ISO 27447:2009

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

ISO 27447:2009 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. ISO 27447:2009 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 an antibacterial effect. Other types of performance of photocatalytic materials, i.e. decomposition of water contaminants, self-cleaning, antifogging and air purification, are not determined by this method. The values expressed in ISO 27447:2009 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• Status: Withdrawn
• Publication Date: 27-May-2009
• Withdrawal Date: 27-May-2009
• ICS: 81.060.30 - Advanced ceramics
• Technical Committee: ISO/TC 206 - Fine ceramics
• Drafting Committee: ISO/TC 206/WG 9 - Photocatalysis
• Current Stage: 9599 - Withdrawal of International Standard
• Completion Date: 17-Jul-2019

Relations

• Revised: ISO 27447:2019 - Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for antibacterial activity of semiconducting photocatalytic materials
• Effective Date: 05-Nov-2015

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 27447
First edition
2009-06-01
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 27447:2009(E)
ISO 2009
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ISO 27447:2009(E)
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ii © ISO 2009 – All rights reserved
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ISO 27447:2009(E)
Contents Page

Foreword ............................................................................................................................................................iv

Introduction.........................................................................................................................................................v

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

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

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

7.1 Adhesive film.........................................................................................................................................6

7.2 Adhesive glass.......................................................................................................................................6

7.3 Moisture preservation glass.................................................................................................................6

7.4 Glass tube or glass rod.........................................................................................................................7

7.5 Black light fluorescent lamp.................................................................................................................7

7.6 Ultraviolet light radiation meter ...........................................................................................................7

7.7 Punched metal sheet.............................................................................................................................7

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

8.1 Film adhesion method ..........................................................................................................................8

8.2 Glass adhesion method ........................................................................................................................8

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

9.1 Film adhesion method ........................................................................................................................10

9.2 Glass adhesion method ......................................................................................................................11

9.3 UV irradiation condition......................................................................................................................12

9.4 Measurement of number of living bacteria .......................................................................................12

10 Calculation...........................................................................................................................................13

10.1 Film adhesion method ........................................................................................................................13

10.2 Glass adhesion method ......................................................................................................................14

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

Annex A (informative) Examples of test results ............................................................................................17

Annex B (informative) Reference data for adhesive films and adhesive glasses ......................................19

Annex C (informative) Reference data for damage of ultraviolet irradiation to bacteria...........................21

Bibliography......................................................................................................................................................23

© ISO 2009 – All rights reserved iii
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ISO 27447:2009(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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

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.

ISO 27447 was prepared by Technical Committee ISO/TC 206, Fine ceramics.
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ISO 27447:2009(E)
Introduction

As a result of continuing efforts to provide test methods for photocatalytic materials, this International

Standard was developed for antibacterial activity. However, for test pieces with permeable or rough surfaces,

etc., the antibacterial activity cannot be measured, 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 the photocatalyst (References [10] and [11] in the Bibliography). 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, paper, etc. 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 and 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 required to provide different evaluation methods for air purification,

water decontamination and self-cleaning.

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

materials produced by either the coating or 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 27447:2009(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 International Standard 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 International Standard 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 an antibacterial effect. Other

types of performance of photocatalytic materials, i.e. decomposition of water contaminants, self-cleaning,

antifogging and air purification, are not determined by this method.

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

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 31-0, Quantities and units — Part 0: General principles
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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, self-cleaning and antifogging actions
3.2
photocatalytic materials

materials in which, or on which, the photocatalyst is added by coating, impregnation, mixing, etc.

NOTE Photocatalytic materials are to be used for building and road construction materials to obtain the functions

mentioned in 3.1.
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ISO 27447:2009(E)
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 adhesion method

difference between the total number of viable bacteria of photocatalytic treated flat surface materials and non-

treated materials after UV irradiation

NOTE This value includes the decrease of the number of bacteria without UV irradiation.

3.5
photocatalyst antibacterial activity value for glass adhesion method

difference between the total number of viable bacteria of photocatalytic treated cloths and standard cloths

after UV irradiation

NOTE This value includes the decrease of the number of bacteria without UV irradiation.

3.6

photocatalyst antibacterial activity value with UV irradiation for film adhesion method

difference between the total number of viable bacteria of photocatalytic treated flat surface materials after UV

irradiation and photocatalytic treated flat surface materials in a dark place
3.7

photocatalyst antibacterial activity value with UV irradiation for glass adhesion method

difference between the total number of viable bacteria of photocatalytic treated cloths after UV irradiation and

photocatalytic treated cloths in a dark place
3.8
film adhesion method

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

3.9
glass adhesion method
test method to evaluate the antibacterial performance of photocatalytic cloths
3.10
fluorescent UV lamp

lamp that provides UV-A irradiation within a wavelength range of 300 nm to 400 nm

NOTE A suitable lamp is the so-called black light blue (BLB) fluorescent lamp, with a maximum at 351 nm, as

described in ISO 4892-3.
4 Symbols

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

B average number of viable bacteria of non-treated specimens, after being kept in a dark place

B average number of viable bacteria of non-treated specimens, after UV irradiation of intensity L

C average number of viable bacteria of photocatalytic treated specimens, after being kept in a dark place

C average number of viable bacteria of photocatalytic treated specimens, after UV irradiation of intensity L

F growth value, after being kept in a dark place
F growth value, after UV irradiation of intensity L
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ISO 27447:2009(E)
L UV irradiation intensity
maximum logarithmic value of viable bacteria
max
L average logarithmic value of viable bacteria for 3 specimens
mean
L minimum logarithmic value of viable bacteria
min
M number of viable bacteria with glass adhesion method

M average logarithmic value of the number of viable bacteria for 3 non-treated specimens, just after

inoculation

M average logarithmic value of the number of viable bacteria for 3 non-treated specimens, after being

kept in a dark place

M average logarithmic value of the number of viable bacteria for 3 non-treated specimens, after UV

irradiation of intensity L

M average logarithmic value of the number of viable bacteria for 3 photocatalytic treated specimens, after

being kept in a dark place

M average logarithmic value of the number of viable bacteria for 3 photocatalytic treated specimens, after

UV irradiation of intensity L
N number of viable bacteria with film adhesion method
P bacteria concentration
R dilution factor

R photocatalyst antibacterial activity value, after irradiation at a constant intensity (L) on a photocatalytic

material
∆R photocatalyst antibacterial activity value with UV irradiation

S photocatalyst antibacterial activity value, after UV irradiation of intensity L

∆S photocatalyst antibacterial activity value with UV irradiation

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

Z average number of colonies in 2 Petri dishes
5 Principle

This International Standard 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

adhesion method is available for flat sheet, board or plate-shaped materials. To avoid warpage in the cloths or

textiles, the glass adhesion method is available for cloths or textiles.

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

adhesive 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 adhesive film or glass. This washout suspension is measured by the

viable bacterial count method.
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ISO 27447:2009(E)
6 Materials
6.1 Bacteria used and preparation for tests
6.1.1 Film adhesion method
a) Staphylococcus aureus
b) Escherichia coli
6.1.2 Glass adhesion 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. The slant

culture must not be used for further storage after 1 month.

NOTE 1 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.
NOTE 2 If necessary, additional tests with other bacteria can be allowed.
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 27447:2009(E)
6.2 Chemicals and implements
6.2.1 1/500 nutrient broth

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

them in a flask and dissolve them thoroughly. When the contents are thoroughly diluted, 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 month

ago.
6.2.2 Nutrient broth

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

them in a flask and dissolve them thoroughly. When the contents are thoroughly diluted, 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 6.2.1). After preparation, if 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.
6.2.3 Nutrient agar

For 1 000 ml of purified water, take 3,0 g of meat extract, 5,0 g of peptone and 15,0 g of agar powder, put

them in a flask and mix. Heat the flask in boiling water to dissolve the contents thoroughly. Use a 0,1 mol/l

solution of sodium hydroxide to bring the pH to (6,8 ± 0,2) at 25 °C. Add a cotton plug and sterilize in an

autoclave (see 6.2.1). 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 the medium temperature between 45 °C and 48 °C

when mixing with a bacterial suspension.
6.2.4 Soybean-casein digest broth with lecithin and polysorbate 80 (SCDLP)

For 1 000 ml of purified water, take 17,0 g of casein peptone, 3,0 g of soybean peptone, 5,0 g of sodium

chloride, 2,5 g of phosphoric acid monopotassium dehydrogenate, 2,5 g of glucose and 1,0 g of lecithin, put

them in a flask and dissolve them. Add 7,0 g of non-ionic surfactant and dissolve it. Use a solution of sodium

hydroxide or hydrochloric acid to bring the pH to (7,0 ± 0,2) at 25 °C. If necessary, dispense it in a test tube,

add a cotton plug and sterilize in an autoclave (see 6.2.1). After preparation, if SCDLP is not used immediately,

store it at 5 °C to 10 °C. Do not use SCDLP medium made more than 1 month ago.
6.2.5 Physiological saline solution

For 1 000 ml of purified water, take 8,5 g of sodium chloride, put it in a flask and dissolve it thoroughly. If

necessary, dispense it in a test tube and sterilize in an autoclave (see 6.2.1). After preparation, if physiological

saline solution is not used immediately, store it at 5 °C to 10 °C. Do not use physiological saline solution made

more than 1 month ago.
6.2.6 Physiological saline solution for washout

For 1 000 ml of purified water, take 8,5 g of sodium chloride, put it in a flask and dissolve it thoroughly. Add

2,0 g of non-ionic surfactant and dilute. If necessary, dispense 20 ml of the solution in a test tube or

Erlenmeyer flask and sterilize in an autoclave (see 6.2.1). After preparation, if physiological saline solution for

washout is not used immediately, store it at 5 °C to 10 °C. Do not use physiological saline solution made more

than 1 month ago for washout.
6.2.7 Non-ionic surfactant
Polyoxyethylene sorbitan monooleate (polysorbate 80).
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ISO 27447:2009(E)
7 Apparatus

The test equipment enables a photocatalytic material to be examined for its antibacterial activity by providing

UV irradiation to activate the photocatalyst. It consists of a light source and a chamber with a test piece. An

example of a test system is shown in Figure 1.
Key
1 light source
2 punched metal
3 glass stick
4 paper filter
5 test piece
6 adhesive film or glass
7 moisture preservation glass
Figure 1 — Schematic diagram of the test equipment
7.1 Adhesive film

The adhesive film is inert and non-water absorbent with good sealing properties, with a transparency rate over

85 % for the 340 nm to 380 nm range. The sheets are cut with dimensions of (40 ± 2) mm.

NOTE Reference data for adhesive films is given in Annex B.
7.2 Adhesive glass

The adhesive glass consists of glass panes with a thickness less than or equal to 1,1 mm, with a transparency

rate over 85 % for the 340 nm to 380 nm range. The panes are cut with dimensions of (40 ± 2) mm.

NOTE Reference data for adhesive glasses is given in Annex B.
7.3 Moisture preservation glass

The moisture preservation glass consists of glass panes with a thickness less than or equal to 1,1 mm, with a

transparency rate over 85 % for the 340 nm to 380 nm range. The panes are cut to fully cover Petri dishes.

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ISO 27447:2009(E)
7.4 Glass tube or glass rod

The glass tube or glass rod is prepared by cutting a tube or rod to a 10 cm to 15 cm length and bending it into

a U-shape or V-shape.
7.5 Black light fluorescent lamp

The fluorescent lamp shall be a BLB (black light blue) lamp that has a peak wavelength of 351 nm with blue

glass for absorbing visible light.
7.6 Ultraviolet light radiation meter

The irradiation intensity shall be measurable at the test sample position. The UV radiation meter shall be

calibrated for the light source to be used or corrected to ascertain sensitivity within the wavelength range to be

absorbed by the photocatalytic test piece.
7.7 Punched metal sheet

When the prescribed intensity cannot be obtained by tuning the light source height, attenuate the intensity by

using a punched metal sheet (see Figures 2 and 3) directly below the lamp.
Dimensions in millimetres
Key
1 lamp position
2 bore (diameter approximately 5 to 15)
Figure 2 — Punched metal sheet for 0,01 mW/cm light intensity
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ISO 27447:2009(E)
Dimensions in millimetres
Key
1 lamp position
2 bore (diameter approximately 5 to 15)
Figure 3 — Punched metal sheet for 0,001 mW/cm light intensity
8 Test piece
8.1 Film adhesion method

Cut a flat portion of the material in a (50 ± 2) mm × (50 ± 2) mm square. The materials should be up to 10 mm

in thickness. Use it as the standardized shaped specimen. Prepare 9 pieces of non-treated specimens and

6 pieces of photocatalytic treated specimens. When non-treated specimens cannot be provided, use glass

panes instead. Take great care to avoid microbial contamination and cross-contamination among specimens.

NOTE When it is difficult or impossible to cut (50 ± 2) mm long (up to 10 mm thickness) squares, it is acceptable to

2 2

use a different specimen size as long as the specimen surface can be covered with a 400 mm to 1 600 mm film. When

the specimen surface is stained with organic contaminant, it is acceptable to first eliminate the contaminant by exposure to

a 1,0 mW/cm light source within the limit of 24 h. If necessary, specimens can be disinfected prior to testing (e.g. by

wiping with ethanol or 70 % ethanol in water).
8.2 Glass adhesion method

Cut the material into (50 ± 2) mm × (50 ± 2) mm squares and use them as specimens. Prepare 9 pieces of

standard cloth and 6 pieces of photocatalytic treated specimens. Take great care to avoid microbial

contamination and cross-contamination among specimens.

Put each of the specimens in a glass Petri dish. Put the dishes in a wire-mesh basket, cover the upper part

with aluminum foil and sterilize them in an autoclave. After autoclaving, take off the aluminum foil, move the

cover of the dishes to a clean bench and dry the specimens for about 60 min.
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ISO 27447:2009(E)
9 Procedure
The flowcharts of test methods are shown in Figures 4 and 5.
Figure 4 — Flowchart of film adhesion method
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ISO 27447:2009(E)
Figure 5 — Flowchart of glass adhesion method
9.1 Film adhesion method

9.1.1 Transfer the stored bacteria to the nutrient agar slant using a platinum loop and incubate at

(37 ± 1) °C for 16 h to 24 h. Transfer the bacteria to a new nutrient agar slant and incubate at (37 ± 1) °C for

16 h to 20 h. Uniformly disperse a small quantity of test bacteria in 1/500 NB with a platinum loop, and

measure the bacteria count using the optical microscope observation method or any other adequate method.

Suitably dilute this bacteria suspension with 1/500 NB to obtain a count of 6,7 × 10 cells/ml to

2,6 × 10 cells/ml and use the result as the bacterial suspension for the test. If the test bacteria suspension is

not to be used immediately, store it at 0 °C and use it within 4 h.

9.1.2 Lay a sterilized moisture control paper filter in the bottom of a sterilized Petri dish, add an adequate

quantity of sterilized water, intercalate a glass tube or glass rod in order to avoid contact between the test

piece and the paper filter, and place the test piece on it wit
...