ISO 18061:2014

Fine Ceramics (Advanced Ceramics, Advanced Technical Ceramics) — Determination of antiviral activity of semiconducting photocatalytic materials — Test method using bacteriophage Q-beta

ISO 18061:2014 specifies a test method for the determination of the antiviral activity of materials that contain photocatalytic materials or have photocatalytic films on the surface, by enumerating the destruction of bacteriophage Q-beta after irradiation of ultraviolet light. It is intended for use with different kinds of semiconducting photocatalytic materials used in construction materials, in flat sheet, board, or plate shape that are the basic forms of materials for various applications. It does not include powder, granular, or porous photocatalytic materials. It is applicable to photocatalytic materials produced for an antiviral application. Other types of performance of photocatalytic materials, i.e. antibacterial activity, antifungal activity, decomposition of water contaminants, self-cleaning, antifogging, and air purification, are not determined by the test method.

Céramiques techniques — Détermination de l'activité antivirale des matériaux photocatalytiques semi-conducteurs — Méthode d'essai utilisant le bactériophage Q-beta

General Information

• Status: Published
• Publication Date: 20-May-2014
• ICS: 81.060.30 - Advanced ceramics
• Technical Committee: ISO/TC 206 - Fine ceramics
• Drafting Committee: ISO/TC 206/WG 9 - Photocatalysis
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 04-Sep-2019

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 18061
First edition
2014-06-01
Fine Ceramics (Advanced Ceramics,
Advanced Technical Ceramics) —
Determination of antiviral activity
of semiconducting photocatalytic
materials — Test method using
bacteriophage Q-beta
Céramiques techniques — Détermination de l’activité antivirale des
matériaux photocatalytiques semi-conducteurs — Méthode d’essai
utilisant le bactériophage Q-beta
Reference number
ISO 18061:2014(E)
©
ISO 2014

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ISO 18061:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Published in Switzerland
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ISO 18061:2014(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 . 3
6.1 Strains and preparation for tests . 3
6.2 Media . 5
7 Apparatus and equipment . 6
7.1 Test equipment . 6
7.2 Cover film . 7
7.3 Moisture preservation glass plate . 7
7.4 Glass tube or glass rod . 7
7.5 Paper filter . 7
7.6 Fluorescent ultraviolet lamp . 8
7.7 UV radiometer . 8
7.8 Punched metal sheet . 8
7.9 Centrifuge . 9
7.10 Sterilized syringe filter unit . 9
8 Test piece . 9
9 Procedure.10
9.1 General .10
9.2 Procedure for preparation of bacteria suspension .10
9.3 Procedure of preparation of test bacteriophage solution .10
9.4 Procedure of test for photocatalytic antiviral activity .11
9.5 UV irradiation condition .11
9.6 Measurement of titre of bacteriophage .12
10 Calculation .13
10.1 General .13
10.2 Calculate titre of bacteriophage .13
10.3 Test requirement fulfilment validation .13
10.4 Photocatalyst antiviral activity value calculation.14
10.5 Antiviral activity value calculation without photocatalyst .15
11 Test report .15
Annex A (informative) Reference data of comparison between influenza virus and
bacteriophage Q-beta .16
Annex B (informative) Comparison of photocatalytic activities determined using ATCC23631-B1
and NBRC20012 .18
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ISO 18061:2014(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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 206, Fine ceramics.
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ISO 18061:2014(E)

Introduction
This International Standard applies to testing the antiviral activity of photocatalytic ceramics and
other materials produced by either coating or mixing of a photocatalyst. The International Standard
for testing the antibacterial activity that use photocatalytic materials has been published as ISO 27447.
The International Standard for testing the antifungal activity that use photocatalytic materials has also
been published as ISO 13125.
The test method for cloths or textiles is not included in this International Standard because of lack of
photocatalytic cloths or textiles with antiviral activity using photocatalytic activity. When photocatalytic
cloths or textiles with antiviral activity using photocatalytic activity have been developed, a test method
for photocatalytic cloths or textiles will be proposed with the glass adhesion method in ISO 27447.
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INTERNATIONAL STANDARD ISO 18061:2014(E)
Fine Ceramics (Advanced Ceramics, Advanced Technical
Ceramics) — Determination of antiviral activity of
semiconducting photocatalytic materials — Test method
using bacteriophage Q-beta
WARNING — Only personnel trained in microbiological techniques should carry out tests.
1 Scope
The test method in this International Standard specifies the determination of the antiviral activity
of materials that contain photocatalytic materials or have photocatalytic films on the surface, by
enumerating the destruction of bacteriophage Q-beta after irradiation of ultraviolet light.
NOTE In this test method, the surrogate microbe is bacteriophage Q-beta, intended as a model for Influenza
viruses.
The test method in this International Standard is intended for use with different kinds of semiconducting
photocatalytic materials used in construction materials, in flat sheet, board, or plate shape that are
the basic forms of materials for various applications. It does not include powder, granular, or porous
photocatalytic materials.
The test method in this International Standard is applicable to photocatalytic materials produced
for an antiviral application. Other types of performance of photocatalytic materials, i.e. antibacterial
activity, antifungal activity, decomposition of water contaminants, self-cleaning, antifogging, and air
purification, are not determined by this test method.
The values expressed in this International Standard are in accordance with the International System of
Units (SI).
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 10677, Fine ceramics (advanced ceramics, advanced technical ceramics) — Ultraviolet light source for
testing semiconducting photocatalytic materials
ISO 27447, Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for antibacterial
activity of 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.
3.1
photocatalyst
substance that carries out many functions based on oxidization and reduction reactions under
photoirradiation, including decomposition and removal of air and water contaminants, deodorization,
and antiviral, antibacterial, antifungal, self-cleaning, and antifogging actions
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ISO 18061:2014(E)

3.2
photocatalytic materials
materials in which, or on which, the photocatalyst is added by coating, impregnation, mixing, etc.
Note 1 to entry: Photocatalytic materials are used for building and road construction materials to obtain the
functions mentioned in 3.1.
3.3
antiviral
condition decreasing the infectivity of viruses on the surface of materials
3.4
bacteriophage
type of virus which infects bacteria
Note 1 to entry: The bacteriophage used in this test method is Q-beta that is one of F-specific RNA phages. The
bacteriophage Q-beta is not pathogenic to humans and animals but serves to simulate Influenza viruses that are
pathogenic to humans.
Note 2 to entry: Example of test results with Influenza virus and bacteriophage Q-beta are given in Annex A.
3.5
plaque
visible, clear area which is theoretically the result of infection and lysis of host cells by a single viable
bacteriophage
3.6
photocatalyst antiviral activity value
difference value between the logarithms of the total number of bacteriophage plaques on photocatalytic
treated materials after UV irradiation and on non-treated materials after UV irradiation
Note 1 to entry: This value includes the decrease of number of bacteriophage plaques without UV irradiation.
3.7
photocatalyst antiviral activity value for UV irradiation
difference between the logarithms of the total number of bacteriophage plaques on photocatalytic
treated materials after UV irradiation and on photocatalytic treated materials kept in the dark
4 Symbols
A average of titre of bacteriophage on non-treated specimens, just after inoculation
B average of titre of bacteriophage on non-treated specimens, after being kept in a dark place
D
B average of titre of bacteriophage on non-treated specimens, after UV irradiation of intensity
L
L
C average of titre of bacteriophage on photocatalytic treated specimens, after being kept in a
D
dark place
C average of titre of bacteriophage on photocatalytic treated specimens, after UV irradiation
L
of intensity L
D dilution factor
F
L UV irradiation intensity
Log maximum logarithmic value of titre of bacteriophage
max
Log average logarithmic value of titre of bacteriophage for three non-treated specimens
mean
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ISO 18061:2014(E)

Log minimum logarithmic value of titre of bacteriophage
min
N titre of bacteriophage (plaque forming unit)
V antiviral activity value without photocatalyst, after being kept in a dark place on a testing
D
material
V photocatalyst antiviral activity value, after irradiation at a constant intensity (L) on a photo-
L
catalytic testing material
ΔV photocatalyst antiviral activity value with UV irradiation
Z average number of plaques in two Petri dishes
5 Principle
This test method is suitable for use in development, comparison, quality assurance, characterization,
reliability, and design data generation of photocatalytic materials. The method is used to obtain the
antiviral activity of photocatalytic materials by the contact of a specimen with bacteriophage under UV
light irradiation. The method is suitable for use with flat sheet, board, or plate-shaped materials.
The specimen of photocatalytic-treated material is inoculated with bacteriophage suspension and
exposed to UV radiation of known intensity for a specified period. Following exposure, the test suspension
is removed and measured by the plaque-forming method with Escherichia coli which is sensitive
to bacteriophage Q-bata. The results obtained are compared with those obtained from inoculated
specimens of non-photocatalytic treated material exposed to UV radiation 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 Strains and preparation for tests
6.1.1 Strains
The strains to be used in the test shall be the same as or equivalent to those described in Table 1 and are
supplied by an entity that is registered under the World Federation for Culture Collections or the Japan
Society for Culture Collections. Aseptic manipulations using microorganisms can be performed in an
appropriate safety cabinet.
Table 1 — Bacteriophage and bacteria strains to be used in test
Species Strain number Organization for the collection
Bacteriophage Q-beta ATCC 23631-B1 American Type Culture Collection
German Collection of Microorganisms
DSM 13768
and Cell Cultures (DSMZ)
NBRC20012 NITE Biological Resource Center
Escherichia coli ATCC 23631 American Type Culture Collection
German Collection of Microorganisms
DSM 5210
and Cell Cultures (DSMZ)
NBRC 106373 NITE Biological Resource Center
NOTE ATCC23631-B1 and NBRC20012 are not strictly the same but they are from the same origin and their
photocatalytic activity effects is equivalent, as shown in Annex B.
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ISO 18061:2014(E)

6.1.2 Bacteria preparation
a) Inoculate E. coli strain into a slant culture medium [6 ml to 10 ml of LB agar (see 6.2.6)], incubate for
16 h to 24 h at (37 ± 1) °C, and then store in refrigerator at 5 °C to 10 °C.
b) Repeat subcultures within 1 month by replicating this process.
c) The slant culture must not be used for further storing after 1 month.
d) The maximum number of subcultures from the original strain transferred by culture collection is
10.
NOTE 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.
6.1.3 Bacteriophage preparation
a) Introduce 25 ml of LB broth with calcium (see 6.2.4) into a conical flask of 300 ml and inoculate with
E. coli strain.
−1 −1
b) Incubate for 18 h ± 2 h at (37 ± 1) °C while shaking at 110 min ± 10 min .
c) Pre-warm 25 ml of LB broth with calcium in a 300 ml conical flask to 35 °C to 37 °C and inoculate
with 0,025 ml of the culture prepared under item b).
8
d) Incubate as above condition until a bacterial concentration will be reached at 2,0 ± 1,0 × 10 cfu/ml.
This procedure should be carried out several times to establish the relationship between turbidity
measurements and colony counts. If sufficient data have been obtained, further work can be based
on turbidity measurements only.
e) Inoculate the bacterial culture with Q-beta from a stock solution to a final concentration of
7
pfu (plaque forming unit)/ml [multiplicity of infection (m.o.i.) is approximately
approximately 2 × 10
0,1].
f) Incubate the inoculated bacterial culture for 4 h as under b).
g) Store the culture overnight at 4 °C ± 2 °C.
h) Pour the culture into centrifuge tubes and centrifuge for 20 min at 4 °C ± 2 °C at 10 000 g.
i) Pipette the supernatant carefully to a sterilized tube.
j) Filter bacteriophage containing supernatant suspension through a sterilized syringe filter unit to
purify the bacteriophage solution.
k) Determine the titre of the bacteriophage stock solution and store at 4 °C ± 2 °C.
l) To check bacterial contamination, mix 1 ml of the bacteriophage stock solution with LB agar (see
6.2.6) and incubate for 24 h at (37 ± 1) °C. Discard the bacteriophage stock solution if any colonies
are detected.
10
m) Do not use the bacteriophage stock solution with less than 1,0 × 10 pfu/ml or contaminated stock
solution.
11 13
NOTE 2 The titre of the phage suspension should be above 1,0 × 10 pfu/ml and might reach up to 1,0 × 10
pfu/ml.
NOTE 3 The titre of the phage stock suspension will slowly decrease over time.
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ISO 18061:2014(E)

6.2 Media
6.2.1 General
Commercial media of same components described below can be used.
Volume of prepared media can be adjusted in accordance with the number of specimens.
6.2.2 1/500 Nutrient broth (1/500 NB)
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 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 by 500 times using purified water, and set the pH to (7,0 ± 0,2) at 25 °C 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 at 5 °C to 10 °C. Do not use 1/500
nutrient broth made more than a week ago.
6.2.3 Calcium solution
For 100 ml of purified water, take 3,0 g of calcium chloride dihydrate, put it in a flask, and dissolve it
thoroughly. Add a cotton plug and sterilize in an autoclave (see 6.2.2). After preparation, if calcium
solution is not used immediately, store at 5 °C to 10 °C. Do not use the calcium solution made more than
6 months ago.
6.2.4 LB broth with calcium
For 1 000 ml of purified water, take 10,0 g of peptone, 5,0 g of yeast extract, and 10,0 g of 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 (7,0 ± 0,2) at 25 °C. Add a cotton
plug and sterilize in an autoclave (see 6.2.2). After autoclaving, add 10 ml of calcium solution to medium
and mix well. After preparation, if LB broth with calcium is not used immediately, store at 5 °C to 10 °C.
Do not use the broth made more than 1 month ago.
6.2.5 Agar powder
2 2
Use agar powder for which the gel strength of 1,5 % agar is from 400 g/cm to 600 g/cm .
6.2.6 LB agar
For 1 000 ml of purified water, take 10,0 g of peptone, 5,0 g of yeast extract, and 10,0 g of sodium
chloride and 10,0 g of agar powder (see 6.2.5), 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 (7,0 ± 0,2) at 25 °C. Add a cotton plug and sterilize in an autoclave (see 6.2.2). After preparation, if
nutrient agar is not used immediately, store at 5 °C to 10 °C. Do not use nutrient agar made more than
1 month ago.
6.2.7 Bottom agar plate (LB agar plate with calcium)
For 1 000 ml of purified water, take 10,0 g of peptone, 5,0 g of yeast extract, and 10,0 g of sodium
chloride and 15,0 g of agar powder (see 6.2.5), 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 (7,0 ± 0,2) at 25 °C. Add a cotton plug and sterilize in an autoclave (see 6.2.2). After autoclaving, add
10 ml of calcium solution to medium and mix well. After preparation, pour 15 ml to 20 ml of medium into
90 mm diameter Petri dish, store at 5 °C to 10 °C. Do not use nutrient agar made more than 2 weeks ago.
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ISO 18061:2014(E)

6.2.8 Top agar
For 1 000 ml of purified water, take 15,0 g of peptone, 7,5 g of yeast extract, and 15,0 g of sodium
chloride and 10,0 g of agar powder (see 6.2.5), 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 (7,0 ± 0,2) at 25 °C. Add a cotton plug and sterilize in an autoclave (see 6.2.2). After autoclaving, add
15 ml of calcium solution to medium and mix well. After preparation, if top agar is not used immediately,
store at 5 °C to 10 °C. Do not use nutrient agar made more than 1 month ago.
NOTE When the top agar is remelted, heat the flask in boiling water, but not autoclaving.
6.2.9 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 dipotassium hydrogenphosphate, 2,5 g of glucose, 1,0 g of lecithin, put them into 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 of (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 6.2.2). After preparation, if SCDLP is not used immediately,
store at 5 °C to 10 °C. Do not use SCDLP medium made more than 1 month ago.
6.2.10 Peptone saline solution
For 1 000 ml of purified water, take 10,0 g of peptone and 8,5 g of sodium chloride, put them into 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 (7,0 ± 0,1) at 25 °C. If necessary, dispense it in a test
tube and sterilize in an autoclave (see 6.2.2). After preparation, if peptone saline solution is not used
immediately, store at 5 °C to 10 °C. Do not use peptone saline solution made more than 1 month ago.
7 Apparatus and equipment
7.1 Test equipment
The test equipment enables a photocatalytic material to be examined for its antiviral activity by
providing UV irradiation to activate the photocatalyst. It consists of a light source and the chamber with
a test piece. A schematic of a testing equipment is shown in Figure 1.
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ISO 18061:2014(E)

1
2
4
3
8
6
5
7
Key
1 light source
2 punched metal sheet
3 cover film
4 moisture preservation glass
5 Petri dish
6 glass tube or glass rod
7 paper filter
8 test piece
Figure 1 — Schematic of the testing equipment
7.2 Cover film
The cover film shall be used as specified in ISO 27447, 7.1. The sheets should be cut in a
(40 ± 2) mm × (40 ± 2) mm square. In case of irregularly sized test piece [see Clause 8,item b), NOTE 1],
prepare cover film with the same shape as the test piece but with dimensions 2,5 mm to 5 mm smaller
than those of the test piece.
NOTE Reference data for cover films is given in ISO 27447, Annex B.
7.3 Moisture preservation glass plate
The moisture preservation glass plate shall be used as specified in ISO 27447, 7.2. The dimensions of the
glass plates should be sufficient to fully cover Petri dishes.
NOTE Reference data for moisture preservation glasses is given in ISO 27447, Annex B.
7.4 Glass tube or glass rod
The glass tube or rod in an approximately 4 mm to 6 mm diameter should be prepared by cutting it to
10 cm to 15 cm in length and bending it into a U- or V-shape.
NOTE The glass tube or glass rod supports the test piece in the Petri dish.
7.5 Paper filter
The cellulose paper filter should be prepared by cutting it to approximately 85 mm in diameter.
NOTE One to four pieces of round-shaped cellulose paper filters per Petri dish are needed, depending on the
amount of water absorption.
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ISO 18061:2014(E)

7.6 Fluorescent ultraviolet lamp
The fl
...