SIST EN ISO 18416:2016

Cosmetics - Microbiology - Detection of Candida albicans (ISO 18416:2015)

This International Standard gives general guidelines for the detection and identification of the specified
microorganism Candida albicans i n c osmetic products. M icroorganisms c onsidered a s s pecified i n t his
International Standard might differ from country to country according to national practices or regulations.
In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate
microbiological risk analysis to determine the types of cosmetic product to which this International
Standard is applicable. Products considered to present a low microbiological (see ISO 29621) risk
include those with low water activity, hydro-alcoholic products, extreme pH values, etc.
The method described in this International Standard is based on the detection of Candida albicans in
a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar medium.
Other methods may be appropriate dependent on the level of detection required.
NOTE For the detection of Candida albicans, subcultures can be performed on non-selective culture media
followed by suitable identification steps (e.g. using identification kits).
Because of the large variety of cosmetic products within this field of application, this method may
not be appropriate in every detail for some products (e.g. certain water immiscible products). Other
International Standards (ISO 18415) may be appropriate. Other methods (e.g. automated) may be
substituted for the tests presented here provided that their equivalence has been demonstrated or the
method has been otherwise shown to be suitable.

Kosmetische Mittel - Mikrobiologie - Nachweis von Candida albicans (ISO 18416:2015)

Diese Internationale Norm gibt allgemeine Anleitungen für den Nachweis und die Identifizierung des festgelegten Mikroorganismus Candida albicans in kosmetischen Mitteln. Die in dieser Internationalen Norm festgelegten Mikroorganismen können in Übereinstimmung mit nationalen Praktiken oder Regelungen von Land zu Land unterschiedlich sein.
Um die Qualität des Produkts und die Sicherheit für Verbraucher sicherzustellen, ist es ratsam, eine geeignete mikrobiologische Risikoanalyse zur Bestimmung der Arten von kosmetischen Mitteln durchzuführen, für die diese Internationale Norm anwendbar ist. Produkte, die ein geringes mikrobiologisches Risiko (siehe ISO 29621) darstellen, umfassen jene mit geringer Wasseraktivität, alkoholisch wässrige Produkte, Produkte mit extremen pH Werten usw.
Das in dieser Internationalen Norm beschriebene Verfahren basiert auf dem Nachweis von Candida albicans in einem nicht selektiven Flüssigmedium (Anreicherungsbouillon), gefolgt von einer Isolation auf einem selek¬tiven Agarmedium. In Abhängigkeit von der erforderlichen Nachweisgrenze können andere Verfahren an¬gemessen sein.
ANMERKUNG   Für den Nachweis von Candida albicans kann eine Subkultivierung auf nicht selektiven Nährmedien erfolgen, gefolgt von geeigneten Schritten zur Identifizierung (z. B. unter Anwendung von Identifizierungskits).
Wegen der großen Vielfalt an kosmetischen Mitteln innerhalb dieses Anwendungsbereichs, ist dieses Verfahren möglicherweise für einige Produkte nicht in allen Einzelheiten geeignet (z. B. bestimmte mit Wasser nicht mischbare Produkte). Andere Internationale Normen (ISO 18415) können geeignet sein. Ersatzweise können für die hier aufgeführten Untersuchungen andere Verfahren (z. B. automatisierte) zur Anwendung kommen, vorausgesetzt, dass deren Gleichwertigkeit nach¬gewiesen oder sich das Verfahren anderweitig als geeignet erwiesen hat.

Cosmétiques - Microbiologie - Détection de Candida albicans (ISO 18416:2015)

ISO 18416:2015 donne des lignes directrices générales pour la détection et l'identification du microorganisme spécifié Candida albicans dans les produits cosmétiques. Les microorganismes considérés comme spécifiés dans la présente Norme internationale peuvent différer d'un pays à l'autre suivant les pratiques ou les réglementations nationales.
Pour garantir la qualité du produit et la sécurité des consommateurs, il est préférable d'effectuer une analyse appropriée du risque microbiologique pour déterminer les types de produits cosmétiques qui relèvent de la présente Norme internationale. Les produits considérés comme présentant un faible risque microbiologique (voir ISO 29621) comprennent ceux ayant une faible activité de l'eau, les produits hydroalcooliques, ceux ayant des valeurs de pH extrêmes, etc.
La méthode décrite dans la présente Norme internationale repose d'abord sur la détection de Candida albicans dans un milieu liquide non sélectif (bouillon d'enrichissement), suivie d'un isolement sur un milieu gélosé sélectif. D'autres méthodes peuvent être appropriées en fonction du niveau de détection exigé.
NOTE          Pour la détection de Candida albicans, il est possible de réaliser des subcultures sur des milieux de culture non sélectifs, puis de procéder aux étapes appropriées d'identification (par exemple en utilisant des kits d'identification).
En raison de la grande variété de produits cosmétiques relevant de ce domaine d'application, il se peut que cette méthode ne soit pas appropriée en tous points à certains produits (par exemple aux produits non miscibles à l'eau). D'autres Normes internationales (ISO 18415) peuvent être appropriées. Il est possible de remplacer les essais présentés ici par d'autres méthodes (par exemple des méthodes automatisées) sous réserve que leur équivalence ait été démontrée ou que la méthode ait été par ailleurs indiquée comme adéquate.

Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti kvasovke Candida albicans (ISO 18416:2015)

Ta mednarodni standard podaja splošne smernice za ugotavljanje prisotnosti in prepoznavanje mikroorganizma Candida albicans v kozmetičnih izdelkih. Mikroorganizmi, ki so upoštevani, kot so določeni v tem mednarodnem standardu, se lahko v različnih državah zaradi državnih praks ali predpisov razlikujejo. Za namen zagotovitve kakovosti in varnosti izdelkov za stranke je priporočljivo izvesti ustrezno mikrobiološko analizo tveganja, s katero se določijo vrste kozmetičnih izdelkov, za katere velja ta mednarodni standard. Izdelki, za katere se meni, da predstavljajo nizko mikrobiološko tveganje (glej ISO 29621), vključujejo tiste z nizko aktivnostjo vode, hidro-alkoholne izdelke, skrajne vrednosti pH itd. Metoda, opisana v tem mednarodnem standardu, temelji na ugotavljanju prisotnosti Candida albicans v neselektivnem tekočem gojišču (obogatitven bujon), ki mu sledi izolacija na selektivnem agarskem gojišču.
Ustrezne so lahko tudi druge metode, odvisno od zahtevane ravni ugotavljanja prisotnosti.
OPOMBA: Za namen ugotavljanja prisotnosti Candida albicans je mogoče precepljene kulture vzgojiti v neselektivnem gojišču kultur, čemur sledijo ustrezni koraki prepoznavanja (npr. uporaba kompletov za prepoznavanje).
Zaradi velike raznolikosti kozmetičnih izdelkov na tem področju uporabe ta metoda morda ni primerna za nekatere izdelke (npr. tiste, ki se ne mešajo z vodo). Uporabljajo se lahko drugi mednarodni standardi (ISO 18415). Druge metode (npr. avtomatske) je mogoče zamenjati za tukaj predstavljene preskuse, če je bila dokazana njihova enakovrednost ali je bila metoda drugače dokazana za primerno.

General Information

• Status: Published
• Public Enquiry End Date: 29-Sep-2015
• Publication Date: 01-Feb-2016
• ICS: 07.100.40 - Cosmetics microbiology
• Technical Committee: KDS - Cosmetics, chemical disinfectants and surface active agents
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 08-Jan-2016
• Due Date: 14-Mar-2016
• Completion Date: 02-Feb-2016
• Mandate: M/426 - Standardisation mandate assigned to CEN concerning the microbiological analysis of cosmetic products
• Ref Project: EN ISO 18416:2015 - Cosmetics - Microbiology - Detection of Candida albicans (ISO 18416:2015, Corrected version 2016-12-15)

Relations

• Replaces: SIST EN ISO 18416:2009 - Cosmetics - Microbiology - Detection of Candida albicans (ISO 18416:2007)
• Effective Date: 01-Mar-2016
• Amended By: SIST EN ISO 18416:2016/A1:2023 - Cosmetics - Microbiology - Detection of Candida albicans - Amendment 1 (ISO 18416:2015/Amd 1:2022)
• Effective Date: 01-Mar-2023

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 18416:2016
01-marec-2016
1DGRPHãþD
SIST EN ISO 18416:2009
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti kvasovke Candida albicans
(ISO 18416:2015)
Cosmetics - Microbiology - Detection of Candida albicans (ISO 18416:2015)

Kosmetische Mittel - Mikrobiologie - Nachweis von Candida albicans (ISO 18416:2015)

Cosmétiques - Microbiologie - Détection de Candida albicans (ISO 18416:2015)
Ta slovenski standard je istoveten z: EN ISO 18416:2015
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 18416:2016 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 18416:2016
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SIST EN ISO 18416:2016
EN ISO 18416
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2015
EUROPÄISCHE NORM
ICS 07.100.99; 71.100.70 Supersedes EN ISO 18416:2009
English Version
Cosmetics - Microbiology - Detection of Candida albicans
(ISO 18416:2015)

Cosmétiques - Microbiologie - Détection de Candida Kosmetische Mittel - Mikrobiologie - Nachweis von

albicans (ISO 18416:2015) Candida albicans (ISO 18416:2015)
This European Standard was approved by CEN on 26 September 2015.

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, Former Yugoslav Republic of Macedonia, 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
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18416:2015 E

worldwide for CEN national Members.
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SIST EN ISO 18416:2016
EN ISO 18416:2015 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 18416:2016
EN ISO 18416:2015 (E)
European foreword

This document (EN ISO 18416:2015) 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 juin 2016, and conflicting national standards shall be

withdrawn at the latest by juin 2016.

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.
This document supersedes EN ISO 18416:2009.

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.

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, Former Yugoslav Republic of Macedonia,

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 18416:2015 has been approved by CEN as EN ISO 18416:2015 without any modification.

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SIST EN ISO 18416:2016
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SIST EN ISO 18416:2016
INTERNATIONAL ISO
STANDARD 18416
Second edition
2015-12-01
Cosmetics — Microbiology —
Detection of Candida albicans
Cosmétiques — Microbiologie — Détection de Candida albicans
Reference number
ISO 18416:2015(E)
ISO 2015
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SIST EN ISO 18416:2016
ISO 18416:2015(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
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SIST EN ISO 18416:2016
ISO 18416:2015(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Diluents and culture media ....................................................................................................................................................................... 2

5.1 General ........................................................................................................................................................................................................... 2

5.2 Diluent for the yeast suspension (tryptone sodium chloride solution) ................................................ 3

5.2.1 General...................................................................................................................................................................................... 3

5.2.2 Composition ......................................................................................................................................................................... 3

5.2.3 Preparation ........................................................................................................................................................................... 3

5.3 Culture media ........................................................................................................................................................................................... 3

5.3.1 General...................................................................................................................................................................................... 3

5.3.2 Agar medium for suitability test (see Clause 11) ................................................................................ 3

5.3.3 Enrichment broth ............................................................................................................................................................ 4

5.3.4 Selective agar medium for isolation of Candida albicans .......................................................... 4

5.3.5 Corn meal agar with 1 % polysorbate 80 ................................................................................................... 5

6 Apparatus and glassware ............................................................................................................................................................................ 5

7 Strains of microorganisms ......................................................................................................................................................................... 5

8 Handling of cosmetic products and laboratory samples ............................................................................................ 6

9 Procedure..................................................................................................................................................................................................................... 6

9.1 General recommendation .............................................................................................................................................................. 6

9.2 Preparation of the initial suspension in the enrichment broth .................................................................... 6

9.2.1 General...................................................................................................................................................................................... 6

9.2.2 Water-miscible products........................................................................................................................................... 6

9.2.3 Water-immiscible products .................................................................................................................................... 6

9.2.4 Filterable products ......................................................................................................................................................... 6

9.3 Incubation of the inoculated enrichment broth ......................................................................................................... 7

9.4 Detection and identification of Candida albicans .................................................................................................. 7

9.4.1 Isolation ................................................................................................................................................................................... 7

9.4.2 Identification of Candida albicans ................................................................................................................. 7

10 Expression of the results (detection of Candida albicans) ........................................................................................ 8

11 Neutralization of the antimicrobial properties of the product ............................................................................ 8

11.1 General ........................................................................................................................................................................................................... 8

11.2 Preparation of inoculum ................................................................................................................................................................. 8

11.3 Suitability of the detection method ....................................................................................................................................... 8

11.3.1 Procedure ............................................................................................................................................................................... 8

11.3.2 Interpretation of suitability test results ...................................................................................................... 9

12 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Other media ...................................................................................................................................................................10

Annex B (informative) Neutralizers of antimicrobial activity of preservatives and

rinsing liquids ......................................................................................................................................................................................................14

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

© ISO 2015 – All rights reserved iii
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SIST EN ISO 18416:2016
ISO 18416:2015(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 217, Cosmetics.

This second edition cancels and replaces the first edition (ISO 18416:2007), of which it constitutes a

minor revision.
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SIST EN ISO 18416:2016
ISO 18416:2015(E)
Introduction

Microbiological examinations of cosmetic products are carried out according to an appropriate

microbiological risk analysis in order to ensure their quality and safety for consumers.

Microbiological risk analysis depends on several parameters such as the following:

— potential alteration of cosmetic products;
— pathogenicity of microorganisms;

— site of application of the cosmetic product (hair, skin, eyes, mucous membranes);

— type of users (adults, children under 3 years).

For cosmetics and other topical products, the detection of skin pathogens such as Staphylococcus

aureus, Pseudomonas aeruginosa and Candida albicans may be relevant because they can cause skin

or eye infections. The detection of other kinds of microorganism might be of interest since these

microorganisms (including indicators of faecal contamination e.g. Escherichia coli) suggest hygienic

failure during the manufacturing process.
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SIST EN ISO 18416:2016
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SIST EN ISO 18416:2016
INTERNATIONAL STANDARD ISO 18416:2015(E)
Cosmetics — Microbiology — Detection of Candida albicans
1 Scope

This International Standard gives general guidelines for the detection and identification of the specified

microorganism Candida albicans in cosmetic products. Microorganisms considered as specified in this

International Standard might differ from country to country according to national practices or regulations.

In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate

microbiological risk analysis to determine the types of cosmetic product to which this International

Standard is applicable. Products considered to present a low microbiological (see ISO 29621) risk

include those with low water activity, hydro-alcoholic products, extreme pH values, etc.

The method described in this International Standard is based on the detection of Candida albicans in

a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar medium.

Other methods may be appropriate dependent on the level of detection required.

NOTE For the detection of Candida albicans, subcultures can be performed on non-selective culture media

followed by suitable identification steps (e.g. using identification kits).

Because of the large variety of cosmetic products within this field of application, this method may

not be appropriate in every detail for some products (e.g. certain water immiscible products). Other

International Standards (ISO 18415) may be appropriate. Other methods (e.g. automated) may be

substituted for the tests presented here provided that their equivalence has been demonstrated or the

method has been otherwise shown to be suitable.
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 21148:2005, Cosmetics — Microbiology — General instructions for microbiological examination

EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the

determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal

(including bacteriophages) activity
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
product
portion of an identified cosmetic product received in the laboratory for testing
3.2
sample

portion of the product (at least 1 g or 1 ml) that is used in the test to prepare the initial suspension

3.3
initial suspension

suspension (or solution) of the sample in a defined volume of an appropriate enrichment broth

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SIST EN ISO 18416:2016
ISO 18416:2015(E)
3.4
sample dilution
dilution of the initial suspension
3.5
specified microorganism

aerobic mesophilic bacterium or yeast that is undesirable in a cosmetic product and is recognized as a

skin pathogen species that may be harmful for human health or as an indication of hygienic failure in

the manufacturing process
3.6
Candida albicans

yeast that form white to beige, creamy and convex colonies on the surface of a selective medium

Note 1 to entry: The main characteristic for identification is the production of germ tube and/or pseudomycelium

and chlamydospore when the test is performed following the method specified in this International Standard.

3.7
enrichment broth

non-selective liquid medium containing suitable neutralizers and/or dispersing agents and

demonstrated to be suitable for the product under test
4 Principle

The first step of the procedure is to perform an enrichment by using a non-selective broth medium to

increase the number of microorganisms without the risk of inhibition by the selective ingredients that

are present in selective/differential growth media.

The second step of the test (isolation) of the test is performed on a selective medium followed by

identification tests.

The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection

[1]

of viable microorganisms. In all cases and whatever the methodology, the neutralization of the

antimicrobial properties of the product shall be checked and demonstrated (see Clause 11).

5 Diluents and culture media
5.1 General

General instructions are given in ISO 21148. When water is mentioned in this International Standard,

use distilled water or purified water as specified in ISO 21148.

The enrichment broth is used to disperse the sample and to increase the initial microbial population. It

may contain neutralizers if the specimen to be tested has antimicrobial properties. The efficacy of the

neutralization shall be demonstrated (see Clause 11). Information relative to suitable neutralizers is

given in Annex B.

The enrichment broth (5.3.3.1), or any of the ones listed in Annex A, is suitable for checking the

presence of Candida albicans in accordance with this International Standard provided that it has been

demonstrated to be suitable in accordance with Clause 11.

Other diluents and culture media may be used if it has been demonstrated that they are suitable for use.

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ISO 18416:2015(E)
5.2 Diluent for the yeast suspension (tryptone sodium chloride solution)
5.2.1 General

The diluent is used for the preparation of yeast suspension used for the suitability test procedure

(see Clause 11).
5.2.2 Composition
— tryptone, pancreatic digest of casein 1,0 g
— sodium chloride 8,5 g
— water 1 000 ml
5.2.3 Preparation

Dissolve the components in water by mixing while heating. Dispense into suitable containers. Sterilize

in the autoclave at 121 °C for 15 min.

After sterilization and cooling of the solution, the pH shall be equivalent to 7,0 ± 0,2 when measured at

room temperature.
5.3 Culture media
5.3.1 General

Culture media may be prepared using the descriptions provided below or from dehydrated culture

media in accordance with the manufacturer’s instructions. The instructions provided by the supplier

of the media should be followed.

NOTE Ready-to-use media can be used when their composition and/or growth yields are comparable to

those of the formulae given herein.
5.3.2 Agar medium for suitability test (see Clause 11)
5.3.2.1 Sabouraud dextrose agar (SDA)
5.3.2.1.1 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
5.3.2.1.2 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.
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SIST EN ISO 18416:2016
ISO 18416:2015(E)
5.3.2.2 Other agar media for suitability test
Other agar media for suitability test may be used as appropriate (see Annex A).
5.3.3 Enrichment broth
5.3.3.1 Eugon LT 100 broth
5.3.3.1.1 General

This medium contains ingredients which neutralize inhibitory substances present in the sample:

lecithin and polysorbate 80, and dispersing agent octoxynol 9.
5.3.3.1.2 Composition
— pancreatic digest of casein 15,0 g
— papaic digest of soybean meal 5,0 g
— L-cystine 0,7 g
— sodium chloride 4,0 g
— sodium sulfite 0,2 g
— glucose 5,5 g
— egg lecithin 1,0 g
— polysorbate 80 5,0 g
— octoxynol 9 1,0 g
— water 1 000 ml
5.3.3.1.3 Preparation

Dissolve the components polysorbate 80, octoxynol 9 and egg lecithin successively in boiling water to

complete dissolution. Dissolve the other components by mixing while heating. Dispense the medium

into suitable containers. Sterilize in the autoclave at 121 °C for 15 min.

After sterilization and cooling of the solution, the pH shall be equivalent to 7,0 ± 0,2 when measured at

room temperature.
5.3.3.2 Other enrichment broths
Other enrichment broths may be used as appropriate (see Annex A).
5.3.4 Selective agar medium for isolation of Candida albicans
5.3.4.1 Sabouraud dextrose chloramphenicol agar
5.3.4.1.1 Composition
— dextrose 40,0 g
— peptic digest of animal tissue 5,0 g
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ISO 18416:2015(E)
— pancreatic digest of casein 5,0 g
— chloramphenicol 0,050 g
— agar 15,0 g
— water 1 000 ml
5.3.4.1.2 Preparation

Dissolve the components (including the chloramphenicol) 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.
5.3.4.2 Other selective agar media
Other selective agar media may be used as appropriate (see Annex A).
5.3.5 Corn meal agar with 1 % polysorbate 80
5.3.5.1 Composition
— infusion from corn meal 50,0 g
— agar 15,0 g
— polysorbate 80 10,0 g
— water 1 000 ml
5.3.5.2 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 6,0 ± 0,2 when measured at room temperature.

6 Apparatus and glassware
Use the laboratory equipment, apparatus and glassware described in ISO 21148.
7 Strains of microorganisms

For the verification of the test conditions suitability, the following representative strain is used:

1) 2) 3) 4)

Candida albicans ATCC 10231 or equivalent strain: IP 48.72 or NCPF 3179 or NBRC 1594 or

KCTC 17205, or other equivalent national collection strain.

The culture should be reconstituted according to the procedures provided by the supplier of the

reference strain.
1) American Type Culture Collection.
2) Institute Pasteur.
3) National Collection of Pathogenic Fungi.
4) National Biological Resource Center.
5) Korean Collection for Type Culture.
© ISO 2015 – All rights reserved 5
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SIST EN ISO 18416:2016
ISO 18416:2015(E)
The strain can be kept in the laboratory in accordance with EN 12353.
8 Handling of cosmetic products and laboratory samples
If necessary, store products to be tested at room temperature.

Do not incubate, refrigerate or freeze products and samples before or after analysis.

Sampling of cosmetic products to be analysed should be carried out as described in ISO 21148. Analyse

samples as described in ISO 21148 and according to the procedure in Clause 9.
9 Procedure
9.1 General recommendation

Use sterile material, equipment and aseptic techniques to prepare the sample, initial suspension and

dilutions. In the case of the preparation of the initial suspension in an appropriate solubilizing agent, the

time which elapses between the end of preparation and the moment the inoculum comes into contact

with the enrichment broth shall not exceed 45 min, unless specifically mentioned in the established

protocols or documents.
9.2 Preparation of the initial suspension in the enrichment broth
9.2.1 General

The enrichment is prepared from a sample (3.2) of at least 1 g or 1 ml of the well-mixed product under

test, which is dispersed in at least 9 ml of enrichment broth.
Note S, the exact weight or volume of the sample.

The method shall be checked to ensure that the composition (neutralizer eventually added) and the

volume of the broth perform satisfactorily (see 11.3).

NOTE In some cases, and when possible, filtration of the cosmetic product through a membrane that is

afterwards immersed in the enrichment broth, facilitates the neutralization of the antimicrobial properties of

the product (see 11.3.)
9.2.2 Water-miscible products

Transfer the sample, S, of product to a suitable container containing an appropriate volume of broth.

9.2.3 Water-immiscible products

Transfer the sample, S, of product to a suitable container containing a suitable quantity of solubilizing

agent (e.g. Polysorbate 80).
Disperse the sample within the solubilizing agent and add an
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