SIST EN ISO 18416:2016
(Main)Cosmetics - Microbiology - Detection of Candida albicans (ISO 18416:2015)
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
Relations
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
2
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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.
3
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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. +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 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
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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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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
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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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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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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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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— 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
5)
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.
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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
...
SLOVENSKI STANDARD
kSIST FprEN ISO 18416:2015
01-september-2015
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti kvasovke Candida albicans
(ISO/FDIS 18416:2015)
Cosmetics - Microbiology - Detection of Candida albicans (ISO/FDIS 18416:2015)
Kosmetische Mittel - Mikrobiologie - Nachweis von Candida albicans (ISO/FDIS
18416:2015)
Cosmétiques - Microbiologie - Détection de Candida albicans (ISO/FDIS 18416:2015)
Ta slovenski standard je istoveten z: FprEN ISO 18416
ICS:
07.100.99 Drugi standardi v zvezi z Other standards related to
mikrobiologijo microbiology
71.100.70 .R]PHWLND7RDOHWQL Cosmetics. Toiletries
SULSRPRþNL
kSIST FprEN ISO 18416:2015 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST FprEN ISO 18416:2015
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kSIST FprEN ISO 18416:2015
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 18416
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: ISIRI
Detection of Candida albicans
Voting begins on:
20150625
Cosmétiques — Microbiologie — Détection de Candida albicans
Voting terminates on:
20150825
Please see the administrative notes on page iii
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
ISO/FDIS 18416:2015(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN
DARDS TO WHICH REFERENCE MAY BE MADE IN
©
NATIONAL REGULATIONS. ISO 2015
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kSIST FprEN ISO 18416:2015
ISO/FDIS 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
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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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kSIST FprEN ISO 18416:2015
ISO/FDIS 18416:2015(E)
ISO/CEN PARALLEL PROCESSING
This final draft has been developed within the International Organization for Standardization (ISO), and pro
cessed under the ISO-lead mode of collaboration as defined in the Vienna Agreement. The final draft was
established on the basis of comments received during a parallel enquiry on the draft.
This final draft is hereby submitted to the ISO member bodies and to the CEN member bodies for a parallel
twomonth approval vote in ISO and formal vote in CEN.
Positive votes shall not be accompanied by comments.
Negative votes shall be accompanied by the relevant technical reasons.
© ISO 2015 – All rights reserved iii
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kSIST FprEN ISO 18416:2015
ISO/FDIS 18416:2015(E)
Contents Page
Foreword .v
Introduction .vi
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) . 2
5.2.1 General. 2
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 . 5
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 Watermiscible products. 6
9.2.3 Waterimmiscible products . 7
9.2.4 Filterable products . 7
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 . 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 .15
Bibliography .16
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kSIST FprEN ISO 18416:2015
ISO/FDIS 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 nongovernmental, 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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kSIST FprEN ISO 18416:2015
ISO/FDIS 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 user (adults, children, including under 3 years).
For cosmetics and other topical products, the detection of 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 those microorganisms (including indicators of
faecal contamination, e.g. Escherichia coli) suggest hygienic failure during the manufacturing process.
vi © ISO 2015 – All rights reserved
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kSIST FprEN ISO 18416:2015
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 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 so as 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 nonselective 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 might not
be suited in every detail to some products (e.g. certain water-immiscible products). Other International
Standards (e.g. ISO 18415) might be appropriate. Other methods (e.g. automated) can be substituted for
the test presented here provided that their equivalence has been demonstrated or the method has been
otherwise validated.
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, mycobactericidal, sporicidal and fungicidal 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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3.4
sample dilution
dilution of the initial suspension
3.5
specified microorganisms
aerobic mesophilic bacteria or yeast that is undesirable in a cosmetic product because it can cause skin
or eye infections or is an indication of hygienic failure
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
nonselective 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 (isolation) of the test is performed on a selective medium followed by identification
tests.
To prevent the possible inhibition of microbial growth by the sample it shall be neutralized to allow the
[1]
detection 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.
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).
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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.
5.3.2.2 Other agar media for suitability test
Other agar media for suitability test may be used as appropriate (see Annex A).
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kSIST FprEN ISO 18416:2015
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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).
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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
— 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:
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kSIST FprEN ISO 18416:2015
ISO/FDIS 18416:2015(E)
1) 2) 3) 4)
Candida albicans ATCC 10231 or equivalent strain: IP 48.72 or NCPF 3179 or NBRC 1594 or
5)
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.
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 (3.1) and samples (3.2) 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 given 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 preparation of the initial suspension in an appropriate solubilizing agent, the
time that elapses between the end of the 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 of at least 1 g or 1 ml of the wellmixed product under test,
which is dispersed in at least 9 ml of enrichment broth.
Note S, the exact mass 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 which 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.
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.
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ISO/FDIS 18416:2015(E)
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 appropriate volume of broth.
9.2.4 Filterable products
Use a membrane filter having a nominal pore size no greater than 0,45 µm.
Transfer the sample, S, on to the membrane in a filtration apparatus (see ISO 21148). Filter immediately
and wash the membrane using defined volumes of water and/or diluent.
Transfer and immerse the membrane into a tube or flask of suitable size containing an appropriate
volume of broth.
9.3 Incubation of the inoculated enrichment broth
Incubate the initial suspension prepared in broth (see 9.2) at 32,5 °C ± 2,5 °C for at least 20 h but not
more than 72 h.
9.4 Detection and identification of Candida albicans
9.4.1 Isolation
Using a sterile loop, streak an aliquot of the incubated enrichment broth on to the surface of Sabouraud
dextrose chloramphenicol agar in order to obtain isolated colonies.
Invert the Petri dish and then incubate at 32,5 °C ± 2,5 °C for 24 h to 48 h.
Check for characteristic colonies (see Table 1).
Table 1 — Morphologic characteristics of Candida albicans on selective agar medium
Selective medium Aspect of the colonies of Candida albicans
Sabouraud dextrose chloramphenicol agar White to beige, creamy and convex
9.4.2 Identification of Candida albicans
9.4.2.1 General
Candida albicans can appear to be dimorphic and is capable of producing pseudohyphae, some true
hyphae, and clusters of round blastoconidia as well as large thick-walled chlamydospores. At low
ambient temperature the culture might express this pseudo-mycelial form; however, it can change to
the unicellular form at higher temperatures.
Proceed to the following tests for the suspect colonies isolated on the Sabouraud dextrose
chloramphenicol agar medium. The presence of Candida albicans may be confirmed by other suitable
cultural and biochemical tests.
9.4.2.2 Gram’s stain
Follow the procedure specified in ISO 21148.
The microscopic observation shall reveal a violet colour, short ovoid or elongated cells, sometimes with
budding cells.
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9.4.2.3 Germ tube production
9.4.2.3.1 Place 0,5 ml to 1 ml of serum (feotal calf or horse serum) in a small test tube.
9.4.2.3.2 Emulsify a small portion of yeast colony to be tested in the serum.
9.4.2.3.3 Incubate in a water bath, at 37 °C ± 1 °C, for 1,5 h to 2 h, or in an incubator at 37 °C ± 2 °C for
3 h.
9.4.2.3.4 Place a drop of serum on a s
...
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