two subsequent dilutions of Na both VC-values of the higher dilution are within the counting
limits and one VC-value of the lower dilution is „more than”, calculate Na as the weighted mean, using
the Formula (3), see c2.
EXAMPLE 5
VC1 VC2 mean x 10

Na =
( > 538 + 320 + 46 + 57 ) × 10 = > 4, 4 × 103
> 4368,18 =
Na 0 > 538 320 > 4290 2, 2 × 100
Na -1 46 57 515
Na -2 < 14 < 14 < 140
c4 If in two subsequent dilutions of Na one of the higher dilution duplicate values shows„< 14”, take
only the lower dilution as result for Na.
EXAMPLE 6
VC1 VC2 mean x 10 Na = > 6145 x 101 = > 6,1 x 104

Na 0 > 660 > 660 > 6600
Na -1 569 > 660 > 6145
Na -2 < 14 26 < 200
5.6.2.5 Calculation of NV, NV0 and NVB
NV is the number of cells per ml in the validation suspension [5.4.1.5 a)]. It is tenfold higher than the
counts in terms of VC-values due to the dilution step of 10-1 [5.4.1.5 b)].
NV0 is the number of cells per ml in the mixtures A, B and C at the beginning of the contact time (time 0)
(5.6.1.1). In the case of neutralizer control B – dilution-neutralization method (5.5.2.4) it is the number
27
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
of cells per ml after 100 fold dilution. NV0 is one-tenth of the mean of the VC-values of NV [5.4.1.6 c)]
taken into account, in case of NVB it is one thousandth.
Calculate NV, NVB and NV0 using the following formulas:
Nv = 10 c / n (4)
NvB = 1000 c / n (5)
Nv0 = c / n (6)
where
5.6.2.6 Calculation of A, B and C
A, B and C are the numbers of survivors in the experimental conditions control A (5.5.2.3 or 5.5.3.3),
neutralizer control B (5.5.2.4) or filtration control (5.5.3.4) and method validation C (5.5.2.5 or 5.5.3.5)
at the end of the contact time t (A) or the defined times 5 min (B) and 30 min (C). They correspond to
the mean of the VC-values of the mixtures A, B and C taken into account.
Calculate A, B and C using the following formula:
A, B, C = c / n (7)
where
5.7 Verification of methodology
5.7.1 General
A test is valid if:

— all results meet the criteria of 5.7.3 and
— it is not invalidated by a result described under 5.6.2.4 c) first special case (c1).
5.7.2 Control of weighted mean counts
For results calculated by weighted mean of two subsequent dilutions (e.g. N), the quotient of the means
of the two results shall be not higher than 15 and not lower than 5. Results below the lower limit are
taken as the lower limit number (14). Results above the respective upper limit [5.6.2.2 b)] are taken as
the upper limit number.
EXAMPLE:
For N: 10-6 dilution: 168 + 215 cfu/ml, 10-7 dilution: 20 +< 14 cfu/ml; (168 + 215) / (20 + 14) = 383/34 = 11,26 =
between 5 and 15.
28
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
NOTE When series of plates for the same dilution (2 or 4) are out of limits fixed for the determination of VC-
values [5.6.2.2b)], the quotient of the mean of the two results is not calculated.
5.7.3 Basic limits
For each test organism check that:

a) N is between 1,5 x 108 and 5,0 x 108 (8,17 ≤ lg N ≤ 8,70)
N (modified method 5.5.4) is between 1,5 x 109 and 5,0 x 109 (9,17 ≤ lg N ≤ 9,70)
N0 is between 1,5 x 107 and 5,0 x 107 (7,17 ≤ lgN0 ≤ 7,70)
b) NV0 is between 30 and 160 (3,0 x 101 and 1,6 x 102)

NV is between 3,0 x 102 and 1,6 x 103
NV (modified method 5.5.4) is between 3,0 x 103 and 1,6 x 104
NVB (5.5.2.4) is between 3,0 x 104 and 1,6 x 105
c) A, B, C are equal to or greater than

0,5 x NV0
B (dil.-neutr.) is equal to or greater than
0,0005 x NVB (half of one
thousandth)
d) Control of weighted mean counts (5.7.2): quotient is not lower than 5 and not higher than 15.
NOTE In case of using the modified method for ready-to-use products (5.5.4) the limits for N and NV are 10
fold higher, but No, Nvo and NVB are unchanged.
5.8 Expression of results and precision
5.8.1 Reduction
The reduction (R = N0/ Na) is expressed in logarithm.

For each test organism record the number of cfu/ml in the test suspension N (5.6.2.3) and in the test Na
(5.6.2.4). Calculate NO (5.6.2.3).
For each product concentration and each experimental condition, calculate and record the decimal log
reduction (lg) separately using the formula:
lg R lg N 0 − lg N a
=
For the controls and validation of the dilution-neutralization method or membrane filtration method,
record NV0 (5.6.2.5), the results of A, B and C (5.6.2.6) and their comparison with NV0 [(5.7.3 c)].
5.8.2 Control of active and non-active product test solution (5.4.2)
At least one concentration per test [5.5.2.2 a) - c) or 5.5.3.2 a) - c) or 5.5.4] shall demonstrate a 5 lg or
more reduction (for !hygienic" handwash products 3 lg or more) and at least one concentration
shall demonstrate a lg reduction of less than 5 (for !hygienic" handwash products less than 3).
29
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.8.3 Limiting test organism and bactericidal concentration
#For each test organism, record the lowest concentration of the product which passes the test
(lg R ≥ 5 or for hygienic handwash products lg R ≥ 3). Record as the limiting test organism the test
organism requiring the highest of these concentrations (it is the least susceptible to the product in the
chosen experimental conditions).$
5.8.4 Precision, repetitions
Taking into account the precision of the methodology determined by a statistical analysis based on data
provided by a collaborative study, repetition of the test [for a precision of ± 1 lg in reduction: 4
repetitions in the best case, 6 repetitions in the worst case is recommended (Annex E)]. The number of
repetitions shall be decided according to the required level of precision, taking into account the
intended use of the test results.
#Repetition means the complete test procedure with test and validation suspensions prepared
specially for each test. The repetitions may be restricted to the limiting test organism. The mean (i.e.
before logarithmation) of the results of the repetitions - not each single result - shall demonstrate at
least a 5 lg reduction (in the case of hygienic handwash products 3 lg) and shall also be calculated and
recorded.$
5.9 Interpretation of results - conclusion
5.9.1 General
According to the chosen experimental conditions the bactericidal concentrations determined according
to this standard may differ (Clause 4). A product can only pass the test if the requirements of 5.8.2 are
fulfilled.
5.9.2 Bactericidal activity for handrub and handwash products
The product shall be deemed to have passed the EN 13727 standard if it demonstrates in a valid test for
handrub and handwash products at 20 °C under the conditions defined by this standard when the test
organisms are Escherichia coli K12, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus
hirae at least a:
a) 5 lg reduction within max. 1 min under clean conditions (hygienic handrub);
b) 5 lg reduction within max. 5 min under clean conditions (surgical handrub);
c) 3 lg reduction within max. 1 min under dirty conditions (hygienic handwash);
d) !5 lg" reduction within max. 5 min under dirty conditions (surgical handwash).
5.9.3 Bactericidal activity for instrument disinfection products
instrument disinfection products at least a 5 lg reduction within max. 60 min at the lowest temperature
recommended by the manufacturer, min. 20 °C and max. 70 °C, with the chosen interfering substance
(clean or dirty conditions) under the conditions defined by this standard when the test organisms are
Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus hirae (In the case of a temperature of
40 °C or higher only E. faecium is used as test organism).
30
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
5.9.4 Bactericidal activity for surface disinfection products
surface disinfection products at least a 5 lg reduction within max. 5 min (or between 6 and 60 min for
products used on surfaces which do not require an action within 5 min or shorter at) min 4 °C and max
30 °C with the chosen interfering substance (clean or dirty conditions) under the conditions defined by
this standard when the test organisms are Pseudomonas aeruginosa, Staphylococcus aureus and
Enterococcus hirae.
5.9.5 Qualification for certain fields of application
See EN 14885.
5.10 Test report
The test report shall refer to this European Standard (EN 13727).
The test report shall state, at least, the following information:
a) identification of the testing laboratory;
b) identification of the client;
c) identification of the sample:
1) name of the product;
2) batch number and — if available — expiry date;
3) manufacturer – if not known: supplier;
4) date of delivery;
5) storage conditions;
6) product diluent recommended by the manufacturer for use;
7) active substance(s) and its/their concentration(s) (optional);
8) appearance of the product;
d) test method and its validation:
1) if the dilution-neutralization method is used, full details of the test for validation of the
neutralizer shall be given;
2) if the membrane filtration method is used, full details of the procedure which was carried out in
order to justify the use of the membrane filtration method shall be given;
e) experimental conditions:
1) date(s) of test (period of analysis);
2) diluent used for product test solution (hard water or distilled water);
3) product test concentrations (= desired test concentrations according to 5.4.2);
31
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
4) appearance of the product dilutions;
5) contact time(s);
6) test temperature(s);
7) interfering substance;
8) stability and appearance of the mixture during the procedure (note the formation of any
precipitate or flocculant);
9) temperature of incubation ;
10) neutralizer or rinsing liquid ;
11) identification of the bacterial strains used;
f) test results:
1) controls and validation;
2) evaluation of bactericidal activity;
3) number of repetitions per test organism;
g) special remarks;
h) conclusion;
i) locality, date and identified signature.
NOTE An example of a typical test report is given in Annex D.
32
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Annex A
(informative)
Referenced strains in national collections
Escherichia coli K12 CIP 54.117

NCIMB 10083
CCUG 46621
NCTC 10538
Pseudomonas aeruginosa ATCC 15442
CIP 103467
DSM 939
NCIMB 10421
CCUG 2080
Staphylococcus aureus ATCC 6538
CIP 4.83
DSM 799
NCTC 10788
NCIMB 9518
CCUG 10778
Enterococcus hirae ATCC 10541
CIP 58.55
DSM 3320
NCIMB 8192
CCUG 32258
Enterococcus faecium ATCC 6057
DSM 2146
33
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Annex B
(informative)
Neutralizers and rinsing liquids
Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics
and of rinsing liquids.
IMPORTANT — Neutralizers of the residual antimicrobial activity of chemical disinfectants and
antiseptics and rinsing liquids shall be validated according to the prescriptions of the standard.
Table B.1 — Neutralizers and rinsing liquids
Chemical compounds able to Examples of suitable neutralizers

Antimicrobial agent neutralize residual and of rinsing liquids (for
antimicrobial activity membrane filtration methods) a
Quaternary ammonium Lecithin, Saponin, Polysorbate — Polysorbate 80, 30 g/l + saponin,
compounds and fatty 80, Sodium dodecyl sulphate, 30 g/l + lecithin, 3 g/l.
amines Ethylene oxide condensate of Polysorbate 80, 30 g/l + sodium
fatty alcohol (non-ionic dodecyl sulphate, 4 g/l + lecithin,
Amphoteric compounds
surfactants) b 3 g/l.
— Ethylene oxide condensate of fatty
alcohol, 3 g/l + lecithin, 20 g/l +
polysorbate 80, 5 g/l.
— Rinsing liquid : tryptone, 1 g/l +
NaCl, 9 g/l; polysorbate 80, 5 g/l.
Biguanides and similar Lecithinc, Saponin, Polysorbate — Polysorbate 80, 30 g/l + saponin,
compounds 80 30 g/l + lecithin, 3 g/l.
Rinsing liquid : tryptone, 1 g/l + NaCl, 9
g/l; polysorbate 80, 5 g/l.
Oxidizing compounds Sodium thiosulphate d — Sodium thiosulphate, 3 g/l to 20
(Chlorine, iodine, g/l + polysorbate 80, 30 g/l +
hydrogen peroxide, Catalase [for hydrogen peroxide lecithin, 3 g/l.
peracetic acid, or products releasing hydrogen
peroxide] — Polysorbate 80, 50 g/l + catalase
hypochlorites, etc…)
0,25 g/l + lecithin 10 g/l.
Rinsing liquid : sodium thiosulphate, 3
g/l.
Aldehydes L – histidine — Polysorbate 80, 30 g/l + lecithin,
3 g/l + L-histidine, 1 g/l (or +
Glycine
glycine, 1 g/l).
— Polysorbate 80, 30 g/l + saponin,
30 g/l + L-histidine, 1 g/l (or +
glycine, 1 g/l).
Rinsing liquid : polysorbate 80, 5 g/l +
L-histidine, 0,5 g/l (or + glycine, 1 g/l).
34
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Phenolic and related Lecithin — Polysorbate 80, 30 g/l + lecithin, 3

compounds: g/l.
Polysorbate 80
orthophenylphenol,
— Ethylene oxide condensate of fatty
phenoxyethanol, Ethylene oxide condensate of
alcohol, 7 g/l + lecithin, 20 g/l, +
triclosan, phenylethanol, fatty alcohol b polysorbate 80, 4 g/l.
etc.
Anilides
Alcohols Lecithin, Saponin, Polysorbate — Polysorbate 80, 30 g/l + saponin,
80 e 30 g/l + lecithin, 3 g/l.
a According to the pH of the tested product, the pH of the neutralizer or the rinsing liquid may be adjusted at a
suitable value or prepared in phosphate buffer [ex: phosphate buffer 0,25 mol/l: potassium dihydrogen
phosphate (KH2PO4) 34 g; distilled water (500 ml); adjusted to pH 7,2 ± 0,2 with sodium hydroxide (NaOH) 1
mol/l; distilled water up to 1 000 ml].
b The carbon chain-length varies from C12 to C18 carbon atoms.
c Egg and soya; egg is preferable.
d The toxic effect of sodium thiosulphate differs from one test organism to another.
e For the neutralization of short chain alcohols (less than C5), simple dilution may be appropriate. Care should
be taken if the alcohol-based -products contain additional antimicrobial agents.
NOTE 1 Other neutralizer mixtures may be required for products containing more than one antimicrobial
agent.
NOTE 2 The concentrations of the various neutralizing compounds or of the neutralizer as such may not be
adequate to neutralize high concentrations of the products.
35
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Annex C
(informative)
Graphical representation of test procedures
Figure C.1 — Dilution – neutralization method – Test procedure (Na)
36
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Figure C.2 — Dilution – neutralization method – Validation
37
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Figure C.3 — Membrane filtration method – Test procedure (Na)
38
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Figure C.4 — Membrane filtration method – Validation
39
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Figure C.5 — Dilution-neutralization method (modified method for ready-to-use products) – Test procedure (Na)
40
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Figure C.6 — Dilution-neutralization method (modified method for ready-to-use products) – Validation
41
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Figure C.7 — Membrane filtration method (modified method for ready-to-use products) – Test procedure (Na)
42
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Figure C.8 — Membrane filtration method (modified method for ready-to-use products) – Validation
43
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Annex D
(informative)
Example of a typical test report
NOTE 1 All names and examples in Annex D are fictitious apart from those used in this European Standard.
NOTE 2 Only the test results of one replicate for Pseudomonas aeruginosa is given as an example.
NOTE 3 The results of a hygienic handrub product tested according to the modified method 5.5.4 is given
without a complete test report as an additional example.
HHQ Laboratories
Antiseptville/Euroland
Tel. ++011.57 83 62-0
Fax ++011-57 83 62-19
e-mail: h.h.Q.lab@net.com
TEST REPORT
EN 13727, BACTERICIDAL ACTIVITY
(minimum and additional conditions)
Client: Cult Formulations Inc., Mannheim/Euroland

Disinfectant-sample
Name of the product: JN (instruments and surfaces)
Batch number: 10-10-76
Manufacturer or – if not known – supplier: MDI Formulations Inc. (manufacturer)
Storage conditions (temp. and other): Room temperature, darkness
Appearance of the product: Liquid, clear, yellowish
Active substance(s) and their concentration(s): Not indicated
Product diluent recommended by the manufacturer for use: Potable water
Period of testing
Date of delivery of the product: 2010-05-01 Dates of tests: see "Test results" (attached)
Experimental conditions
Product diluent: hard water; concentrations of the product tested: see "Test results" (attached)
test-organisms: Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538,
Enterococcus hirae ATCC 10541, Enterococcus faecium ATCC 6057
test temperature: 25 °C; contact time: 45 min; (+ 50 °C; 60 min)
interfering substance: 0,3 g/l bovine albumin = clean conditions; (+ 3,0 g/l bovine albumin plus 3,0
ml/l erythrocytes = dirty conditions)
44
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
incubation temperature: 36 °C
test results: see attached sheets
Special remarks regarding the results:
1. All controls and validation were within the basic limits
2. At least one concentration of the product demonstrated a lg reduction of less than 5 lg.
3. No precipitate during the test procedure (test mixtures were homogeneous).
Conclusion:
For the product JN (batch 10-10-76), the bactericidal concentration for instrument and surface
disinfection determined according to the EN 13727 standard under clean conditions at 25 °C within
45 min is:
1,0 % (v/v)
(the mean reduction of six repetitions with the limiting test organism Pseudomonas aeruginosa was
1,2 x 105. Staphylococcus aureus and Enterococcus hirae were tested once and showed a 5 lg reduction
or more at a lower concentration than Pseudomonas aeruginosa).
For the product JN (batch 10-10-76), the bactericidal concentration for specific instrument disinfection
determined according to the EN 13727 standard at 40 °C, with 60 min contact time under dirty
conditions, using Enterococcus faecium ATCC 6057 as test organism is:
0,50 % (v/v)
Antiseptville, 2010-11-11
Alexander May, MD, PhD, Scientific Director
45
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Test results (bactericidal quantitative suspension test)
EN....13727….(Phase 2, step 1)Product-name: ..JN..(Instruments and surfaces)...Batch No.: .. 10-10-76.

Remarks:
Dilution neutralization X Pour plate Spread plate x Number of plates .....2 / ml
method
Neutralizer:.....Lecithin 3,0 g/l in diluent.................................................................................................................
Membrane filtration method Rinsing liquid:........................................................................................................
Test temperature: 25 °C interfering substance:….bovine albumin: 0,3 g/l ………………………………..
Test organism: ....Pseudomonas aeruginosa ATCC 15442...........................Incubation temperature: 36°C
Internal lab. No :...QS Date of test:.2010-06-06 Responsible person: ...Fang....... Signature: Fang.
68/00 ....
Diluent used for product test solutions: ...water..Appearance of the product test solutions: ........clear.......
Validation and controls
Validation Experimental Neutralizer or filtration Method validation (C)

suspension (Nvo) conditions control (A) control (B) Product conc.: 10 ml/l
VC1 86 VC1 79 VC1 86 x= VC1 75
(40 + 46) x= (43 + 36) x= (42 + 44) 88,5 (35 + 40) x=
VC2 92 89 VC2 84 81,5 VC2 91 VC2 87 81
(47 + 45) (39 + 45) (43 + 48) (41 + 46)
30 ≤ x of Nvo ≤ 160 ? x of A is ≥ 0,5x x of Nvo ? x of B is ≥ 0,5x x of Nvo (or x of C is ≥ 0,5x x of Nvo?
yes no yes no NVB/1000) yes no yes no
Validation suspension (NVB) VC1 99 VC2 71 x= 85 30 ≤ x of NVB/1000 ≤ 160 ? yes no
(49 + 50) (39 + 32)
Test suspension and Test Test-suspension N VC1 VC2 x wm =193,64 x 106 ; lg N =8,29
(N and No): 10-6 168 213 No = N/10 ; lgNO = 7,29
10-7 20 25 7,17 ≤ lgNo ≤ 7,70? yes no
Real conc. of the Dilution VC1 VC2 Na (= x or lg Na lg R Contact

product % step x wm x 10) (lgNo = 7,29) time (min)
100 >660 >660
0,50 8100 3,91 3,38 45
10-1 77 85
100 122 154
0,75 1395,5 3,15 4,14 45
10-1 14 17
100 7 0
1,00 <140 <2,15 >5,14 45
10-1 0 0
Countings per plate for

N = 10-6: 80+88; 105+108 10-7: 9+11; 15+10
Na = 0,50% 10-1: 42+35; : 44+41 ; 0,75% 100 : 66+56 ; : 71+83 10-1 : 3 + 11 10+7
1,00% 100 : 1+6
Explanations:
x wm = weighted mean of x
VC = count per ml (one plate or more) R = reduction (lg R = lgN0 – lgNa)
x = average of VC1 and VC2 (1. + 2. duplicate)
46
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Test results (bactericidal suspension test) The test results for the two other
concentrations tested are not included in this example.
EN....13727…....(Phase 2, step 1) Product-name: ..BU..(hygienic handrub) Batch No.: 26-01-48.....

Remarks: Ready-to-use product see 5.5.4
Dilution neutralization X Pour plate Spread plate x Number of plates .....2 / ml

method
Neutralizer:.....Lecithin 3,0 g/l in diluent..................................................................................................................
Membrane filtration method Rinsing liquid:.........................................................................................................
Test temperature: 20 °C interfering substance:….bovine albumin: 0,3 g/L ………………………………..
Test organism: ....Pseudomonas aeruginosa ATCC 15442...........................Incubation temperature: 36°C
Internal lab. No :...QS 68/12.... Date of test:.2010-05-27 Responsible person: ...Fang....... Signature: Fang.
Diluent used for product test solutions: ..hard.water..Appearance of the product test
solutions: ........clear.......
Validation and controls

Validation Experimental Neutralizer or filtration control Method validation (C)
suspension (Nvo) conditions control (A) (B) Product conc.: undiluted
VC1 86 x= VC1 30s 79 x =81,5 VC1 86 x =88,5 VC1 30s 75(35+40) x =81
VC2 30s 84 VC2 30s 87(41+46)
(40 + 46) 89 (42 + 44)
VC2 92 VC1 1min 75 x = VC2 91 VC1 1min 82(37+45) x =
(47 + 45) VC2 1min 82 78,5 (43 + 48) VC2 1min 79(35+44) 80,5
30 ≤ x of Nvo ≤ 160 ? x of A is ≥ 0,5x x of Nvo ? x of B is ≥ 0,5x x of Nvo x of C is ≥ 0,5x x of Nvo ?
yes no yes no (or NVB/1000) yes no yes no
Validation suspension (NVB) VC1 99 VC2 71 x= 30 ≤ x of NVB/1000 ≤ 160 ? yes no
(49 + 50) (39 + 42) 85
Test suspension and Test Test-suspension N VC1 VC2 x wm = 193,64 x 107 ; lg N =9,29
(N and No): 10-7 168 213 No = N/100 ; lgNo = 7,29
10-8 20 25 7,17 ≤ lgNo ≤ 7,70? yes no
Real conc. of Dilution VC1 VC2 Na = ( x or lg Na lg R (lgN0 Contact

the step x wm x 10) = 7,29) time (min)
product %
100 >660 >660 0,5
8100 3,91 3,38
10-1 77 85 (30s)
97 100 7 0
<140 <2,15 >5,14 1
10-1 0 0
Countings per plate for:
N 10-7: 80+88; 105+108 10-8: 9+11; 15+10 Na 0,5min: 42+35; 44+41 1min: 1+6;
A 30s: 43+36; 39+45 1min: 45+30; 43+39
Explanations:
x wm = weighted mean of x
VC = count per ml (one plate or more) R = reduction (lg R = lgN0 – lg Na)
x = average of VC1 and VC2 (1. + 2. duplicate)
47
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Annex E
(informative)
Precision of the test result
The scope of the study is to determine the precision of the test method within and between a random
sample of laboratories. The sample size necessary to achieve a precision of the reduction of ± 1 lg is
determined.
In order to determine the required sample size it is necessary to quantify the residual, unexplained
variance in different experiments. This variance is estimated by the help of a statistical mixed model
including the random effect of laboratories and the fixed effect of the combination of a specified test
organism, a product and a contact time. Reduction is determined with different concentrations of the
products labeled R_1, R_2, R_3 (and in that case R_4). Separate statistical analyses are performed for
each reduction.
The statistical analyses are done using different data sets. All data available are used in the
“unrestricted” case in contrast to the “restricted” case, where a measured reduction is excluded from
the statistical analysis if it is below 0 or above 5.
This study involved ten laboratories from different European countries and was carried out in the years
2000-2002. Each laboratory repeated the test at least three times.
Beside the random effect of laboratories, a possible influence of the test organism, the product and the
contact time on the independent variable reduction is analyzed.
The experimental design is not balanced, i.e. not all possible combinations have been tested. Therefore a
new variable called Influence was created which combines each of the three variables (test organism,
product and contact time) with their different possible appearances, e.g. P. aeruginosa – Glutaraldehyde
– 5 min into the one Influence “1” (see Table E.1).
Table E.1 — Number of repetitions (unrestricted data set) for a given test organism, product,
contact time and laboratory and independent variable R_1, R_2 and R_3. Each cell shows the
number of the repetitions for the given reductions R_1, R_2 and R_3 separated by semicolon
Test Contact Influ- Laboratory ID

Product tested
organism time ence 3 6 9 10 11 14 20 26 30 34
P. aeruginosa Glutaraldehyd
e 5 1 -;-;- 6;6;- 6;6;- -;5;- 6;6;- 4;4;- 3;6;- -;-;- -;-;- 6;6;-
S. aureus Peracetic acid 2 -;-;- 12;12;12 12;12;12 5;6;6 6;6;- 6;6;6 -;-;- 6;-;6 7;8;8 6;6;6
E. hirae 3 -;-;- 6;6;- 12;12;- 6;6;- 6;6;- 6;6;- 6;6;- 4;4;- 6;6;- 6;6;-
Glutaraldehyd
P. aeruginosa 60 4 -;-;- 6;6;- 12;12;- 6;6;- 6;6;- 5;5;- 5;3;- 6;6;- 6;6;- 6;-;-
e
S. aureus 5 6;6;- 6;6;- 12;12;- 6;6;- 6;6;- 6;6;6 5;6;- 6;6;- 5;5;- 6;6;6
Table E.1 shows that the combination Glutaraldehyde, 60 min and E. hirae, P. aeruginosa and S. aureus
was tested in all labs, except lab 3. The latter is excluded from the statistical analysis, since the
estimation of the variance components requires full rank data sets.
Table E.1 also clarifies that the labs did not perform all required tests, especially in the case of the
“Influence 1” (Pseudomonas aeruginosa / Glutaraldehyde / contact time 5 min). Nevertheless, the new
design is the best way to estimate the residual variance needed for the calculation of the precision of the
test results and its dependency on the number of repetitions.
48
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Results
Unrestricted and restricted mixed effects models using R_1 to R_3 as dependent variable and influence
as fixed and laboratory as random effect are used to estimate the variance components.
Table E.2 — Results of statistical analyses (“Est.Var.”: estimated variance; “SE”: standard error;
“Z”: test value; “Pr Z”: significance; N = number of results included)
Unrestricted Restricted
Variance Est.Var. SE Z Pr Z Variance Est. SE Z Pr Z

component component Var.
R_1 Laboratory 0,5109 0,3244 1,57 0,0576 Laboratory 0,4725 0,3026 1,56 0,0592
Residual 0,1963 0,0186 10,58 < 0,0001 Residual 0,1782 0,0171 10,41 < 0,0001
N = 265 N = 285
Variance Est. SE Z Pr Z Variance Est. SE Z Pr Z

component Var. component Var.
N = 268 N = 237
Variance Est. SE Z Pr Z Variance Est. SE Z Pr Z

component Var. component Var.
N = 62 N = 52
“Laboratory” in Table E.2 means the interlaboratory variance and “Residual” means the unexplained
residual intralaboratory variance.
The fixed effect Influence on the reduction cannot be estimated for R_3 because of insufficient data. For
R_1 and R_2, this effect is significant except for R_2 using the restricted data set.
The estimated residual variances given in the table E.2 are used to determine the precision of the test
results. The formula used for the calculation of the precision of the test results can be found in
EN 1040:2005, Annex E. The worst case (largest variance) is derived from “unrestricted” R_3 Var.Est.
0,8657 and the best case (smallest variance) is derived from “restricted” R_1 Var.Est. 0,1782.
As seen from the Figure E.1 below, the number of repetitions of four (best case) or six (worst case) or
more repetitions gives a precision of the reduction of ± 1 lg or less.
49
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Key:
X Number of repetitions
Y Precision [in lg terms]
● Worst case
○ Best case
Figure E.1 — Dependency of the precision of the reduction (y-axis) on the sample size (x-axis)
for worst and best case
50
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Annex ZA
(informative)
Relationship between this European Standard and the Essential

Requirements of EU Directive 93/42/EEC
This European Standard has been prepared under a mandate given to CEN by the European
Commission and the European Free Trade Association to provide a means of conforming to Essential
Requirements of the New Approach Directive 93/42/EEC.
Once this standard is cited in the Official Journal of the European Union under that Directive and has
been implemented as a national standard in at least one Member State, compliance with the clauses of
this standard given in Table ZA.1 confers, within the limits of the scope of this standard, a presumption
of conformity with the corresponding Essential Requirements of that Directive and associated EFTA
regulations.
Table ZA.1 — Correspondence between this European Standard and Directive 93/42/EEC
Clauses of this EN Essential Requirements (ERs) of Qualifying remarks/Notes

Directive 93/42/EEC
1, 2, 3 and 4 6a (in connection with annex X, This standard is intended for
1.1d) products whose main efficacy claim
is of a microbicidal nature, e.g.
chemical disinfectants and
antiseptics. Complying with the
requirements of this standard
demonstrates the bactericidal
activity of the product. Generally at
least one additional European
Standard (phase 2, step 2) has to be
complied with to demonstrate a
sufficient performance evaluation of
such products.
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling
within the scope of this standard.
51
BS EN 13727:2012+A2:2015
EN 13727:2012+A2:2015 (E)
Bibliography
[1] EUROPEAN PHARMACOPOEIA (EP). Edition 1997 supplement 2000, Water for injections
52
This page deliberately left blank
NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW
British Standards Institution (BSI)

BSI is the national body responsible for preparing British Standards and other
standards-related publications, information and services.
BSI is incorporated by Royal Charter. British Standards and other standardization
products are published by BSI Standards Limited.
About us Revisions
We bring together business, industry, government, consumers, innovators Our British Standards and other publications are updated by amendment or revision.
and others to shape their combined experience and expertise into standards We continually improve the quality of our products and services to benefit your
-based solutions. business. If you find an inaccuracy or ambiguity within a British Standard or other
The knowledge embodied in our standards has been carefully assembled in BSI publication please inform the Knowledge Centre.
a dependable format and refined through our open consultation process.
Organizations of all sizes and across all sectors choose standards to help Copyright
them achieve their goals. All the data, software and documentation set out in all British Standards and
other BSI publications are the property of and copyrighted by BSI, or some person
Information on standards or entity that owns copyright in the information used (such as the international
We can provide you with the knowledge that your organization needs standardization bodies) and has formally licensed such information to BSI for
to succeed. Find out more about British Standards by visiting our website at commercial publication and use. Except as permitted under the Copyright, Designs
bsigroup.com/standards or contacting our Customer Services team or and Patents Act 1988 no extract may be reproduced, stored in a retrieval system
Knowledge Centre. or transmitted in any form or by any means – electronic, photocopying, recording
or otherwise – without prior written permission from BSI. Details and advice can
Buying standards be obtained from the Copyright & Licensing Department.
You can buy and download PDF versions of BSI publications, including British
and adopted European and international standards, through our website at Useful Contacts:
bsigroup.com/shop, where hard copies can also be purchased. Customer Services
If you need international and foreign standards from other Standards Development Tel: +44 845 086 9001
Organizations, hard copies can be ordered from our Customer Services team. Email (orders): orders@bsigroup.com
Email (enquiries): cservices@bsigroup.com
Subscriptions
Subscriptions
Our range of subscription services are designed to make using standards
Tel: +44 845 086 9001
easier for you. For further information on our subscription products go to
Email: subscriptions@bsigroup.com
bsigroup.com/subscriptions.
With British Standards Online (BSOL) you’ll have instant access to over 55,000 Knowledge Centre
British and adopted European and international standards from your desktop. Tel: +44 20 8996 7004
It’s available 24/7 and is refreshed daily so you’ll always be up to date. Email: knowledgecentre@bsigroup.com
You can keep in touch with standards developments and receive substantial
Copyright & Licensing
discounts on the purchase price of standards, both in single copy and subscription
format, by becoming a BSI Subscribing Member. Tel: +44 20 8996 7070
Email: copyright@bsigroup.com
PLUS is an updating service exclusive to BSI Subscribing Members. You will
automatically receive the latest hard copy of your standards when they’re
revised or replaced.
To find out more about becoming a BSI Subscribing Member and the benefits
of membership, please visit bsigroup.com/shop.
With a Multi-User Network Licence (MUNL) you are able to host standards
publications on your intranet. Licences can cover as few or as many users as you
wish. With updates supplied as soon as they’re available, you can be sure your
documentation is current. For further information, email bsmusales@bsigroup.com.
BSI Group Headquarters

389 Chiswick High Road London W4 4AL UK

EN 13727-2012 Plus A2-2015

Uploaded by

Copyright:

Available Formats

EN 13727-2012 Plus A2-2015

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

EN 13727-2012 Plus A2-2015

Uploaded by

Copyright:

Available Formats

BS EN 13727:2012+A1:2013

BSI Standards Publication

Chemical disinfectants and

ICS 11.080.20 Supersedes EN 13727:2012+A1:2013

Chemical disinfectants and antiseptics - Quantitative

EUROPEAN COMMITTEE FOR STANDARDIZATION

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

Annex C (informative) Graphical representation of test procedures ....................................................... 36

This document supersedes #EN 13727:2012+A1:2013$.

— in clinics of schools, of kindergartens and of nursing homes;

NOTE 2 This method corresponds to a phase 2 step 1 test.

3 Terms and definitions

Table 1 — Minimum and additional test conditions

Test Hygienic handrub Surgical handrub Instrument Surface

additional clean or dirty; clean or dirty; any relevant any relevant

5.2 Materials and reagents

5.2.1 Test organisms

The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3).

5.2.2 Culture media and reagents

All specified pH values are measured at 20 °C ± 1 °C.

5.2.2.3 Tryptone Soya Agar (TSA)

Tryptone soya agar, consisting of:

Tryptone sodium chloride solution, consisting of:

5.2.2.6 Rinsing liquid (for membrane filtration)

5.2.2.7 Hard water for dilution of products

For the preparation of 1 l of hard water, the procedure is as follows:

5.2.2.8 Interfering substance

5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)

5.2.2.9 Defibrinated sheep blood

b) by dry heat, in the hot air oven [5.3.2.1 b)].

5.3.2 Usual microbiological laboratory equipment 2)

and, in particular, the following:

5.3.2.3 Incubator, capable of being controlled either at 36 °C ± 1 °C or 37 °C ± 1 °C (5.2.1). The same

a) Electromechanical agitator, e.g. Vortex® mixer 3);

5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.

2) Disposable sterile equipment is an acceptable alternative to reusable glassware.

5.3.2.11 Glass beads (Diameter 3 mm to 4 mm).

5.3.2.12 Volumetric flasks.

5.3.2.13 Centrifuge (800 gN).

5.4 Preparation of test organism suspensions and product test solutions

5.4.1 Test organism suspensions (test and validation suspension)

4) cfu/ml = colony forming unit(s) per millilitre.

Mix [5.3.2.6 a)].

For incubation and counting see 5.4.1.6.

5.4.2 Product test solutions

5.5.1.1 Experimental conditions

The temperatures to be tested are specified in Clause 4, Table 1.

The contact times to be tested are specified in Clause 4, Table 1.

5.5.1.3 General instructions for validation and control procedures

5.5.1.5 Precautions for manipulation of test organisms

The procedure for determining bactericidal concentrations is as follows:

For incubation and counting, see 5.5.2.6.$

For incubation and counting, see 5.5.2.6.

To verify the absence of toxicity of the neutralizer, the procedure is as follows:

For incubation and counting, see 5.5.2.6.

To validate the dilution neutralization method, the procedure is as follows:

For incubation and counting, see 5.5.2.6.

5.5.3 Membrane filtration method 6)

#The procedure for determining the bactericidal concentrations is as follows: