Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for antibacterial activity of semiconducting photocatalytic materials

This document specifies a test method for the determination of the antibacterial activity of materials that contain a photocatalyst or have photocatalytic films on the surface, by measuring the enumeration of bacteria under irradiation of ultraviolet light. This document is intended for use with different kinds of semiconducting photocatalytic materials used in construction materials in flat sheet, board, plate shape or textiles that are the basic forms of materials for various applications. It does not include powder, granular or porous photocatalytic materials. This test method is usually applicable to photocatalytic materials produced for antibacterial effect. Other types of performance of photocatalytic materials, i.e. antifungal activity, antiviral activity, decomposition of water contaminants, self-cleaning, antifogging and air purification, are not determined by this method. The values expressed in this document are in accordance with the International System of Units (SI).

Céramiques techniques — Méthode d'essai de l'activité antibactérienne des matériaux photocatalytiques semiconducteurs

General Information

Status
Published
Publication Date
16-Jul-2019
Technical Committee
Current Stage
6060 - International Standard published
Start Date
17-Jul-2019
Completion Date
17-Jul-2019
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INTERNATIONAL ISO
STANDARD 27447
Second edition
2019-07
Fine ceramics (advanced ceramics,
advanced technical ceramics) — Test
method for antibacterial activity
of semiconducting photocatalytic
materials
Céramiques techniques — Méthode d'essai de l'activité
antibactérienne des matériaux photocatalytiques semiconducteurs
Reference number
ISO 27447:2019(E)
ISO 2019
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ISO 27447:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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.
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Published in Switzerland
ii © ISO 2019 – All rights reserved
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ISO 27447:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ..............................................................................................................................................................................................................................vii

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Symbols .......................................................................................................................................................................................................................... 2

5 Principle ........................................................................................................................................................................................................................ 3

6 Materials ....................................................................................................................................................................................................................... 4

6.1 Bacteria used and preparation for tests ............................................................................................................................ 4

6.1.1 Film cover method.......................................................................................................................................................... 4

6.1.2 Glass cover method ........................................................................................................................................................ 4

6.1.3 Bacteria preparation..................................................................................................................................................... 4

6.2 Chemicals and implements .......................................................................................................................................................... 5

6.2.1 General...................................................................................................................................................................................... 5

6.2.2 1/500 nutrient broth ................................................................................................................................................... 5

6.2.3 Nutrient broth .................................................................................................................................................................... 5

6.2.4 Nutrient agar ....................................................................................................................................................................... 5

6.2.5 Soybean-casein digest broth with lecithin and polysorbate 80 (SCDLP)........................ 5

6.2.6 Physiological saline solution ................................................................................................................................. 5

6.2.7 Physiological saline solution for washout .................................................................................................. 5

6.2.8 Non-ionic surfactant ..................................................................................................................................................... 6

7 Apparatus ..................................................................................................................................................................................................................... 6

7.1 Test equipment ....................................................................................................................................................................................... 6

7.2 Cover film ..................................................................................................................................................................................................... 6

7.3 Cover glass .................................................................................................................................................................................................. 6

7.4 Moisture preservation glass ........................................................................................................................................................ 7

7.5 Glass tube or glass rod ...................................................................................................................................................................... 7

7.6 Paper filter .................................................................................................................................................................................................. 7

7.7 Light source ............................................................................................................................................................................................... 7

7.8 UV radiometer ......................................................................................................................................................................................... 7

7.9 Punched metal sheet .......................................................................................................................................................................... 7

8 Test piece ...................................................................................................................................................................................................................... 9

8.1 Film cover method ............................................................................................................................................................................... 9

8.2 Glass cover method ............................................................................................................................................................................. 9

9 Procedure..................................................................................................................................................................................................................... 9

9.1 General ........................................................................................................................................................................................................... 9

9.2 Film cover method ............................................................................................................................................................................11

9.3 Glass cover method ..........................................................................................................................................................................11

9.4 UV irradiation condition ..............................................................................................................................................................12

9.5 Measurement of the number of living bacteria ........................................................................................................13

10 Calculation ...............................................................................................................................................................................................................14

10.1 General ........................................................................................................................................................................................................14

10.2 Film cover method ............................................................................................................................................................................14

10.3 Glass cover method ..........................................................................................................................................................................15

11 Test report ................................................................................................................................................................................................................16

Annex A (informative) Examples of test results .....................................................................................................................................18

Annex B (informative) Reference data of cover films and cover glasses ......................................................................20

Annex C (informative) Reference data of damage caused by ultraviolet to bacteria ......................................22

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ISO 27447:2019(E)

Bibliography .............................................................................................................................................................................................................................24

iv © ISO 2019 – All rights reserved
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ISO 27447:2019(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 (see 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 (see 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.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 206, Fine ceramics. This second edition

cancels and replaces the first edition (ISO 27447:2009), which has been technically revised. The main

changes to the previous edition are as follows:
— updating of reference document and cross-references;
— replacement of “adhesive” with “cover” throughout;

— clarification of definition of “photocatalyst antibacterial activity value” (3.4, 3.5, 3.6, 3.7) in Clause 3;

— deletion of a definition of “fluorescent UV lamp” in Clause 3 due to updating of the reference document

ISO 10677;

— inclusion of a statement in Clause 5 regarding treatment of results measured by the viable bacterial

count method;
— NOTE 1 changed to body text in 6.1.3;

— revision of “storage period of 1/500 nutrient broth” from 1 month ago to 1 week ago in 6.2.2

(formerly 6.2.1);

— addition of a new subclause, 6.2.1, renumbering of subsequent subclauses and updating of cross-

references in Clause 6;

— addition of a new subclause, 7.1, and renumbering of subsequent subclauses in Clause 7;

— revision of Figure 1, Figure 4 and Figure 5;
— addition of “paper filter” apparatus in 7.6;

— replacement of “black light fluorescent lamp” with “light source” in 7.7 (formerly 7.5) and revision of

a statement in 7.7 that the light source shall be 351BLB or 351BL as specified in ISO 10677;

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ISO 27447:2019(E)

— replacement of “ultraviolet light radiation mater” with “UV radiometer” in 7.8 (formerly 7.6) and

inclusion of a statement in 7.8 that the UV radiometer shall be used as specified in ISO 10677;

— NOTE changed to body text in 8.1;

— addition of a new subclause, 9.1, and renumbering of subsequent subclauses in Clause 9;

— revision of storage time of “the bacteria suspension in case of not using immediately” from 4 h to 2 h

in 9.2.1(formerly 9.1.1);
— NOTE 2 changed to body text in 9.2.2 (formerly 9.1.2);
— NOTE 2 and NOTE 3 changed to body text in 9.3.2 (formerly 9.2.2);
— addition of the test environment temperature (25 °C ± 3 °C) in 9.4.1;

— addition of a new subclause, 10.1, and renumbering of subsequent subclauses in Clause 10.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
vi © ISO 2019 – All rights reserved
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ISO 27447:2019(E)
Introduction

This document was developed for antibacterial activity as a result of continuing efforts to provide

test methods for photocatalytic materials. However, antibacterial activity cannot be measured for test

pieces with permeable or rough surfaces, so other test methods are required.

Under the irradiation of photons, photocatalysts show diverse functions, such as the decomposition

of air and water contaminants, as well as deodorization, self-cleaning, antifogging and antibacterial

actions. These functions of photocatalysts are generally based on the action of active oxygen species

such as hydroxyl (OH) radicals formed on the surface of a photocatalyst (References [14] and [15]). The

energy- and labour-saving nature of photocatalysis has attracted keen interest when the photocatalyst

is activated by sunlight (or artificial lighting).

Practical applications of photocatalysts for both indoor and outdoor use have rapidly expanded in recent

years. Many kinds of photocatalytic materials have been proposed or are already commercialized,

based on ceramics, glass, concrete, plastics or paper. Such materials are produced by either the coating

or mixing of a photocatalyst; in most cases, titanium dioxide (TiO ).

However, the effect of photocatalysis is not easily inspected visually, and no appropriate or official

evaluation methods have been available to date. Some confusion has thus arisen as photocatalytic

materials have been introduced. Furthermore, the above-mentioned diverse functions of photocatalysts

cannot be evaluated with a single method; thus, it is necessary to provide different evaluation methods

for air purification, water decontamination and self-cleaning.

This document applies to the testing of the antibacterial activity of photocatalytic ceramics and other

materials produced by either the coating or the mixing of a photocatalyst. Standards for testing the

antifungal activity that use photocatalytic materials will be developed separately.

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INTERNATIONAL STANDARD ISO 27447:2019(E)
Fine ceramics (advanced ceramics, advanced technical
ceramics) — Test method for antibacterial activity of
semiconducting photocatalytic materials

WARNING — Handling and manipulation of microorganisms that are potentially hazardous

requires a high degree of technical competence. Only personnel trained in microbiological

techniques should carry out tests.
1 Scope

This document specifies a test method for the determination of the antibacterial activity of materials

that contain a photocatalyst or have photocatalytic films on the surface, by measuring the enumeration

of bacteria under irradiation of ultraviolet light.

This document is intended for use with different kinds of semiconducting photocatalytic materials used

in construction materials in flat sheet, board, plate shape or textiles that are the basic forms of materials

for various applications. It does not include powder, granular or porous photocatalytic materials.

This test method is usually applicable to photocatalytic materials produced for antibacterial effect.

Other types of performance of photocatalytic materials, i.e. antifungal activity, antiviral activity,

decomposition of water contaminants, self-cleaning, antifogging and air purification, are not

determined by this method.

The values expressed in this document are in accordance with the International System of Units (SI).

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 10677, Fine ceramics (advanced ceramics, advanced technical ceramics) — Ultraviolet light source for

testing semiconducting photocatalytic materials
ISO 80000-1, Quantities and units — Part 1: General
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
photocatalyst

substance that carries out many functions based on oxidization and reduction reactions under

ultraviolet (UV) irradiation, including decomposition and removal of air and water contaminants,

deodorization, and antibacterial, antifungal, antiviral, self-cleaning and antifogging actions

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ISO 27447:2019(E)
3.2
photocatalytic material

material in which or on which the photocatalyst is added by coating, impregnation or mixing

Note 1 to entry: Photocatalytic materials are to be used for building and road construction materials to obtain

the functions mentioned in 3.1.
3.3
antibacterial

condition inhibiting the growth of bacteria on the surface of flat surface materials or cloths

3.4
photocatalyst antibacterial activity value for film cover method

difference between the logarithms of the total number of viable bacteria on photocatalytic treated

materials after UV irradiation and on non-treated materials after UV irradiation

Note 1 to entry: This value includes the decrease in the number of bacteria without UV irradiation.

3.5
photocatalyst antibacterial activity value for glass cover method

difference between the logarithms of the total number of viable bacteria on photocatalytic treated

cloths after UV irradiation and on standard cloths after UV irradiation

Note 1 to entry: This value includes the decrease in the number of bacteria without UV irradiation.

3.6

photocatalyst antibacterial activity value with UV irradiation for film cover method

difference between the logarithms of the total number of viable bacteria on photocatalytic treated

materials after UV irradiation and on photocatalytic treated materials kept in a dark place

3.7

photocatalyst antibacterial activity value with UV irradiation for glass cover method

difference between the logarithms of the total number of viable bacteria on photocatalytic treated

cloths after UV irradiation and on photocatalytic treated cloths kept in a dark place

3.8
film cover method

test method to evaluate the antibacterial performance of photocatalytic flat surface materials

3.9
glass cover method
test method to evaluate antibacterial performance of photocatalytic cloths
4 Symbols

A average number of viable bacteria on non-treated specimens, just after inoculation

B average number of viable bacteria on non-treated specimens,
after being kept in a dark place
B average number of viable bacteria on non-treated specimens,
after UV irradiation of intensity L
C average number of viable bacteria on photocatalytic treated specimens,
after being kept in a dark place
C average number of viable bacteria on photocatalytic treated specimens,
after UV irradiation of intensity L
F growth value, after being kept in a dark place
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ISO 27447:2019(E)
F growth value, after UV irradiation of intensity L
L UV irradiation intensity
L maximum logarithmic value of viable bacteria
max
L average logarithmic value of viable bacteria for three specimens
mean
L minimum logarithmic value of viable bacteria
min
M number of viable bacteria with glass cover method

M average logarithmic value of the number of viable bacteria for three non-treated specimens,

just after inoculation

M average logarithmic value of the number of viable bacteria for three non-treated specimens,

after being kept in a dark place

M average logarithmic value of the number of viable bacteria for three non-treated specimens,

after UV irradiation of intensity L

M average logarithmic value of the number of viable bacteria for three photocatalytic treated

specimens, after being kept in a dark place

M average logarithmic value of the number of viable bacteria for three photocatalytic treated

specimens, after UV irradiation of intensity L
N number of viable bacteria with film cover method
P bacteria concentration
D dilution factor

R photocatalyst antibacterial activity value for film cover method, after irradiation at a con-

stant intensity L

ΔR photocatalyst antibacterial activity value with UV irradiation for film cover method

S photocatalyst antibacterial activity value for glass cover method, after UV irradiation of in-

tensity L

ΔS photocatalyst antibacterial activity value with UV irradiation for glass cover method

V volume of soybean casein digest broth with lecithin and polysorbate 80 medium for washout

Z average number of colonies in two Petri dishes
5 Principle

This document is for the development, comparison, quality assurance, characterization, reliability

and design data generation of photocatalytic materials. The method is used to obtain the antibacterial

activity of photocatalytic materials by the contact of a specimen with bacteria, under UV light

irradiation. The film cover method is available for flat sheet, board or plate-shaped materials. To avoid

warpage in the cloths or textiles, the glass cover method is available for the cloths or textiles.

The specimen is laid in a Petri dish and the bacterial suspension is dripped onto the specimen. Then

the cover film or glass is placed on the suspension and the moisture conservation glass is placed on

top of the Petri dish. The Petri dish containing the specimen is exposed to light. After exposure, the

test bacteria are washed out of the specimen and the cover film or glass. This washout suspension is

measured by the viable bacterial count method. The results obtained are compared with those obtained

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ISO 27447:2019(E)

from inoculated specimens of non-photocatalytic treated material exposed to UV irradiation under

identical conditions to the treated material, and to those obtained from inoculated specimens of both

photocatalytic treated and non-treated material kept in the dark for the same period of time.

6 Materials
6.1 Bacteria used and preparation for tests
6.1.1 Film cover method
a) Staphylococcus aureus
b) Escherichia coli
6.1.2 Glass cover method
a) Staphylococcus aureus
b) Klebsiella pneumoniae
6.1.3 Bacteria preparation

The bacteria strains to be used in the test are equivalent to those described in Table 1 and are stored by

entities that are registered under the World Federation for Culture Collections or the Japan Society for

Culture Collections.

Aseptic manipulations using microorganisms can be performed in an adequate safety cabinet. Inoculate

each strain into a slant culture medium (nutrient agar medium), incubate for 16 h to 24 h at 37 °C ± 1 °C,

and then store in a refrigerator at 5 °C to 10 °C. Repeat subcultures within 1 month by replicating this

process. The maximum number of subcultures from the original strain transferred by culture collection

is 10 times. In the case of bacteria stored in a deep freezer, the maximum number of subcultures from

the original strain transferred by culture collection is 10. The slant culture shall not be used for further

storing after 1 month.
NOTE If necessary, additional tests with other bacteria can be carried out.
Table 1 — Bacteria strains to be used in test
Bacteria species Strain number Organization for the collection
Staphylococcus aureus ATCC 6538P American Type Culture Collection
DSM 346 German Collection of Microorganisms and
Cell Cultures (DSMZ)
NBRC 12732 NITE Biological Resource Center
Escherichia coli ATCC 8739 American Type Culture Collection
DSM 1576 German Collection of Microorganisms and
Cell Cultures (DSMZ)
NBRC 3972 NITE Biological Resource Center
Klebsiella pneumoniae ATCC 4352 American Type Culture Collection
DSM 789 German Collection of Microorganisms and
Cell Cultures (DSMZ)
NBRC 13277 NITE Biological Resource Center
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ISO 27447:2019(E)
6.2 Chemicals and implements
6.2.1 General
Commercial media with the components described as follows may be used.

The volume of prepared media may be adjusted in accordance with the number of specimens.

6.2.2 1/500 nutrient broth

For 1 000 ml of purified water, take 3,0 g meat extract, 10,0 g peptone and 5,0 g sodium chloride, put

them in a flask and dissolve them thoroughly. When the contents are thoroughly dissolved, use a solution

of sodium hydroxide or hydrochloric acid to bring the pH to (7,1 ± 0,1) at 25 °C. Dilute this medium

500 times using purified water, and set the pH to (7,0 ± 0,2) using hydrochloric acid solution or sodium

hydroxide solution. Sterilize in an autoclave at 121 °C ± 2 °C for at least 15 min. After preparation, if

1/500 nutrient broth is not used immediately, store it at 5 °C to 10 °C. Do not use 1/500 nutrient broth

made more than 1 week ago.
6.2.3 Nutrient broth

For 1 000 ml of purified water, take 3,0 g meat extract, 10,0 g peptone and 5,0 g sodium chloride,

put them in a flask and dissolve them thoroughly. When the contents are thoroughly dissolved, use a

solution of sodium hydroxide or hydrochloric acid to bring the pH to (7,1 ± 0,1) at 25 °C. If necessary,

dispense the contents in a test tube, add a cotton plug and sterilize in an autoclave (see 6.2.2). After

preparation, if the nutrient broth is not used immediately, store it at 5 °C to 10 °C. Do not use nutrient

broth made more than 1 month ago.
6.2.4 Nutrient agar

For 1 000 ml of purified water, take 3,0 g meat extract and 5,0 g peptone, put them in a flask and dissolve

them thoroughly. When the contents are thoroughly dissolved, use a solution of sodium hydroxide or

hydrochloric acid to bring the pH to (6,8 ± 0,2) at 25 °C. Add 15,0 g agar powder to this medium and

heat the flask in boiling water to dissolve agar powder thoroughly. Add a cotton plug and sterilize in an

autoclave (see 6.2.2). After preparation, if nutrient agar is not used immediately, store it at 5 °C to 10 °C.

Do not use nutrient agar made more than 1 month ago. Keep
...

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