Cosmetics - Microbiology - Enumeration of yeast and mould (ISO 16212:2008)

This International Standard gives general guidelines for enumeration of yeast and mould present in cosmetics by counting the colonies on selective agar medium after aerobic incubation. 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 products to which this International Standard is applicable. Products considered to present a low microbiological risk include those with low water activity, hydro-alcoholic products, products with extreme pH values, etc.
Because of the large variety of cosmetic products within this field of application, this method might not be suited to some products in every detail (e.g. certain water-immiscible products). Other methods (e.g. automated) can be used for the test presented here provided that their equivalence has been demonstrated or the method has been otherwise validated. Yeast enumerated can be identified using suitable identification tests, for example tests described in the standards listed in the Bibliography. Mould enumerated can be identified by other appropriate methods, if necessary.

Kosmetik - Mikrobiologie - Zählung von Hefen und Schimmelpilzen (ISO 16212:2008)

Diese Internationale Norm gibt allgemeine Anleitungen für die Zählung von in Kosmetika vorhandenen Hefen und Schimmelpilzen durch Zählen der Kolonien auf einem selektiven Agarmedium nach aerober Bebrütung.
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, auf die diese Internationale Norm anwendbar ist. Produkte, die ein geringes mikrobiologisches Risiko darstellen, um-fassen Produkte mit geringer Wasseraktivität, alkoholisch wässrige Produkte, Produkte mit extremen pH Wer-ten usw.
Wegen der großen Vielfalt an kosmetischen Mitteln innerhalb dieses Anwendungsbereiches ist dieses Verfahren möglicherweise für einige Produkte nicht in allen Einzelheiten geeignet (z. B. bestimmte mit Wasser nicht mischbare Produkte). Für die hier aufgeführten Untersuchungen können andere Verfahren (z. B. automatisierte) zur Anwendung kommen, vorausgesetzt, dass deren Gleichwertigkeit nachgewiesen oder das Verfahren anderweitig validiert wurde.
Ausgezählte Hefen können mit geeigneten Identifizierungsprüfungen bestimmt werden, zum Beispiel Prüfungen, die in den unter den Literaturhinweisen aufgeführten Normen beschrieben sind. Falls notwendig, können ausgezählte Schimmelpilze nach anderen geeigneten Verfahren identifiziert werden.

Cosmétiques - Microbiologie - Dénombrement des levures et des moisissures (ISO 16212:2008)

L'ISO 16212:2008 donne des lignes directrices générales pour le dénombrement des levures et des moisissures présentes dans les cosmétiques par comptage des colonies en milieu gélosé sélectif après une incubation aérobie.
Afin de garantir la qualité du produit et la sécurité des consommateurs, il est conseillé d'effectuer une analyse de risque microbiologique pour déterminer les types de produits cosmétiques auxquels l'ISO 16212:2008 s'applique. Les produits considérés comme présentant un faible risque microbiologique comprennent ceux ayant une faible activité de l'eau, les produits hydro-alcooliques, les produits ayant des valeurs de pH extrêmes, etc.

Kozmetika - Mikrobiologija - Ugotavljanje števila kvasovk in plesni (ISO 16212:2008)

Ta mednarodni standard podaja splošne smernice za ugotavljanje števila kvasovk in plesni, prisotnih v kozmetiki, s štetjem kolonij na selektivnem agarskem gojišču po aerobni inkubaciji. Za zagotavljanje kakovosti izdelka in varnosti za potrošnike je priporočljivo opraviti ustrezno analizo mikrobiološkega tveganja, da se določijo vrste kozmetičnih izdelkov, za katere velja ta mednarodni standard. Izdelki, za katere velja, da predstavljajo nizko mikrobiološko tveganje, vključujejo izdelke z nizko aktivnostjo vode, hidroalkoholne izdelke, izdelke z ekstremnimi vrednostmi pH itd. Zaradi velike raznolikosti kozmetičnih izdelkov v tem okviru uporabe ta metoda za nekatere izdelke morda ni primerna v vsaki podrobnosti (npr. za nekatere izdelke, ki se ne mešajo z vodo). Pri tu predstavljenem preskusu se lahko uporabijo druge metode (npr. avtomatske), pod pogojem, da je bila dokazana njihova enakovrednost ali da je bila metoda kako drugače validirana. Preštete kvasovke se lahko identificirajo s primernimi identifikacijskimi preskusi, na primer s preskusi, opisanimi v standardih, navedenih v bibliografiji. Preštete plesni se lahko po potrebi identificirajo z drugimi ustreznimi metodami.

General Information

Status
Withdrawn
Public Enquiry End Date
19-Feb-2011
Publication Date
15-Sep-2011
Withdrawal Date
18-Sep-2017
Current Stage
9900 - Withdrawal (Adopted Project)
Start Date
18-Sep-2017
Due Date
11-Oct-2017
Completion Date
19-Sep-2017

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SLOVENSKI STANDARD
SIST EN ISO 16212:2011
01-oktober-2011
Kozmetika - Mikrobiologija - Ugotavljanje števila kvasovk in plesni (ISO
16212:2008)
Cosmetics - Microbiology - Enumeration of yeast and mould (ISO 16212:2008)
Kosmetik - Mikrobiologie - Zählung von Hefen und Schimmelpilzen (ISO 16212:2008)
Cosmétiques - Microbiologie - Dénombrement des levures et des moisissures (ISO
16212:2008)
Ta slovenski standard je istoveten z: EN ISO 16212:2011
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 16212:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16212:2011

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SIST EN ISO 16212:2011


EUROPEAN STANDARD
EN ISO 16212

NORME EUROPÉENNE

EUROPÄISCHE NORM
June 2011
ICS 71.100.70
English Version
Cosmetics - Microbiology - Enumeration of yeast and mould
(ISO 16212:2008)
Cosmétiques - Microbiologie - Dénombrement des levures Kosmetik - Mikrobiologie - Zählung von Hefen und
et des moisissures (ISO 16212:2008) Schimmelpilzen (ISO 16212:2008)
This European Standard was approved by CEN on 12 May 2011.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16212:2011: E
worldwide for CEN national Members.

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SIST EN ISO 16212:2011
EN ISO 16212:2011 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 16212:2011
EN ISO 16212:2011 (E)
Foreword
The text of ISO 16212:2008 has been prepared by Technical Committee ISO/TC 217 “Cosmetics” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 16212:2011 by
Technical Committee CEN/TC 392 “Cosmetics” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by December 2011, and conflicting national standards shall be withdrawn
at the latest by December 2011.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 16212:2008 has been approved by CEN as a EN ISO 16212:2011 without any modification.

3

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SIST EN ISO 16212:2011

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SIST EN ISO 16212:2011

INTERNATIONAL ISO
STANDARD 16212
First edition
2008-10-15

Cosmetics — Microbiology —
Enumeration of yeast and mould
Cosmétiques — Microbiologie — Dénombrement des levures et des
moisissures




Reference number
ISO 16212:2008(E)
©
ISO 2008

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SIST EN ISO 16212:2011
ISO 16212:2008(E)
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©  ISO 2008
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
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Published in Switzerland

ii © ISO 2008 – All rights reserved

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SIST EN ISO 16212:2011
ISO 16212:2008(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principles. 2
4.1 General. 2
4.2 Plate count. 2
4.3 Membrane filtration. 2
5 Diluents, neutralizers and culture media. 3
5.1 General. 3
5.2 Neutralizing diluents and diluents . 3
5.3 Diluent for yeast suspension (tryptone sodium chloride solution). 4
5.4 Culture media . 4
6 Apparatus and glassware . 5
7 Strain 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. 6
9.3 Counting methods . 6
10 Counting of colonies (plate counts and membrane filtration methods). 7
11 Expression of results . 8
11.1 Method of calculation for plate count. 8
11.2 Interpretation. 8
12 Neutralization of the antifungicidal properties of the product. 10
12.1 General. 10
12.2 Preparation of inoculum . 10
12.3 Validation of counting methods. 11
13 Test report . 12
Annex A (informative) Other neutralizing diluents . 13
Annex B (informative) Other diluents. 15
Annex C (informative) Other culture media . 16
Annex D (informative) Neutralizers of antifungicidal activity of preservatives and rinsing liquids . 18
Bibliography . 19

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SIST EN ISO 16212:2011
ISO 16212:2008(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 16212 was prepared by Technical Committee ISO/TC 217, Comestics.

iv © ISO 2008 – All rights reserved

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SIST EN ISO 16212:2011
INTERNATIONAL STANDARD ISO 16212:2008(E)

Cosmetics — Microbiology — Enumeration of yeast and mould
1 Scope
This International Standard gives general guidelines for enumeration of yeast and mould present in cosmetics
by counting the colonies on selective agar medium after aerobic incubation.
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 products to which this International
Standard is applicable. Products considered to present a low microbiological risk include those with low water
activity, hydro-alcoholic products, products with extreme pH values, etc.
Because of the large variety of cosmetic products within this field of application, this method might not be
suited to some products in every detail (e.g. certain water-immiscible products). Other methods (e.g.
automated) can be used for the test presented here provided that their equivalence has been demonstrated or
the method has been otherwise validated.
Yeast enumerated can be identified using suitable identification tests, for example tests described in the
standards listed in the Bibliography. Mould enumerated can be identified by other appropriate methods, if
necessary.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 21148, 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
yeast
single-cell fungus, which multiplies mainly vegetatively by budding, able to grow under the test conditions
specified in this International Standard
3.2
mould
mycelium forming microfungus, including spores and conidia, able to grow under the test conditions specified
in this International Standard
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SIST EN ISO 16212:2011
ISO 16212:2008(E)
3.3
product
portion of an identified cosmetic product received in the laboratory for testing
3.4
sample
portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension
3.5
initial suspension
suspension (or solution) of a sample in a defined volume of an appropriate liquid (diluent, neutralizer, broth or
combination thereof)
3.6
sample dilution(s)
dilution(s) of the initial suspension
4 Principles
4.1 General
This method involves enumeration of colonies on a selective agar medium. The possible inhibition of fungal
growth by the sample shall be neutralized to allow the detection of viable microorganism (see Reference [2]).
In all cases and whatever the methodology, the neutralization of the antifungicidal properties of the product
shall be checked and validated (see References [3], [5], [6]).
4.2 Plate count
Plate count consists of the following steps.
a) Preparation of poured plates or spread plates, using a specified culture medium, and inoculation of the
plates using a defined quantity of the initial suspension or dilution of the product.
b) Aerobic incubation of the plates at 25 °C ± 2,5 °C for 3 d to 5 d.
c) Counting of the number of colony-forming units (CFU) and calculation of the amount of yeast and mould
per millilitre or per gram of product.
NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C for 5 d to 7 d using the culture medium without
antibiotic.
4.3 Membrane filtration
Membrane filtration consists of the following steps.
a) Transfer of a suitable amount of the sample, prepared by a valid method, in the filtration apparatus,
wetted with a small volume of an appropriate sterile diluent. Immediate filtration and washing according to
the validated procedure. Transfer of the membrane filter on to the surface of the specified agar medium
as specified in ISO 21148.
b) Aerobic incubation of the membranes at 25 °C ± 2,5 °C for 3 d to 5 d.
c) Counting of the number of colony-forming units (CFU) and calculation of the amount of yeast and mould
per millilitre or per gram of product.
NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C for 5 d to 7 d using the culture medium without
antibiotic.
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SIST EN ISO 16212:2011
ISO 16212:2008(E)
5 Diluents, neutralizers and culture media
5.1 General
General specifications are given in ISO 21148. When water is mentioned in a formula, use distilled water or
purified water as specified in ISO 21148.
The following diluents, neutralizers and culture media are suitable for enumeration of yeasts and moulds.
Other diluents, neutralizers and culture media may be used if they have been demonstrated to be suitable for
use.
5.2 Neutralizing diluents and diluents
5.2.1 General
The diluent is used to disperse the sample. It may contain neutralizers if the sample to be tested has
antifungicidal properties. The efficacy of the neutralization shall be demonstrated before the determination of
the count (see Clause 12). Information relative to suitable neutralizers is given in Annex D.
5.2.2 Neutralizing diluent
5.2.2.1 Fluid casein digest-soy lecithin-polysorbate 20 medium (SCDLP 20 broth)
5.2.2.1.1 Composition
⎯ pancreatic digest of casein 20,0 g
⎯ soy lecithin 5,0 g
⎯ polysorbate 20 40 ml
⎯ water 960 ml
5.2.2.1.2 Preparation
Dissolve the polysorbate 20 in 960 ml of water by mixing while heating in a water bath at 49 °C ± 2 °C. Add
pancreatic digest of casein and soy lecithin. Heat for about 30 min to effect solution. Mix and dispense the
medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall
be equivalent to 7,3 ± 0,2 when measured at room temperature.
5.2.2.2 Other neutralizing diluents
Other neutralizing diluents may be used as appropriate (see Annex A and Annex D).
5.2.3 Diluent
5.2.3.1 Fluid A
5.2.3.1.1 Composition
⎯ peptic digest of animal tissue 1,0 g
⎯ water 1 000 ml
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SIST EN ISO 16212:2011
ISO 16212:2008(E)
5.2.3.1.2 Preparation
Dissolve 1 g of peptone in water to make 1 l. Heat with frequent agitation. Dispense into suitable containers.
Sterilize in the autoclave at 121 °C for 15 min. After sterilization the pH shall be equivalent to 7,1 ± 0,2 when
measured at room temperature.
5.2.3.2 Other diluents
Other diluents may be used as appropriate (see Annex B).
5.3 Diluent for yeast suspension (tryptone sodium chloride solution)
5.3.1 Composition
⎯ tryptone, pancreatic digest of casein 1,00 g
⎯ sodium chloride 8,50 g
⎯ water 1 000 ml
5.3.2 Preparation
Dissolve the components in the water by mixing while heating. Dispense into suitable containers. Sterilize in
the autoclave at 121 °C for 15 min. After sterilization the pH shall be equivalent to 7,0 ± 0,2 when measured at
room temperature.
5.4 Culture media
5.4.1 General
Culture media may be prepared as follows, or from dehydrated culture media according to the manufacturer's
instructions. Ready-to-use media may be used when their composition and/or growth yields are comparable to
those of the formulae given herein.
5.4.2 Sabouraud dextrose chloramphenicol agar medium (SDCA)
5.4.2.1 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
5.4.2.2 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.
NOTE For known and non-contaminated products (with bacteria), the media are used without chloramphenicol.
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SIST EN ISO 16212:2011
ISO 16212:2008(E)
5.4.3 Other media
Other media may be used as appropriate (see Annex C).
5.4.4 Agar medium for cultivation of reference strain: Sabouraud dextrose agar medium (SDA)
5.4.4.1 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
5.4.4.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating. Dispense
the medium into suitable containers. Sterilize in an autoclave at 121 °C for 15 min. After sterilization the pH
shall be equivalent to 5,6 ± 0,2 when measured at room temperature.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strain of microorganisms
For testing the efficacy of neutralizers, one yeast reference 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) 6)
KCTC 17205 or TISTR 5779) or other equivalent national collection strain.
The selected yeast strain being considered more susceptible to antifungicidal activity than moulds may be
accepted as representative of fungi (yeast and mould) for the validation of the methodology. However, in case
of specific needs, the test for the efficacy of neutralizers may be performed with an additional mould reference
[11]
strain, using a suitable protocol for the preparation of a calibrated inoculum (e.g. see EN 13624:2003 ,
5.4.1.4).
The culture should be reconstituted according to the procedures provided by the supplier of reference strain.
The strain may be kept in the laboratory in accordance with EN 12353.

1) ATCC = American Type Culture Collection.
2) IP = Institut Pasteur.
3) NCPF = National Collection of Pathogenic Fungi.
4) NBRC = National Biological Resource Center.
5) KCTC = Korean Collection for Type Culture.
6) TISTR = Thailand Institute of Scientific and Technological Research.
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SIST EN ISO 16212:2011
ISO 16212:2008(E)
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 (3.3) and samples (3.4) 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 described 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, the time that elapses between the end of the
preparation and the moment the inoculum comes into contact with the culture medium shall not exceed 45 min,
unless specifically mentioned in the established protocols or documents.
9.2 Preparation of the initial suspension
9.2.1 General
The initial suspension is prepared from a sample of at least 1 g or 1 ml of the well-mixed product under test.
Note S, the exact mass or volume of the sample.
The initial suspension is usually a 1:10 dilution. Larger volumes of diluent may be required if high levels of
contamination are expected and/or if antifungicidal properties are still present in the 1:10 dilution.
9.2.2 Water-miscible products
Transfer the sample, S, of product to an appropriate volume (e.g. 9 ml) of neutralizing diluent (see 5.2.2) or
diluent (see 5.2.3).
Note d, the dilution factor.
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 solution). Disperse the sample within the solubilizing agent and add an appropriate
volume (e.g. 9 ml) of neutralizing diluent (see 5.2.2) or diluent (see 5.2.3).
Note d, the dilution factor.
9.3 Counting methods
9.3.1 Dilutions for counting methods
Usually, the initial suspension is the first counted dilution. If needed, additional serial dilutions (e.g. 1:10
dilution) may be performed from the initial suspension using the same diluent (according to the expected level
of contamination of the product).
Generally, counting is performed using at least two Petri dishes, but it is possible to use only one Petri dish in
case of routine testing, or if counts are performed on successive dilutions of the same sample or according to
previous results.
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SIST EN ISO 16212:2011
ISO 16212:2008(E)
9.3.2 Plate-count methods
9.3.2.1 Pour-plate method
In Petri dishes of 85 mm to 100 mm in diameter, add 1 ml of the initial suspension and/or sample dilution
prepared as validated (see Clause 12) and pour 15 ml to 20 ml of the melted agar medium (see 5.4.2) kept in
a water bath at no more than 48 °C. If larger Petri dishes are used, the amount of agar medium is increased
accordingly.
Mix the initial suspension and/or sample dilution with the medium, carefully rotating or tilting the plates
sufficiently to disperse them. Allow the mixture in the Petri dishes to solidify on a horizontal surface at room
temperature.
9.3.2.2 Surface spread method
In Petri dishes of 85 mm to 100 mm in diameter, put 15 ml to 20 ml of the melted agar medium (see 5.4.2)
kept in a water bath at no more than 48 °C. If larger Petri dishes are used, the volume of the agar is increased
accordingly.
Allow plates to cool and solidify, for example in a microbiological cabinet or in an incubator. Spread over the
surface of the medium a measured volume of not less than 0,1 ml of the initial suspension and/or sample
dilution prepared as validated (see Clause 12).
9.3.2.3 Membrane filtration method
Use a membrane having a nominal pore size u 0,45 µm.
Transfer a suitable amount of the initial suspension or of the sample dilution prepared as validated (preferably
representing at least 1 g or 1 ml of the product) on to the membrane.
Filter immediately and wash the membrane (follow the procedure developed during the validation,
see Clause 12).
Transfer the membrane on to the surface of the agar medium (see 5.4.2).
9.3.2.4 Incubation
Unless otherwise stated, invert the inoculated dishes and place them in the incubator set at 25 °C ± 2,5 °C for
3 d to 5 d or use the alternative condition (see Notes in 4.2 and 4.3). After incubation, the dishes shall, if
possible, be examined immediately. Alternatively, they may be stored, unless otherwise specified, for up to a
maximum of 24 h in the refrigerator at 5 °C ± 3 °C.
NOTE 1 In certain cases, where there is a potential for confusing particles from the product with counted colonies, it
can be useful to prepare duplicate dishes containing the same sample dilutions and agar medium which are stored in the
refrigerator for comparison with incubated dishes.
NOTE 2 An intermediate check can be performed where both yeast and mould are suspected.
10 Counting of colonies (plate counts and membrane filtration methods)
After incubation, count the colonies:
⎯ in Petri dishes containing 15 colonies to 150 colonies; if less than 15 colonies are counted, see 11.2.3;
⎯ on membranes containing 15 colonies to 150 colonies; if less than 15 colonies are counted, see 11.2.3.
© ISO 2008 – All rights reserved 7

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SIST EN ISO 16212:2011
ISO 16212:2008(E)
11 Expression of results
11.1 Method of calculation for plate count
Calculate the number, N, of microorganisms present in the sample, S, using:
⎯ m, the arithmetic mean of the counts obtained from the duplicates (1),
⎯ c, the number of colonies counted on a single plate (2) or
⎯ wm, the weighted mean of the counts obtained from two successive dilutions (3),
according to the following formulae:
N = m/(V × d) (1)
N = c/(V × d) (2)
N = wm/(V × d) (3)
where:
m is the arithmetic mean of the counts obtained from the duplicates;
V is the volume of inoculum applied to each dish, in millilitres;
d is the dilution factor corresponding to the dilution made for the preparation of the initial suspension
(see 9.2) or for the first counted dilution;
c is the
...

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Kosmetik - Mikrobiologie - Zählung von Hefen und Schimmelpilzen (ISO 16212:2008)Cosmétiques - Microbiologie - Dénombrement des levures et des moisissures (ISO 16212:2008)Cosmetics - Microbiology - Enumeration of yeast and mould (ISO 16212:2008)71.100.70SULSRPRþNLCosmetics. Toiletries07.100.99Drugi standardi v zvezi z mikrobiologijoOther standards related to microbiologyICS:Ta slovenski standard je istoveten z:FprEN ISO 16212kSIST FprEN ISO 16212:2011en,de01-januar-2011kSIST FprEN ISO 16212:2011SLOVENSKI
STANDARD



kSIST FprEN ISO 16212:2011



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
FINAL DRAFT
FprEN ISO 16212
November 2010 ICS 07.100.99; 71.100.70 English Version
Cosmetics - Microbiology - Enumeration of yeast and mould (ISO 16212:2008)
Cosmétiques - Microbiologie - Dénombrement des levures et des moisissures (ISO 16212:2008)
Kosmetik - Mikrobiologie - Zählung von Hefen und Schimmelpilzen (ISO 16212:2008) This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical Committee CEN/TC 392.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
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 supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. FprEN ISO 16212:2010: EkSIST FprEN ISO 16212:2011



FprEN ISO 16212:2010 (E) 2 Contents Page Foreword .3 kSIST FprEN ISO 16212:2011



FprEN ISO 16212:2010 (E) 3 Foreword The text of ISO 16212:2008 has been prepared by Technical Committee ISO/TC 217 “Cosmetics” of the International Organization for Standardization (ISO) and has been taken over as FprEN ISO 16212:2010 by Technical Committee CEN/TC 392 “Cosmetics” the secretariat of which is held by NEN. This document is currently submitted to the Unique Acceptance Procedure. Endorsement notice The text of ISO 16212:2008 has been approved by CEN as a FprEN ISO 16212:2010 without any modification.
kSIST FprEN ISO 16212:2011



kSIST FprEN ISO 16212:2011



Reference numberISO 16212:2008(E)© ISO 2008
INTERNATIONAL STANDARD ISO16212First edition2008-10-15Cosmetics — Microbiology — Enumeration of yeast and mould Cosmétiques — Microbiologie — Dénombrement des levures et des moisissures
kSIST FprEN ISO 16212:2011



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ISO 2008 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body in the country of the requester. ISO copyright office Case postale 56 • CH-1211 Geneva 20 Tel.
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ii © ISO 2008 – All rights reserved
kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) © ISO 2008 – All rights reserved iiiContents Page Foreword.iv 1 Scope.1 2 Normative references.1 3 Terms and definitions.1 4 Principles.2 4.1 General.2 4.2 Plate count.2 4.3 Membrane filtration.2 5 Diluents, neutralizers and culture media.3 5.1 General.3 5.2 Neutralizing diluents and diluents.3 5.3 Diluent for yeast suspension (tryptone sodium chloride solution).4 5.4 Culture media.4 6 Apparatus and glassware.5 7 Strain 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.6 9.3 Counting methods.6 10 Counting of colonies (plate counts and membrane filtration methods).7 11 Expression of results.8 11.1 Method of calculation for plate count.8 11.2 Interpretation.8 12 Neutralization of the antifungicidal properties of the product.10 12.1 General.10 12.2 Preparation of inoculum.10 12.3 Validation of counting methods.11 13 Test report.12 Annex A (informative)
Other neutralizing diluents.13 Annex B (informative)
Other diluents.15 Annex C (informative)
Other culture media.16 Annex D (informative)
Neutralizers of antifungicidal activity of preservatives and rinsing liquids.18 Bibliography.19
kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) iv © ISO 2008 – All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 16212 was prepared by Technical Committee ISO/TC 217, Comestics.
kSIST FprEN ISO 16212:2011



INTERNATIONAL STANDARD ISO 16212:2008(E) © ISO 2008 – All rights reserved 1Cosmetics — Microbiology — Enumeration of yeast and mould 1 Scope This International Standard gives general guidelines for enumeration of yeast and mould present in cosmetics by counting the colonies on selective agar medium after aerobic incubation. 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 products to which this International Standard is applicable. Products considered to present a low microbiological risk include those with low water activity, hydro-alcoholic products, products with extreme pH values, etc. Because of the large variety of cosmetic products within this field of application, this method might not be suited to some products in every detail (e.g. certain water-immiscible products). Other methods (e.g. automated) can be used for the test presented here provided that their equivalence has been demonstrated or the method has been otherwise validated. Yeast enumerated can be identified using suitable identification tests, for example tests described in the standards listed in the Bibliography. Mould enumerated can be identified by other appropriate methods, if necessary. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 21148, 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 yeast single-cell fungus, which multiplies mainly vegetatively by budding, able to grow under the test conditions specified in this International Standard 3.2 mould mycelium forming microfungus, including spores and conidia, able to grow under the test conditions specified in this International Standard kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) 2 © ISO 2008 – All rights reserved 3.3 product portion of an identified cosmetic product received in the laboratory for testing 3.4 sample portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension 3.5 initial suspension suspension (or solution) of a sample in a defined volume of an appropriate liquid (diluent, neutralizer, broth or combination thereof) 3.6 sample dilution(s) dilution(s) of the initial suspension 4 Principles 4.1 General This method involves enumeration of colonies on a selective agar medium. The possible inhibition of fungal growth by the sample shall be neutralized to allow the detection of viable microorganism (see Reference [2]). In all cases and whatever the methodology, the neutralization of the antifungicidal properties of the product shall be checked and validated (see References [3], [5], [6]). 4.2 Plate count Plate count consists of the following steps. a) Preparation of poured plates or spread plates, using a specified culture medium, and inoculation of the plates using a defined quantity of the initial suspension or dilution of the product. b) Aerobic incubation of the plates at 25 °C ± 2,5 °C for 3 d to 5 d. c) Counting of the number of colony-forming units (CFU) and calculation of the amount of yeast and mould per millilitre or per gram of product. NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C for 5 d to 7 d using the culture medium without antibiotic. 4.3 Membrane filtration Membrane filtration consists of the following steps. a) Transfer of a suitable amount of the sample, prepared by a valid method, in the filtration apparatus, wetted with a small volume of an appropriate sterile diluent. Immediate filtration and washing according to the validated procedure. Transfer of the membrane filter on to the surface of the specified agar medium as specified in ISO 21148. b) Aerobic incubation of the membranes at 25 °C ± 2,5 °C for 3 d to 5 d. c) Counting of the number of colony-forming units (CFU) and calculation of the amount of yeast and mould per millilitre or per gram of product. NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C for 5 d to 7 d using the culture medium without antibiotic. kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) © ISO 2008 – All rights reserved 35 Diluents, neutralizers and culture media 5.1 General General specifications are given in ISO 21148. When water is mentioned in a formula, use distilled water or purified water as specified in ISO 21148. The following diluents, neutralizers and culture media are suitable for enumeration of yeasts and moulds. Other diluents, neutralizers and culture media may be used if they have been demonstrated to be suitable for use. 5.2 Neutralizing diluents and diluents 5.2.1 General The diluent is used to disperse the sample. It may contain neutralizers if the sample to be tested has antifungicidal properties. The efficacy of the neutralization shall be demonstrated before the determination of the count (see Clause 12). Information relative to suitable neutralizers is given in Annex D. 5.2.2 Neutralizing diluent 5.2.2.1 Fluid casein digest-soy lecithin-polysorbate 20 medium (SCDLP 20 broth) 5.2.2.1.1 Composition ⎯ pancreatic digest of casein 20,0 g ⎯ soy lecithin 5,0 g ⎯ polysorbate 20 40 ml ⎯ water 960 ml 5.2.2.1.2 Preparation Dissolve the polysorbate 20 in 960 ml of water by mixing while heating in a water bath at 49 °C ± 2 °C. Add pancreatic digest of casein and soy lecithin. Heat for about 30 min to effect solution. Mix and dispense the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent to 7,3 ± 0,2 when measured at room temperature. 5.2.2.2 Other neutralizing diluents Other neutralizing diluents may be used as appropriate (see Annex A and Annex D). 5.2.3 Diluent 5.2.3.1 Fluid A 5.2.3.1.1 Composition ⎯ peptic digest of animal tissue 1,0 g ⎯ water 1 000 ml kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) 4 © ISO 2008 – All rights reserved 5.2.3.1.2 Preparation Dissolve 1 g of peptone in water to make 1 l. Heat with frequent agitation. Dispense into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization the pH shall be equivalent to 7,1 ± 0,2 when measured at room temperature. 5.2.3.2 Other diluents Other diluents may be used as appropriate (see Annex B). 5.3 Diluent for yeast suspension (tryptone sodium chloride solution) 5.3.1 Composition ⎯ tryptone, pancreatic digest of casein 1,00 g ⎯ sodium chloride 8,50 g ⎯ water 1 000 ml 5.3.2 Preparation Dissolve the components in the water by mixing while heating. Dispense into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization the pH shall be equivalent to 7,0 ± 0,2 when measured at room temperature. 5.4 Culture media 5.4.1 General Culture media may be prepared as follows, or from dehydrated culture media according to the manufacturer's instructions. Ready-to-use media may be used when their composition and/or growth yields are comparable to those of the formulae given herein. 5.4.2 Sabouraud dextrose chloramphenicol agar medium (SDCA) 5.4.2.1 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 5.4.2.2 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. NOTE For known and non-contaminated products (with bacteria), the media are used without chloramphenicol. kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) © ISO 2008 – All rights reserved 55.4.3 Other media Other media may be used as appropriate (see Annex C). 5.4.4 Agar medium for cultivation of reference strain: Sabouraud dextrose agar medium (SDA) 5.4.4.1 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 5.4.4.2 Preparation Dissolve the components or the dehydrated complete medium in the water by mixing while heating. Dispense the medium into suitable containers. Sterilize in an autoclave at 121 °C for 15 min. After sterilization the pH shall be equivalent to 5,6 ± 0,2 when measured at room temperature. 6 Apparatus and glassware The laboratory equipment, apparatus and glassware are described in ISO 21148. 7 Strain of microorganisms For testing the efficacy of neutralizers, one yeast reference strain is used. Candida albicans ATCC1) 10231 or equivalent strain (IP2) 48.72 or NCPF3) 3179 or NBRC4) 1594 or KCTC5) 17205 or TISTR6) 5779) or other equivalent national collection strain. The selected yeast strain being considered more susceptible to antifungicidal activity than moulds may be accepted as representative of fungi (yeast and mould) for the validation of the methodology. However, in case of specific needs, the test for the efficacy of neutralizers may be performed with an additional mould reference strain, using a suitable protocol for the preparation of a calibrated inoculum (e.g. see EN 13624:2003[11], 5.4.1.4). The culture should be reconstituted according to the procedures provided by the supplier of reference strain. The strain may be kept in the laboratory in accordance with EN 12353.
1) ATCC = American Type Culture Collection. 2) IP = Institut Pasteur. 3) NCPF = National Collection of Pathogenic Fungi. 4) NBRC = National Biological Resource Center. 5) KCTC = Korean Collection for Type Culture. 6) TISTR = Thailand Institute of Scientific and Technological Research. kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) 6 © ISO 2008 – All rights reserved 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 (3.3) and samples (3.4) 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 described 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, the time that elapses between the end of the preparation and the moment the inoculum comes into contact with the culture medium shall not exceed 45 min, unless specifically mentioned in the established protocols or documents. 9.2 Preparation of the initial suspension 9.2.1 General The initial suspension is prepared from a sample of at least 1 g or 1 ml of the well-mixed product under test. Note S, the exact mass or volume of the sample. The initial suspension is usually a 1:10 dilution. Larger volumes of diluent may be required if high levels of contamination are expected and/or if antifungicidal properties are still present in the 1:10 dilution. 9.2.2 Water-miscible products Transfer the sample, S, of product to an appropriate volume (e.g. 9 ml) of neutralizing diluent (see 5.2.2) or diluent (see 5.2.3). Note d, the dilution factor. 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 solution). Disperse the sample within the solubilizing agent and add an appropriate volume (e.g. 9 ml) of neutralizing diluent (see 5.2.2) or diluent (see 5.2.3). Note d, the dilution factor. 9.3 Counting methods 9.3.1 Dilutions for counting methods Usually, the initial suspension is the first counted dilution. If needed, additional serial dilutions (e.g. 1:10 dilution) may be performed from the initial suspension using the same diluent (according to the expected level of contamination of the product). Generally, counting is performed using at least two Petri dishes, but it is possible to use only one Petri dish in case of routine testing, or if counts are performed on successive dilutions of the same sample or according to previous results. kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) © ISO 2008 – All rights reserved 79.3.2 Plate-count methods 9.3.2.1 Pour-plate method In Petri dishes of 85 mm to 100 mm in diameter, add 1 ml of the initial suspension and/or sample dilution prepared as validated (see Clause 12) and pour 15 ml to 20 ml of the melted agar medium (see 5.4.2) kept in a water bath at no more than 48 °C. If larger Petri dishes are used, the amount of agar medium is increased accordingly. Mix the initial suspension and/or sample dilution with the medium, carefully rotating or tilting the plates sufficiently to disperse them. Allow the mixture in the Petri dishes to solidify on a horizontal surface at room temperature. 9.3.2.2 Surface spread method In Petri dishes of 85 mm to 100 mm in diameter, put 15 ml to 20 ml of the melted agar medium (see 5.4.2) kept in a water bath at no more than 48 °C. If larger Petri dishes are used, the volume of the agar is increased accordingly. Allow plates to cool and solidify, for example in a microbiological cabinet or in an incubator. Spread over the surface of the medium a measured volume of not less than 0,1 ml of the initial suspension and/or sample dilution prepared as validated (see Clause 12). 9.3.2.3 Membrane filtration method Use a membrane having a nominal pore size u 0,45 µm. Transfer a suitable amount of the initial suspension or of the sample dilution prepared as validated (preferably representing at least 1 g or 1 ml of the product) on to the membrane. Filter immediately and wash the membrane (follow the procedure developed during the validation, see Clause 12). Transfer the membrane on to the surface of the agar medium (see 5.4.2). 9.3.2.4 Incubation Unless otherwise stated, invert the inoculated dishes and place them in the incubator set at 25 °C ± 2,5 °C for 3 d to 5 d or use the alternative condition (see Notes in 4.2 and 4.3). After incubation, the dishes shall, if possible, be examined immediately. Alternatively, they may be stored, unless otherwise specified, for up to a maximum of 24 h in the refrigerator at 5 °C ± 3 °C. NOTE 1 In certain cases, where there is a potential for confusing particles from the product with counted colonies, it can be useful to prepare duplicate dishes containing the same sample dilutions and agar medium which are stored in the refrigerator for comparison with incubated dishes. NOTE 2 An intermediate check can be performed where both yeast and mould are suspected. 10 Counting of colonies (plate counts and membrane filtration methods) After incubation, count the colonies: ⎯ in Petri dishes containing 15 colonies to 150 colonies; if less than 15 colonies are counted, see 11.2.3; ⎯ on membranes containing 15 colonies to 150 colonies; if less than 15 colonies are counted, see 11.2.3. kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) 8 © ISO 2008 – All rights reserved 11 Expression of results 11.1 Method of calculation for plate count Calculate the number, N, of microorganisms present in the sample, S, using: ⎯ m, the arithmetic mean of the counts obtained from the duplicates (1), ⎯ c, the number of colonies counted on a single plate (2) or ⎯ wm, the weighted mean of the counts obtained from two successive dilutions (3), according to the following formulae: N = m/(V × d) (1) N = c/(V × d) (2) N = wm/(V × d) (3) where: m is the arithmetic mean of the counts obtained from the duplicates; V is the volume of inoculum applied to each dish, in millilitres; d is the dilution factor corresponding to the dilution made for the preparation of the initial suspension (see 9.2) or for the first counted dilution; c is the number of colonies counted on a single plate. The weighted mean of the colonies counted from two successive dilutions, cx, is given by: ()120,1ccxnn=+ where Σc is the sum of colonies counted on all the dishes retained from two successive dilutions; n1 is the number of dishes counted for the initial suspension (or for the first counted dilution); n2 is the number of dishes counted for the 1:10 dilution of the initial suspension (or for the second counted dilution). Round off the result calculated to two significant figures. For this, if the last figure is below 5, the preceding figure is not modified; if the last figure is 5 or more, the preceding figure is increased by one unit. Proceed stepwise until two significant figures are obtained. Note the number, N, obtained. 11.2 Interpretation 11.2.1 The inherent variability of plate count should be taken into account. Two results should only be considered different if the difference exceeds 50 % or, when expressed logarithmically, the difference exceeds 0,3 log. kSIST FprEN ISO 16212:2011



ISO 16212:2008(E) © ISO 2008 – All rights reserved 9For a count to be precise, only plates or membranes with more than 15 colonies and less than 150 colonies shall be taken into account. Check that the counts are obtained from dilutions validated according to the chosen method (see Clause 12). 11.2.2 Where the number of CFU is more than 15 and less than 150 on plates or membranes, express the result as follows: ⎯ if S is at least 1 g or 1 ml, and V is at least 1 ml, the number of yeast and mould per millilitre or per gram of the sample = N/S; ⎯ if S is less than 1 g or 1 ml, and/or V is less than 1 ml, the number of yeast and mould in the sample (note the tested quantity of sample taking into account S and V) = N; where S is the mass or the volume of the sample (see 9.2). Express the result as a number between 1,0 and 9,9 multiplied by the appropriate power of 10 (see examples). 11.2.3 Where the number of CFU is less than
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