SIST EN ISO 21149:2017
(Main)Cosmetics - Microbiology - Enumeration and detection of aerobic mesophilic bacteria (ISO 21149:2017)
Cosmetics - Microbiology - Enumeration and detection of aerobic mesophilic bacteria (ISO 21149:2017)
This document gives general guidelines for enumeration and detection of aerobic mesophilic 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 shown to be suitable.
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 to determine the types of cosmetic products to which this document is
applicable. Products considered to present a low microbiological risk (see ISO 29621) include those
with low water activity, hydro-alcoholic products, extreme pH values, etc.
Kosmetische Mittel - Mikrobiologie - Zählung und Nachweis von aeroben mesophilen Bakterien (ISO 21149:2017)
Dieses Dokument gibt allgemeine Anleitungen für die Zählung und zum Nachweis von in Kosmetischen Mitteln vorhandenen mesophilen aeroben Bakterien,
durch Zählen der Kolonien auf einem Agarmedium nach aerober Bebrütung oder
durch Überprüfen des Ausbleibens von bakteriellem Wachstum nach Anreicherung.
Wegen der großen Vielfalt an kosmetischen Mitteln innerhalb dieses Anwendungsbereiches kann dieses Verfahren nicht zwangsläufig bis in alle Einzelheiten auf bestimmte Mittel anwendbar sein (z. B. bestimmte mit Wasser nicht mischbare Produkte). Ersatzweise dürfen für die hier aufgeführten Untersuchungen andere Verfahren (z. B. automatisierte) zur Anwendung kommen, vorausgesetzt, dass deren Gleichwertigkeit nach¬gewiesen oder das Verfahren anderweitig validiert wurde.
Falls erforderlich, dürfen ausgezählte oder nachgewiesene Mikroorganismen mit geeigneten Identifizierungsprüfungen bestimmt werden, die in den unter den Literaturhinweisen aufgeführten Normen beschrieben sind.
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 dieses Dokument anwendbar ist. Produkte, die ein geringes mikrobiologisches Risiko darstellen, um¬fassen jene mit geringer Wasseraktivität, alkoholisch wässrige Produkte, Produkte mit extremen pH Wer¬ten usw.
Cosmétiques - Microbiologie - Dénombrement et détection des bactéries aérobies mésophiles (ISO 21149:2017)
ISO 21149:2017 donne des lignes directrices générales pour le dénombrement et la détection des bactéries aérobies mésophiles présentes dans les cosmétiques
- par dénombrement des colonies en milieu gélosé après une incubation aérobie, ou
- en vérifiant l'absence de croissance bactérienne après enrichissement.
En raison de la grande variété de produits cosmétiques entrant dans ce domaine d'application, la présente méthode peut ne pas être, en tout point, applicable à certains produits (par exemple à certains produits non miscibles dans l'eau). Il est possible de remplacer les essais présentés ici par d'autres méthodes (automatisées, par exemple), sous réserve que leur équivalence ait été démontrée ou que la méthode ait été validée par ailleurs.
Au besoin, les micro-organismes dénombrés ou détectés peuvent être identifiés à l'aide d'essais d'identification appropriés, décrits dans les normes indiquées en Bibliographie.
Pour garantir la qualité du produit et la sécurité des consommateurs, il est conseillé d'effectuer une analyse de risque microbiologique appropriée, afin de déterminer les types de produits cosmétiques qui relèvent du présent document. 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 prisotnosti in števila aerobnih mezofilnih bakterij (ISO 21149:2017)
Ta dokument podaja splošne smernice za ugotavljanje prisotnosti in števila aerobnih mezofilnih bakterij v kozmetičnih izdelkih:
– s štetjem kolonij na agarskem gojišču po aerobni inkubaciji, ali
– s preverjanjem odsotnosti bakterijske rasti po obogatitvi.
Zaradi velike raznolikosti kozmetičnih izdelkov na tem področju uporabe ta metoda morda ni v celoti primerna za nekatere izdelke (npr. tiste, ki se ne mešajo z vodo). 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.
Po potrebi se lahko za ugotavljanje prisotnosti in števila mikroorganizmov uporabijo ustrezni identifikacijski preskusi, opisani v standardih, navedenih v bibliografiji.
Za zagotovitev kakovosti in varnosti izdelkov za stranke je priporočljivo izvesti ustrezno mikrobiološko analizo tveganja, s katero se določijo vrste kozmetičnih izdelkov, za katere se uporablja ta dokument. Izdelki, ki po ocenah predstavljajo nizko mikrobiološko tveganje (glej ISO 29621), vključujejo izdelke z nizko aktivnostjo vode ali s skrajnimi vrednostmi pH, hidro-alkoholne izdelke itd.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 21149:2017
01-november-2017
1DGRPHãþD
SIST EN ISO 21149:2009
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti in števila aerobnih mezofilnih
bakterij (ISO 21149:2017)
Cosmetics - Microbiology - Enumeration and detection of aerobic mesophilic bacteria
(ISO 21149:2017)
Kosmetische Mittel - Mikrobiologie - Zählung und Nachweis von aeroben mesophilen
Bakterien (ISO 21149:2017)
Cosmétiques - Microbiologie - Dénombrement et détection des bactéries aérobies
mésophiles (ISO 21149:2017)
Ta slovenski standard je istoveten z: EN ISO 21149:2017
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 21149:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST EN ISO 21149:2017
---------------------- Page: 2 ----------------------
SIST EN ISO 21149:2017
EN ISO 21149
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2017
EUROPÄISCHE NORM
ICS 71.100.70; 07.100.99 Supersedes EN ISO 21149:2009
English Version
Cosmetics - Microbiology - Enumeration and detection of
aerobic mesophilic bacteria (ISO 21149:2017)
Cosmétiques - Microbiologie - Dénombrement et Kosmetische Mittel - Mikrobiologie - Zählung und
détection des bactéries aérobies mésophiles (ISO Nachweis von aeroben mesophilen Bakterien (ISO
21149:2017) 21149:2017)
This European Standard was approved by CEN on 26 April 2017.
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, Serbia, 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
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21149:2017 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 21149:2017
EN ISO 21149:2017 (E)
Contents Page
European foreword . 3
2
---------------------- Page: 4 ----------------------
SIST EN ISO 21149:2017
EN ISO 21149:2017 (E)
European foreword
This document (EN ISO 21149:2017) 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 December 2017, and conflicting national standards
shall be withdrawn at the latest by December 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 21149:2009.
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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 21149:2017 has been approved by CEN as EN ISO 21149:2017 without any modification.
3
---------------------- Page: 5 ----------------------
SIST EN ISO 21149:2017
---------------------- Page: 6 ----------------------
SIST EN ISO 21149:2017
INTERNATIONAL ISO
STANDARD 21149
Second edition
2017-06
Cosmetics — Microbiology —
Enumeration and detection of aerobic
mesophilic bacteria
Cosmétiques — Microbiologie — Dénombrement et détection des
bactéries aérobies mésophiles
Reference number
ISO 21149:2017(E)
©
ISO 2017
---------------------- Page: 7 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, 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 2017 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(E)
Contents Page
Foreword .v
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 . 7
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
9.2.1 General. 7
9.2.2 Water-miscible products. 8
9.2.3 Water-immiscible products . 8
9.3 Counting methods . 8
9.3.1 Dilutions for counting methods . 8
9.3.2 Plate-count methods . 8
9.4 Enrichment . 9
9.4.1 General. 9
9.4.2 Incubation of the sample . 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 .11
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 Suitability of counting methods .14
13.3.1 Principle .14
13.3.2 Suitability test of the pour-plate method .14
13.3.3 Suitability of the surface spread method .14
13.3.4 Suitability of the membrane filtration method .14
13.4 Suitability of the detection method by enrichment .15
13.4.1 Procedure .15
13.4.2 Interpretation of results .15
13.5 Interpretation of suitability test results .15
14 Test report .16
© ISO 2017 – All rights reserved iii
---------------------- Page: 9 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(E)
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
iv © ISO 2017 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(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 voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 21149:2006), of which it constitutes a
minor revision with the following changes:
— in the Scope, “validated” has been changed to “shown to be suitable”;
— in the Scope, “see ISO 29621” has been added and the reference has been added to the Bibliography;
— in 4.1, “validated” has been changed to “demonstrated”;
— in 4.3, “validated” has been changed to “described”;
— in 5.1, “specifications” has been changed to “instructions”;
— in 9.3.2.1, 9.3.2.2 and 9.3.2.3, “validated” has been changed to “described”;
— in 9.3.2.3, “procedure developed during the validation” has been changed to “suitability test
procedure”;
— in 9.4.1, “validation” has been changed to “suitability test”;
— in 12.2.1, “validated according to the chosen method” has been changed to “demonstrated to be
suitable for the chosen method”;
— in 13.3 and 13.4, “validation” has been changed to “suitability”;
— in 13.3.2, 13.3.3 and 13.3.4, “validation” has been changed to “suitability”;
— in 13.3.2, 13.3.3 and 13.3.4, “if the validation count is at least 50 % (0,3 log) of the control count” has
been changed to “if the count is at least 50 % of the control”;
— in 13.4.1, instances of “validation test” have been changed to “suitability test”;
© ISO 2017 – All rights reserved v
---------------------- Page: 11 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(E)
— in 13.4.2, instances of “validation plate” have been changed to “suitability test plate”;
— in 13.5, “validation results” has been changed to “suitability test results” and “validation plates” has
been changed to “suitability test plates”;
— in Clause 14 f), “validation of the method” has been changed to “demonstration of the suitability”;
— in A.1, B.1 and C.1, “validated” has been changed to “demonstrated to be suitable”.
vi © ISO 2017 – All rights reserved
---------------------- Page: 12 ----------------------
SIST EN ISO 21149:2017
INTERNATIONAL STANDARD ISO 21149:2017(E)
Cosmetics — Microbiology — Enumeration and detection
of aerobic mesophilic bacteria
1 Scope
This document gives general guidelines for enumeration and detection of aerobic mesophilic 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 shown to be suitable.
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 to determine the types of cosmetic products to which this document is
applicable. Products considered to present a low microbiological risk (see ISO 29621) include those
with low water activity, hydro-alcoholic products, extreme pH values, etc.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 21148:2017, 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.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
aerobic mesophilic bacterium
mesophilic bacterium growing aerobically under the conditions specified in this document
Note 1 to entry: In the described conditions, other types of microorganisms (e.g. yeast, mould) can be detected.
© ISO 2017 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(E)
3.2
product
portion of an identified cosmetic product received in the laboratory for testing
3.3
sample
portion of the product (3.2) (at least 1 g or 1 ml) which is used in the test to prepare the initial
suspension (3.4)
3.4
initial suspension
suspension (or solution) of a sample (3.3) in a defined volume of an appropriate liquid (diluent,
neutralizer, broth or combination of them)
3.5
sample dilution
dilution of the initial suspension (3.4)
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
[7]
shall be neutralized to allow the detection of viable microorganisms . In all cases and whatever the
methodology, the neutralization of the antimicrobial properties of the product shall be checked and
[8][9][10]
demonstrated (see Clause 13) .
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 32,5 °C ± 2,5 °C for 72 h ± 6 h.
c) 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.
a) Transfer a suitable amount of the sample prepared as described in Clause 13 in the filtration
apparatus wetted with a small volume of an appropriate sterile diluent, filter immediately and
wash according to the described procedure (see 13.3.4). Transfer the membrane filter onto the
surface of the specified agar medium as specified in ISO 21148.
b) Aerobic incubation of the membranes at 32,5 °C ± 2,5 °C for 72 h ± 6 h.
c) Counting the number of colony forming units (CFU) and calculation of the number of aerobic
mesophilic bacteria per millilitre or per gram of product.
2 © ISO 2017 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(E)
4.4 Detection of bacteria by enrichment
Detection of bacteria by enrichment consists of the following steps.
a) 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.
b) Transfer of a defined quantity of the previous suspension on non-selective solid agar medium.
c) Aerobic incubation at 32,5 °C ± 2,5 °C for 48 h to 72 h.
d) 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 instructions are given in ISO 21148. When water is mentioned in a document, 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.
© ISO 2017 – All rights reserved 3
---------------------- Page: 15 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(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.
4 © ISO 2017 – All rights reserved
---------------------- Page: 16 ----------------------
SIST EN ISO 21149:2017
ISO 21149:2017(E)
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
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 use
...
SLOVENSKI STANDARD
oSIST prEN ISO 21149:2017
01-februar-2017
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti in števila aerobnih mezofilnih
bakterij (ISO/FDIS 21149:2017)
Cosmetics - Microbiology - Enumeration and detection of aerobic mesophilic bacteria
(ISO/FDIS 21149:2017)
Kosmetische Mittel - Mikrobiologie - Zählung und Nachweis von aeroben mesophilen
Bakterien (ISO/FDIS 21149:2017)
Cosmétiques - Microbiologie - Dénombrement et détection des bactéries aérobies
mésophiles (ISO/FDIS 21149:2017)
Ta slovenski standard je istoveten z: prEN ISO 21149
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
oSIST prEN ISO 21149:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN ISO 21149:2017
---------------------- Page: 2 ----------------------
oSIST prEN ISO 21149:2017
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 21149
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: ISIRI
Enumeration and detection of aerobic
Voting begins on:
20170102 mesophilic bacteria
Voting terminates on:
Cosmétiques — Microbiologie — Dénombrement et détection des
20170327
bactéries aérobies mésophiles
ISO/CEN PARALLEL PROCESSING
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 21149:2017(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 2017
---------------------- Page: 3 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, 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
CH1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
Contents Page
Foreword .v
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 . 2
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 . 7
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
9.2.1 General. 7
9.2.2 Watermiscible products. 8
9.2.3 Waterimmiscible products . 8
9.3 Counting methods . 8
9.3.1 Dilutions for counting methods . 8
9.3.2 Platecount methods . 8
9.4 Enrichment . 9
9.4.1 General. 9
9.4.2 Incubation of the sample . 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 .11
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 Suitability of counting methods .14
13.3.1 Principle .14
13.3.2 Suitability of the pour-plate method .14
13.3.3 Suitability of the surface spread method .14
13.3.4 Suitability of the membrane filtration method .14
13.4 Suitability of the detection method by enrichment .15
13.4.1 Procedure .15
13.4.2 Interpretation of results .15
13.5 Interpretation of suitability test results .15
14 Test report .16
© ISO 2017 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
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
iv © ISO 2017 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(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 World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 21149:2006), of which it constitutes a
minor revision with the following changes:
— in 4.1, “validated” has been changed to “demonstrated”;
— in 4.3, “validated” has been changed to “described”;
— in 9.3.2.1, 9.3.2.2 and 9.3.2.3, “validated” has been changed to “described”;
— in 9.3.2.3, “procedure developed during the validation” has been changed to “suitability test
procedure”;
— in 9.4.1, “validation” has been changed to “suitability test”;
— in 12.2.1, “validated according to the chosen method” has been changed to “demonstrated to be
suitable for the chosen method”;
— in 13.3 and 13.4, “validation” has been changed to “suitability”;
— in 13.3.2, 13.3.3 and 13.3.4, “validation” has been changed to “suitability”;
— in 13.3.2, 13.3.3 and 13.3.4, “if the validation count is at least 50 % (0,3 log) of the control count” has
been changed to “if the count is at least 50 % of the control”;
— in 13.4.1, instances of “validation test” have been changed to “suitability test”;
— in 13.4.2, instances of “validation plate” have been changed to “suitability test plate”;
— in 13.5, “validation results” has been changed to “suitability test results” and “validation plates” has
been changed to “suitability test plates”;
— in Clause 14 f), “validation of the method” has been changed to “demonstration of the suitability”;
© ISO 2017 – All rights reserved v
---------------------- Page: 7 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
— in A.1, B.1 and C.1, “validated” has been changed to “demonstrated to be suitable”.
vi © ISO 2017 – All rights reserved
---------------------- Page: 8 ----------------------
oSIST prEN ISO 21149:2017
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 21149:2017(E)
Cosmetics — Microbiology — Enumeration and detection
of aerobic mesophilic bacteria
1 Scope
This document 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 document
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 documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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.
1)
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 (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.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
aerobic mesophilic bacteria
mesophilic bacteria growing aerobically under the conditions specified in this document
Note 1 to entry: In the described conditions, other types of microorganisms (e.g. yeast, mould) can be detected.
1) To be published.
© ISO 2017 – All rights reserved 1
---------------------- Page: 9 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
3.2
product
portion of an identified cosmetic product received in the laboratory for testing
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
[7]
sample shall be neutralized to allow the detection of viable microorganism . In all cases and whatever
the methodology, the neutralization of the antimicrobial properties of the product shall be checked and
[8][9][10]
demonstrated (see Clause 13) .
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 32,5 °C ± 2,5 °C for 72 h ± 6 h.
c) 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.
a) Transfer a suitable amount of the sample prepared as described in Clause 13 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.
b) Aerobic incubation of the membranes at 32,5 °C ± 2,5 °C for 72 h ± 6 h.
c) Counting the number of colony forming units (CFU) and calculation of the number of aerobic
mesophilic bacteria per millilitre or per gram of product.
4.4 Detection of bacteria by enrichment
Detection of bacteria by enrichment consists of the following steps.
a) Incubation at 32,5 °C ± 2,5 °C for at least 20 h of a defined quantity of the initial suspension in a
nonselective liquid medium containing suitable neutralizers and/or dispersing agents.
2 © ISO 2017 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
b) Transfer of a defined quantity of the previous suspension on non-selective solid agar medium.
c) Aerobic incubation at 32,5 °C ± 2,5 °C for 48 h to 72 h.
d) 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.
5.2.2.2 Other neutralizing diluents
Other neutralizing diluents may be used as appropriate (see Annex A and Annex D).
© ISO 2017 – All rights reserved 3
---------------------- Page: 11 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
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.
4 © ISO 2017 – All rights reserved
---------------------- Page: 12 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
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
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, and
— 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
© ISO 2017 – All rights reserved 5
---------------------- Page: 13 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
Glucose 5,5 g
Egg lecithin 1,0 g
Polysorbate 80 5,0 g
Octoxynol 9 1,0 g
Water 1 000 ml
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 © ISO 2017 – All rights reserved
---------------------- Page: 14 ----------------------
oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strains of microorganisms
For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram
[8][11]
positive microorganisms , respectively, are used:
2) 3) 4) 5)
— Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626 or NBRC
6)
13275 or KCTC 2513 or other equivalent national collection strain);
— Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518 or NBRC 13276 or
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 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
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.