prEN ISO 4973

Cosmetics - Microbiology - Quality control of culture media and diluents used in Cosmetics standards (ISO/DIS 4973:2022)

Develop a standard to explain what tests need to be performed on microbiology culture media in order to demonstrate their ability to detect microorganisms in order to ensure reliability of the methods described in the ISO Cosmetics Microbiology Standards:
ISO 21449 Enumeration and detection of aerobic and mesophilic bacteria
ISO 21150 Detection of Escherichia coliISO 22717 Detection of Pseudomonas aeruginosa
ISO 22718 Detection of Staphylococcus aureus
ISO 18416 Detection of Candida albicans
ISO 16212 Enumeration of yeasts and molds
ISO 18415 Detection of specified and non-specified micro-organisms
ISO 11930 Evaluation of the preservation of a cosmetic product

Kosmetische Mittel - Mikrobiologie - Qualitätskontrolle von Nährböden, die in den Normen für die kosmetische Mikrobiologie beschrieben sind (ISO/DIS 4973:2022)

Cosmétiques - Microbiologie - Contrôle qualité des milieux de culture et des diluants utilisés dans les normes relatives aux cosmétiques (ISO/DIS 4973:2022)

Kozmetika - Mikrobiologija - Kontrola kakovosti gojišč in razredčil, ki se uporabljajo v standardih za kozmetiko (ISO/DIS 4973:2022)

General Information

• Status: Not Published
• Publication Date: 05-Nov-2024
• ICS: 07.100.40 - Cosmetics microbiology
• Technical Committee: CEN/TC 392 - Cosmetics
• Drafting Committee: CEN/TC 392/WG 2 - Microbiological methods
• Current Stage: 4599 - Dispatch of FV draft to CMC - Finalization for Vote
• Start Date: 13-Apr-2023
• Due Date: 14-Nov-2023
• Completion Date: 13-Apr-2023
• Ref Project: oSIST prEN ISO 4973:2022 - Cosmetics - Microbiology - Quality control of culture media and diluents used in Cosmetics standards (ISO/DIS 4973:2022)

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN ISO 4973:2022
01-december-2022
Kozmetika - Mikrobiologija - Kontrola kakovosti gojišč in razredčil, ki se
uporabljajo v standardih za kozmetiko (ISO/DIS 4973:2022)
Cosmetics - Microbiology - Quality control of culture media and diluents used in
Cosmetics standards (ISO/DIS 4973:2022)

Kosmetische Mittel - Mikrobiologie - Qualitätskontrolle von Nährböden, die in den

Normen für die kosmetische Mikrobiologie beschrieben sind (ISO/DIS 4973:2022)

Cosmétiques - Microbiologie - Contrôle qualité des milieux de culture et des diluants

utilisés dans les normes relatives aux cosmétiques (ISO/DIS 4973:2022)
Ta slovenski standard je istoveten z: prEN ISO 4973
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
oSIST prEN ISO 4973:2022 en,fr,de

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

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oSIST prEN ISO 4973:2022
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oSIST prEN ISO 4973:2022
DRAFT INTERNATIONAL STANDARD
ISO/DIS 4973
ISO/TC 217 Secretariat: INSO
Voting begins on: Voting terminates on:
2022-10-12 2023-01-04
Cosmetics — Microbiology — Quality control of culture
media and diluents used in Cosmetics standards
ICS: 07.100.40
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
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USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 4973:2022(E)
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. © ISO 2022
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 4973
ISO/TC 217 Secretariat: INSO
Voting begins on: Voting terminates on:
Cosmetics — Microbiology — Quality control of culture
media and diluents used in Cosmetics standards
ICS: 07.100.40
This document is circulated as received from the committee secretariat.
COPYRIGHT PROTECTED DOCUMENT
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2022
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY

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NOT BE REFERRED TO AS AN INTERNATIONAL

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TECHNOLOGICAL, COMMERCIAL AND
ISO copyright office
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CH-1214 Vernier, Geneva
POTENTIAL TO BECOME STANDARDS TO
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WHICH REFERENCE MAY BE MADE IN
Reference number
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NATIONAL REGULATIONS.
Website: www.iso.org ISO/DIS 4973:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
Published in Switzerland
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
© ISO 2022 – All rights reserved
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction .............................................................................................................................................................................................................................. vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 3

4.1 General Information .......................................................................................................................................................................... 3

4.2 pH ....................................................................................................................................................................................................................... 4

4.3 Absence of microbial growth ..................................................................................................................................................... 4

4.4 Growth promotion............................................................................................................................................................................... 4

4.5 Selective and indicative properties...................................................................................................................................... 4

5 Diluents, neutralizers and culture media ................................................................................................................................. 4

5.1 General ........................................................................................................................................................................................................... 4

5.2 Diluents and neutralizers ............................................................................................................................................................. 4

5.3 Culture media .......................................................................................................................................................................................... 4

6 Apparatus and glassware ...........................................................................................................................................................................5

7 Strains of microorganisms ........................................................................................................................................................................ 5

8 Procedure ....................................................................................................................................................................................................................5

8.1 General recommendation ............................................................................................................................................................. 5

8.2 Preparation of strains ...................................................................................................................................................................... 6

8.2.1 General ........................................................................................................................................................................................ 6

8.2.2 Preparation of bacterial and Candida albicans suspensions ........................................................ 6

8.2.3 Preparation of Aspergillus brasiliensis spore stock suspension ................................................ 7

8.2.4 Microbial suspension for inoculation for Growth promotion and

Selective Properties ................................... ...................................................................................................................... 7

8.3 Absence of microbial growth ..................................................................................................................................................... 8

8.3.1 Solid Media .............................................................................................................................................................................. 8

8.3.2 Liquid media ........................................................................................................................................................................... 8

8.4 Growth Promotion .............................................................................................................................................................................. 8

8.4.1 Solid Media .............................................................................................................................................................................. 8

8.4.2 Liquid media ........................................................................................................................................................................... 8

8.5 Selective properties ........................................................................................................................................................................... 8

8.5.1 Solid media .............................................................................................................................................................................. 8

9 Expression of results ....................................................................................................................................................................................... 9

9.1 Absence of microbial growth ..................................................................................................................................................... 9

9.1.1 Solid Media .............................................................................................................................................................................. 9

9.1.2 Liquid media ........................................................................................................................................................................... 9

9.2 Growth promotion............................................................................................................................................................................... 9

9.2.1 Solid Media .............................................................................................................................................................................. 9

9.2.2 Liquid media ........................................................................................................................................................................... 9

9.3 Selective and indicative properties...................................................................................................................................... 9

9.3.1 Solid Media .............................................................................................................................................................................. 9

10 Interpretation and acceptance criteria ....................................................................................................................................... 9

10.1 Absence of microbial growth ..................................................................................................................................................... 9

10.2 Growth Promotion ........................................................................................................................................................................... 10

10.3 Selective properties ........................................................................................................................................................................ 10

Annex A (informative) Shortest time mentioned in all the ISO standards for Growth

Promotion test (all temperature conditions) – Medium time for indicative

properties – Longest time for Inhibition properties ..................................................................................................11

iii
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oSIST prEN ISO 4973:2022
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Annex B (informative) Table B.1 - Example of Culture Media Quality Control Report for

Culture media used in Cosmetics Microbiology standards .................................................................................16

Bibliography .............................................................................................................................................................................................................................19

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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(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 of 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

www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 217, Cosmetics.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www.iso.org/members.html.
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)
Introduction

The quality of culture media used in the current standards for cosmetic microbiology is an essential

part of microbiological analysis reliability and need to be verified.

This standard is intended to provide methods to assess the quality of the media used in cosmetics

microbiology standards and define the minimum acceptance criteria required to ensure their

performance.
This applies to:
a) Commercially prepared ready to use and dehydrated media;

b) Culture media prepared from dehydrated media plus additional ingredients, or only ingredients.

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oSIST prEN ISO 4973:2022
DRAFT INTERNATIONAL STANDARD ISO/DIS 4973:2022(E)
Cosmetics — Microbiology — Quality control of culture
media and diluents used in Cosmetics standards
1 Scope

This document specifies the minimum requirements for quality control of microbiology culture media

and diluents in order to demonstrate their ability to detect microorganisms and to ensure reliability of

the microbiological test methods described in the ISO Cosmetics Microbiology Standards.

Checking different parameters of media such as growth promotion, absence of microbial growth for

non-inoculated media, physical characteristics, and batch contamination can help to assess their

quality.

This standard describes mainly growth promotion and microbial control tests and will apply to

both commercially ready-to-use media and culture media prepared from dehydrated media or basic

constituents in the user’s laboratory.

Other methods may be substituted provided that their equivalence has been demonstrated.

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 11930, Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product

ISO 16212, Cosmetics — Microbiology — Enumeration of yeast and mould

ISO 18415, Cosmetics — Microbiology — Detection of specified and non-specified microorganisms

ISO 18416, Cosmetics — Microbiology — Detection of Candida albicans

ISO 21149, Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria

ISO 21150, Cosmetics — Microbiology — Detection of Escherichia coli
ISO 22717, Cosmetics — Microbiology — Detection of Pseudomonas aeruginosa
ISO 22718, Cosmetics — Microbiology — Detection of Staphylococcus aureus

ISO 21322, Cosmetics — Microbiology — Testing of impregnated or coated wipes and masks

ISO 21148, Cosmetics — Microbiology — General instructions for microbiological examination

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:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)
3.1
culture medium

a culture medium is a formulation of ingredients, in liquid or solid form intended to support the growth

of microorganisms under specific conditions

There are different types of media suitable for growing different types of microorganisms depending on

different included nutrients and chemicals present in the formulation.
3.1.1
batch or lot of culture medium

a batch or lot of culture medium is an a homogenous and fully traceable unit of medium referring to a

defined amount of bulk, which has been produced within one defined production period, having been

assigned the same batch or lot number
3.1.2
ready-to-use medium

ready to use medium is a sterilized liquid or solid medium that is supplied in plates, tubes, or other

containers in ready to use form
3.1.3
liquid medium

liquid culture medium consisting in aqueous solution of one or more constituents, such as peptone

water or nutrient broth
Liquid media in tubes, flask or bottles are commonly called “broths”.

Enrichment media are generally liquid medium which, due to its composition, provides particularly fa-

vourable conditions for microorganisms’ multiplication.
3.1.4
solid medium

liquid medium containing solidifying substances (e.g. agar, gelatin) in different concentrations

3.1.5
non-selective culture medium

a non-selective culture medium is a medium allowing the growth of most aerobic mesophilic

microorganisms in the range of incubation temperature

This medium may be a liquid or solid form and may contain neutralizing agents to inactivate antimicro-

bial agents such as preservatives.
3.1.6
selective culture medium

selective culture medium is a medium which allows specifically the growth of a selected microorganism

while inhibiting partially or totally the growth of microorganisms which could be in the product to be

tested

Generally, the medium has indicative properties with growth of characteristic aspect of colonies.

3.2
diluent

diluent is a liquid phase designed to separate microorganisms from a solid medium and to reduce their

concentration by dilution without multiplication or inhibition during the time of contact

Diluent could contain neutralizing agent to inactivate the antimicrobial properties of the product.

3.3
strains
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)
3.3.1
reference strains

reference strains should come from a reliable source, directly from a reference culture collection which

is a member of the World Federation of Culture Collections (WFCC)
World Data Center Microorganisms (WDCM) is the data center for WFCC.

The reference strains are subcultured to make “stock cultures,” which are subcultured weekly or monthly

to make the “working cultures”.
3.3.2
stored strains

the strains are stored in a seed lot system (e.g single-use vial or bead) to maintain reference strains in

laboratories
3.3.3
stock culture
the stock culture is the primary subculture from a stored strain
3.3.4
working culture

the working culture is a subculture from a stock culture and is often kept as slants, used for preparation

of calibrated microbial suspension
3.3.5
subculture
a subculture is a passage

“One passage is defined as the transfer of organisms from a viable culture to fresh medium with growth

of the microorganisms. Any form of subculturing is considered to be a transfer/passage.”

USP<1117> Microbiological Best Laboratory Practices.
4 Principle
4.1 General Information
The quality control of culture media refers to different parameters such as:
— pH;
— Absence of microbial growth;
— Growth promotion;
— Selective and indicative properties (when relevant).

The above parameters are the key parameters to ensure and control the quality of the culture media.

However, particular attention should also be paid to:
— manufacturer’s instructions;
— preparing conditions (volume, weighing, water quality);
— sterilization conditions (cycle time, packaging);
— storage conditions.
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)

Failure to comply with these instructions and conditions may affect appearance and functional

characteristics provided in manufacturer’s guidance such as color, gel consistency, clear and

homogeneity.

NOTE 1 For water quality, sterilization conditions, etc… follow the general instructions given in ISO 21148.

NOTE 2 The user’s laboratory should ensure their own preparation process is accurate.

4.2 pH
pH is an essential physical parameter of all culture media.

The target pH value should be reached after autoclaving when culture media are prepared from

dehydrated media or basic constituents in the user’s laboratory.
4.3 Absence of microbial growth

The purpose of this test is to check that the medium does not contain any microbial contamination

which could interfere with microbial tests results.
4.4 Growth promotion

The purpose of this test is to ensure the ability of microorganisms to grow on the culture media.

4.5 Selective and indicative properties

The purpose of this test is to verify the ability of the culture medium to allow the growth of target

microorganisms and/or to confirm its colony morphology within the range of incubation time and

temperature.
5 Diluents, neutralizers and culture media
5.1 General

The diluents, neutralizers and culture media suitable for enumeration and detection of microorganisms

are described in the standard listed as normative references. Other diluents, neutralizers and culture

media may be used if they have been demonstrated to be suitable for use.

Use the general instructions given in ISO 21148. When water is mentioned in this international

standard, use water as specified in ISO 21148.
5.2 Diluents and neutralizers

Diluents may be used for preparation and dilutions of calibrated microbial suspensions and to disperse

the samples. In this case it is required that it contain neutralizers if the sample to be tested has

antimicrobial properties or contains preservatives. Diluent and neutralizers are described in ISO 21149,

ISO 21150, ISO 16212, ISO 22717, ISO 22718, ISO 18416, ISO 11930 and ISO 21322.
5.3 Culture media

Culture media for enumeration and/or detection of micro-organisms are described in ISO 21149,

ISO 21150, ISO 22717, ISO 22718, ISO 18416, ISO 16212, ISO 11930 and ISO 21322.

Culture media may be prepared from dehydrated culture media using specific standards instructions

or the instructions provided by the manufacturer of the media.
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strains of microorganisms

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

reference strain. The strains may be stored as described in EN 12353 or according to another suitable

method.

The following strains are used for the growth promotion test. The relationship between the medium to

be checked and the recommended test microorganisms is described in the Normative Annex A1.

— Staphylococcus aureus WDCM 00032 ;
— Pseudomonas aeruginosa WDCM 00026;
— Escherichia coli WDCM 00012;
— Candida albicans WDCM 00054;
— Aspergillus brasiliensis (formerly known as Aspergillus niger) WDCM 00053.

NOTE Other relevant strains or in-house isolates may be used as needed to supplement the strains cited

above.
8 Procedure
8.1 General recommendation

For culture media prepared from dehydrated media or basic constituents in the users’ laboratories,

follow the general instructions given in ISO 21148. The supplier’s certificate of analysis has to be

checked that it complies with specification.

For commercially ready-to-use media, the supplier’s certificate of analysis has to be checked that it

complies with specification.

It is recommended to conduct the tests described below for each batch of culture media, both ready to

use or prepared in the users’ laboratory.
— pH

For culture media prepared from dehydrated media or basic constituents in the users’ laboratories, pH

shall be measured according to ISO 21148.

For commercially ready-to-use media, the indication given by the supplier’s certificate of analysis is

sufficient.
— Absence of microbial growth

For checking the absence of microbial growth of the media, a fraction of media is incubated for specified

time and temperatures.
— Growth promotion test

For performing a growth promotion test a fraction of media is inoculated with a known level of

microorganisms. After incubation for specified time and temperatures, the colonies are counted and

compared with the theoretical inoculum or a control conducted with a previously approved batch.

1) WDCM: World Data Center for Microorganisms.
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oSIST prEN ISO 4973:2022
ISO/DIS 4973:2022(E)
Each media is tested with only one microorganism at a time.

The test organisms recommended are listed in the Normative Annex A.1. They also may be selected

from the medium manufacturer's recommendation or may include representative environmental

isolates.
— Selective and indicative properties

In addition to the properties of growth promotion, qualitative assay can be performed to assess

selective properties by inoculating with the target and non-target micro-organisms. Growth of the

target organism with the absence of the non-target organism should be observed.

Indicative properties are assessed by the morphology of the target micro- organism.

8.2 Preparation of strains

Commercially prepared strains ready for use can be used for microbiological control of the performance

of culture media.
8.2.1 General

To perform the tests, use the strains stored in the laboratory to obtain stock cultures and the working

cultures.

The stock culture is a confluent culture obtained by streaking slant tubes or plates with the stored

strain (see 3.3.2). After incubation, the stock culture can be kept between 2 °C and 8 °C for up to two

months depending on the microorganisms.

The working culture, prepared when needed to perform test, is used to obtain a calibrated suspension

of known microbial content (inoculum).
Microorganisms sh
...