ISO 27447:2009
(Main)Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for antibacterial activity of semiconducting photocatalytic materials
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
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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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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
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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 ).
2
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
D
B average number of viable bacteria of non-treated specimens, after UV irradiation of intensity L
L
C average number of viable bacteria of photocatalytic treated specimens, after being kept in a dark place
D
C average number of viable bacteria of photocatalytic treated specimens, after UV irradiation of intensity L
L
F growth value, after being kept in a dark place
BD
F growth value, after UV irradiation of intensity L
BL
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ISO 27447:2009(E)
L UV irradiation intensity
maximum logarithmic value of viable bacteria
L
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
BA
inoculation
M average logarithmic value of the number of viable bacteria for 3 non-treated specimens, after being
BD
kept in a dark place
M average logarithmic value of the number of viable bacteria for 3 non-treated specimens, after UV
BL
irradiation of intensity L
M average logarithmic value of the number of viable bacteria for 3 photocatalytic treated specimens, after
D
being kept in a dark place
M average logarithmic value of the number of viable bacteria for 3 photocatalytic treated specimens, after
L
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
L
material
∆R photocatalyst antibacterial activity value with UV irradiation
S photocatalyst antibacterial activity value, after UV irradiation of intensity L
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)
2
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)
2
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
2
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.
5
Suitably dilute this bacteria suspension with 1/500 NB to obtain a count of 6,7 × 10 cells/ml to
6
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
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