ISO 19635:2016

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

ISO 19635:2016 specifies a test method for evaluating anti-algal activities in outdoor structures, specifically flat photocatalytic materials (for example, window panes and water tank glasses, films, guardrails, etc.) under irradiation of ultraviolet light. It does not include powder, granular or porous photocatalytic materials.

Céramiques techniques — Méthode d'essai pour l'activité algicide des matériaux photocatalytiques semi-conducteurs

General Information

• Status: Published
• Publication Date: 31-Mar-2016
• ICS: 81.060.30 - Advanced ceramics
• Technical Committee: ISO/TC 206 - Fine ceramics
• Drafting Committee: ISO/TC 206/WG 9 - Photocatalysis
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 07-Sep-2021

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 19635
First edition
2016-03-15
Fine ceramics (advanced ceramics,
advanced technical ceramics) —
Test method for antialgal activity
of semiconducting photocatalytic
materials
Céramiques techniques — Méthode d’essai pour l’activité algicide des
matériaux photocatalytiques semi-conducteurs
Reference number
ISO 19635:2016(E)
©
ISO 2016

---------------------- Page: 1 ----------------------
ISO 19635:2016(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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2016 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 19635:2016(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Algae used in the test . 2
5 Preparation for the test . 2
5.1 Conditions for handling of algae . 2
5.2 Chemicals, materials, and apparatus . 2
5.3 Sterilization and filtration methods . 3
5.3.1 Hot-air sterilization . 3
5.3.2 Hot-steam sterilization . 3
5.3.3 Flaming . . 4
5.3.4 Sterilization with alcohol . 4
5.3.5 Sterilization through filtration . 4
5.4 Culture media . 4
5.4.1 General. 4
5.4.2 C culture medium . 5
5.4.3 Slant medium . 5
5.4.4 Washing solution . 5
6 Cultivation of algae . 5
6.1 Transplantation and cultivation of algae . 5
6.2 Preparation of test solution . 6
7 Photoirradiation method . 6
7.1 Measurement of UV light intensity and preparation of the test piece installation location 6
7.2 Photoirradiation conditions . 6
8 Test . 7
8.1 Preparation of test piece . 7
8.2 Cleaning and installation of test pieces . 7
8.2.1 Cleaning of test pieces . 7
8.2.2 Installation of test pieces . 7
8.3 Inoculation of test solution. 8
8.4 Washing of test solution immediately after inoculation of test solution . 9
8.5 Washing of test solution after test. 9
8.6 Measurement according to the three-point absorbance method . . 9
8.7 Storage in the dark of test piece on which the test solution has been inoculated .10
9 Test results .10
9.1 General .10
9.2 Calculation of antialgal activity on the basis of absorption spectrum method for
photocatalytic antialgal materials .11
10 Recording of test results .11
Bibliography .12
© ISO 2016 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO 19635:2016(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 WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information.
The committee responsible for this document is ISO/TC 206, Fine ceramics.
iv © ISO 2016 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 19635:2016(E)

Introduction
Under ultraviolet (UV) light illumination, photocatalysts have diverse functions, such as prevention
of fouling, antifogging effects, antibacterial effects, deodorization, and decomposition of air and
water contaminants, and their applications have grown in recent years. Since products utilizing these
photocatalytic functions are commercialized in large quantities, a method to evaluate and determine
photocatalytic effects is required. This International Standard is intended to provide a method
for objective evaluation of activity in controlling algae, which are a primary producer of microbial
contamination (in the form of environmental biofilms) of outdoor structures, under illumination
with ultraviolet light to simulate the outdoor environment and to contribute, via control of algae, to
conservation of urban landscapes, prevention of member corrosion, and prevention of fouling of water
tank window materials.
© ISO 2016 – All rights reserved v

---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 19635:2016(E)
Fine ceramics (advanced ceramics, advanced technical
ceramics) — Test method for antialgal activity of
semiconducting photocatalytic materials
1 Scope
This International Standard specifies a test method for evaluating anti-algal activities in outdoor
structures, specifically flat photocatalytic materials (for example, window panes and water tank
glasses, films, guardrails, etc.) under irradiation of ultraviolet light. It does not include powder, granular
or porous photocatalytic materials.
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 27447, Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for antibacterial
activity of semiconducting photocatalytic materials
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
photocatalyst
substance with many functions based on oxidization and reduction reactions under photoirradiation
(3.3), including decomposition and removal of contaminants, deodorization, antibacterial actions, and
prevention of fouling
3.2
photocatalytic materials
materials in which or on which the photocatalyst (3.1) is added by coating, impregnation, mixing, etc.
Note 1 to entry: Photocatalytic materials are to be used for building and road construction materials to obtain
the above-mentioned functions.
3.3
photoirradiation
irradiation of UV light at wavelength 300 nm to 380 nm
3.4
antialgal activity
activity in suppressing growth of algae over a material surface
3.5
photocatalytic antialgal treatment
support of a photocatalyst (3.1) by means of various methods, including coating, impregnation, blending,
and others, for utilization of the antialgal activity of a photocatalyst
© ISO 2016 – All rights reserved 1

---------------------- Page: 6 ----------------------
ISO 19635:2016(E)

3.6
photocatalytic antialgal activity based on three-point absorbance spectrum method
photocatalytic antialgal activity (3.4) derived from the ratio of the absorbance of material treated
with photocatalyst (3.1) after photoirradiation (3.3) of UV light to that of non-treated material after
photoirradiation
4 Algae used in the test
The type of algae to be used in the test with photocatalytic-treated antialgal materials shall be Chlorella
vulgaris (NIES-227). The strain of algae to be used in the test shall be the same strain stored in agencies
affiliated with the World Federation for Culture Collections or the Japan Society for Culture Collection.
5 Preparation for the test
5.1 Conditions for handling of algae
The test shall be performed in a laboratory with equipment needed to prevent mixing with other
microorganisms.
5.2 Chemicals, materials, and apparatus
Unless otherwise specified, the chemicals, materials, and apparatus to be used in the test shall be as
follows. Test tubes, flasks, pipettes, tweezers, etc. shall be carefully cleaned with alkaline or neutral
detergent, rinsed thoroughly with water, dried, and sterilized by hot air or high-pressure steam
before use.
5.2.1 Purified water, the water used for the preparation of all solutions and culture media and for all
determinations shall be distilled or deionized and shall have a conductivity of <1 μS/cm.
5.2.2 Ethanol for disinfection, solution whose ethanol concentration has been adjusted to 77 % to
82 % in volume fraction by means of purified water.
5.2.3 Autoclave, capable of maintaining the temperature at 121 °C ± 1 °C (equivalent to a pressure of
103 kPa).
5.2.4 Dry sterilizer, capable of maintaining the temperature at 160 °C to 180 °C.
5.2.5 Spectrophotometer, capable of measurement within the wavelength range of 400 nm to 800 nm.
5.2.6 Refrigerator, capable of maintaining the temperature at 4 °C ± 1 °C.
5.2.7 Platinum loop, with an end loop of approximately 4 mm.
5.2.8 Glass plate, made from material not affecting the growth of microorganisms.
5.2.9 Cotton plug, plug of non-degreased cotton, or silicon plug, metal plug, or molten plug.
5.2.10 Test tube mixer, for microorganism test.
5.2.11 Contact film, film of material not affecting the growth of microorganisms, which is free of
water-absorbing properties and features satisfactory adherence, and in which transmittance in the
range of 340 nm to 380 nm, as measured according to the method specified in ISO 27447, accounts for
85 % or more.
2 © ISO 2016 – All rights reserved

---------------------- Page: 7 ----------------------
ISO 19635:2016(E)

5.2.12 Moisture retention glass, glass with thickness of 1,1 mm or less, in which transmittance of
340 nm to 380 nm, as measured according to the method specified in ISO 27447, accounts for 85 % or
more and which has been cut to a size capable of covering the entire surface of a Petri dish.
5.2.13 Storage Petri dish, dish with the inside diameter of about 90 mm.
5.2.14 Moisture-conditioning filter paper, filter paper not affecting the growth of microorganisms,
which has been cut to a size appropriate for placement in the container in which the test piece is to be set.
5.2.15 Disposable cuvette, plastic cuvettes (about 12 mm × 12 mm × 45 mm) appropriate for placement
in the storage container.
5.2.16 Tooth brush, soft type of brush made of polyamide.
5.2.17 UV fluorescent lamp, 20-W black-light blue type UV fluorescent lamp (BLB lamp) that provides
UV-A ranging from 300 nm to 400 nm with a peak emission at 351 nm.
5.2.18 Fluorescent lamp, 20-W starter and straight-type white (symbol: W) lamp.
5.2.19 UV irradiator, system comprised of one UV fluorescent lamp, which can shield light from the
surrounding region.
5.2.20 UV light radiometer, radiometer with a detector whose sensitivity peak is at 351 nm and
provide limit, e.g. ±10 nm, shall be used. The radiometer shall be calibrated for the light source to be used
or corrected to ascertain sensitivity within the wavelength range to be absorbed by the photocatalytic
test piece with suitable approach.
5.2.21 Illuminometer, general type AA-grade illuminometer.
5.2.22 Filter unit, in which the membrane filter is made from a less-absorbent material, such as
hydrophilic polyvinylidene fluoride, hydrophilic polytetrafluoroethylene, etc., and the filter pore size is
0,22 μm or 0,45 μm.
5.2.23 Centrifuge, capable of the centrifugal force to 1 500 × g for preparation of test solutions.
5.3 Sterilization and filtration methods
5.3.1 Hot-air sterilization
The apparatus to be sterilized shall be placed in a hot-air sterilizer at 160 °C to 180 °C and held at this
temperature for 30 min to 60 min. If the sterilized sample exhibits any wet cotton plug or package
water after completion of hot-air sterilization, the apparatus shall not be used.
5.3.2 Hot-steam sterilization
An autoclave shall be filled with water and the apparatus to be sterilized shall be placed in a wire cage
and placed on the autoclave shelf. The autoclave shall be closed with the lid and heated and held at a
temperature of 121 °C (equivalent to a pressure of 103 kPa) for 15 min to 20 min. After discontinuing
heating and allowing the temperature to cool to 100 °C or less, the exhaust valve shall be opened to
bleed steam and the lid shall be opened. The apparatus sterilized in this fashion shall be taken out and,
if necessary, allowed to cool in the safety cabinet.
In order to maintain cleanness to prevent contamination by culture media and processing chemicals,
the autoclave shall be cleaned with neutral detergent as required and rinsed with water
...

DRAFT INTERNATIONAL STANDARD
ISO/DIS 19635
ISO/TC 206 Secretariat: JISC
Voting begins on: Voting terminates on:
2015-09-14 2015-12-14
Fine ceramics (advanced ceramics, advanced technical
ceramics) — Test method for antialgal activity of
semiconducting photocatalytic materials
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 19635:2015(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 19635:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, 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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2015 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/DIS 19635
Contents Page
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Algae used in the test . 2
5 Preparation for the test . 2
5.1 Conditions for handling of algae . 2
5.2 Chemicals, materials, and apparatus . 2
5.3 Sterilization and filtration methods . 3
5.4 Culture media, etc. . 4
6 Cultivation of algae . 5
6.1 Transplantation and cultivation of algae . 5
6.2 Preparation of test solution . 6
7 Photoirradiation method . 6
7.1 Measurement of UV light intensity and preparation of the test piece installation location . 6
7.2 Photoirradiation conditions. 6
8 Test . 7
8.1 Preparation of test piece. 7
8.2 Cleaning and installation of test pieces . 7
8.3 Inoculation of test solution. 8
8.4 Washing of test solution immediately after inoculation of test solution . 9
8.5 Washing of test solution after test . 9
8.6 Measurement according to the three-point absorbance method . 9
8.7 Storage in the dark of test piece on which the test solution has been inoculated . 10
9 Test results . 10
9.1 Calculation of antialgal activity on the basis of absorption spectrum method for
photocatalytic antialgal materials . 10
10 Recording of test results . 11

© ISO 2015 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO/DIS 19635
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 19635 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.
iv © ISO 2015 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/DIS 19635
Introduction
Under ultraviolet (UV) light illumination, photocatalysts have diverse functions, such as prevention of fouling,
antifogging effects, antibacterial effects, deodorization, and decomposition of air and water contaminants, and
their applications have grown in recent years. Since products utilizing these photocatalytic functions are
commercialized in large quantities, a method to evaluate and determine photocatalytic effects is required. This
standard is intended to provide a method for objective evaluation of activity in controlling algae, which are a
primary producer of microbial contamination (in the form of environmental biofilms) of outdoor structures,
under illumination with ultraviolet light to simulate the outdoor environment and to contribute, via control of
algae, to conservation of urban landscapes, prevention of member corrosion, and prevention of fouling of
water tank window materials.

© ISO 2015 – All rights reserved v

---------------------- Page: 5 ----------------------
DRAFT INTERNATIONAL STANDARD ISO/DIS 19635

Fine ceramics (advanced ceramics, advanced technical
ceramics) — Test method for antialgal activity of
semiconducting photocatalytic materials
1 Scope
This standard specifies a test method for evaluating anti-algal activities in outdoor structures, specifically flat
photocatalytic materials (for example, window panes and water tank glasses, films, gurdrails, etc.) under
irradiation of ultraviolet light. It does not include powder, granular or porous photocatalytic materials.
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.
ISO31-0, 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 13125:2013 Fine ceramics (advanced ceramics, advanced technical ceramics) -- Test method for
antifungal activity of semiconducting photocatalytic materials
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
photocatalyst
A substance with many functions based on oxidization and reduction reactions under photoirradiation,
including decomposition and removal of contaminants, deodorization, antibacterial actions, and prevention of
fouling.
3.2
photocatalytic materials
Materials in which or on which the photocatalyst is added by coating, impregnation, mixing, etc. Photocatalytic
materials are to be used for building and road construction materials to obtain the above-mentioned functions.
3.3
photoirradiation
Irradiation of UV light at wavelength 300 - 380 nm.
3.4
antialgal activity
Activity in suppressing growth of algae over a material surface
© ISO 2015 – All rights reserved 1

---------------------- Page: 6 ----------------------
ISO/DIS 19635
3.5
photocatalytic antialgal treatment
Support of a photocatalyst by means of various methods, including coating, impregnation, blending, and
others, for utilization of the antialgal activty of a photocatalyst
3.6
photocatalytic antialgal activity based on three-point absorbance spectrum method
Photocatalytic antialgal activity derived from the ratio of the absorbance of material treated with photocatalyst
after photoirradiation of UV light to that of non-treated material after photoirradiation.
4 Algae used in the test
The type of algae to be used in the test with photocatalytic-treated antialgal materials shall be Chlorella
vulgaris (NIES-227). The strain of algae to be used in the test shall be the same strain stored in agencies
affiliated with the World Federation for Culture Collections or the Japan Society for Culture Collection.
5 Preparation for the test
5.1 Conditions for handling of algae
The test shall be performed in a laboratory with equipment needed to prevent mixing with other
microorganisms.
5.2 Chemicals, materials, and apparatus
Unless otherwise specified, the chemicals, materials, and apparatus to be used in the test shall be as follows.
Test tubes, flasks, pipettes, tweezers, etc. shall be carefully cleaned with alkaline or neutral detergent, rinsed
thoroughly with water, dried, and sterilized by hot air or high-pressure steam before use.
5.2.1 Purified water: The water used for the preparation of all solutions and culture media and for all
determinations shall be distilled or deionized and shall have a conductivity of < 1μS/cm.
5.2.2  Ethanol for disinfection: Solution whose ethanol concentration has been adjusted to 77 to 82% in
volume fraction by means of purified water
5.2.3  Autoclave: Capable of maintaining the temperature at 121ºC ± 1ºC (equivalent to a pressure of 103
kPa)
5.2.4  Dry sterilizer: Capable of maintaining the temperature at 160 to 180ºC
5.2.5  Spectrophotometer: Capable of measurement within the wavelength range of 400 to 800 nm
5.2.6  Refrigerator: Capable of maintaining the temperature at 4ºC ± 1ºC.
5.2.7  Platinum loop: With an end loop of approximately 4 mm
5.2.8  Glass plate: Made from material not affecting the growth of microorganisms
5.2.9  Cotton plug: Plug of non-degreased cotton, or silicon plug, metal plug, or molten plug
5.2.10  Test tube mixer: For microorganism test
5.2.11  Contact film: Film of material not affecting the growth of microorganisms, which is free of water-
absorbing properties and features satisfactory adherence, and in which transmittance in the range of 340 to
380 nm as measured according to the method specified in ISO 27447 accounts for 85% or more
2 © ISO 2015 – All rights reserved

---------------------- Page: 7 ----------------------
ISO/DIS 19635
5.2.12  Moisture retention glass: Glass with thickness of 1.1 mm or less, in which transmittance of 340 to
380 nm as measured according to the method specified in ISO 27447 accounts for 85% or more and which
has been cut to a size capable of covering the entire surface of a Petri dish
5.2.13  Storage Petri dish: Dish with the inside diameter of about 90 mm
5.2.14  Moisture-conditioning filter paper: Filter paper not affecting the growth of microorganisms , which
has been cut to a size appropriate for placement in the container in which the test piece is to be set
5.2.15  Disposable cuvette: Plastic cuvettes (about 12×12×45 mm) appropriate for placement in the
storage container
5.2.16  Tooth brush: Soft type of brush made of polyamide
5.2.17  UV fluorescent lamp: 20-W black-light blue type UV fluorescent lamp (BLB lamp) that provides UV-A
ranging from 300 nm to 400 nm with a peak emission at 351 nm.
5.2.18  Fluorescent lamp: 20-W starter and straight-type white (symbol: W) lamp
5.2.19  UV irradiator: System comprised of one UV fluorescent lamp, which can shield light from the
surrounding region
5.2.20  UV light radiometer: A radiometer with a detector whose sensitivity peak is at 351 nm and provide
limit, e.g. ±10 nm shall be used. The radiometer shall be calibrated for the light source to be used or corrected
to ascertain sensitivity within the wavelength range to be absorbed by the photocatalytic test piece with
suitable approach.
5.2.21  Illuminometer: General type AA-grade illuminometer
5.2.22  Filter unit: Filter unit, in which the membrane filter is made from a less-absorbent material, such as
hydrophilic polyvinylidene-fluoride, hydrophilic polytetrafluoro-ethylene, etc., and the filter pore size is 0.22 or
0.45 μm
5.2.23  Centrifuge: Capable of the centrifugal force to 1500  g for preparation of test solutions
5.3 Sterilization and filtration methods
5.3.1 Hot-air sterilization
The apparatus to be sterilized shall be placed in a hot-air sterilizer at 160 to 180ºC and held at this
temperature for 30 to 60 minutes. If the sterilized sample exhibits any wet cotton plug or package water after
completion of hot-air sterilization, the apparatus shall not be used.
5.3.2 Hot-steam sterilization
An autoclave shall be filled with water and the apparatus to be sterilized shall be placed in a wire cage and
placed on the autoclave shelf. The autoclave shall be closed with the lid and heated and held at a temperature
of 121ºC (equivalent to a pressure of 103 kPa) for 15 to 20 minutes. After discontinuing heating and allowing
the temperature to cool to 100ºC or less, the exhaust valve shall be opened to bleed steam and the lid shall
be opened. The apparatus sterilized in this fashion shall be taken out and, if necessary, allowed to cool in the
safety cabinet.
In order to maintain cleanness to prevent contamination by culture media and processing chemicals, the
autoclave shall be cleaned with neutral detergent as required and rinsed with water thoroughly.
5.3.3 Flaming
© ISO 2015 – All rights reserved 3

---------------------- Page: 8 ----------------------
ISO/DIS 19635
The apparatus to be sterilized shall be subjected to gas or alcohol flame treatment. Platinum loops shall be
subjected to flame treatment until they become red hot, while test tubes shall be subjected to flame treatment
for two to three seconds.
5.3.4 Sterilization with alcohol
Absorbent cotton or gauze shall be soaked with ethanol for disinfection and squeezed lightly, for use in wiping
both hands.
5.3.5 Sterilization through filtration
Liquid to be sterilized shall be filtered with the filter unit
Liquid to be sterilized shall be filtered with the filter unit
5.4 Culture media, etc.
Culture media to be used shall be of the composition shown in Tables 1 - 3.
Table 1 -- C culture medium
Tris (hydroxymethyl) aminomethane 500 mg
Ca(NO )･4H O 150 mg
3 2 2
KNO 100 mg
3
Glyceric acid disodium 5.5 hydrate  50 mg
MgSO･7H O  40 mg
4 2
PIV metallic salt solution  3 ml
Cyanocobalamin (vitamin B ) solution  1 ml
12
Biotin solution  1 ml
Thiamine hydrochloride (vitamin B ) solution  1 ml
1
Vitamin mixture   0.1 ml
Distilled water (purified water) 994 ml
The contents shall be thoroughly dissolved.
Table 2 -- PIV metallic salt solution
Ethylene diamine tetra-acetic acid dihydrogen disodium dihydrate
...