INTERNATIONAL ISO

STANDARD 21148

First edition
2005-10-15

Cosmetics — Microbiology — General

instructions for microbiological
examination
Cosmétiques — Microbiologie — Instructions générales pour les
examens microbiologiques

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Reference number
ISO 21148:2005(E)

© ISO 2005
ISO 21148:2005(E)

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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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Contents Page

Foreword............................................................................................................................................................ iv
Introduction ........................................................................................................................................................ v
1 Scope ......................................................................................................................................................1
2 Terms and definitions ...........................................................................................................................1
3 Premises .................................................................................................................................................1
4 Equipment ..............................................................................................................................................3
5 Strains of microorganisms ...................................................................................................................6
6 Personnel................................................................................................................................................6
7 Preparation of the apparatus and glassware......................................................................................6
8 Preparation and sterilization of culture media and reagents............................................................8
9 Laboratory samples.............................................................................................................................10
10 Operating practices .............................................................................................................................12
11 iTeh STANDARD PREVIEW
Expression of results ..........................................................................................................................13
12 (standards.iteh.ai)
Neutralization of the antimicrobial properties of the product.........................................................13
Annex A (informative) Basic identification techniques .................................................................................14
ISO 21148:2005
Annex B (informative) Basic techniques for counting and plating ..............................................................19
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Annex C (informative) Preparation and calibration of inoculums................................................................20
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ISO 21148:2005(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 21148 was prepared by Technical Committee ISO/TC 217, Cosmetics.

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Introduction
The purpose of this International Standard is to help ensure that the general techniques used for conducting
cosmetic microbiological examinations are the same in other laboratories that adopt these standards, to help
achieve homogeneous results in different laboratories and to contribute towards the protection of the health of
the laboratory personnel by preventing risk of infection.

When conducting microbiological examinations for cosmetic products, it is especially important that:

⎯ only those microorganisms which are present in the samples be isolated or enumerated;

⎯ the microorganisms do not contaminate the environment.

In order to achieve this, it is necessary to pay attention to personal hygiene and to use working techniques
which ensure, as far as possible, exclusion of extraneous contamination.

Since, in this International Standard it is possible to give only a few examples of the precautions to be taken
during microbiological examinations, a thorough knowledge of the microbiological techniques and of the
microorganisms involved is essential. It is important that the analyses be conducted as accurately as possible,
including calculation of the number of microorganisms.
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A large number of manipulations can, for example, unintentionally lead to cross-contamination and the analyst
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should always verify the accuracy of the results given by his/her technique. It is necessary to take special
precautions, not only for reasons of hygiene, but also to ensure good reproducibility of the results. It is not
possible to specify all the precautions to be taken in all circumstances, but this International Standard at least
ISO 21148:2005
provides the main measures to be taken when preparing, sterilizing and storing the media and the equipment.
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The given recommendations will allowabf8fe138133/iso-21148-2005
enumeration and detection of mesophilic microorganisms which may
grow under aerobic conditions.

The recommendations are applicable to the determination of the absence of, or limited occurrence of specified
microorganisms that are of interest for cosmetic products.

The test methods are described in the individual standards. Alternative microbiological procedures can be
used provided that their equivalence has been demonstrated or the method has been otherwise validated.
The choice of a specific method, or combination of methods mentioned in these International Standards will
depend on the purpose for performing the test and it is for the user to decide which approach is best for
his/her application.

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INTERNATIONAL STANDARD ISO 21148:2005(E)

Cosmetics — Microbiology — General instructions for

microbiological examination

1 Scope
This International Standard gives general instructions for carrying out microbiological examinations of
cosmetic products, in order to ensure their quality and safety, in accordance with an appropriate risk analysis
(e.g. low water activity, hydro-alcoholic, extreme pH values).

Because of the large variety of products and potential uses within this field of application, these instructions
might not be appropriate for some products in every detail (e.g. certain water-immiscible products).

2 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

2.1
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product (standards.iteh.ai)
portion of an identified cosmetic product received in the laboratory for testing
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portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension

2.3
initial suspension
suspension (or solution) of a sample in a defined volume of an appropriate liquid (diluent, neutralizer, broth or
combination of them)

2.4
sample dilution
dilution of the initial suspension

3 Premises

3.1 Test areas

The areas required for the specific operation of a microbiology laboratory are as follows:

⎯ receipt, storage, preparation and processing of the samples;

⎯ preparation and sterilization of culture media, apparatus and glassware;

⎯ performance of analyses: weighing, dilutions, inoculations, subculturing, incubation, maintenance of the

strain, etc.;

⎯ decontamination and cleaning of apparatus, glassware, and processing of the analysis waste.

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3.2 Additional areas

The areas included in this category are, for example:

⎯ entrances, corridors, stairways, lifts;

⎯ administrative areas (e.g. secretarial, offices, documentation rooms, etc.);

⎯ cloakrooms and toilets;

⎯ archive rooms;

⎯ stores.

3.3 Location of the premises

The environment within which the microbiological analyses are carried out shall not affect the reliability of the
analyses.

Care shall be taken to locate the premises so as to avoid risk of cross-contamination.

Care shall be taken to ensure protection against extreme conditions such as excess temperature, dust,
humidity, steam, noise, vibration, exposure to direct sunlight, etc.

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The surface area shall be sufficiently large to keep the work areas clean and orderly.

During the course of the tests, care shall(standards.iteh.ai)

be taken to limit access to the test areas to only those persons
required to conduct the tests.
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Separate rooms and/or separate areas and/or specific enclosures should be provided for the following:
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⎯ receipt, storage and preparation of samples;

⎯ manipulation of microbial cultures;

⎯ preparation of culture media, apparatus and glassware;

⎯ decontamination and washing area;

⎯ sterilization;

⎯ incubators, refrigerators and freezers.

3.4 Equipping the premises

3.4.1 The test premises shall be fitted out in the following ways in order to reduce the risks of contamination
by dust and therefore by microorganisms:

⎯ walls, ceilings and floors should be smooth, non-porous, easy to clean, and resistant to detergents and
disinfectants used in laboratories;

⎯ overhead pipes conveying fluids should not cross the premises unless they are hermetically enclosed;

⎯ sun-protection systems, when used, shall be installed on the outside of the windows, where practicable;

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⎯ windows and doors shall be able to be closed when conducting the test in order to minimize draughts.
Furthermore, they shall be designed so as to avoid the formation of dust traps and hence, to facilitate the
cleaning.

3.4.2 The ambient temperature and air quality (microorganism content, humidity, dust-spreading rate, etc.)
shall be compatible with carrying out the tests.

According to needs, a filter-ventilation and/or a microbiological cabinet are recommended for this purpose.

3.4.3 The laboratory bench tops and furniture shall be made of smooth, non-porous impermeable materials,
which are easy to clean and disinfect. Cabinet and equipment tops should be accessible for cleaning.

Non-fixed laboratory furniture shall be designed so as to facilitate cleaning the floors.

It is desirable that documents or books that are not frequently used be kept outside the test areas.

3.5 Maintenance

The floors, walls, ceiling, laboratory bench tops and furniture shall be maintained in good order to avoid cracks
where dirt might particularly accumulate and thus cause a source of contamination.

Regular cleaning and, when relevant, disinfection shall be carried out in order to keep the premises in a
condition suitable for conducting tests.

The ventilation systems and their filters shall be regularly maintained and filters changed when necessary.
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4 Equipment
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In general, all equipment shall be kept clean and in proper working condition.

Maintenance operations should be monitored. The measurement instruments and apparatus shall be regularly
verified according to an appropriate timetable and results recorded.

4.2 Microbiological cabinets

Cabinets are of two types:

a) clean-air cabinets, which are intended to protect the product from extraneous contamination and to
minimize contamination due to the operator;

b) safety cabinets, which are intended to protect the product from extraneous contamination, and also to
protect the operator and the environment.

Either cabinet can be used. Safety cabinets should be used for all work involving risk for the operator.

A cabinet is a dust-free workstation equipped with vertical laminar airflow. In microbiology, a safety cabinet is
used to retain the microorganisms on filters.

4.3 Balances

A microbiology laboratory for analyses of cosmetic products should be equipped with balances of the required
range and accuracy for the different products to be weighed. Generally, the accuracy required for weighing the
samples to be analysed and some components of the culture media and reagents, is ± 0,01 g.

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4.4 Homogenizer

This equipment (e.g. blender, stomacher, etc.) may be used to prepare the initial suspension from the test
samples of non-liquid products.

4.5 pH–meter

The pH-meter should be capable of measuring to an accuracy of ± 0,1 pH units and its minimum measuring
threshold shall be 0,01 pH units.

4.6 Autoclave

The autoclave shall be kept in good operating condition and shall regularly be inspected by the competent
departments in accordance with the manufacturer’s instructions and proper documentation should be
recorded.

The autoclave shall not be used to sterilize both clean materials and also to decontaminate used materials at
the same time. Wherever possible, separate autoclaves for these two processes should be used.

4.7 Incubator

Incubators shall be equipped with a regulation system which allows the temperature to be kept even and
stable over their entire working volume.

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If the ambient temperature is close to, or higher than, that of the incubator, use an incubator with a cooling
system.
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Incubators should be protected from direct sunlight.
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If possible, incubators should not be completely filled in one single operation because the culture media will
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take a long time to equilibrate to temperature, whatever type of incubator is used (forced-air convection or
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otherwise).

The temperature shall be checked and recorded at least every working day.

4.8 Water baths

Water baths are of two types:

⎯ thermostatically-controlled baths, suitable for incubation of inoculated culture media, for identification
tests, etc.;

⎯ temperature-controlled water baths for maintenance of sterile agar media in a molten state for later use in
specified procedures.

The required temperature and accuracy are stipulated in each method of application.

4.9 Refrigerator or cold-storage room

The temperature, unless otherwise specified, shall be 5 °C ± 3 °C.

4.10 Freezer

The temperature, unless otherwise specified, shall be below − 18 °C.

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4.11 Sterilizing oven

A sterilizing oven is a chamber which allows the destruction of microorganisms by dry heat.

The temperature shall be evenly distributed within the chamber.

The oven shall be equipped with:

⎯ a thermostat;

⎯ a thermometer or a recording thermocouple;

⎯ a duration indicator or a programmer/timer.

4.12 Colony-counting device

A colony-counting device may be used.

4.13 Other equipment

WARNING — Volumetric glassware shall not be sterilized in a sterilizing oven.

Other equipment and apparatus for everyday use, include the following:

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filtration apparatus (see below);

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glass or plastic containers (test tubes, flasks, bottles);

c) glass or plastic Petri dishes (most commonly between 85 mm and 100 mm in diameter);
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d) glass or plastic pipettes (10 ml, 2 ml, 1 ml), automatic pipettes;
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e) sampling instruments;

f) wires and loops (of nickel/ chromium, platinum/ iridium or disposable plastic, etc.);

g) optical microscope;

h) gas burner or wire incinerator;

i) dispenser for culture media and reagents;

j) mechanical stirrer.

If the membrane filtration method is used, the equipment shall also include:

⎯ a membrane filtration system or filtration apparatus constructed of a suitable material, with a filter holder
of at least 50 ml, and suitable for use of filters with diameter 47 mm to 50 mm and not more than 0,45 µm
pore size;

⎯ the type of membrane material is chosen in such a way that the bacteria are not affected by the residual
components of the sample to be investigated;

⎯ a vacuum source able to give an even filtration flow rate (the device shall be set as to obtain the filtration
of 100 ml of liquid in less than 2 min).

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