SIST EN ISO 21149:2009

SLOVENSKI STANDARD
SIST EN ISO 21149:2009
01-oktober-2009
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti in števila aerobnih mezofilnih
bakterij (ISO 21149:2006)
Cosmetics - Microbiology - Enumeration and detection of aerobic mesophilic bacteria
(ISO 21149:2006)
Kosmetik - Mikrobiologie - Zählung und Nachweis von aeroben mesophilen Bakterien
(ISO 21149:2006)
Cosmétiques - Microbiologie - Dénombrement et détection des bactéries aérobies
mésophiles (ISO 21149:2006)
Ta slovenski standard je istoveten z: EN ISO 21149:2009
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 21149:2009 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 21149:2009

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SIST EN ISO 21149:2009
EUROPEAN STANDARD
EN ISO 21149
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2009
ICS 07.100.99; 71.100.70
English Version
Cosmetics - Microbiology - Enumeration and detection of
aerobic mesophilic bacteria (ISO 21149:2006)
Cosmétiques - Microbiologie - Dénombrement et détection Kosmetik - Mikrobiologie - Zählung und Nachweis von
des bactéries aérobies mésophiles (ISO 21149:2006) aeroben mesophilen Bakterien (ISO 21149:2006)
This European Standard was approved by CEN on 23 May 2009.
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 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 Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21149:2009: E
worldwide for CEN national Members.

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SIST EN ISO 21149:2009
EN ISO 21149:2009 (E)
Contents Page
Foreword .3

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SIST EN ISO 21149:2009
EN ISO 21149:2009 (E)
Foreword
The text of ISO 21149:2006 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 21149:2009 by
Technical Committee CEN/SS H99 “Products for household and leisure use - Undetermined” the secretariat of
which is held by CMC.
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 2009, and conflicting national standards shall be withdrawn
at the latest by December 2009.
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, 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 21149:2006 has been approved by CEN as a EN ISO 21149:2009 without any modification.

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SIST EN ISO 21149:2009

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SIST EN ISO 21149:2009

INTERNATIONAL ISO
STANDARD 21149
First edition
2006-03-01


Cosmetics — Microbiology —
Enumeration and detection of aerobic
mesophilic bacteria
Cosmétiques — Microbiologie — Dénombrement et détection des
bactéries aérobies mésophiles





Reference number
ISO 21149:2006(E)
©
ISO 2006

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SIST EN ISO 21149:2009
ISO 21149:2006(E)
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©  ISO 2006
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 2006 – All rights reserved

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SIST EN ISO 21149:2009
ISO 21149:2006(E)
Contents Page
Foreword. iv
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
4 Principle. 2
4.1 General. 2
4.2 Plate count. 2
4.3 Membrane filtration. 2
4.4 Detection of bacteria by enrichment. 3
5 Diluents, neutralizers and culture media. 3
5.1 General. 3
5.2 Neutralizing diluents and diluents . 3
5.3 Diluent for the bacterial suspension (tryptone sodium chloride solution). 4
5.4 Culture media . 4
6 Apparatus and glassware. 6
7 Strains of microorganisms . 7
8 Handling of cosmetic products and laboratory samples . 7
9 Procedure. 7
9.1 General recommendation . 7
9.2 Preparation of the initial suspension. 7
9.3 Counting methods . 8
9.4 Enrichment. 9
10 Counting of colonies (plate counts and membrane filtration methods). 9
11 Detection of growth (enrichment method) . 9
12 Expression of results. 10
12.1 Method of calculation for plate count. 10
12.2 Interpretation. 10
12.3 Examples. 11
12.4 Detection after enrichment . 13
13 Neutralization of the antimicrobial properties of the product. 13
13.1 General. 13
13.2 Preparation of inoculum . 14
13.3 Validation of counting methods. 14
13.4 Validation of the detection method by enrichment . 15
13.5 Interpretation of validation results. 15
14 Test report. 16
Annex A (informative) Other neutralizing diluents . 17
Annex B (informative) Other diluents. 19
Annex C (informative) Other culture media . 20
Annex D (informative) Neutralizers of antimicrobial activity of preservatives and rinsing liquids . 23
Bibliography . 24

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SIST EN ISO 21149:2009
ISO 21149:2006(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 21149 was prepared by Technical Committee ISO/TC 217, Cosmetics.

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SIST EN ISO 21149:2009
INTERNATIONAL STANDARD ISO 21149:2006(E)

Cosmetics — Microbiology — Enumeration and detection
of aerobic mesophilic bacteria
1 Scope
This International Standard gives general guidelines for enumeration and detection of mesophilic aerobic
bacteria present in cosmetics,
⎯ by counting the colonies on agar medium after aerobic incubation, or
⎯ by checking the absence of bacterial growth after enrichment.
Because of the large variety of cosmetic products within this field of application, this method may not be
appropriate for some products in every detail (e.g. certain water immiscible products). Other 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 validated.
If needed, microorganisms enumerated or detected may be identified using suitable identification tests
described in the standards given in the Bibliography.
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, extreme pH values, etc.
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 21148:2005, Cosmetics — Microbiology — General instructions for microbiological examination
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
aerobic mesophilic bacteria
mesophilic bacteria growing aerobically under the conditions specified in this International Standard
NOTE In the described conditions, other types of microorganisms (e.g. yeast, mould) can be detected.
3.2
product
portion of an identified cosmetic product received in the laboratory for testing
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SIST EN ISO 21149:2009
ISO 21149:2006(E)
3.3
sample
portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension
3.4
initial suspension
suspension (or solution) of a sample in a defined volume of an appropriate liquid (diluent, neutralizer, broth or
combination of them)
3.5
sample dilution
dilution of the initial suspension
4 Principle
4.1 General
This method involves enumeration of colonies on a non-selective agar medium or by the presence or absence
of bacterial growth after enrichment. The possible inhibition of microbial growth by the sample shall be
[1]
neutralized to allow the detection of viable microorganism . In all cases and whatever the methodology, the
[2] [3] [4]
neutralization of the antimicrobial properties of the product shall be checked and validated .
4.2 Plate count
Plate count consists of the following steps.
⎯ 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.
⎯ Aerobic incubation of the plates at 32,5 °C ± 2,5 °C for 72 h ± 6 h.
⎯ Counting the number of colony forming units (CFU) and calculation of the number of aerobic mesophilic
bacteria per millilitre or per gram of product.
4.3 Membrane filtration
Membrane filtration consists of the following steps.
⎯ Transfer a suitable amount of the sample prepared as validated in the filtration apparatus wetted with a
small volume of an appropriate sterile diluent, filter immediately and wash according to the validated
procedure (see 13.3.4). Transfer the membrane filter onto the surface of the specified agar medium as
specified in ISO 21148.
⎯ Aerobic incubation of the membranes at 32,5 °C ± 2,5 °C for 72 h ± 6 h.
⎯ Counting the number of colony forming units (CFU) and calculation of the number of aerobic mesophilic
bacteria per millilitre or per gram of product.
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SIST EN ISO 21149:2009
ISO 21149:2006(E)
4.4 Detection of bacteria by enrichment
Detection of bacteria by enrichment consists of the following steps:
⎯ Incubation at 32,5 °C ± 2,5 °C for at least 20 h of a defined quantity of the initial suspension in a
non-selective liquid medium containing suitable neutralizers and/or dispersing agents.
⎯ Transfer of a defined quantity of the previous suspension on non-selective solid agar medium.
⎯ Aerobic incubation at 32,5 °C ± 2,5 °C for 48 h to 72 h.
⎯ Detection of growth and expression of results as “presence/absence” of aerobic mesophilic bacteria per
sample S of product.
5 Diluents, neutralizers and culture media
5.1 General
General specifications are given in ISO 21148. When water is used 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 and detection of aerobic
mesophilic bacteria. 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 specimen to be tested has
antimicrobial properties. The efficacy of the neutralization shall be demonstrated before the determination of
the count (see Clause 13). Information relative to suitable neutralizers is given in Annex D.
5.2.2 Neutralizing diluents
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 40,0 ml
Water 960,0 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 obtain 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.
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SIST EN ISO 21149:2009
ISO 21149:2006(E)
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
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 the bacterial suspension (tryptone sodium chloride solution)
5.3.1 Composition
Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride 8,5 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 instructions of
the manufacturer. Ready-to-use media may be used when their composition and/or growth yields are
comparable to those of the formulas given herein.
5.4.2 Culture media for counting
5.4.2.1 Soybean–casein digest agar medium (SCDA) or tryptic soy agar (TSA)
5.4.2.1.1 Composition
Pancreatic digest of casein 15,0 g
Papaic digest of soybean meal 5,0 g
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SIST EN ISO 21149:2009
ISO 21149:2006(E)
Sodium chloride 5,0 g
Agar 15,0 g
Water 1 000 ml
5.4.2.1.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating. Dispense
the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization and
cooling down, the pH shall be equivalent to 7,3 ± 0,2 when measured at room temperature.
5.4.2.2 Other media for counting
Other media may be used as appropriate (see Annex C).
5.4.3 Culture media for detection
5.4.3.1 General
When chosen, an enrichment broth and an agar medium shall be used for bacterial detection.
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.
5.4.3.2 Enrichment broth: Eugon LT 100 broth
5.4.3.2.1 General
This medium contains ingredients
⎯ which neutralize inhibitory substances present in the sample: lecithin and polysorbate 80,
⎯ dispersing agent: octoxynol 9.
5.4.3.2.2 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
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SIST EN ISO 21149:2009
ISO 21149:2006(E)
5.4.3.2.3 Preparation
Dissolve successively polysorbate 80, octoxynol 9 and egg lecithin into boiling water until their complete
dissolution. Dissolve the other components by mixing while heating. Dispense the medium into suitable
containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent to
7,0 ± 0,2 when measured at room temperature.
5.4.3.3 Agar media for detection
5.4.3.3.1 Eugon LT 100 agar medium
5.4.3.3.1.1 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
Agar 15,0 g
Water 1 000 ml
5.4.3.3.1.2 Preparation
Dissolve successively polysorbate 80, octoxynol 9 and egg lecithin into boiling water until their complete
dissolution. Dissolve the other components by mixing while heating. Mix gently to avoid foam. Dispense the
medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization and cooling
down, the pH shall be equivalent to 7,0 ± 0,2 when measured at room temperature.
5.4.3.3.2 Other agar media for detection
Other media may be used as appropriate (see Annex C).
5.4.4 Agar medium for cultivation of reference strains
Use soybean-casein digest agar medium (SCDA) or tryptic soy agar (TSA) (5.4.2.1).
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
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SIST EN ISO 21149:2009
ISO 21149:2006(E)
7 Strains of microorganisms
For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram positive
[2] [5]
microorganisms , respectively, are used:
⎯ Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626 or NBRC 13275 or
KCTC 2513 or other equivalent national collection strain);
1) 2) 3) 4)
⎯ Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518 or NBRC 13276 or
5)
KCTC 1916 or other equivalent national collection strain).
An alternative to the Gram negative strain may be: Escherichia coli ATCC 8739 (equivalent strain: CIP 53.126
or NCIMB 8545 or NBRC 3972 or KCTC 2571 or other equivalent national collection strain).
The culture should be reconstituted according to the procedures provided by the supplier of reference strain.
The strains may be kept in the laboratory according to the 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
(3.2) and samples (3.3) before or after analysis.
Sampling of cosmetic products to be analysed should be carried out, as described in ISO 21148. Analyse
samples as specified in ISO 21148 and in accordance with the following procedure.
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 an initial suspension, the time which 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 (3.3) of at least 1 g or 1 ml of the well-mixed product (3.2)
under test.
Note S the exact mass or volume of the sample.
The initial suspension is usually 1:10 dilution. Larger volumes of diluent or enrichment broth may be required if
high levels of contamination are expected and/or if anti-microbial properties are still present in 1:10 dilution.

1) ATCC = American Type Culture Collection.
2) CIP = Institut Pasteur Collection.
3) NCIMB = National Collection of Industrial and Marine Bacteria.
4) NBRC = National Biological Resource Center.
5) KCTC = Korean Collection for Type Culture.
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SIST EN ISO 21149:2009
ISO 21149:2006(E)
9.2.2 Water-miscible products
Transfer the sample S of product to an appropriate volume (e.g. 9 ml) of neutralizing diluent (5.2.2) or diluent
(5.2.3) or enrichment broth (5.4.3.2), depending on the method used (9.3 or 9.4).
Note the dilution factor d.
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 (e.g.
9 ml) of neutralizing diluent (5.2.2) or diluent (5.2.3) or enrichment broth (5.4.3.2), depending on the method
used (9.3 or 9.4).
Note the dilution factor d.
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.
9.3.2 Plate-count methods
9.3.2.1 Pour-plate method
In Petri dishes 85 mm to 100 mm in diameter, add 1 ml of the initial suspension and/or sample dilution
prepared as validated (see Clause 13) and pour 15 ml to 20 ml of the melted agar medium (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
...