Cosmetics - Microbiology - Detection of Staphylococcus aureus (ISO 22718:2015)

This International Standard gives general guidelines for the detection and identification of the
specified microorganism Staphylococcus aureus 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 Staphylococcus aureus
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 Staphylococcus aureus, 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 for some products in every detail (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 Staphylococcus aureus (ISO 22718:2015)

Diese Internationale Norm gibt allgemeine Anleitungen für den Nachweis und die Identifizierung des festgelegten Mikroorganismus Staphylococcus aureus 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 Staphylococcus aureus in einem nicht selektiven Flüssigmedium (Anreicherungsbouillon), gefolgt von einer Isolation auf einem selektiven Agarmedium. In Abhängigkeit von der erforderlichen Nachweisgrenze können andere Verfahren angemessen sein.
ANMERKUNG   Für den Nachweis von Staphylococcus aureus kann eine Subkultivierung auf nicht selektiven Nähr-medien 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 Staphylococcus aureus (ISO 22718:2015)

ISO 22718:2015 donne des lignes directrices générales pour la détection et l'identification du microorganisme spécifié Staphylococcus aureus 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 afin de 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 Staphylococcus aureus 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 Staphylococcus aureus, 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 adaptée 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 bakterije Staphylococcus aureus (ISO 22718:2015)

Ta mednarodni standard podaja splošne smernice za ugotavljanje prisotnosti in prepoznavanje bakterije Staphylococcus aureus 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, ki po ocenah predstavljajo nizko mikrobiološko (glejte ISO 29621) tveganje, vključujejo tiste z nizko aktivnostjo vode, hidro-alkoholne izdelke, skrajne vrednosti pH itd.
Metoda, opisana v tem mednarodnem standardu, temelji na ugotavljanju prisotnosti Staphylococcus aureus 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 Staphylococcus aureus 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
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
08-Jan-2016
Due Date
14-Mar-2016
Completion Date
02-Feb-2016

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SLOVENSKI STANDARD
SIST EN ISO 22718:2016
01-marec-2016
1DGRPHãþD
SIST EN ISO 22718:2009
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti bakterije Staphylococcus
aureus (ISO 22718:2015)
Cosmetics - Microbiology - Detection of Staphylococcus aureus (ISO 22718:2015)
Kosmetische Mittel - Mikrobiologie - Nachweis von Staphylococcus aureus (ISO
22718:2015)

Cosmétiques - Microbiologie - Détection de Staphylococcus aureus (ISO 22718:2015)

Ta slovenski standard je istoveten z: EN ISO 22718:2015
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 22718:2016 en,fr,de

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

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SIST EN ISO 22718:2016
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SIST EN ISO 22718:2016
EN ISO 22718
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2015
EUROPÄISCHE NORM
ICS 07.100.99; 71.100.70 Supersedes EN ISO 22718:2009
English Version
Cosmetics - Microbiology - Detection of Staphylococcus
aureus (ISO 22718:2015)

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

Staphylococcus aureus (ISO 22718:2015) Staphylococcus aureus (ISO 22718:2015)
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 22718:2015 E

worldwide for CEN national Members.
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SIST EN ISO 22718:2016
EN ISO 22718:2015 (E)
Contents Page

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

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SIST EN ISO 22718:2016
EN ISO 22718:2015 (E)
European foreword

This document (EN ISO 22718:2015) 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 June 2016, and conflicting national standards shall be

withdrawn at the latest by June 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 22718:2009.

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 22718:2015 has been approved by CEN as EN ISO 22718:2015 without any modification.

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SIST EN ISO 22718:2016
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SIST EN ISO 22718:2016
INTERNATIONAL ISO
STANDARD 22718
Second edition
2015-12-01
Cosmetics — Microbiology —
Detection of Staphylococcus aureus
Cosmétiques — Microbiologie — Détection de Staphylococcus aureus
Reference number
ISO 22718:2015(E)
ISO 2015
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SIST EN ISO 22718:2016
ISO 22718:2015(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. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved
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SIST EN ISO 22718:2016
ISO 22718:2015(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 bacterial 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 the suitability test (see Clause 11) [soybean-casein

digest agar medium (SCDA) or tryptic soy agar (TSA)] ................................................................. 3

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

5.3.4 Selective agar medium for isolation of Staphylococcus aureus ............................................ 5

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

7 Strains of microorganisms ......................................................................................................................................................................... 6

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

9 Procedure..................................................................................................................................................................................................................... 7

9.1 General recommendation .............................................................................................................................................................. 7

9.2 Preparation of the initial suspension in the enrichment broth .................................................................... 7

9.2.1 General...................................................................................................................................................................................... 7

9.2.2 Water-miscible products........................................................................................................................................... 7

9.2.3 Water-immiscible products .................................................................................................................................... 7

9.2.4 Filterable products ......................................................................................................................................................... 7

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

9.4 Detection and identification of Staphylococcus aureus .................................................................................... 7

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

9.4.2 Identification of Staphylococcus aureus ................................................................................................... 8

10 Expression of the results (detection of Staphylococcus aureus) ......................................................................... 8

11 Neutralization of the antimicrobial properties of the product ............................................................................ 9

11.1 General ........................................................................................................................................................................................................... 9

11.2 Preparation of inoculum ................................................................................................................................................................. 9

11.3 Suitability of the detection method ....................................................................................................................................... 9

11.3.1 Procedure ............................................................................................................................................................................... 9

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

12 Test report ................................................................................................................................................................................................................10

Annex A (informative) Other media ...................................................................................................................................................................11

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 22718:2016
ISO 22718:2015(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 22718:2006), of which it constitutes a

minor revision.
iv © ISO 2015 – All rights reserved
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SIST EN ISO 22718:2016
ISO 22718:2015(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 22718:2016
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SIST EN ISO 22718:2016
INTERNATIONAL STANDARD ISO 22718:2015(E)
Cosmetics — Microbiology — Detection of Staphylococcus
aureus
1 Scope

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

specified microorganism Staphylococcus aureus 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 Staphylococcus aureus

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 Staphylococcus aureus, 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 for some products in every detail (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

© ISO 2015 – All rights reserved 1
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SIST EN ISO 22718:2016
ISO 22718:2015(E)
3.3
initial suspension

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

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
Staphylococcus aureus

gram-positive cocci, mainly aggregated in grape-like clusters, smooth colonies generally pigmented in

yellow

Note 1 to entry: The main characteristics for identification are: growth on specific selective medium, catalase

positive, coagulase positive.

Note 2 to entry: Staphylococcus aureus is an opportunistic pathogen for humans that can also be present on

the skin of healthy people without causing disorder for them. It is undesirable in cosmetic products due to its

potential pathogenicity.
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) 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 Staphylococcus aureus in accordance with this International Standard provided that it has

been demonstrated to be suitable in accordance with Clause 11.
2 © ISO 2015 – All rights reserved
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SIST EN ISO 22718:2016
ISO 22718:2015(E)

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

5.2 Diluent for the bacterial suspension (tryptone sodium chloride solution)
5.2.1 General

The diluent is used for the preparation of bacterial 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 down, 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 according to the instructions from the manufacturer. 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 the suitability test (see Clause 11) [soybean-casein digest agar medium

(SCDA) or tryptic soy agar (TSA)]
5.3.2.1 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
5.3.2.2 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.

After sterilization and cooling down, the pH shall be equivalent to 7,3 ± 0,2 when measured at room

temperature.
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SIST EN ISO 22718:2016
ISO 22718:2015(E)
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 into boiling water

until their 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 down, 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).
4 © ISO 2015 – All rights reserved
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SIST EN ISO 22718:2016
ISO 22718:2015(E)
5.3.4 Selective agar medium for isolation of Staphylococcus aureus
5.3.4.1 Baird Parker agar medium
5.3.4.1.1 Base medium
5.3.4.1.1.1 Composition
— pancreatic digest of casein 10,0 g
— yeast extract 1,0 g
— meat extract 5,0 g
— sodium pyruvate 10,0 g
— L-glycine 12,0 g
— lithium chloride 5,0 g
— agar 12 g to 22 g
— water to a final volume of 950 ml
5.3.4.1.1.2 Preparation

Dissolve the components or the complete dehydrated base in the water by boiling. Transfer the medium

in quantities of 100 ml to flasks or bottles of appropriate capacity. Sterilize the medium in the autoclave

at 121 °C for 15 min.

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

room temperature.
5.3.4.1.2 Potassium tellurite solution
5.3.4.1.2.1 Composition
— potassium tellurite (K TeO ) 1,0 g
2 3
— water 100 ml
5.3.4.1.2.2 Preparation
Dissolve the potassium tellurite completely in the water with minimal heating.

Sterilize by filtration using 0,22 µm pore size membranes. The solution may be stored at the maximum

for one month at 3 °C ± 2 °C. Discard the solution if a white precipitate forms.

The solid should be readily soluble. If a white insoluble material is present in the water, the powder

should be discarded.

5.3.4.1.3 Egg yolk emulsion (concentration approximately 20 % or according to the

manufacturer’s instructions)
If a commercial preparation is not available, prepare the medium as follows.

Use fresh hens’ eggs, the shells being intact. Clean the eggs with a brush using a liquid detergent. Rinse

them under running water, then disinfect the shells either by immersing them in 70 % (volume fraction)

ethanol for 30 s and allow them to dry in the air, or by spraying them with alcohol followed by flame

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SIST EN ISO 22718:2016
ISO 22718:2015(E)

sterilization. Proceeding under aseptic conditions, break each egg and separate the yolk from its white

by repeated transfer of the yolk from one half of the shell to the other. Place the yolks in a sterile flask

and add four times their volume of sterile water. Mix thoroughly. Heat the mixture at 47 °C for 2 h and

leave for 18 h to 24 h at 3 °C ± 2 °C to allow a precipitate to form. Aseptically collect the supernatant

liquid in a fresh sterile flask for use.
The emulsion may be stored at 3 °C ± 2 °C for a maximum of 72 h.
5.3.4.1.4 Complete medium
5.3.4.1.4.1 Composition
— base medium (5.3.4.1.1) 100 ml
— potassium tellurite solution (5.3.4.1.2) 1,0 ml
— egg yolk emulsion (5.3.4.1.3) 5,0 ml
5.3.4.1.4.2 Preparation

Melt the base medium (5.3.4.1.1) then cool it to approximately 47 °C. Add, under aseptic conditions, the

two other solutions (5.3.4.1.2 and 5.3.4.1.3), each of them previously warmed at 47 °C, mixing well after

each addition.
5.3.4.2 Other selective agar media
Other selective agar media may be used (see Annex A).
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)
Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518).

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

reference strain.
The strain may 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.
1) ATCC = American Type Culture Collection.
2) CIP = Institut Pasteur Collection.
3) NCIMB = National Collection
...

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