SIST EN ISO 11930:2012
(Main)Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic product (ISO 11930:2012)
Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic product (ISO 11930:2012)
This International Standard comprises: - a preservation efficacy test; - a procedure for evaluating the overall antimicrobial protection of a cosmetic product which is not considered low risk, based on a risk assessment described in ISO 29621. This International Standard provides a procedure for the interpretation of data generated by the preservation efficacy test or by the microbiological risk assessment, or both.
Kosmetische Mittel - Mikrobiologie - Bewertung des mikrobiellen Schutzes eines kosmetischen Produktes (ISO 11930:2012)
1.1 Allgemeines
Diese Norm umfasst:
die Prüfung der Wirksamkeit der Konservierung;
das Verfahren für die Bewertung des allgemeinen antimikrobiellen Schutzes eines kosmetischen Produktes, das aufgrund einer Risikobewertung nach ISO 29621 nicht als risikoarm gilt.
Diese Internationale Norm liefert ein Verfahren zur Interpretation der Daten, die durch die Prüfung der Wirksamkeit der Konservierung oder der mikrobiologischen Risikobewertung, oder beidem, ermittelt wurden.
1.2 Prüfung der Wirksamkeit der Konservierung
Diese Prüfung ist ein Referenzverfahren, das zur Beurteilung der Konservierung von kosmetischen Rezepturen angewendet werden kann. Es gilt für kosmetische Produkte auf dem Markt.
Diese Prüfung ist für die kosmetischen Produkte nicht erforderlich, bei denen das mikrobiologische Risiko als niedrig bestimmt wurde (siehe Anhang A und ISO 29621).
Diese Prüfung ist vor allem für wasserlösliche oder wassermischbare kosmetische Mittel bestimmt, und kann eine Anpassung erfordern, z. B. bei Prüfprodukten, in denen sich Wasser in der internen Phase befindet. Die in dieser Norm beschriebene Prüfung beinhaltet bei jedem Prüfmikroorganismus das Inkontaktbringen der Rezeptur mit einem kalibrierten Inoculum, und die Messung der Veränderungen in der Mikroorganismenzahl bei festgelegten Zeitintervallen für eine festgelegte Zeitdauer und bei einer festgelegten Temperatur.
ANMERKUNG Diese Prüfung kann als Leitfaden für die Entwicklung einer hausinternen Methode für den Entwicklungsprozess eines kosmetischen Mittels angewendet werden. In diesem Fall kann die Prüfung modifiziert oder erweitert werden, oder beides, z. B. zur Berücksichtigung von vorherigen Daten und verschiedenen Variablen (mikrobielle Stämme, Medien, Einwirkungszeit der Bebrütungsbedingungen usw.). Die Übereinstimmungskriterien können den spezifischen Zielvorgaben angepasst werden. In der Entwicklungsphase von kosmetischen Produkten können gegebenenfalls andere Verfahren zur Bestimmung der Wirksamkeit der Konservierungsrezepturen angewendet werden.
1.3 Verfahren für die Bewertung des antimikrobiellen Schutzes eines kosmetischen Produktes
Dieses Verfahren basiert auf der sorgfältigen Berücksichtigung folgender Punkte:
- Ergebnisse der Prüfung der Wirksamkeit der Konservierung. Nicht alle kosmetischen Produkte werden eine Prüfung der Wirksamkeit der Konservierung erfordern (siehe Anhang A und ISO 29621).
- Rezeptureigenschaften und Daten, die sich aus der mikrobiologischen Risikobewertung (siehe ISO 29621) ergeben. Die Analyse der mikrobiologischen Risikobewertung erfolgt auf der Grundlage einer allgemeinen Herangehensweise. Die Analyse fasst Variablen zusammen, z. B. die Eigenschaften und die Zusammensetzung der Rezeptur, deren Herstellungsbedingungen, die Eigenschaften der Verpackung, in der die Rezeptur auf den Markt gebracht wird, Empfehlungen für die Anwendung des kosmetischen Produktes, und falls von Bedeutung, den Anwendungsbereich und die Verbraucher¬zielgruppe (siehe Anhang D).
Cosmétiques - Microbiologie - Évaluation de la protection antimicrobienne d'un produit cosmétique (ISO 11930:2012)
L'ISO 11930:2012 comprend un essai d'efficacité de la protection antimicrobienne et une procédure permettant d'évaluer la protection antimicrobienne globale d'un produit cosmétique qui n'est pas identifié comme étant à faible risque microbiologique.
L'ISO 11930:2012 fournit une procédure pour l'interprétation des données résultant de l'essai d'efficacité de la protection antimicrobienne ou de l'évaluation du risque microbiologique, ou des deux.
Kozmetika - Mikrobiologija - Vrednotenje protimikrobne zaščite kozmetičnih izdelkov (ISO 11930:2012)
Ta mednarodni standard vključuje naslednje: – preizkus ohranjanja učinkovitosti; – postopek za ocenjevanje celotne antimikrobske zaščite kozmetičnega izdelka, ki ne velja za izdelek z nizkim tveganjem, na podlagi ocene tveganja, navedene v standardu ISO 29621. Ta mednarodni standard zagotavlja postopek za interpretacijo podatkov, pridobljenih s preizkusom ohranjanja učinkovitosti ali z oceno mikrobiološkega tveganja ali obojega.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 11930:2012
01-december-2012
.R]PHWLND0LNURELRORJLMD9UHGQRWHQMHSURWLPLNUREQH]DãþLWHNR]PHWLþQLK
L]GHONRY ,62
Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic
product (ISO 11930:2012)
Kosmetische Mittel - Mikrobiologie - Bewertung des mikrobiellen Schutzes eines
kosmetischen Produktes (ISO 11930:2012)
Cosmétiques - Microbiologie - Évaluation de la protection antimicrobienne d'un produit
cosmétique (ISO 11930:2012)
Ta slovenski standard je istoveten z: EN ISO 11930:2012
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
71.100.70 .R]PHWLND7RDOHWQL Cosmetics. Toiletries
SULSRPRþNL
SIST EN ISO 11930:2012 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 11930:2012
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SIST EN ISO 11930:2012
EUROPEAN STANDARD
EN ISO 11930
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2012
ICS 07.100.99; 71.100.70
English Version
Cosmetics - Microbiology - Evaluation of the antimicrobial
protection of a cosmetic product (ISO 11930:2012)
Cosmétiques - Microbiologie - Évaluation de la protection Kosmetische Mittel - Mikrobiologie - Bewertung des
antimicrobienne d'un produit cosmétique (ISO 11930:2012) mikrobiellen Schutzes eines kosmetischen Produktes (ISO
11930:2012)
This European Standard was approved by CEN on 31 March 2012.
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-CENELEC 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-CENELEC Management Centre has the same
status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, 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, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11930:2012: E
worldwide for CEN national Members.
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SIST EN ISO 11930:2012
EN ISO 11930:2012 (E)
Contents Page
Foreword .3
2
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SIST EN ISO 11930:2012
EN ISO 11930:2012 (E)
Foreword
This document (EN ISO 11930:2012) has been prepared by Technical Committee ISO/TC 217 "Cosmetics" in
collaboration with Technical Committee CEN/TC 392 “Cosmetics” the secretariat of which is held by AFNOR.
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 October 2012, and conflicting national standards shall be withdrawn at
the latest by October 2012.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
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, Croatia, 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, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11930:2012 has been approved by CEN as a EN ISO 11930:2012 without any modification.
3
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SIST EN ISO 11930:2012
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SIST EN ISO 11930:2012
INTERNATIONAL ISO
STANDARD 11930
First edition
2012-04-01
Cosmetics — Microbiology — Evaluation
of the antimicrobial protection of a
cosmetic product
Cosmétiques — Microbiologie — Évaluation de la protection
antimicrobienne d’un produit cosmétique
Reference number
ISO 11930:2012(E)
©
ISO 2012
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
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 2012 – All rights reserved
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
Contents Page
Foreword .iv
Introduction . v
1 Scope . 1
1.1 General . 1
1.2 Preservation efficacy test . 1
1.3 Procedure for evaluating the antimicrobial protection of the cosmetic product . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
5 Preservation efficacy test . 3
5.1 General . 3
5.2 Materials, apparatus, reagents and culture media . 3
5.3 Microbial strains . 6
5.4 Preparation and enumeration of inocula . 7
5.5 Demonstration of the neutralizer efficacy . 8
5.6 Determination of the preservation efficacy of the formulation .10
5.7 Interpretation of test results and conclusions .12
5.8 Test report .13
6 Overall evaluation of the antimicrobial protection of the cosmetic product .14
6.1 General .14
6.2 Case 1 — The preservation efficacy test has been performed on the formulation .14
6.3 Case 2 — The preservation efficacy test has not been performed on the formulation .14
Annex A (normative) Decision diagram .16
Annex B (normative) Evaluation criteria for the preservation efficacy test (see 5.7) .17
Annex C (informative) Examples of neutralizers for the antimicrobial activity of preservatives and
washing liquids .18
Annex D (informative) Packaging characteristics.19
Bibliography .20
© ISO 2012 – All rights reserved iii
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SIST EN ISO 11930:2012
ISO 11930:2012(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 11930 was prepared by Technical Committee ISO/TC 217, Cosmetics.
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
Introduction
This International Standard is to be used in the overall evaluation of the antimicrobial protection of a
cosmetic product.
The antimicrobial protection of a product can come from many sources:
— chemical preservation;
— inherent characteristics of the formulation;
— package design;
— manufacturing process.
This International Standard defines a series of steps to be taken when assessing the overall antimicrobial
protection of a cosmetic product. A reference method for a preservation efficacy test (challenge test) along with
evaluation criteria is also described in this International Standard.
The data generated by the risk assessment (see ISO 29621) or by the preservation efficacy test, or both, are
to be used to establish the level of antimicrobial protection required to minimize user risk.
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SIST EN ISO 11930:2012
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SIST EN ISO 11930:2012
INTERNATIONAL STANDARD ISO 11930:2012(E)
Cosmetics — Microbiology — Evaluation of the antimicrobial
protection of a cosmetic product
1 Scope
1.1 General
This International Standard comprises:
— a preservation efficacy test;
— a procedure for evaluating the overall antimicrobial protection of a cosmetic product which is not considered
low risk, based on a risk assessment described in ISO 29621.
This International Standard provides a procedure for the interpretation of data generated by the preservation
efficacy test or by the microbiological risk assessment, or both.
1.2 Preservation efficacy test
This test is a reference method that is to be used to evaluate the preservation of a cosmetic formulation. It
applies to cosmetic products in the market place.
This test is not required for those cosmetic products for which the microbiological risk has been determined to
be low (see Annex A and ISO 29621).
This test is primarily designed for water-soluble or water-miscible cosmetic products and can require adaptation,
for example to test products in which water is the internal phase. The test described in this International Standard
involves, for each test micro-organism, placing the formulation in contact with a calibrated inoculum, and then
measuring the changes in the micro-organism count at set time intervals for a set period and at a set temperature.
NOTE This test can be used as a guideline to develop an in-house method during the development cycle of cosmetic
products. In this case, the test can be modified or extended, or both, for example to make allowance for prior data
and different variables (microbial strains, media, incubation conditions exposure time, etc.). Compliance criteria can be
adapted to specific objectives. During the development stage of cosmetic products, other methods, where relevant, can
be used to determine the preservation efficacy of formulations.
1.3 Procedure for evaluating the antimicrobial protection of the cosmetic product
This procedure is based on careful consideration of the following points.
— Results of the preservation efficacy test. Not all cosmetic products will require a preservation efficacy test
(see Annex A and ISO 29621).
— Formulation characteristics and data provided by the microbiological risk assessment (see ISO 29621).
The analysis of the microbiological risk assessment is based on an overall approach. In particular, it
integrates variables such as characteristics and composition of the formulation, its production conditions,
the characteristics of the packaging in which the formulation will be delivered to the market place,
recommendations for use of the cosmetic product and, when relevant, the area of application and the
targeted user population (see Annex D).
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.
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
ISO 16212, Cosmetics — Microbiology — Enumeration of yeast and mould
ISO 18415, Cosmetics — Microbiology — Detection of specified and non‑specified microorganisms
ISO 21148, Cosmetics — Microbiology — General instructions for microbiological examination
ISO 21149, Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria
ISO 22716, Cosmetics — Good Manufacturing Practices (GMP) — Guidelines on Good Manufacturing Practices
ISO 29621, Cosmetics — Microbiology — Guidelines for the risk assessment and identification of microbiologically
low‑risk products
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 21148 and the following apply.
3.1
cosmetic formulation
preparation of raw materials with a qualitatively and quantitatively defined composition
3.2
cosmetic product
finished cosmetic product that has undergone all stages of production, including packaging in its final
container for shipment
3.3
antimicrobial protection of a cosmetic product
ability of a cosmetic product to overcome microbial contamination that might present a potential risk to the user
NOTE The overall antimicrobial protection includes preservation of the formulation, the specific manufacturing
process and protective packaging.
3.4
preservation of a cosmetic formulation
set of means used to avoid microbial proliferation in a cosmetic formulation
EXAMPLES Preservatives, multifunctional compounds, hostile raw materials, extreme pH, low water-activity values.
3.5
reference method
method applied by interested parties to assess a product on the market and in case of dispute
3.6
development method
in-house method
method used during the development stage of a product before the product is put on the market
3.7
consumer
end user of a cosmetic product
4 Principle
The evaluation of the antimicrobial protection of a cosmetic product combines the following elements (see Annex A).
a) The characteristics of its formulation (see ISO 29621) or the results of the preservation efficacy test (if
performed), or both.
The preservation efficacy test is described in 5.1.
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
b) The characteristics of the cosmetic product in conjunction with the production conditions (see ISO 22716 and
ISO 29621), the packaging materials and, if justified, recommendations for use of the product (see ISO 29621).
This International Standard describes a procedure for the interpretation of data generated by the preservation
efficacy test (if appropriate) and by the microbiological risk assessment.
5 Preservation efficacy test
5.1 General
The evaluation of the preservation of a cosmetic formulation is based on inoculation of the formulation with
calibrated inocula (prepared from relevant strains of micro-organisms). The number of surviving micro-organisms
is measured at defined intervals during a period of 28 days. For each time and each strain, the log reduction
value is calculated and compared to the minimum values required for evaluation criteria A or B (see Annex B).
When used as a reference method, procedures shall be strictly followed in order to avoid variability in results. To
determine the preservation efficacy of a formulation during product development, other suitable development
methods may be used (see 1.2).
Prior to the test, the microbiological quality of the product shall be determined in accordance with ISO 21149
and ISO 16212, or with ISO 18415.
NOTE The micro-organisms present in the test sample should not interfere with the recovery of the test organisms.
In the test, the neutralization of the possible antimicrobial activity of the tested sample shall be checked and
demonstrated (see 5.5).
5.2 Materials, apparatus, reagents and culture media
General specifications and instructions are given in ISO 21148. When water is used in a formula, use distilled
water or purified water as specified in ISO 21148:2005, 8.2.
5.2.1 Materials
Use usual microbiology laboratory equipment (see ISO 21148) and:
5.2.1.1 Glass beads, 3 mm to 4 mm in diameter.
5.2.1.2 Sintered glass filter, of porosity 2 (40 µm to 100 µm).
5.2.1.3 Roux flasks.
5.2.1.4 Sterile glass containers with closures, of suitable volumes.
5.2.1.5 Centrifuge, capable of a centrifugal force of 2 000 g.
5.2.2 Diluents, neutralizers and culture media
5.2.2.1 General
Unless otherwise specified, all reagents shall be equilibrated at ambient temperature before use. When
available, ready-to-use reagents and media may be used.
5.2.2.2 Diluent
5.2.2.2.1 Composition
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
Pancreatic digest of casein 1,0 g
Sodium chloride 8,5 g
Water 1 000 ml
5.2.2.2.2 Preparation
Dissolve the components in the water by mixing while heating. Dispense into suitable containers. Sterilize in
the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent to 7,0 ± 0,2 when measured at
room temperature.
5.2.2.2.3 Polysorbate solution (for preparation of A. brasiliensis spore suspension)
Prepare a solution of polysorbate 80 (0,5 g/l). Dissolve by mixing while heating until complete dissolution is
achieved. Dispense the solution into suitable containers. Sterilize in the autoclave at 121 °C for 15 min.
5.2.2.3 Neutralizer
5.2.2.3.1 General
The suitability and effectiveness of the neutralizing agent with respect to the test strains used and to the tested
formulation shall be demonstrated as specified in 5.5.
The neutralizer described in 5.2.2.3.2 is frequently used. Examples of other suitable neutralizers are given in Annex C.
5.2.2.3.2 Eugon LT 100 liquid broth
5.2.2.3.2.1 General
This medium contains ingredients which neutralize inhibitory substances present in the sample (lecithin
®1)
and polysorbate 80) and dispersing agent octoxynol 9 (Triton X100 ). It may be prepared as described in
5.2.2.3.2.2, or from dehydrated culture medium, according to the manufacturer’s instructions. A ready-to-use
medium may also be used.
5.2.2.3.2.2 Composition
Pancreatic digest of casein 15 g
Papaic digest of soybean meal 5 g
Sodium chloride 4 g
L-cystine 0,7 g
Sodium sulphite 0,2 g
Glucose 5,5 g
Egg lecithin 1 g
Polysorbate 80 5 g
Octoxynol 9 1 g
Water 1 000 ml
1) Triton X100® is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of this product.
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
5.2.2.3.2.3 Preparation
Dissolve successively into boiling water polysorbate 80, octoxynol 9 and egg lecithin until they are completely
dissolved. Dissolve the other components by mixing while heating. Dispense the medium into suitable
containers. Sterilize in the autoclave at 121 °C for 15 min. Mix well after sterilization while the liquid is still hot
to redissolve settled substances. After sterilization, the pH shall be equivalent to 7,0 ± 0,2 when measured at
room temperature.
5.2.2.4 Culture media
5.2.2.4.1 General
Culture media may be prepared as in 5.2.2.4.2, or from dehydrated culture media according to the manufacturer’s
instructions. Ready-to-use media may be used when their composition and/or growth yields are comparable to
those of the formulae given in 5.2.2.4.2.1.
5.2.2.4.2 Culture medium for bacteria: tryptic soy agar (TSA) or soybean casein digest agar medium
5.2.2.4.2.1 Composition
Pancreatic digest of casein 15,0 g
Papaic digest of soybean meal 5,0 g
Sodium chloride 5,0 g
Agar 15,0 g
Water 1 000 ml
5.2.2.4.2.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating. Dispense
the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. Mix well after sterilization
while the liquid is still hot to redissolve settled substances. After sterilization and cooling down, the pH shall be
equivalent to 7,3 ± 0,2 when measured at room temperature.
5.2.2.4.3 Culture medium for C. albicans: Sabouraud dextrose agar medium (SDA)
5.2.2.4.3.1 Composition
Dextrose 40,0 g
Peptic digest of animal tissue 5,0 g
Pancreatic digest of casein 5,0 g
Agar 15,0 g
Water 1 000 ml
5.2.2.4.3.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating. Dispense
the medium into suitable containers. Sterilize in an autoclave at 121 °C for 15 min. After sterilization, the pH
shall be equivalent to 5,6 ± 0,2 when measured at room temperature.
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
5.2.2.4.4 Culture medium for A. brasiliensis: potato dextrose agar (PDA)
5.2.2.4.4.1 Composition
Potato infusion (see 5.2.2.4.4.2, Note 1) 200,0 g
Dextrose 20,0 g
Agar (see 5.2.2.4.4.2, Note 2) 20,0 g
Water 1 000 ml
5.2.2.4.4.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by heating. Dispense the medium
into suitable containers. Sterilize in an autoclave at 121 °C for 15 min. After sterilization, the pH shall be
equivalent to 5,6 ± 0,2 when measured at room temperature.
NOTE 1 To prepare potato infusion, use a commercial dehydrated form or boil 200 g sliced, unpeeled potatoes in 1 l of
water for 30 min. Filter through a cheesecloth, saving the effluent.
NOTE 2 Commercially available dehydrated medium powders which contain less than 20 g/l of agar can be supplemented
with extra agar to the final concentration of 20 g/l if necessary.
5.3 Microbial strains
The test shall be run using the following strains as test micro-organisms:
® TM2) ® TM3) ® TM4)
— Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626 or
® TM5) ® TM6)
NBRC 13275 or KCTC 2513 or other equivalent national collection strain);
® TM ® TM ® TM
— Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518 or
® TM ® TM ® TM7)
NBRC 13276 or KCTC 3881 or NCTC 10788 or other equivalent national collection strain);
® TM ® TM ® TM ® TM
— Escherichia coli ATCC 8739 (equivalent strain: CIP 53.126 or NCIMB 8545 or NBRC 3972 or
® TM ® TM
KCTC 2571 or NCTC 12923 or other equivalent national collection strain);
® TM TM8) ® TM9) ® TM
— Candida albicans ATCC 10231 (equivalent strain: IP 48.72 or NCPF 3179 or NBRC 1594
® TM
or KCTC 7965 or other equivalent national collection strain);
® TM ® TM10)
— Aspergillus brasiliensis (previously A. niger) ATCC 16404 (equivalent strain: IP 1431 or IMI 149007
® TM ® TM
or NBRC 9455 or KCTC 6196 or other equivalent national collection strain).
The culture should be reconstituted according to the procedures provided by the supplier of the reference strain.
The strains should be stored in a laboratory conforming to EN 12353 or according to another suitable method.
®
2) ATCC : American Type Culture Collection
®
3) CIP : Collection de l’Institut Pasteur
®
4) NCIMB : National Collection of Industrial Marine Bacteria
®
5) NBRC : NITE Biological Resource Center, JP
®
6) KCTC : Korean Collection for Type Cultures
®
7) NCTC : National Collection of Type Cultures
8) IP: Institut Pasteur
®
9) NCPF : National Collection of Pathogenic Fungi
10) IMI: International Mycological Institute, UK
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SIST EN ISO 11930:2012
ISO 11930:2012(E)
5.4 Preparation and enumeration of inocula
5.4.1 General
To perform the tests, use the strains stored in the laboratory (see 5.3) to obtain the stock cultures and the
working cultures.
The stock culture is a confluent culture obtained by streaking slant tubes or plates with the stored strain (single-
use vial or bead). After incubation, the stock culture can be kept between 2 °C and 8 °C for two months and is
used to obtain the working cultures.
The working culture, prepared when needed to perform a test, is used to obtain the calibrated suspension (inoculum).
The same growth conditions (agar media and incubation) are used for both stock cultures and working cultures
(see 5.4.2 and 5.4.3).
NOTE 1 A limited number of serial subcultures and the use of confluent cultures instead of isolated colonies lower
the risk of change in the susceptibility of strains. The standardization of growth conditions and of inoculum preparation
improves the reproducibility of the test.
NOTE 2 Changes in susceptibility of strains stored by freezing may be observed (due to repeated heat shocks) when
multidose containers are used (for example, containers with several beads brought out of the freezer to take one bead,
then replaced in the freezer).
5.4.2 Preparation of bacterial and Candida albicans suspensions
5.4.2.1 To prepare the working culture of the test micro-organism, prepare a subculture from the stock culture
by streaking slant tubes or plates (TSA for bacteria, SDA for C.albicans) in order to obtain a confluent culture.
Incubate at (32,5 ± 2,5) °C for 18 h to 24 h.
Prepare in the same way a second subculture, starting from the first, and incubate at (32,5 ± 2,5) °C for 18 h to
24 h. A third subculture can be grown in the same way, starting from the second. The second culture and the
third one (if it was carried out) form the working culture.
If the second subculture cannot be carried out in a timely manner, then the first subculture can be kept for up to
48 h in the incubator (32,5 ± 2,5) °C and used to prepare the second subculture. In this case, prepare the third
18 h to 24 h subculture and use this in the test.
It is recommended that a fourth subculture not be prepared from the initial stock culture.
5.4.2.2 Take 10 ml of diluent (5.2.2.2) and place in a suitable sterile container with approximately 5 g of sterile
glass beads. Transfer loopfuls of the cells harvested from the
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