EN ISO 20743:2013

SLOVENSKI STANDARD
01-december-2013
1DGRPHãþD
SIST EN ISO 20743:2007
Tekstilije - Ugotavljanje protibakterijske aktivnosti na tekstilnih izdelkih (ISO
20743:2013)
Textiles - Determination of antibacterial activity of textile products (ISO 20743:2013)
Textilien - Bestimmung der antibakteriellen Wirksamkeit von textilen Produkten (ISO
20743:2013)
Textiles - Détermination de l'activité antibactérienne des produits textiles (ISO
20743:2013)
Ta slovenski standard je istoveten z: EN ISO 20743:2013
ICS:
07.100.99 Drugi standardi v zvezi z Other standards related to
mikrobiologijo microbiology
59.080.01 Tekstilije na splošno Textiles in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 20743
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2013
ICS 07.100.99; 59.080.01 Supersedes EN ISO 20743:2007
English Version
Textiles - Determination of antibacterial activity of textile
products (ISO 20743:2013)
Textiles - Détermination de l'activité antibactérienne des Textilien - Bestimmung der antibakteriellen Wirkung
produits textiles (ISO 20743:2013) antibakteriell behandelter Erzeugnisse (ISO 20743:2013)
This European Standard was approved by CEN on 22 June 2013.

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

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20743:2013: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 20743:2013) has been prepared by Technical Committee ISO/TC 38 "Textiles" in
collaboration with Technical Committee CEN/TC 248 “Textiles and textile products” the secretariat of which is
held by BSI.
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 January 2014, and conflicting national standards shall be withdrawn at
the latest by January 2014.
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 supersedes EN ISO 20743:2007.
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.
Endorsement notice
The text of ISO 20743:2013 has been approved by CEN as EN ISO 20743:2013 without any modification.
INTERNATIONAL ISO
STANDARD 20743
Second edition
2013-07-15
Textiles — Determination of
antibacterial activity of textile products
Textiles — Détermination de l’activité antibactérienne des produits
textiles
Reference number
ISO 20743:2013(E)
©
ISO 2013
ISO 20743:2013(E)
© ISO 2013
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved

ISO 20743:2013(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Safety precaution . 2
5 Apparatus . 2
6 Reagents and culture media . 4
6.1 Water . 4
6.2 Tryptone soya broth (TSB) . 4
6.3 Tryptone soya agar (TSA) . 4
6.4 Agar for transfer . 5
6.5 Nutrient broth (NB) . 5
6.6 Peptone salt solution . 5
6.7 Physiological saline . 5
6.8 SCDLP medium . 6
6.9 Dilution buffer for shake-out bacterial suspension. 6
6.10 Neutralizing solution . 6
6.11 Enumeration agar (EA) . 7
6.12 Agar for printing . 7
6.13 Cryoprotective solution for bacterial species . 7
6.14 Stock solution of ATP standard reagent . 7
6.15 Buffer solution for ATP luminescent reagent . 8
6.16 ATP luminescent reagent . 8
6.17 ATP extracting reagent . 8
6.18 ATP eliminating reagent . 8
6.19 SCDLP or other medium for preparing ATP reference solution . 9
6.20 Shake-out physiological saline . 9
7 Reference strains . 9
7.1 Strains . 9
7.2 Storage of strains .10
8 Test procedures .11
8.1 Absorption method (see Annex E) .11
8.2 Transfer method (see Annex E) .15
8.3 Printing method (see Annex E) .18
9 Test report .22
Annex A (normative) Strain numbers .23
Annex B (normative) Shaking method .24
Annex C (normative) Quantitative measurement by plate count method .25
Annex D (normative) Quantitative measurement by luminescence method .26
Annex E (informative) Testing examples .28
Annex F (informative) Efficacy of antibacterial activity .31
Bibliography .32
ISO 20743:2013(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2. www.iso.org/directives
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received. www.iso.org/patents
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
The committee responsible for this document is ISO/TC 38, Textiles.
This second edition cancels and replaces the first edition (ISO 20743:2007), which has been
technically revised.
iv © ISO 2013 – All rights reserved

ISO 20743:2013(E)
Introduction
Speciality products of antibacterial-treated textiles have been introduced in the market and are expanding
year by year in various applications. Those textiles certainly meet the consumer’s requirement to seek
prevention and protection from the negative effects caused by bacteria and to secure the quality of life.
In this situation, the test methods to determine the antibacterial activity for antibacterial textile products
were expected to be established in order to address the substantial need for an International Standard.
The test method for antibacterial activity was developed as ISO 20645 which was a qualitative test
method. There are no testing standards for the quantitative method which gives more objective
information for the antibacterial activity of the textile products.
There are several practical test methods to determine the quantitative antibacterial activity specified
in this International Standard. The test methods are composed of 2 major steps, such as inoculation of
bacteria and quantitative measurement of bacteria.
The methods for the inoculation of bacteria specified in this International Standard are the absorption
method, transfer method and printing method.
The methods of the quantitative measurement of bacteria specified in this International Standard are
colony plate count method and ATP luminescence methods.
Although there are 6 ways for the combination of inoculation methods and quantitative measurements
to execute this test, the choice of the ways depends on the user’s availability and consensus between the
concerned parties.
INTERNATIONAL STANDARD ISO 20743:2013(E)
Textiles — Determination of antibacterial activity of
textile products
1 Scope
This International Standard specifies quantitative test methods to determine the antibacterial activity
of all antibacterial textile products including nonwovens.
This International Standard is applicable to all textile products, including cloth, wadding, thread and
material for clothing, bedclothes, home furnishings and miscellaneous goods, regardless of the type of
antibacterial agent used (organic, inorganic, natural or man-made) or the method of application (built-
in, after-treatment or grafting).
Based on the intended application and on the environment in which the textile product is to be used
and also on the surface properties of the textile properties, the user can select the most suitable of the
following three inoculation methods on determination of antibacterial activity:
a) absorption method (an evaluation method in which the test bacterial suspension is inoculated
directly onto specimens);
b) transfer method (an evaluation method in which test bacteria are placed on an agar plate and
transferred onto specimens);
c) printing method (an evaluation method in which test bacteria are placed on a filter and printed
onto specimens).
The colony plate count method and the ATP (ATP = Adenosine Tri-phosphate) luminescence method are
also specified for measuring the enumeration of bacteria.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 6330, Textiles — Domestic washing and drying procedures for textile testing
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
control fabric
fabric used to validate the growth condition of test bacteria and validate the test
Note 1 to entry: The same fabric as the fabric to be tested but without antibacterial treatment or a 100 % cotton
fabric without fluorescent brighteners or other finish can be used.
3.2
antibacterial agent
product designed to prevent or mitigate the growth of bacteria, to reduce the number of bacteria or to
kill bacteria
ISO 20743:2013(E)
3.3
antibacterial finish
treatment designed to prevent or mitigate the growth of bacteria, to reduce the number of bacteria or
to kill bacteria
3.4
antibacterial activity
activity of an antibacterial finish used to prevent or mitigate the growth of bacteria, to reduce the
number of bacteria or to kill bacteria
3.5
plate count method
method in which the number of bacteria present after incubation is calculated by counting the number
of colonies according to a ten-time dilution method
Note 1 to entry: The results are expressed in “CFU (Colony Forming Unit)”.
3.6
luminescence method
method in which the amount of ATP contained in bacterial cells is measured
Note 1 to entry: The results are expressed in “moles of ATP”.
3.7
neutralizer
chemical agents used to inactivate, neutralize or quench the antibacterial properties of antibacterial agents
4 Safety precaution
The test methods specified in this International Standard require the use of bacteria.
These tests should be carried out by persons with training and experience in the use of microbiological
techniques.
Appropriate safety precautions should be observed with due consideration given to country-specific
regulations.
5 Apparatus
Usual laboratory apparatus and, in particular, the following.
5.1 Spectrophotometer, capable of measuring at a 620 nm to 660 nm wavelength, or McFarland’s
nephelometer.
5.2 Incubator, capable of maintaining a constant temperature of 37 °C ± 2 °C.
5.3 Water baths, one capable of maintaining a constant temperature of 46 °C ± 2 °C and another
capable of maintaining a temperature of 70 °C to 90 °C.
5.4 Mixer, producing a vortex shaking action.
5.5 Stomacher, capable of speeds of 6 blows per second to 8 blows per second, with the corresponding
disposable containers.
5.6 Clean bench, for microbial test.
5.7 Washing machine, in accordance with the specifications of ISO 6330.
2 © ISO 2013 – All rights reserved

ISO 20743:2013(E)
5.8 Humidity chamber, tropical chamber or other container capable of maintaining a high-humidity
more than 70 %RH atmospheric condition.
−12 −7
5.9 Luminescence photometer, capable of measuring ATP of 10 mol/l to 10 mol/l at 300 nm to
650 nm with a luminescence-measuring reagent.
5.10 Printing apparatus, capable of applying a 4 N load to a test specimen and rotating the specimen
180° in one direction for a period of 3,0 s.
5.11 Refrigerator, capable of maintaining a temperature of between 2 °C and 8 °C.
5.12 Freezers, one adjustable to a temperature below −70 °C and another to a temperature below −20 °C.
5.13 Balance, which can be read to the nearest 0,01 g.
5.14 Filtering apparatus, consisting of an upper container equipped with a membrane filter and a
lower container equipped with a suction opening.
5.15 Pipette, having the most suitable volume for each use, with a tip made of glass or plastic, and with
a tolerance of 0,5 % or less.
5.16 Vials, 30 ml glass bottles, with screw openings, polytetrafluoroethylene or silicone packing and
caps made of polypropylene, polycarbonate or another suitable material.
5.17 Petri dishes, that have been sterilized, made of glass or plastic, in diameter sizes of 90 mm to
100 mm or 55 mm to 60 mm.
5.18 Glass rod, with a diameter of approximately 18 mm.
5.19 Anti-bumping granules (glass beads), with a diameter of 3 mm to 4 mm.
5.20 Erlenmeyer flask, of capacity 100 ml.
5.21 Cutting template, made of a sterilizable material (stainless steel or glass) with a diameter of
3,8 cm ± 0,1 cm.
5.22 Disposable plastic bags, sterile bags suitable for containing food products, to be used for one of
the shaking methods of the specimens.
5.23 Tweezers, made of a material which can be sterilized.
5.24 Stainless-steel cylinder, with a mass of 200 g ± 10 g and a diameter of 3,5 cm ± 0,1 cm.
5.25 Metal wire basket, for autoclaving.
5.26 Aluminium foil.
5.27 Reciprocal incubation shaker.
5.28 Autoclave, capable of sterilizing at 121 °C ± 2 °C and 103 kPa ± 5 kPa.
ISO 20743:2013(E)
6 Reagents and culture media
Reagents used in tests shall be of analytical quality and/or suited for microbiological purposes.
Dehydrated products available on the commercial market are recommended for use in preparing the culture
media. The manufacturer’s instructions for the preparation of these products should be strictly followed.
6.1 Water
Water used in tests shall be analytical-grade water for microbiological media preparation, which is
freshly distilled and/or ion-exchanged and/or ultra-filtered and/or filtered with RO (reverse osmosis).
It shall be free from all toxic or bacteria inhibitory substances.
6.2 Tryptone soya broth (TSB)
Tryptone, pancreatic digest of casein       17 g
Soya peptone, papain digest of soya        3 g
Sodium chloride (NaCl)        5 g
Glucose      2,5 g
Dipotassium hydrogen phosphate      2,5 g
Water    1 000 ml
Mix well and adjust pH,       7,2 ± 0,2
then sterilize by autoclave (5.28).
6.3 Tryptone soya agar (TSA)
Tryptone, pancreatic digest of casein     15 g
Soya peptone, papain digest of soya      5 g
Sodium chloride (NaCl)      5 g
Agar     15 g
Water   1 000 ml
Mix well and adjust pH,    7,2 ± 0,2
then sterilize by autoclave (5.28).
4 © ISO 2013 – All rights reserved

ISO 20743:2013(E)
6.4 Agar for transfer
Tryptone, pancreatic digest of casein    0,75 g
Soya peptone, papain digest of soya    0,25 g
Sodium chloride (NaCl)      5 g
Agar     15 g
Water   1 000 ml
Mix well and adjust pH,     7,2 ± 0,2
then sterilize by autoclave (5.28).
6.5 Nutrient broth (NB)
Beef extract      3 g
Peptone      5 g
Water   1 000 ml
Mix well and adjust pH, then sterilize by
autoclave (5.28).
pH    6,9 ± 0,2
6.6 Peptone salt solution
Peptone, pancreatic digest of 1 g
casein
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml
Mix well and adjust pH, 6,9 ± 0,2
then sterilize by autoclave
(5.28).
6.7 Physiological saline
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml
Mix well, then sterilize by autoclave (5.28).
ISO 20743:2013(E)
6.8 SCDLP medium
Peptone, digest of casein      17 g
Peptone, digest of soybean       3 g
Sodium chloride (NaCl)       5 g
Dipotassium hydrogenphosphate      2,5 g
Glucose      2,5 g
Lecithin       1 g
Polysorbate 80       7 g
Water   1 000 ml
Mix well and adjust pH,    7,2 ± 0,2
then sterilize by autoclave (5.28).
If the neutralizing power is insufficient, the content of polysorbate 80 or lecithin may be adjusted or
another neutralizing agent may be added. The use of any unspecified neutralizer shall be recorded along
with the name and concentration.
6.9 Dilution buffer for shake-out bacterial suspension
This buffer solution consists of 0,005 mol/l sodium dihydrogenphosphate containing 0,037 % sucrose.
pH   7,2 ± 0,2
6.10 Neutralizing solution
The composition of the standard neutralizing solution shall be as follows.
Polysorbate 80     30 g
Egg-yolk lecithin      3 g
Histidine hydrochloride      1 g
Meat or casein peptone      1 g
Sodium chloride (NaCl)    4,3 g
Monopotassium phosphate    3,6 g
Disodium phosphate dihydrate    7,2 g
Water   1 000 ml
Mix well and sterilize by autoclave (5.28).
If the neutralizing power is insufficient, the content of polysorbate 80 or lecithin may be adjusted or
another neutralizing agent may be added. The use of any unspecified neutralizer shall be recorded along
with the name and concentration.
6 © ISO 2013 – All rights reserved

ISO 20743:2013(E)
6.11 Enumeration agar (EA)
Dehydrated yeast extract    2,5 g
Casein tryptone    5,0 g
Glucose    1,0 g
Agar    12 g to 18 g (depending on the gel strength of the product)
Water   1 000 ml
Mix well and adjust pH,   7,2 ± 0,2
then sterilize by autoclave (5.28).
6.12 Agar for printing
Agar     20 g
Water   1 000 ml
Mix well and sterilize by autoclave (5.28).
6.13 Cryoprotective solution for bacterial species
For freezing, a cryoprotective solution containing 150 g/l of glycerol or 100 g/l of dimethylsulfoxide
shall be used and prepared as follows,
TSB (6.2) or NB (6.5): 1 000 ml
Add,
Glycerol: 150 g
or
dimethylsulfoxide: 100 g
Mix well and sterilize by autoclave (5.28).
For solutions containing glycerol, sterilize the mixed solution by autoclave (5.28). For solutions
containing dimethylsulfoxide, sterilize the mixed solution by using 0,22 µm membrane filter.
NOTE Any commercially available product may be used as long as it is a cryoprotective solution or preserving
system that contains glycerol or dimethylsulfoxide and allows preservation of the strains in the same manner as
the specified solutions.
6.14 Stock solution of ATP standard reagent
−4
The concentration of ATP standard reagent is 1 X 10 mol/l which is obtained by the following mixing.
Adenosine-disodium 5’-triphosphate trihydrate 60,5 mg
Water 1 000 ml (final volume)
After preparation, the solution shall be placed in a tightly sealed container and cryopreserved at a temperature
of −20 °C or lower. The solution shall be used no later than 6 months from the date of preparation.
NOTE The suitable amount of adenosine-disodium 5’-triphosphate trihydrate may be calculated from the
ATP content of each commercial product.
ISO 20743:2013(E)
6.15 Buffer solution for ATP luminescent reagent
N-[Tris (hydroxymethyl) methyl] glycine 1 117 mg
Ethylenediamine disodium tetraacetatedehydrate 183 mg
Magnesium acetate tetrahydrate 808 mg
DL-dithiothreitol 6,7 mg
Dextrin 25 000 mg
Sucrose 925 mg
Water 250 ml (final volume)
pH 7,5 ± 0,2
6.16 ATP luminescent reagent
Luciferase (EC: 1.13.12.7) 16,0 mg
D-luciferin 12,6 mg
Bovine serum albumin 56 mg
Buffer solution (6.15)   30 ml
Once fully dissolved, let sit at room temperature for 15 min before use. Use within 3 h of preparation.
When a different ATP luminescent reagent is used, its composition shall be recorded.
6.17 ATP extracting reagent
N-[Tris (hydroxymethyl) methyl] glycine 45 mg
10 % aqueous benzalkonium chloride 0,2 ml
Water 9,8 ml
pH 12,0 ± 0,5
When a different ATP extraction reagent is used, its composition shall be recorded.
6.18 ATP eliminating reagent
−13
An agent to reduce the ATP in NB (6.5) to less than 10 mol/l within 15 min.
8 © ISO 2013 – All rights reserved

ISO 20743:2013(E)
Use within 8 h of preparation.
Apyrase (EC: 3.6.1.5) 4,6 international units/ml
Adenosine phosphate deaminase (EC: 3.5.4.6 or 46 international units/ml
EC 3.5.4.17)
Sucrose 37 mg
Bovine serum albumin 20 mg
0,05 mol/l buffer solution of 2-morpholinoethanesul- 10 ml
fonicacid, monohydrate
pH 6,0 ± 0,5
When a different eliminating reagent is used, its composition shall be recorded.
NOTE Commercially available reagent.
6.19 SCDLP or other medium for preparing ATP reference solution
SCDLP (6.8) or other medium 10 ml
ATP eliminating agent (6.18) 1 ml
After mixing, maintain at 30 °C to 37 °C for 1 h to prevent microbiological contamination.
Next, transfer to a hot-water bath at 70 °C to 90 °C for 1 h and cool down to room temperature.
Preserve the solution under refrigeration and use within 24 h.
An ATP reference solution should be prepared if the addition of neutralizing agents causes the ATP
−11
content in the shake-out solution to exceed 10 mol/l.
6.20 Shake-out physiological saline
Sodium chloride (NaCl): 8,5 g
Polysorbate 80: 2,0 g
Water:    1 000 ml
Mix well and sterilize by autoclave (5.28).
7 Reference strains
7.1 Strains
The following strains shall be used in all antibacterial activity tests as the details are described in Annex A.
— Staphylococcus aureus
— Klebsiella pneumoniae
ISO 20743:2013(E)
7.2 Storage of strains
7.2.1 General
The strains shall be stored in accordance with the supplier’s recommendations.
7.2.2 Ceramic bead method
Obtain a sample of the freeze-dried bacterial strain following the recommendations supplied with the
culture and resuspend it in 5 ml of TSB (6.2). Obtain a sample of the suspension and isolate it in a Petri
dish (5.17) containing TSA (6.3). Incubate the cultures for 18 h to 24 h at 37 °C ± 2 °C.
After incubation, use the culture isolated in the Petri dish to verify the purity of the strain.
After verification, prepare the stock cultures.
Sample 0,7 ml of the broth culture and spread it over the surface of the Petri dish containing the TSA.
Incubate the culture on plates for 18 h to 24 h under the conditions specified for the strain in the standard.
Add 10 ml of cryoprotective solution (6.13) to the surface of the TSA plate culture and resuspend the
cells in the solution using a sterile glass spreader. Sample the suspended cells from the surface of the
agar, dilute them in 100 ml of cryoprotective solution and incubate for 30 min at 20 °C.
Using a pipette (5.15), sample 1 ml of the suspension and transfer it to a cryogenic vial (5.16) containing
the beads (5.19). Shake the vial in order to spread the suspended cells around the beads.
— Where a cryoprotective solution containing dimethylsulfoxide is used, do not let it stand longer
than 1 min at ambient temperature.
— Where a cryoprotective solution containing glycerol is used, let it stand for 30 min at 20 °C.
— Withdraw the excess cryoprotective solution with a sterile pipette. Place the cryogenic vials in a
freezer (5.12) set at −70 °C or lower.
−6 −7
Prepare 10 and 10 dilutions of the suspension using the serial dilution method. Take a 1,0 ml sample
of each dilution and transfer it to separate Petri dishes. Add 12 ml to 15 ml of nutritive solution, cooled
down to 45 °C ± 1 °C. Incubate for 18 h to 24 h under the conditions specified for the strain. Enumerate
the plate cultures and confirm that the suspension contains less than 5 × 10 CFU/ml.
Store the cryogenic vials in a freezer at a temperature below −70 °C.
7.2.3 Glycerol suspension method
Inoculate a 15 ml culture tube containing 5 ml of appropriate medium with a freshly grown isolated
colony. Incubate usually for 5 h to overnight at 37 °C until the bacteria culture seems to reach the late
logarithm or stationary phase in the growth curve.
For each strain to be stored below −70 °C, for the archives, prepare a sterile, labelled cryogenic vial.
Place 225 μl of sterile 80 % glycerol in a cryogenic vial. Add 1,0 ml of the bacterial culture (frozen stock
shall be 15 % glycerol). Mix well using the vortex mixer (5.4) and store in a tube at −70 °C or lower.
For each strain to be stored at −20 °C, as liquid glycerol working stock, pipette equal volumes of 80 %
glycerol and bacterial culture into a labelled polypropylene tube. Mix the contents well to avoid formation
of ice crystals that will decrease the viability of the cells. Place the tube in a freezer at −20 °C. Check the
viability of the cells after 1 week if possible.
To recover a strain from the glycerol stock stored below −70 °C, use a sterile toothpick to scrape pieces
of the solid substance, then streak the cells onto the appropriate medium. Do not thaw the frozen stock
because each freeze–thaw cycle will result in a 50 % loss in cell viability.
To use the −20 °C working stock, pipette 50 μl to 100 μl as inoculum for a 5 ml overnight culture.
10 © ISO 2013 – All rights reserved

ISO 20743:2013(E)
8 Test procedures
8.1 Absorption method (see Annex E)
8.1.1 Incubation
8.1.1.1 Pick up the preserved stock bacteria from the storage container using an inoculating loop.
Streak onto the plate of EA (6.11) and incubate at 37 °C ± 2 °C for 24 h to 48 h.
NOTE The plate is kept at 5 °C to 10 °C and used within 1 week after the date of preparation.
8.1.1.2 Pour 20 ml of NB (6.5) or TSB (6.2) into a 100 ml Erlenmeyer flask. Apply an inoculating loop to
pick one colony up from the incubation as specified in 8.1.1.1 and inoculate it in the broth. Incubate under
the following conditions:
Temperature: 37 °C ± 2 °C
−1
Rate of shaking: 110 min and 3 cm width by reciprocal incubation shaker (5.27)
Incubation time: 18 h to 24 h
8.1.1.3 Pour 20 ml of NB (6.5) or TSB (6.2) into a 100 ml Erlenmeyer flask. Add 0,4 ml of the inoculum
8 8
from the incubation as specified in 8.1.1.2 that contains 1 × 10 CFU/ml to 3 × 10 CFU/ml in bacteria
−6 −6
concentration or an ATP concentration of 1 × 10 mol/l to 3 × 10 mol/l to the flask and incubate under
the following conditions:
Temperature: 37 °C ± 2 °C
−1
Rate of shaking: 110 min and 3 cm width by reciprocal incubation shaker (5.27)
Incubation time: 3 h ± 1 h
7 −7
Target CFU or ATP concentration after incubation: 10 CFU/ml or 10 mol/l.
NOTE The prepared inoculum is preserved by ice-cooling and used within 8 h.
8.1.2 Preparation of test inoculum
5 5
Adjust the bacteria to a concentration of 1 × 10 CFU/ml to 3 × 10 CFU/ml by a spectrophotometer or
McFarland’s nephelometer (5.1).
or
−9 −9
adjust the ATP to a concentration of 1 × 10 mol/l to 3 × 10 mol/l by the luminescence method using
NB (6.5) or TSB (6.2) after it has been diluted 20 times with water at room temperature.
NOTE The prepared inoculum is preserved by ice-cooling and used within 4 h.
8.1.3 Preparation of test specimens
8.1.3.1 Mass and shape of test specimens
8.1.3.1.1 Obtain test specimens with a mass of 0,40 g ± 0,05 g and cut to a suitable size for test specimens.
8.1.3.1.2 Obtain six test specimens of the antibacterial testing sample and six test specimens of the
control fabric or untreated fabric if available.
ISO 20743:2013(E)
NOTE Three of the control specimens and three of the antibacterial testing specimens are used for zero time,
immediately after inoculation. The remaining six specimens are used for the contact time after 18 h to 24 h incubation.
8.1.3.2 Setting the test specimen
Place each of the test specimens in separate vials by selecting the following method appropriate to the
nature of the test sample.
a) If specimens tends to curl easily, or if it contains wadding or down, place a glass rod (5.18) onto the
specimen in the vial. Alternatively, lace up both ends of the specimen with thread.
b) If the specimen is yarn, arrange the yarn in a bundle and place a glass rod onto the specimen in the vial.
c) If the specimen is from a carpet or a similar construction, cut the pile and place a glass rod onto the
specimen in the vial.
When necessary, test samples may be washed in accordance with ISO 6330 or another suitable method,
and after the final washing, the samples are rinsed with water to eliminate the washing detergent. The
use of an unspecified method shall be recorded.
8.1.3.3 Sterilization
When contamination is suspected or was found, sterilize test specimens by autoclave (5.28) according
to the following procedure.
8.1.3.3.1 Wrap the opening of vials containing specimens with aluminium foil (5.26).
8.1.3.3.2 Place the wrapped vials in a metal wire basket (5.25) for autoclaving.
8.1.3.3.3 Wrap the vial caps with aluminium foil and place them in the wire basket.
8.1.3.3.4 Sterilize the caps and the vials containing the test specimens by autoclave for 15 min to 20 min.
8.1.3.3.5 After sterilization, remove the aluminium foil and allow the specimens in the vials to dry for
60 min or more by placing them on a clean bench (5.6) or any other place where there is no risk of
airborne contamination.
8.1.3.3.6 Cap the vials securely.
NOTE When autoclaving is not possible, sterilization may be accomplished by ethylene oxide gas, γ -ray or
another suitable method. The use of alternative methods shall be recorded.
8.1.4 Test operation
8.1.4.1 Inoculation of test specimens
Accurately pipette 0,2 ml of the inoculum prepared in 8.1.2 at several points on each test specimen
prepared in 8.1.3.2 to ensure that no inoculum touches the surface of the vial and cap the vials.
8.1.4.2 Shake-out after inoculation
Immediately after the inoculation of 8.1.4.1, add 20 ml of SCDLP medium (6.8) or the neutralizing solution
(6.10) or the shake-out physiological saline (6.20) into each of the six vials in which a control specimen
and an antibacterial testing specimen have been placed, cap the vials and shake out as specified in
Annex B by hand or mixer (5.4).
12 © ISO 2013 – All rights reserved

ISO 20743:2013(E)
8.1.4.3 Incubation
Incubate the six vials (three control specimens, three testing specimens) at 37 °C ± 2 °C for 18 h to 24 h.
8.1.4.4 Shake-out after incubation
After the incubation of 8.1.4.3, add 20 ml of SCDLP medium (6.8) or of the neutralizing solution (6.10) or
the shake-out physiological saline (6.20) to each of the six vials, cap the vials and shake out as specified
in Annex B by hand or mixer (5.4).
8.1.4.5 Calculation of number of bacteria or amount of ATP
8.1.4.5.1 General
Obtain the number of bacteria or amount of the ATP as specified in 8.1.4.2 and 8.1.4.4 from the bacteria
concentration or the ATP concentration obtained by the quantitative measurement methods in Annex C
or Annex D according to the following formulae:
8.1.4.5.2 Number of bacteria
Mc=×20
B
where
M is the number of bacteria per specimen;
c is the bacteria concentration obtained in Annex C;
B
20 is the volume of the shake-out solution, in millilitres (ml).
8.1.4.5.3 Amount of ATP
Mc'=×20
ATP'
where
M’ is the amount of ATP per specimen;
cATP’is the ATP concentration obtained in Annex D;
20 is the volume of the shake-out solution, in millilitres (ml).
8.1.5 Test results
8.1.5.1 Judgement of test effectiveness with the control specimen
When the conditions of a), b) and c) or a), b) and d) are satisfied, the test is judged to be effective. When
the test is judged to be ineffective, a retest shall be carried out.
5 5
a) The test inoculum of 8.1.2 shall be 1 × 10 CFU/ml to 3 × 10 CFU/ml or the ATP concentration shall
−9 −9
be 1 × 10 mol/l to 3 × 10 mol/l.
b) The difference in common logarithm in extremes of the number of bacteria, or the amount of ATP for
the three control specimens immediately after inoculation and after incubation, respectively, shall
be less than one .
ISO 20743:2013(E)
c) The growth value obtained according to the following formula shall be equal or more than 1,0 in the
plate count method.
d) The growth value obtained according to the following formula shall be equal or more than 0,5 in the
luminescence method.
FC=−lg lgC
t 0
where
F is the growth value on the control specimen;
lg C is the common logarithm of arithmetic average of the numbers of bacteria, or the amount
t
of ATP, obtained from three control specimens after an 18 h to 24 h incubation;
lg C is the common logarithm of arithmetic average of the numbers of bacteria, or the amount
o
of ATP, obtained from three control specimens immediately after inoculation.
8.1.5.2 Calculation of antibacterial activity value
When the condition in the next paragraph is satisfied, the test is judged to be effective. When the test is
judged to be ineffective, a retest shall be carried out.
The difference in common logarithm in extreme of the number of bacteria, or the amount of ATP for the
three antibacterial testing specimens immediately after inoculation and after incubation, respectively,
shall be less than two.
To validate the test, it is necessary that the difference in lo
...