ISO 16187:2013

INTERNATIONAL ISO
STANDARD 16187
First edition
2013-08-01
Footwear and footwear
components — Test method to assess
antibacterial activity
Chaussure et composants de chaussure — Méthode d’essai pour
évaluer l’activité antibactérienne
Reference number
©
ISO 2013
© ISO 2013
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ii © ISO 2013 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Safety . 1
5 Apparatus and materials. 2
6 Reagents and culture medium . 2
7 Test microorganisms . 4
7.1 Test strains . 4
7.2 Storage of strains . 4
8 Preparation of test inoculums . 4
9 Preparation of test samples . 4
9.1 General . 4
9.2 Test specimen . 5
9.3 Pre-treatment of the test specimen . 5
10 Test procedure . 5
11 Expression of results . 5
12 Test report . 6
Annex A (normative) Static challenge test . 7
Annex B (normative) Film contact method . 9
Annex C (normative) Dynamic challenge test .12
Annex D (informative) Summarized results of round robin tests .14
Bibliography .16
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
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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
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constitute an endorsement.
The committee responsible for this document is ISO/TC 216, Footwear.
iv © ISO 2013 – All rights reserved

INTERNATIONAL STANDARD ISO 16187:2013(E)
Footwear and footwear components — Test method to
assess antibacterial activity
CAUTION — Test methods specified herein require the use of bacteria. These tests are only to
be carried out in facilities with containment techniques for handling microorganisms and by
persons with training and experience in the use of microbiological techniques. Appropriate safety
precautions are to be observed with due consideration given to country-specific regulations.
1 Scope
This International Standard specifies quantitative test methods to evaluate the antibacterial activity of
footwear and components.
This International Standard is applicable to all types of footwear and components employing non-
diffusing antibacterial treatments.
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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 19952, Footwear — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 19952 and the following apply.
3.1
antibacterial activity
efficacy of a material or finish used to prevent or mitigate the growth of bacteria, to reduce the number
of bacteria or to kill bacteria
3.2
control sample
material identical to the test material but without antibacterial treatment
4 Safety
Handling of microorganisms which are potentially hazardous requires a high degree of technical
competence and can be subject to current national legislation and regulations. Only personnel trained
in microbiological techniques should carry out such tests. Codes of practice for disinfection, sterilization
and personal hygiene shall be strictly observed.
NOTE It is recommended that workers consult IEC 60068–2-10, appendix A “Danger to personnel”, and ISO 7218.
5 Apparatus and materials
5.1 General
Standard laboratory equipment and the following.
5.2 Biological safety cabinet.
5.3 Incubator, capable of maintaining a temperature of (37 ± 2) °C.
5.4 Autoclave.
5.5 Humidity chamber, capable of maintaining a temperature of (37 ± 2) °C and a relative humidity
not less than 90 %.
5.6 Ultraviolet lamp.
5.7 Wide mouth jars, with cap, 100 ml, capable of being used with an autoclave (5.4).
5.8 Cover film that does not affect bacterial growth or absorb water, which can be made of either
polyethylene, polypropylene or polyester [poly (ethylene terephthalate)]. Film that is 0,05 mm to
0,10 mm thick is recommended. For example, disposal bag suitable for use with an autoclave (5.4).
5.9 Vortex mixer.
5.10 Dimensional shaker, two dimensional or three dimensional, capable of adjusting to 50 rpm.
5.11 Shaking incubator, capable of maintaining a temperature of (37 ± 2) °C and a rotational
frequency of (120 ± 10) rpm.
6 Reagents and culture medium
6.1 Principle
The preparation and test shall be freshly prepared in order to ensure the culture quality.
NOTE This can be done according to ISO/TS 11133-1, ISO/TS 11133-2, or according to national standards
or regulations.
Reagents used in tests shall be of analytical grade and/or suited for microbiological purposes.
Use only water Grade 3 according to ISO 3696.
6.2 Nutrient broth (NB)
6.2.1 Composition
Beef extract, 3,0 g.
Peptone, 5,0 g.
Sodium chloride (NaCl), 5,0 g.
Water, 1 000 ml.
6.2.2 Preparation
Stir and adjust pH to (7,2 ± 0,2) (at room temperature). Heat with stirring on a hotplate or in a boiling-water
bath until the components are completely dissolved. Sterilize with autoclave (5.4) at (121 ± 2) °C for 15 min.
6.3 Nutrient agar (NA)
2 © ISO 2013 – All rights reserved

6.3.1 Composition
Beef extract, 5,0 g.
Peptone, 10,0 g.
Sodium chloride (NaCl), 5,0 g.
Agar, 15,0 g.
Water, 1 000 ml.
NOTE If solidification is insufficient, 15 g to 18 g of agar can be used.
6.3.2 Preparation
Stir and adjust pH to (7,2 ± 0,2) (at room temperature). Heat with stirring on a hotplate or in a boiling-
water bath until the components are completely dissolved. Sterilize with autoclave (5.4) at (121 ± 2) °C
for 15 min. Cool and shake solution well, then pour into the Petri dishes.
6.4 Soybean casein digest broth with lecithin and polyoxyethylene medium (SCDLP)
6.4.1 Composition
Peptone, digest of casein, 17,0 g.
Peptone, digest of soybean, 3,0 g.
Sodium chloride (NaCl), 5,0 g.
Potassium dihydrogen phosphate, 2,5 g.
Glucose, 2,5 g.
Lecithin, 1,0 g.
Polysorbate 80, 7,0 g.
Water, 1 000 ml.
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.
NOTE Information about selection and evaluation of alternative antibacterial neutralizing agents can be
found in ASTM E 1054 and EN 1040.
6.4.2 Preparation
After mixing well, adjust pH to (7,2 ± 0,2) (at room temperature) and sterilize with autoclave (5.4) at
(121 ± 2) °C for 15 min.
6.5 Sodium chloride solution (physiological saline)
6.5.1 Composition
Sodium chloride (NaCl), 8,5 g.
Water, 1 000 ml.
6.5.2 Preparation
After mixing well, adjust pH to (6,9 ± 0,2) (at room temperature) and sterilize at (121 ± 2) °C for 15 min.
7 Test microorganisms
7.1 Test strains
The following species shall be used in all antibacterial activity tests.
a) Staphylococcus aureus AS 1.89 or ATCC 6538.
b) Klebsiella pneumoniae AS 1.1736 or ATCC 4352.
NOTE 1 If required, other species or other strains can be used. However, the selected organisms should contain
at least one gram-positive and one gram-negative organism as the antibacterial agents may have different activities.
Test strains shall be obtained from agencies of the World Federation of Culture Collection (WFCC).
The bacteria species and their supply sources shall be included in the test report.
NOTE 2 AS refers to the China General Microbiological Culture Collection Centre (CGMCC), ATCC is the American
Type Culture Collection.
7.2 Storage of strains
Inoculate the strains to the nutrient agar (NA) (6.3), and incubate at (37 ± 2) °C for 24 h. Store at (5 ± 3) °C
(maximum one month) and keep it as stock culture of the strains. Transfer and incubate one time each month.
Strains can be preserved in accordance with the supplier’s direction or EN 12353.
8 Preparation of test inoculums
Using a sterile inoculating loop, transfer one colony (7.2) into 20 ml of nutrient broth (NB) (6.2) and
incubate in the shaking incubator (5.11) at (37 ± 2) °C for about 16 h (overnight culture). Estimate the
number of bacteria with microscopic observation or other methods. Prepare physiological saline (6.5)
with 1 % nutrient broth (NB) (6.2). Use this media to prepare a suspension with a bacterial concentration
of (2,5 a
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