SIST EN ISO 20743:2007
(Main)Textiles - Determination of antibacterial activity of antibacterial finished products (ISO 20743:2007)
Textiles - Determination of antibacterial activity of antibacterial finished products (ISO 20743:2007)
This International Standard specifies quantitative test methods to determine the antibacterial activity of antibacterial finished textile products including nonwovens. This International Standard is applicable to all textile products, including cloth, wadding, thread and material for clothing, home furnishings and miscellaneous goods regardless of the type of antibacterial agent used (organic, inorganic, natural or man-made) or the method of application (built-in, after-treatment or grafting). Based on the intended application and on the environment in which the textile product is to be used, the user can select the most suitable of the following three methods on determination of antibacterial activity: a) absorption method (an evaluation method in which test bacterial suspension is inoculated directly onto samples); b) transfer method (an evaluation method in which test bacteria are placed on an agar plate and transferred onto samples); c) printing method (an evaluation method in which test bacteria are placed on a filter and printed onto samples). The colony plate count method and the ATP (ATP = Adenosine Tri-phosphate) luminescence method are also specified for measuring the enumeration of bacteria.
Textilien - Bestimmung der antibakteriellen Wirkung antibakteriell behandelter Erzeugnisse (ISO 20743:2007)
Die vorliegende Internationale Norm legt quantitative Prüfverfahren zur Bestimmung der antibakteriellen Wirkung antibakteriell behandelter textiler Erzeugnisse, einschließlich Vliesstoffe fest.
Diese Internationale Norm ist anwendbar auf alle textilen Erzeugnisse, einschließlich Stoffe, Wattierung, Garn und Materialien für Bekleidung, Heimtextilien sowie verschiedene Waren, ungeachtet des Typs des verwendeten antibakteriellen Mittels (organisch, anorganisch, natürlich oder künstlich) oder des Anwendungsverfahrens (eingebaut, Nachbehandlung oder Pfropfen).
Auf der Grundlage der beabsichtigten Anwendung und der Umgebung, für die das textile Erzeugnis zu verwenden ist, kann der Anwender aus den folgenden drei Verfahren zur Bestimmung der antibakteriellen Wirkung das geeignetste auswählen.
a) Absorptionsverfahren (ein Bewertungsverfahren, bei dem die bakterielle Prüfsuspension direkt auf Proben geimpft wird);
b) Übertragungsverfahren (ein Bewertungsverfahren, bei dem Prüfbakterien auf eine Agarplatte aufgebracht und auf Proben übertragen werden);
c) Stempelverfahren (ein Bewertungsverfahren, bei dem Prüfbakterien auf ein Filter aufgebracht und auf Proben gestempelt werden).
Das Kolonie-Plattenzählverfahren und das ATP-Lumineszenzverfahren (ATP = Adenosintriphosphat) werden auch zum Messen der Bakterienzahl festgelegt.
Textiles - Détermination de l'activité antibactérienne des produits finis antibactériens (ISO 20743:2007)
L'ISO 20743:2007 spécifie des méthodes d'essai quantitatives permettant de déterminer l'activité antibactérienne des produits textiles finis antibactériens, y compris les non-tissés.
L'ISO 20743:2007 s'applique à tous les produits textiles, y compris le tissu, le rembourrage, le fil et les matériaux utilisés dans la confection de vêtements, de tissus d'ameublements et d'articles divers, quel que soit le type d'agent antibactérien utilisé (organique, inorganique, naturel ou synthétique) ou quelle que soit la méthode d'application (intégration, post-traitement ou greffage).
Tenant compte de l'application prévue et de l'environnement dans lequel le produit textile est destiné à être utilisé, l'utilisateur peut choisir la plus adaptée des trois méthodes suivantes de détermination de l'activité antibactérienne: a) méthode par absorption (méthode d'évaluation dans laquelle la suspension bactérienne à l'essai est ensemencée directement sur des échantillons); b) méthode par transfert (méthode d'évaluation dans laquelle les bactéries soumises à l'essai sont placées sur une boîte de milieu gélosé, puis transférées sur des échantillons); c) méthode par impression (méthode d'évaluation dans laquelle les bactéries soumises à l'essai sont placées sur un filtre, puis imprimées sur des échantillons);
La technique de dénombrement et la méthode de mesure de luminescence ATP (triphosphate d'adénosine) sont également spécifiées pour le dénombrement des bactéries.
Tekstilije - Ugotavljanje protibakterijske aktivnosti na izdelkih s protibakterijsko apreturo (ISO 20743:2007)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 20743:2007
01-julij-2007
Tekstilije - Ugotavljanje protibakterijske aktivnosti na izdelkih s protibakterijsko
apreturo (ISO 20743:2007)
Textiles - Determination of antibacterial activity of antibacterial finished products (ISO
20743:2007)
Textilien - Bestimmung der antibakteriellen Wirkung antibakteriell behandelter
Erzeugnisse (ISO 20743:2007)
Textiles - Détermination de l'activité antibactérienne des produits finis antibactériens
(ISO 20743:2007)
Ta slovenski standard je istoveten z: EN ISO 20743:2007
ICS:
59.080.01 Tekstilije na splošno Textiles in general
SIST EN ISO 20743:2007 en;fr
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
EUROPEAN STANDARD
EN ISO 20743
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2007
ICS 59.080.01; 07.100.99
English Version
Textiles - Determination of antibacterial activity of antibacterial
finished products (ISO 20743:2007)
Textiles - Détermination de l'activité antibactérienne des Textilien - Bestimmung der antibakteriellen Wirkung
produits finis antibactériens (ISO 20743:2007) antibakteriell behandelter Erzeugnisse (ISO 20743:2007)
This European Standard was approved by CEN on 16 May 2007.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20743:2007: E
worldwide for CEN national Members.
---------------------- Page: 2 ----------------------
EN ISO 20743:2007 (E)
Foreword
This document (prEN ISO 20743:2007) has been prepared by Technical Committee ISO/TC 38
"Textiles" in collaboration with Technical Committee CEN/TC 248 "Textiles and textile products",
the secretariat of which is held by BSI.
This European Standard shall be given the status of a national standard, either by publication of
an identical text or by endorsement, at the latest by December 2007, and conflicting national
standards shall be withdrawn at the latest by December 2007.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United
Kingdom.
Endorsement notice
The text of ISO 20743:2007 has been approved by CEN as EN ISO 20743:2007 without any
modifications.
2
---------------------- Page: 3 ----------------------
INTERNATIONAL ISO
STANDARD 20743
First edition
2007-06-01
Textiles — Determination of antibacterial
activity of antibacterial finished products
Textiles — Détermination de l'activité antibactérienne des produits finis
antibactériens
Reference number
ISO 20743:2007(E)
©
ISO 2007
---------------------- Page: 4 ----------------------
ISO 20743:2007(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
COPYRIGHT PROTECTED DOCUMENT
© ISO 2007
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2007 – All rights reserved
---------------------- Page: 5 ----------------------
ISO 20743:2007(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Safety precaution. 2
5 Apparatus . 2
6 Reagents and culture media. 4
7 Reference strains. 8
7.1 Strains. 8
7.2 Storage of strains . 8
8 Quantitative measurement. 10
8.1 Plate count method. 10
8.2 Luminescence method. 11
9 Shaking method. 12
9.1 Shaking by vortex mixer . 12
9.2 Shaking by hand . 12
9.3 Shaking by Stomacher . 12
10 Test procedures . 12
10.1 Absorption method. 12
10.2 Transfer method. 17
10.3 Printing method . 21
Bibliography . 27
© ISO 2007 – All rights reserved iii
---------------------- Page: 6 ----------------------
ISO 20743:2007(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 20743 was prepared by Technical Committee ISO/TC 38, Textiles.
iv © ISO 2007 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 20743:2007(E)
Introduction
These test methods were established in order to address the substantial need for an International Standard to
determine antibacterial activity for antibacterial finished textile products.
© ISO 2007 – All rights reserved v
---------------------- Page: 8 ----------------------
INTERNATIONAL STANDARD ISO 20743:2007(E)
Textiles — Determination of antibacterial activity of
antibacterial finished products
1 Scope
This International Standard specifies quantitative test methods to determine the antibacterial activity of
antibacterial finished textile products including nonwovens.
This International Standard is applicable to all textile products, including cloth, wadding, thread and material
for clothing, home furnishings and miscellaneous goods regardless of the type of antibacterial agent used
(organic, inorganic, natural or man-made) or the method of application (built-in, after-treatment or grafting).
Based on the intended application and on the environment in which the textile product is to be used, the user
can select the most suitable of the following three methods on determination of antibacterial activity:
a) absorption method (an evaluation method in which test bacterial suspension is inoculated directly onto
samples);
b) transfer method (an evaluation method in which test bacteria are placed on an agar plate and transferred
onto samples);
c) printing method (an evaluation method in which test bacteria are placed on a filter and printed onto
samples).
The colony plate count method and the ATP (ATP = Adenosine Tri-phosphate) luminescence method are
also specified for measuring the enumeration of bacteria.
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 6330, Textiles — Domestic washing and drying procedures for textile testing
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
textile
fabric
general term employed for designating textile surfaces, woven fabrics, knitted fabrics, etc., formed by the
interlocking of textile materials having a certain cohesion and which are generally intended for clothing or
furniture applications
NOTE Often includes certain types of nonwovens.
© ISO 2007 – All rights reserved 1
---------------------- Page: 9 ----------------------
ISO 20743:2007(E)
3.2
control fabric
fabric used to validate the growth condition of test bacteria
NOTE The same fabric as the fabric to be tested but without antibacterial treatment. If this is not possible, then
100 % cotton fabric without fluorescent brighteners or other finish.
3.3
antibacterial agent
product designed to prevent or mitigate the growth of bacteria, to reduce the number of bacteria or to kill
bacteria
3.4
antibacterial finish
treatment designed to prevent or mitigate the growth of bacteria, to reduce the number of bacteria or to kill
bacteria
3.5
antibacterial activity
activity of an antibacterial finish used to prevent or mitigate the growth of bacteria, to reduce the number of
bacteria or to kill bacteria
3.6
plate count method
method in which the number of bacteria present after incubation is calculated by counting the number of
colonies according to a ten-time dilution method
NOTE The results are expressed in “CFU (Colony Forming Unit)”.
3.7
luminescence method
method in which the amount of ATP contained in bacterial cells is measured
NOTE The results are expressed in “mol of ATP”.
3.8
neutralizer
chemical agents used to inactivate, neutralize, or quench the antibacterial properties of antibacterial agents
4 Safety precaution
Test methods specified herein require the use of bacteria.
These tests should be carried out by persons with training and experience in the use of microbiological
techniques.
Appropriate safety precautions should be observed with due consideration given to country-specific
regulations.
5 Apparatus
Usual laboratory apparatus and, in particular, the following.
5.1 Spectrophotometer, capable of measuring at a 620 nm to 660 nm wavelength, or McFarland’s
nephelometer.
5.2 Incubator, capable of maintaining a constant temperature of 37 °C ± 2 °C.
2 © ISO 2007 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 20743:2007(E)
5.3 Water baths, one capable of maintaining a constant temperature of 46 °C ± 2 °C and another capable
of maintaining a temperature of 70 °C to 90 °C.
5.4 Mixer, producing a vortex shaking action.
5.5 Stomacher, capable of speeds of 6 blows/s to 8 blows/s, with the corresponding disposable containers.
5.6 Clean bench, for microbial test.
5.7 Washing machine, in accordance with the specifications of ISO 6330.
5.8 Humidity chamber, tropical chamber or other container capable of maintaining a high-humidity
atmospheric condition.
−13 −7
5.9 Luminescence photometer, capable of measuring ATP of 10 mol/l to 10 mol/l at 300 nm to
650 nm with a luminescence-measuring reagent.
5.10 Printing apparatus, capable of applying a 4 N load to a test piece and rotating the piece 180° in one
direction for a period of 3,0 s.
5.11 Refrigerator, capable of maintaining a temperature of between 2 °C and 8 °C.
5.12 Freezers, one adjustable to a temperature below −70 °C and another to a temperature below −20 °C.
5.13 Balance, which can be read to the nearest 0,01 g.
5.14 Filtering apparatus, consisting of an upper container equipped with a membrane filter and a lower
container equipped with a suction opening.
5.15 Pipette, having the most suitable volume for each use, with a tip made of glass or plastic, and with a
tolerance of 0,5 % or less.
5.16 Vials, 30 ml glass bottles, with screw openings, polytetrafluoroethylene or silicone packing and caps
made of polypropylene, polycarbonate, or other suitable material.
5.17 Petri dishes, that have been sterilized, made of glass or plastic, in diameter sizes of 90 mm to 100 mm
and 55 mm to 60 mm.
5.18 Glass rod, with a diameter of approximately 18 mm.
5.19 Anti-bumping granules (glass beads), with a diameter of 3 mm to 4 mm.
5.20 Erlenmeyer flask, capacity 100 ml.
5.21 Cutting template, made of a sterilizable material (stainless steel or glass) with a diameter of
3,8 cm ± 0,1 cm.
5.22 Disposable plastic bags, suitable for containing food products, to be used for storage of samples.
5.23 Tweezers, made of a material which can be sterilized.
5.24 Stainless steel cylinder, with a mass of 200 g ± 10 g and a diameter of 3,5 cm ± 0,1 cm.
5.25 Metal wire basket, for autoclaving.
5.26 Aluminium foil.
© ISO 2007 – All rights reserved 3
---------------------- Page: 11 ----------------------
ISO 20743:2007(E)
6 Reagents and culture media
Reagents used in tests shall be of analytical quality and/or suited for microbiological purposes.
Dehydrated products available on the commercial market are recommended for use in preparing the culture
media. The manufacturer’s instructions for the preparation of these products should be strictly followed.
6.1 Water.
Water used in tests shall be analytical-grade water for microbiological media preparation which is freshly
distilled and/or ion-exchanged and/or ultra-filtered and/or filtered with RO (reverse osmosis). It shall be free
from all toxic or bacteria inhibitory substances.
6.2 Tryptone soya broth (TSB).
This solution shall be used for the resuscitation of the freeze-dried bacterial strains.
Tryptone, pancreatic digest of casein 15 g
Soya peptone, papain digest of soya 5 g
Sodium chloride (NaCl) 5 g
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
6.3 Tryptone soya agar (TSA).
Tryptone, pancreatic digest of casein 15 g
Soya peptone, papain digest of soya 5 g
Sodium chloride (NaCl) 5 g
Agar 15 g
Water 1 000 ml (final volume)
pH after sterilization 7,2 ± 0,2
6.4 Agar for transfer.
Tryptone, pancreatic digest of casein 0,75 g
Soya peptone, papain digest of soya 0,25 g
Sodium chloride (NaCl) 5 g
Agar 15 g
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
4 © ISO 2007 – All rights reserved
---------------------- Page: 12 ----------------------
ISO 20743:2007(E)
6.5 Nutrient broth (NB).
Beef extract 3 g
Peptone 5 g
Water 1 000 ml (final volume)
pH after sterilization
6,9 ± 0,2
6.6 Peptone-salt solution.
Tryptone, pancreatic digest of casein 1 g
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml (final volume)
pH after sterilization
6,9 ± 0,2
6.7 Physiological saline.
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml (final volume)
6.8 SCDLP medium.
Peptone, digest of casein 17 g
Peptone, digest of soybean 3 g
Sodium chloride (NaCl) 5 g
Potassium dihydrogenphosphate 2,5 g
Glucose 2,5 g
Lecithin 1 g
Polysorbate 80 7 g
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
If the neutralizing power is insufficient, the content of polysorbate 80 or lecithin may be adjusted or another
neutralizing agent may be added. The use of any unspecified neutralizer shall be recorded along with the
name and concentration.
6.9 Dilution buffer for shake-out bacterial suspension.
This buffer solution consists of 0,005 mol/l sodium dihydrogenphosphate containing 0,037 % sucrose.
pH
7,2 ± 0,2
© ISO 2007 – All rights reserved 5
---------------------- Page: 13 ----------------------
ISO 20743:2007(E)
6.10 Neutralizing solution.
The composition of the standard neutralizing solution shall be as follows.
Polysorbate 80 30 g
Egg yolk lecithin 3 g
Histidine hydrochloride 1 g
Meat or casein peptone 1 g
Sodium chloride (NaCl) 4,3 g
Monopotassium phosphate 3,6 g
Disodium phosphate dihydrate 7,2 g
Water 1 000 ml (final volume)
When sufficient neutralizing power cannot be achieved, the content of polysorbate 80 or lecithin may be
adjusted or another neutralizing agent may be added. The use of any unspecified neutralizer shall be
recorded along with the name and concentration.
6.11 Enumeration agar (EA).
Dehydrated yeast extract 2,5 g
Casein tryptone 5,0 g
Glucose 1,0 g
Agar 12 g to 18 g (depending on the gel strength of the product)
Water 1 000 ml (final volume)
pH after sterilization
7,2 ± 0,2
6.12 Agar for printing.
Agar 20 g
Water 1 000 ml (final volume)
6.13 Cryoprotective solution for bacterial species.
For freezing, a cryoprotective solution containing 150 g/l of glycerol or 100 g/l of dimethylsulfoxide shall be
used.
For solutions containing glycerol, prepare the nutritive solution and add 150 g of glycerol per litre prior to
sterilizing.
For solutions containing dimethylsulfoxide, sterilize the dimethylsulfoxide by means of a filtering system
equipped with a 0,22 µm membrane filter. Prepare the nutritive solution and, after sterilization, add 100 g of
dimethylsulfoxide per litre.
6 © ISO 2007 – All rights reserved
---------------------- Page: 14 ----------------------
ISO 20743:2007(E)
NOTE Any commercially available product may be used as long as it is a cryoprotective solution or preserving
system that contains glycerol or dimethylsulfoxide and allows preservation of the strains in the same manner as the
specified solutions.
Reagents used in tests shall be of analytical quality and/or suited for microbiological purposes.
6.14 Stock solution of ATP standard reagent.
Adenosine-disodium 5’triphosphate trihydrate 59,7 mg
Water 1 000 ml (final volume)
After preparation, the solution shall be placed in a tightly sealed container and cryopreserved at a temperature
of −20 °C or lower. The solution shall be used no later than 6 months from the date of preparation.
6.15 Buffer solution for ATP luminescence reagent.
Tris(hydroxymethyl)amino methane 760 mg
Ethylenediamine disodium tetraacetate dihydrate 370 mg
Magnesium acetate tetrahydrate 800 mg
DL-dithiothreitol 8 mg
Water 250 ml (final volume)
pH
7,5 ± 0,2
This solution shall be used no later than 8 h after preparation.
6.16 ATP luminescence reagent.
Luciferase (EC: 1.13.12.7) 10 mg
D-luciferin 15 mg
Bovine serum albumin 60 mg
Buffer solution of 6.15 30 ml (final volume)
After dissolving, the reagent shall be kept at room temperature for a minimum of 15 min before use and shall
be used within 3 h of preparation.
6.17 ATP extracting reagent.
10 % aq. Benzalkonium chloride 1 ml
Water 9 ml
Use of any unspecified extracting agent and the recipe shall be recorded.
© ISO 2007 – All rights reserved 7
---------------------- Page: 15 ----------------------
ISO 20743:2007(E)
6.18 ATP eliminating agent.
−13
An agent to reduce the ATP in nutrient broth to less than 10 mol/l within 15 min.
Apyrase (EC: 3.6.1.5) 5 international units
Adenosine deaminase (EC: 3.5.1.4) 5 international units
0,005 mol/l buffer solution of sodium dihydrogenphoshate 10 ml (final volume)
containing 0,037 % sucrose
pH
7,2 ± 0,2
Use of any unspecified eliminating agent and the recipe shall be recorded.
6.19 SCDLP or other medium for preparing ATP reference solution.
SCDLP of 6.8 or other medium 10 ml
ATP eliminating agent of 6.18 1 ml
After mixing, maintain at 30 °C to 37 °C for 1 h to prevent microbiological contamination.
Next, transfer to a hot water bath at 70 °C to 90 °C for 1 h and cool down to room temperature.
Preserve the solution under refrigeration and use within 24 h.
An ATP reference solution should be prepared if the addition of neutralizing agents causes the ATP content in
−11
the shake-out solution to exceed 10 mol/l.
7 Reference strains
7.1 Strains
The following strains shall be used in all antibacterial activity tests:
⎯ Staphylococcus aureus ATCC 6538, CIP 4.83, DSM 799, NBRC 13276 or NCIMB 9518;
⎯ Klebsiella pneumoniae ATCC 4352, CIP 104216, DSM 789, NBRC 13277 or NCIMB 10341.
NOTE ATCC is the American Type Culture Collection (USA); CIP is the Pasteur Institute Collection (France); DSM is
the German Collection of Microorganism and Cell Cultures (Germany); NBRC is the NITE Biological Resource Center
(Japan); and NCIMB is the National Collection of Industrial Bacteria (UK).
In lieu of the specified strains, equivalent bacteria strains obtained from agencies of the World Federation of
Culture Collection (WFCC) may be used by agreement between the interested parties. The strains used in the
test shall be stated in the test report together with their supply sources.
7.2 Storage of strains
7.2.1 General
The strains shall be stored in accordance with the supplier’s recommendations or EN 12353.
The identification and origin (culture collection) of the strains as well as the laboratory storage method shall be
recorded.
8 © ISO 2007 – All rights reserved
---------------------- Page: 16 ----------------------
ISO 20743:2007(E)
7.2.2 Ceramic bead method
Obtain a sample of the freeze-dried bacterial strain following the recommendations supplied with the culture
and resuspend in 5 ml of TSB (6.2). Obtain a sample of the suspension and isolate it in a Petri dish (5.17)
containing TSA (6.3). Incubate the cultures for 18 h to 24 h at 37 °C ± 2 °C.
After incubation, use the culture isolated in the Petri dish to verify the purity of the strain.
After verification, prepare the stock cultures.
Sample 0,7 ml of the broth culture and spread it over the surface of the Petri dish containing the TSA.
Incubate the culture on plates for 18 h to 24 h under the conditions specified for the strain in the standard.
Add 10 ml of cryoprotective solution (6.13) to the surface of the TSA plate culture and resuspend the cells in
the solution using a sterile glass spreader. Sample the suspended cells from the surface of the agar, dilute
them in 100 ml of cryoprotective solution and incubate for 30 min at 20 °C.
Using a pipette (5.15), sample 1 ml of the suspension and transfer it to a cryogenic vial (5.16) containing the
beads (5.19). Shake the vial in order to spread the suspended cells around the beads.
⎯ Where a cryoprotective solution containing dimethylsulfoxide is used, do not let it stand longer than 1 min
at ambient temperature.
⎯ Where a cryoprotective solution containing glycerol is used, let it stand for 30 min at 20 °C.
⎯ Withdraw the excess cryoprotective solution with a sterile pipette. Place the cryogenic vials in a freezer
(5.12) set at −70 °C or lower.
−6 −7
Prepare 10 and 10 dilutions of the suspension using the serial dilution method. Take a 1,0 ml sample of
each dilution and transfer it to separate Petri dishes. Add 12 ml to 15 ml of nutritive solution, cooled down to
45 °C ± 1 °C. Incubate for 18 h to 24 h under the conditions specified for the strain. Enumerate the plate
8
cultures and confirm that the suspension contains less than 5 × 10 CFU/ml.
Store the cryogenic vials in a freezer at a temperature below −70 °C.
7.2.3 Glycerol suspension method
Inoculate a 15 ml culture tube containing 5 ml of appropriate medium with a freshly grown isolated colony.
Incubate at 37 °C until the culture is in the late log or stationary phase (usually 5 h to overnight).
For each strain to be stored below −70 °C, for the archives, prepare a sterile, labelled cryogenic vial. Place
225 µl of sterile 80 % glycerol in a cryogenic vial. Add 1,0 ml of the bacterial culture (frozen stock shall be
15 % glycerol). Mix well using the vortex mixer (5.4) and store in a tube at −70 °C or lower.
For each strain to be stored at −20 °C, as liquid glycerol working stock, pipette equal volumes of 80 % glycerol
and bacterial culture into a labelled polypropylene tube. Mix the contents well to avoid formation of ice crystals
that will decrease the viability of the cells. Place the tube in a freezer at −20 °C. Check the viability of the cells
after 1 week if possible.
To recover a strain from the below −70 °C glycerol stock, use a sterile toothpick to scrape pieces of the solid
substance, then streak the cells onto the appropriate medium. Do not thaw the frozen stock because each
freeze-thaw cycle will result in a 50 % loss in cell viability.
To use the −20 °C working stock, pipette 50 µl to 100 µl as inoculum for a 5 ml overnight culture.
© ISO 2007 – All rights reserved 9
---------------------- Page: 17 ----------------------
ISO 20743:2007(E)
7.2.4 Remarks
The identity of the strain shall be verified in accordance with commonly accepted identification methods.
For each microbial strain, the following information shall be recorded:
a) the name of the national culture collection from which the freeze-dried strain was obtained;
b) the taxonomic name and reference number of the freeze-dried strain;
c) the batch number of the freeze-dried sample obtained from the culture collection;
d) the date of the resuscitated freeze-dried sample;
e) the date of prepared stock culture;
f) the stock culture laboratory code.
Details of the methods used for verifying the purity and identity of the
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.