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

General Information

Status
Published
Publication Date
14-Jul-2016
Technical Committee
Current Stage
9093 - International Standard confirmed
Completion Date
03-Dec-2021
Ref Project

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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
---------------------- Page: 2 ----------------------
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
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ISO 18071:2016(E)

Bibliography .............................................................................................................................................................................................................................15

iv © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
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
---------------------- Page: 5 ----------------------
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
---------------------- Page: 6 ----------------------
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 Scope

This 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 environment

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
© 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

bacteriophage
3.8
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 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 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

B average of titre of bacteriophage on non-treated specimens, after indoor light illumination

F-L
of intensity L under condition F

C average of titre of bacteriophage on indoor-light-active photocatalytic treated specimens,

after being kept in a dark place
2 © 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-L
after 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 bacteriophage
N 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 material

V indoor-light-active photocatalyst antiviral activity value, after indoor light illumination at

F-L
a 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 dishes
5 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 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 safety cabinet.
© ISO 2016 – All rights reserved 3
---------------------- Page: 9 ----------------------
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 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 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 2
400 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 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 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 DOCUMENT

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

9.2 Procedure for preparation of bacteria suspension ............................................................................ 9

© ISO 2015, Published in Switzerland

9.3 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 401
CH-1214 Vernier, Geneva, Switzerland

11 Test report ............................................................................................................................................ 13

Tel. +41 22 749 01 11

Bibliography ...................................................................................................................................................... 15

Fax +41 22 749 09 47
copyright@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.

© ISO 2015 – All rights reserved v
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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 Scope

This 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 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 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 materials

ISO 14605, Fine Ceramics (advanced ceramics, advanced technical ceramics) -- Visible light source of testing

semiconducting photocatalytic materials
3 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.

3.4
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 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

bacteriophage
3.8
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.

3.9

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 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-L
under 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 place

C average of titre of bacteriophage on indoor-light-active photocatalytic treated specimens, after indoor

F-L
light 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 bacteriophage
N 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 material

V indoor-light-active photocatalyst antiviral activity value, after indoor light illumination at a constant

F-L
intensity (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 dishes
5 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

safety cabinet.
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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 preparation

a) 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.
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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 13

NOTE1 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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