ISO 18071:2016
(Main)Fine ceramics (advanced ceramics, advanced technical ceramics) — Determination of antiviral activity of semiconducting photocatalytic materials under indoor lighting environment — Test method using bacteriophage Q-beta
Fine ceramics (advanced ceramics, advanced technical ceramics) — Determination of antiviral activity of semiconducting photocatalytic materials under indoor lighting environment — Test method using bacteriophage Q-beta
ISO 18071:2016 specifies the determination of the antiviral activity of materials that contain indoor-light-active photocatalytic materials or have indoor-light-active photocatalytic films on the surface by a test method that measures the infectivity titre of bacteriophage Q-beta after illumination with indoor light. NOTE In the test method, the surrogate microbe is bacteriophage Q-beta, intended as a model for influenza viruses. It is intended for use with different kinds of indoor-light-active 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 indoor-light-active photocatalytic materials. It is applicable to indoor-light-active photocatalytic materials produced for an antiviral applications. Other types of performance of indoor-light-active photocatalytic materials, i.e. antibacterial activity, antifungal activity, decomposition of water contaminants, self-cleaning, antifogging and air purification, are not determined by this method.
Céramiques techniques — Détermination de l'activité antivirale des matériaux photocatalytiques semi-conducteurs dans un environnement d'éclairage intérieur — Méthode d'essai utilisant un bactériophage Q-béta
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INTERNATIONAL ISO
STANDARD 18071
First edition
2016-07-15
Fine ceramics (advanced ceramics,
advanced technical ceramics) —
Determination of antiviral activity
of semiconducting photocatalytic
materials under indoor lighting
environment — Test method using
bacteriophage Q-beta
Céramiques techniques — Détermination de l’activité antivirale
des matériaux photocatalytiques semi-conducteurs dans un
environnement d’éclairage intérieur — Méthode d’essai utilisant un
bactériophage Q-béta
Reference number
ISO 18071:2016(E)
ISO 2016
---------------------- Page: 1 ----------------------
ISO 18071:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, 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.ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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ISO 18071:2016(E)
Contents Page
Foreword ..........................................................................................................................................................................................................................................v
Introduction ................................................................................................................................................................................................................................vi
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
6.1.1 Strains ....................................................................................................................................................................................... 3
6.1.2 Bacteria preparation..................................................................................................................................................... 4
6.1.3 Bacteriophage preparation ..................................................................................................................................... 4
6.2 Media ............................................................................................................................................................................................................... 5
6.2.1 General...................................................................................................................................................................................... 5
6.2.2 1/500 Nutrient broth (1/500 NB) .................................................................................................................... 5
6.2.3 Calcium solution ............................................................................................................................................................... 5
6.2.4 LB broth with calcium ................................................................................................................................................. 5
6.2.5 Agar powder ........................................................................................................................................................................ 5
6.2.6 LB agar ...................................................................................................................................................................................... 5
6.2.7 Bottom agar plate (LB agar plate with calcium) ................................................................................... 6
6.2.8 Top agar ................................................................................................................................................................................... 6
6.2.9 Soybean-casein digest broth with lecithin and polysorbate 80 (SCDLP)........................ 6
6.2.10 Peptone saline solution .............................................................................................................................................. 6
7 Apparatus and equipment .......................................................................................................................................................................... 6
7.1 Test equipment ....................................................................................................................................................................................... 6
7.2 Cover film ..................................................................................................................................................................................................... 7
7.3 Moisture preservation glass ........................................................................................................................................................ 7
7.4 Glass tube or glass rod ...................................................................................................................................................................... 7
7.5 Paper filter .................................................................................................................................................................................................. 7
7.6 Light source ............................................................................................................................................................................................... 7
7.7 UV sharp cut-off filter ........................................................................................................................................................................ 8
7.7.1 Condition A (under 400 nm cut-off condition) ...................................................................................... 8
7.7.2 Condition B (under 380 nm cut-off condition) ...................................................................................... 8
7.8 Illuminance meter ................................................................................................................................................................................ 8
7.9 Centrifuge .................................................................................................................................................................................................... 8
7.10 Sterilized syringe filter unit ......................................................................................................................................................... 8
8 Test piece ...................................................................................................................................................................................................................... 8
9 Procedure..................................................................................................................................................................................................................... 8
9.1 General ........................................................................................................................................................................................................... 8
9.2 Procedure for preparation of bacteria suspension .................................................................................................. 9
9.3 Procedure of preparation of test bacteriophage solution .................................................................................. 9
9.4 Procedure of test for indoor-light-active photocatalytic antiviral activity .......................................10
9.5 Indoor lighting condition ............................................................................................................................................................10
9.6 Measurement of titre of bacteriophage ..........................................................................................................................11
10 Calculation ...............................................................................................................................................................................................................12
10.1 General ........................................................................................................................................................................................................12
10.2 Test requirement fulfilment validation ...........................................................................................................................12
10.3 Indoor-light-active photocatalyst antiviral activity value calculation ..................................................13
10.4 Antiviral activity value calculation without indoor-light-active photocatalyst ............................13
11 Test report ................................................................................................................................................................................................................14
© ISO 2016 – All rights reserved iii---------------------- Page: 3 ----------------------
ISO 18071:2016(E)
Bibliography .............................................................................................................................................................................................................................15
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ISO 18071:2016(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 World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 206, Fine ceramics.© ISO 2016 – All rights reserved v
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ISO 18071:2016(E)
Introduction
This International Standard applies to testing the antiviral activity of indoor-light-active photocatalytic
ceramics and other materials produced by either coating or mixing of a light-active photocatalyst.
The International Standard for testing the antibacterial activity of photocatalytic materials has been
published as ISO 27447 and the International Standard for testing the antibacterial activity of indoor-
light-active photocatalytic materials has been published as ISO 17094. The International Standard for
determination of antiviral activity of semiconducting photocatalytic materials has also been published
as ISO 18061.The test method for cloths or textiles is not included in this International Standard because of lack of
indoor-light-active photocatalytic cloths or textiles. When the indoor-light-active photocatalytic cloths
or textiles with antiviral activity using indoor-light-active photocatalytic activity have been developed,
a test method for indoor-light-active photocatalytic cloths or textiles will be proposed with the glass
adhesion method in ISO 27447.vi © ISO 2016 – All rights reserved
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INTERNATIONAL STANDARD ISO 18071:2016(E)
Fine ceramics (advanced ceramics, advanced technical
ceramics) — Determination of antiviral activity of
semiconducting photocatalytic materials under
indoor lighting environment — Test method using
bacteriophage Q-beta
WARNING — Only personnel trained in microbiological techniques should carry out tests.
1 ScopeThis International Standard specifies the determination of the antiviral activity of materials that
contain indoor-light-active photocatalytic materials or have indoor-light-active photocatalytic films
on the surface by a test method that measures the infectivity titre of bacteriophage Q-beta after
illumination with indoor light.NOTE In the test method, the surrogate microbe is bacteriophage Q-beta, intended as a model for influenza
viruses.This International Standard is intended for use with different kinds of indoor-light-active 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 indoor-light-active
photocatalytic materials.This International Standard is applicable to indoor-light-active photocatalytic materials produced for
an antiviral applications. Other types of performance of indoor-light-active photocatalytic materials,
i.e. antibacterial activity, antifungal activity, decomposition of water contaminants, self-cleaning,
antifogging and air purification, are not determined by this method.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 14605, Fine ceramics (advanced ceramics, advanced technical ceramics) — Light source for testing
semiconducting photocatalytic materials used under indoor lighting environmentISO 27447, Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for antibacterial
activity of semiconducting photocatalytic materialsISO 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© ISO 2016 – All rights reserved 1
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ISO 18071:2016(E)
3.2
indoor-light-active photocatalyst
substance that reacts with artificial light source for general lighting service (i.e. indoor lighting
environment)3.3
indoor lighting environment
environment with artificial light source for general lighting service
Note 1 to entry: Does not include sunlight.
3.4
indoor-light-active photocatalytic materials
materials in which or on which the indoor-light-active photocatalyst is added by coating, impregnation,
mixing, etc.3.5
antiviral
condition decreasing the infectivity of viruses on the surface of materials
3.6
bacteriophage
type of virus which infects bacteria
Note 1 to entry: The bacteriophage used in this International Standard 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.3.7
plaque
visible, clear area which is theoretically the result of infection and lysis of host cells by a single viable
bacteriophage3.8
indoor-light-active photocatalyst antiviral activity value
difference between the logarithms of the total number of bacteriophage plaques on photocatalytic
treated materials after indoor light illumination and on non-treated materials after indoor light
illuminationNote 1 to entry: This value includes the decrease of number of bacteriophage plaques without indoor light
illumination.3.9
indoor-light-active photocatalyst antiviral activity value for indoor light illumination
difference between the logarithms of the total number of bacteriophage plaques on photocatalytic treated
materials after indoor light illumination and on photocatalytic treated materials kept in a dark place
4 SymbolsA 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
B average of titre of bacteriophage on non-treated specimens, after indoor light illumination
F-Lof intensity L under condition F
C average of titre of bacteriophage on indoor-light-active photocatalytic treated specimens,
after being kept in a dark place2 © ISO 2016 – All rights reserved
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ISO 18071:2016(E)
C average of titre of bacteriophage on indoor-light-active photocatalytic treated specimens,
F-Lafter indoor light illumination of intensity L under condition F
D dilution factor
F type of UV cut-off condition (condition A or condition B)
L illuminance of indoor light
Logmax maximum logarithmic value of titre of bacteriophage
Logmean average logarithmic value of titre of bacteriophage for three non-treated specimens
Logmin minimum logarithmic value of titre of bacteriophageN titre of bacteriophage (plaque forming unit)
V antiviral activity value without indoor-light-active photocatalyst, after being kept in a dark
place on a testing materialV indoor-light-active photocatalyst antiviral activity value, after indoor light illumination at
F-La constant intensity (F-L) on an indoor-light-active photocatalytic material
ΔV indoor-light-active photocatalyst antiviral activity value with indoor light illumination
Z average number of plaques in two Petri dishes5 Principle
The test method is suitable for use in development, comparison, quality assurance, characterization,
reliability and design data generation of indoor-light-active photocatalytic materials. The method is
used to obtain the antiviral activity of indoor-light-active photocatalytic materials by the contact of a
specimen with bacteriophage under indoor lighting condition. The method is suitable for use with flat
sheet, board or plate-shaped materials.The specimen of indoor-light-active photocatalytic treated material is inoculated with bacteriophage
suspension and exposed to light 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-beta. The results obtained are compared with those obtained from inoculated
specimens of non-photocatalytic treated material exposed to light 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.NOTE This International Standard is adapted from the common methodological concept for ISO 18061.
Namely, the same apparatus without light source (see 7.6), UV sharp cut-off filter (see 7.7), and test piece size,
similar procedure and calculation are adapted between this International Standard and ISO 18061. Therefore,
ISO 18061 is recommended to be used as reference during actual test of this International Standard.
6 Materials6.1 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
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.© ISO 2016 – All rights reserved 3
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ISO 18071:2016(E)
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 and
DSM 13768
Cell Cultures (DSMZ)
NBRC 20012 NITE Biological Resource Center
Escherichia coli ATCC 23631 American Type Culture Collection
German Collection of Microorganisms and
DSM 5210
Cell Cultures (DSMZ)
NBRC 106373 NITE Biological Resource Center
NOTE ATCC23631-B1 and NBRC20012 are not strictly same, but they are from the same origin.
6.1.2 Bacteria preparationa) 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 the refrigerator at 5 °C to 10 °C.b) Repeat subcultures within 1 month by replicating this process.
c) The slant culture shall 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 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.b) Incubate for (18 ± 2) h at (37 ± 1) °C while shaking at (110 ± 10) min .
c) Pre-warm 25 ml of LB broth with calcium (see 6.2.4) in a 300 ml conical flask to 35 °C to 37 °C and
inoculate with 0,025 ml of the culture prepared under b).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 approximately 2 × 10 pfu (plaque forming unit)/ml [multiplicity of infection (m.o.i.) is
approximately 0,1].f) Incubate the inoculated bacterial culture for 4 h as under b).
g) Store the culture overnight at (4 ± 2) °C.
h) Pour the culture into centrifuge tubes and centrifuge for 20 min at (4 ± 2) °C at 10 000g.
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 (see 9.6) and store at (4 ± 2) °C.
4 © ISO 2016 – All rights reserved---------------------- Page: 10 ----------------------
ISO 18071:2016(E)
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.m) Do not use the bacteriophage stock solution with less than 1,0 × 10 pfu/ml or contaminated stock
solution.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 The titre of the phage stock suspension will slowly decrease over time.
6.2 Media
6.2.1 General
Commercial media of same components described below may be used.
Volume of prepared media may be adjusted in accordance with the number of specimens.
6.2.2 1/500 Nutrient broth (1/500 NB)For 100 ml of purified water, take 0,30 g of meat extract, 1,0 g of peptone and 0,50 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. Dilute 1,0 ml of
this medium by 500 times adding 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 ± 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
Use agar powder of microbiological grade. The gel strength measured using 1,5 % agar is between
2 2400 g/cm and 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
© ISO 2016 – All rights reserved 5---------------------- Page: 11 ----------------------
ISO 18071:2016(E)
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.
6.2.8 Top agarFor 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 autoclav
...DRAFT INTERNATIONAL STANDARD
ISO/DIS 18071
ISO/TC 206 Secretariat: JISC
Voting begins on: Voting terminates on:
2015-11-26 2016-02-26
Fine Ceramics (Advanced Ceramics, Advanced Technical
Ceramics) — Determination of antiviral activity of
semiconducting photocatalytic materials under indoor
lighting environment — Test method using
bacteriophage Q-beta
Titre manque
ICS: 81.060.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 18071:2015(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2015
---------------------- Page: 1 ----------------------
ISO/DIS 18071:2015(E) ISO/DIS 18071
Contents Page
Foreword ............................................................................................................................................................ iv
Introduction ......................................................................................................................................................... v
1 Scope ...................................................................................................................................................... 1
2 Normative references ............................................................................................................................ 1
3 Terms and definitions ........................................................................................................................... 1
4 Symbols (and abbreviated terms) ........................................................................................................ 2
5 Principle.................................................................................................................................................. 3
6 Materials ................................................................................................................................................. 3
6.1 Strains and preparation for tests ......................................................................................................... 3
6.1.1 Strains..................................................................................................................................................... 3
6.1.2 Bacteria preparation .............................................................................................................................. 4
6.1.3 Bacteriophage preparation ................................................................................................................... 4
6.2 Media ....................................................................................................................................................... 5
6.2.1 General ................................................................................................................................................... 5
6.2.2 1/500 Nutrient broth (1/500 NB) ............................................................................................................ 5
6.2.3 Calcium solution .................................................................................................................................... 5
6.2.4 LB broth with calcium ........................................................................................................................... 5
6.2.5 Agar powder ........................................................................................................................................... 5
6.2.6 LB agar ................................................................................................................................................... 5
6.2.7 Bottom agar plate (LB agar plate with calcium) ................................................................................. 6
6.2.8 Top agar.................................................................................................................................................. 6
6.2.9 Soybean-casein digest broth with lecithin and polysorbate 80 (SCDLP) ........................................ 6
6.2.10 Peptone saline solution ........................................................................................................................ 6
7 Apparatus and equipment .................................................................................................................... 6
7.1 Test equipment ...................................................................................................................................... 6
7.2 Cover film ............................................................................................................................................... 7
7.3 Moisture preservation glass ................................................................................................................. 7
7.4 Glass tube or glass rod ......................................................................................................................... 7
7.5 Paper filter .............................................................................................................................................. 7
7.6 Light source ........................................................................................................................................... 7
7.7 UV sharp cut-off filter ............................................................................................................................ 8
7.8 Illuminance meter .................................................................................................................................. 8
7.9 Centrifuge ............................................................................................................................................... 8
7.10 Sterilized syringe filter unit .................................................................................................................. 8
8 Test piece ............................................................................................................................................... 8
9 Procedure ............................................................................................................................................... 9
COPYRIGHT PROTECTED DOCUMENT9.1 General ................................................................................................................................................... 9
9.2 Procedure for preparation of bacteria suspension ............................................................................ 9
© ISO 2015, Published in Switzerland9.3 Procedure of preparation of test bacteriophage solution ............................................................... 10
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
9.4 Procedure of test for indoor-light-active photocatalytic antiviral activity ..................................... 10
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
9.5 Indoor lighting condition .................................................................................................................... 10
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
9.6 Measurement of titre of bacteriophage ............................................................................................. 11
the requester.ISO copyright office
10 Calculation ........................................................................................................................................... 12
Ch. de Blandonnet 8 • CP 401CH-1214 Vernier, Geneva, Switzerland
11 Test report ............................................................................................................................................ 13
Tel. +41 22 749 01 11Bibliography ...................................................................................................................................................... 15
Fax +41 22 749 09 47copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved
© ISO 2015 – All rights reserved iii
---------------------- Page: 2 ----------------------
ISO/DIS 18071
Contents Page
Foreword ............................................................................................................................................................ iv
Introduction ......................................................................................................................................................... v
1 Scope ...................................................................................................................................................... 1
2 Normative references ............................................................................................................................ 1
3 Terms and definitions ........................................................................................................................... 1
4 Symbols (and abbreviated terms) ........................................................................................................ 2
5 Principle.................................................................................................................................................. 3
6 Materials ................................................................................................................................................. 3
6.1 Strains and preparation for tests ......................................................................................................... 3
6.1.1 Strains..................................................................................................................................................... 3
6.1.2 Bacteria preparation .............................................................................................................................. 4
6.1.3 Bacteriophage preparation ................................................................................................................... 4
6.2 Media ....................................................................................................................................................... 5
6.2.1 General ................................................................................................................................................... 5
6.2.2 1/500 Nutrient broth (1/500 NB) ............................................................................................................ 5
6.2.3 Calcium solution .................................................................................................................................... 5
6.2.4 LB broth with calcium ........................................................................................................................... 5
6.2.5 Agar powder ........................................................................................................................................... 5
6.2.6 LB agar ................................................................................................................................................... 5
6.2.7 Bottom agar plate (LB agar plate with calcium) ................................................................................. 6
6.2.8 Top agar.................................................................................................................................................. 6
6.2.9 Soybean-casein digest broth with lecithin and polysorbate 80 (SCDLP) ........................................ 6
6.2.10 Peptone saline solution ........................................................................................................................ 6
7 Apparatus and equipment .................................................................................................................... 6
7.1 Test equipment ...................................................................................................................................... 6
7.2 Cover film ............................................................................................................................................... 7
7.3 Moisture preservation glass ................................................................................................................. 7
7.4 Glass tube or glass rod ......................................................................................................................... 7
7.5 Paper filter .............................................................................................................................................. 7
7.6 Light source ........................................................................................................................................... 7
7.7 UV sharp cut-off filter ............................................................................................................................ 8
7.8 Illuminance meter .................................................................................................................................. 8
7.9 Centrifuge ............................................................................................................................................... 8
7.10 Sterilized syringe filter unit .................................................................................................................. 8
8 Test piece ............................................................................................................................................... 8
9 Procedure ............................................................................................................................................... 9
9.1 General ................................................................................................................................................... 9
9.2 Procedure for preparation of bacteria suspension ............................................................................ 9
9.3 Procedure of preparation of test bacteriophage solution ............................................................... 10
9.4 Procedure of test for indoor-light-active photocatalytic antiviral activity ..................................... 10
9.5 Indoor lighting condition .................................................................................................................... 10
9.6 Measurement of titre of bacteriophage ............................................................................................. 11
10 Calculation ........................................................................................................................................... 12
11 Test report ............................................................................................................................................ 13
Bibliography ...................................................................................................................................................... 15
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ISO/DIS 18071
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 18071 was prepared by Technical Committee ISO/TC 206, Fine ceramics.This second/third/... edition cancels and replaces the first/second/... edition (), [clause(s) / subclause(s) /
table(s) / figure(s) / annex(es)] of which [has / have] been technically revised.
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ISO/DIS 18071
Introduction
This standard applies to testing the antiviral activity of indoor-light-active photocatalytic ceramics and other
materials produced by either coating or mixing of a light-active photocatalyst. The standard for testing the
antibacterial activity of photocatalytic materials have been published as ISO 27447 and the standard for
testing the antibacterial activity of indoor-light-active photocatalytic materials have been published as
ISO17094. The standard for determination of antiviral activity of semiconducting photocatalytic materials have
also been published as ISO18061.The test method for cloths or textiles is not included in this draft, because of lack of indoor-light-active
photocatalytic cloths or textiles. When the indoor-light-active photocatalytic cloths or textiles with antiviral
activity using indoor-light-active photocatalytic activity have been developed, test method for indoor-light-
active photocatalytic cloths or textiles will be proposed with the glass adhesion method in ISO27447.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 18071
Fine Ceramics (Advanced Ceramics, Advanced Technical
Ceramics) — Determination of antiviral activity of
semiconducting photocatalytic materials under indoor lighting
environment - Test method using bacteriophage Q-beta
WARNING –Only personnel trained in microbiological techniques should carry out tests.
1 ScopeThis test method specifies the determination of the antiviral activity of materials that contain indoor-light-active
photocatalytic materials or have indoor-light-active photocatalytic films on the surface, by measuring the
infectivity titre of bacteriophage Q-beta after illumination with indoor light.NOTE In this test method, the surrogate microbe is bacteriophage Q-beta, intended as a model for Influenza viruses.
This test method is intended for use with different kinds of indoor-light-active 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 indoor-light-active photocatalytic materials.
This test method is applicable to indoor-light-active photocatalytic materials produced for an antiviral
applications. Other types of performance of indoor-light-active photocatalytic materials, i.e., antibacterial
activity, antifungal activity, decomposition of water contaminants, self-cleaning, antifogging and air purification,
are not determined by this method.The values expressed in this standard are in accordance with the International System of Units (SI).
2 Normative referencesThe 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 80000-1, Quantities and units -- Part 0: General principles
ISO 27447, Fine ceramics (advanced ceramics, advanced technical ceramics) -- Test method for antibacterial
activity of semiconducting photocatalytic materialsISO 14605, Fine Ceramics (advanced ceramics, advanced technical ceramics) -- Visible light source of testing
semiconducting photocatalytic materials3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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ISO/DIS 18071
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.3.2
indoor-light-active photocatalyst
of the photocatalyst, substance that reacts with artificial light source for general lighting service (i.e. indoor
lighting environment).3.3
indoor lighting environment
the environment with artificial light source for general lighting service. Not include sunlight.
3.4indoor-light-active photocatalytic materials
materials in which or on which the indoor-light-active photocatalyst is added by coating, impregnation, mixing,
etc.3.5
antiviral
condition decreasing the infectivity of viruses on the surface of materials
3.6
bacteriophage
type of virus which infects bacteria
NOTE 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.3.7
plaque
visible, clear area which is theoretically the result of infection and lysis of host cells by a single viable
bacteriophage3.8
indoor-light-active photocatalyst antiviral activity value
difference between the logarithms of the total number of bacteriophage plaques on photocatalytic treated
materials after indoor light illumination and on non-treated materials after indoor light illumination
NOTE This value includes the decrease of number of bacteriophage plaques without Indoor light illumination.
3.9indoor-light-active photocatalyst antiviral activity value for Indoor light illumination
difference between the logarithms of the total number of bacteriophage plaques on photocatalytic treated
materials after indoor light illumination and on photocatalytic treated materials kept in the dark place
4 Symbols (and abbreviated terms)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
B average of titre of bacteriophage on non-treated specimens, after indoor light illumination of intensity L
F-Lunder condition F
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ISO/DIS 18071
C average of titre of bacteriophage on indoor-light-active photocatalytic treated specimens, after being
kept in a dark placeC average of titre of bacteriophage on indoor-light-active photocatalytic treated specimens, after indoor
F-Llight illumination of intensity L under condition F
Di dilution factor
F type of UV cut-off condition (condition A or condition B)
L illuminance of indoor light
Logmax maximum logarithmic value of titre of bacteriophage
Logmean average logarithmic value of titre of bacteriophage for 3 non-treated specimens
Logmin minimum logarithmic value of titre of bacteriophageN titre of bacteriophage (plaque forming unit)
V antiviral activity value without indoor-light-active photocatalyst, after being kept in a dark place on a
testing materialV indoor-light-active photocatalyst antiviral activity value, after indoor light illumination at a constant
F-Lintensity (F-L) on a indoor-light-active photocatalytic material
V indoor-light-active photocatalyst antiviral activity value with indoor light illumination
Z average number of plaques in 2 Petri dishes5 Principle
This test method is suitable for use in development, comparison, quality assurance, characterization, reliability,
and design data generation of indoor-light-active photocatalytic materials. The method is used to obtain the
antiviral activity of indoor-light-active photocatalytic materials by the contact of a specimen with bacteriophage
under indoor lighting condition. The method is suitable for use with flat sheet, board or plate shaped materials.
The specimen of indoor-light-active photocatalytic treated material is inoculated with bacteriophage
suspension and exposed to light 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-beta.
The results obtained are compared with those obtained from inoculated specimens of non-photocatalytic
treated material exposed to light 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
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
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ISO/DIS 18071
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)
NBRC 20012 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 same, but they are from same origin.
6.1.2 Bacteria preparationa) Inoculate E. coli strain into a slant culture medium (6 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 1 In the case of bacteria stored in a deep freezer, the maximum number of subcultures from 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 (see 6.2.4) in a 300 ml conical flask to 35 ˚C to 37 ˚C and
inoculate with 0,025 ml of the culture prepared under b).d) Incubate as above condition until a bacterial concentration will be reached at 2,0 ± 1,0 x 10 cfu/ml.
NOTE 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 approximately 2 x
10 pfu (plaque forming unit)/ml [multiplicity of infection (m.o.i.) is approximately 0,1].
f) Incubate the inoculated bacterial culture for 4 h as under b).g) Store the culture for 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.4 © ISO 2015 – All rights reserved
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ISO/DIS 18071
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 (see 9.6) 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.
m) Do not use the bacteriophage stock solution with less than 1,0 x 10 pfu/ml or contaminated stock solution.
11 13NOTE1 The titre of the phage suspension should be above 1,0 x 10 pfu/ml and might reach up to 1,0 x 10 pfu/ml
NOTE2 The titre of the phage stock suspension will slowly decrease over time.6.2 Media
6.2.1 General
Commercial media of same components described below may be used.
Volume of prepared media may be adjusted in accordance with the number of specimens.
6.2.2 1/500 Nutrient broth (1/500 NB)For 100 m
...
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