Bsi bs en 13727 2012 + a2 2015

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 controlle

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)

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is withdrawn.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

Amendments/corrigenda issued since publication

30 November 2015 Implementation of CEN amendment A2:2015

NORME EUROPÉENNE

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

quantitatif de suspension pour l'évaluation de l'activité

bactéricide en médecine - Méthode d'essai et

prescriptions (Phase 2, Étape 1)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung im humanmedizinischen Bereich

- 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

C OMITÉ E URO PÉEN DE N ORMA LI SA TIO N 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

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

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

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

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$

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

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$

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

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

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

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

Table 1 — Minimum and additional test conditions Test

Conditions Hygienic handrub and handwash Surgical handrub and handwash disinfection Instrument disinfection Surface Minimum

spectrum of test organisms

0,3 g/l bovine albumin solution and/or

0,3 g/l bovine albumin solution and/or dirty

plus 3,0 ml/l erythrocytes (hygienic handwash)

c

3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes (surgical handwash)

c

3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes

3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes

any relevant substance

clean or dirty;

any relevant substance

any relevant

NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the minimum test conditions

a The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of the product The recommended contact time for the use of the product is within the responsibility of the manufacturer 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.

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

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

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

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

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:

5.2.2.4 Diluent

Tryptone sodium chloride solution, consisting of:

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

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:

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;

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

produces different final water hardness in each test tube In any case, the final hardness expressed as calcium carbonate (CaCO 3 ) 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

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:

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;

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

produces different final water hardness in each test tube In any case, the final hardness expressed as calcium

carbonate (CaCO 3 ) 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

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)]

5.3.2 Usual microbiological laboratory equipment 2)

and, in particular, the following:

5.3.2.1 Apparatus for sterilization (moist and dry heat)

0

holding time of 15 min;

0

) °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)

b) Mechanical shaker

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

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

5.3.2 Usual microbiological laboratory equipment 2)

and, in particular, the following:

5.3.2.1 Apparatus for sterilization (moist and dry heat)

0

holding time of 15 min;

0

) °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

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

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

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

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

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

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

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 10 3 cfu/ml to 1,6 x10 4 cfu/ml

b) To prepare the validation suspension for the neutralizer control NVB (5.5.2.4) (NVB) dilute the test

c) Maintain and use these validation suspensions (NV and NVB) the same way as the test suspension

[5.4.1.4 b)]

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

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

a) To prepare the validation suspension (NV), dilute the test suspension (5.4.1.4) with the diluent

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 10 3 cfu/ml to 1,6 x10 4 cfu/ml

b) To prepare the validation suspension for the neutralizer control NVB (5.5.2.4) (NVB) dilute the test

c) Maintain and use these validation suspensions (NV and NVB) the same way as the test suspension

[5.4.1.4 b)]

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

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):

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

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

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)

5.5.2.1 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

inoculate using the pour plate or spread plate technique

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

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

the beginning of the addition, mix [5.3.2.6 a)] Transfer 0,5 ml of this mixture into a tube containing

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 $

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

5.5.2.5 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

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

the beginning of the addition, mix [5.3.2.6 a)] Transfer 0,5 ml of this mixture into a tube containing

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 $

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

5.5.2.5 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

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)]

For incubation and counting, see 5.5.2.6

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

b) Note for each plate the exact number of colonies but record > 330 for any counts higher than 330

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

5.5.3.2 Test Na – determination of the bactericidal concentrations

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

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)

For incubation and counting, see 5.5.3.6

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)

For incubation and counting, see 5.5.3.6

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

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)

For incubation and counting, see 5.5.3.6

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

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)

For incubation and counting, see 5.5.3.6

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)

For incubation and counting, see 5.5.3.6

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)

For incubation and counting, see 5.5.3.6

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

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

b) Note for each plate the exact number of colonies but record > 165 for any counts higher than 165

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

5.5.4.1 General

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

and/or verification of the absence of any lethal effect in the test 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

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)]

For incubation and counting, see 5.5.2.6

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

and/or verification of the absence of any lethal effect in the test 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)

For incubation and counting, see 5.5.3.6

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)]

For incubation and counting, see 5.5.2.6

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

and/or verification of the absence of any lethal effect in the test 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)

For incubation and counting, see 5.5.3.6

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

ml in the bacterial suspensions

Number of cells per

ml in the test mixtures at the beginning of the contact time (time 0 )

Number of survivors per

ml in the test mixtures at the end of the contact time

NVB

Validation suspension for

per 1,0 ml sample;

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

Na, NV, NV0, NVB, A, B and C The third step is the calculation of the reduction R (5.8)

5.6.2.2 Determination of VC -values

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)

-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

-value as “more than sum of the counts,” e.g for “>330, 310, 302”, report “ > 942”

case of Na)

above

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:

1 0,1 2 10

c N

n n −

=

where

! NOTE 1 For the modified method (5.5.4), the lower dilution is 10-7 and the higher dilution is 10-8."

5.6.2.2 Determination of VC -values

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)

-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

-value as “more than sum of the counts,” e.g for “>330, 310, 302”, report “ > 942”

case of Na)

above

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:

1 0,1 2 10

c N

n n −

=

where

! NOTE 1 For the modified method (5.5.4), the lower dilution is 10-7 and the higher dilution is 10-8."

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

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

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

0, 1, 2 10 /

a a a

where

c is the sum of VC-values taken into account;

n is the number of VC-values taken into account

results as “less than” or “more than”

a1 duplicate VC-values Na -1 : 2, 16

1 a

( 14 16) 10 1

10 2

a2 duplicate VC-values Na -2 (membrane filtration): >165, >165

2 a

10 2

N− = > + > × × = > 1650 × 102 = > 165 000 = > 1,65 × 105$

a3 duplicate VC-values (two spread plates per 1,0 ml sample): > 660, 600

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

#b2 If all subsequent dilutions of Na show mean values of „less than”, take only the lowest dilution

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