EN ISO 21148:2017

Cosmetics - Microbiology - General instructions for microbiological examination (ISO 21148:2017)

ISO 21148:2017 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).

Kosmetische Mittel - Mikrobiologie - Allgemeine Anleitungen zur mikrobiologischen Untersuchung (ISO 21148:2017)

Dieses Dokument gibt allgemeine Anleitungen für die Durchführung von mikrobiologischen Unter-suchungen kosmetischer Mittel, damit deren Qualität und Sicherheit in Übereinstimmung mit einer entsprechenden Risikoanalyse (z. B. geringe Wasseraktivität, hydroalkoholischer Anteil, extreme pH Werte) sichergestellt ist.
Wegen der großen Vielfalt an kosmetischen Mitteln und potentiellen Anwendungsmöglichkeiten innerhalb dieses Anwendungsbereichs sind diese Anleitungen möglicherweise für einige Produkte nicht in allen Einzelheiten geeignet (z. B. bestimmte mit Wasser nicht mischbare Produkte).

Cosmétiques - Microbiologie - Instructions générales pour les examens microbiologiques (ISO 21148:2017)

ISO 21148:2017 donne des règles générales pour la réalisation d'examens microbiologiques sur des produits cosmétiques afin de garantir leur qualité et leur innocuité, conformément à une analyse de risque appropriée (par exemple faible activité de l'eau, produit hydro-alcoolique, valeurs de pH extrêmes).
En raison de la grande variété de produits entrant dans le champ d'application de la présente norme et de leurs usages potentiels, ces règles peuvent ne pas être adaptées en tout point à certains produits (par exemple produits non miscibles à l'eau).

Kozmetika - Mikrobiologija - Splošna navodila za mikrobiološko preskušanje (ISO 21148:2017)

V tem dokumentu so podana splošna navodila za izvajanje mikrobioloških pregledov kozmetičnih izdelkov, da se zagotovi njihova kakovost in varnost v skladu z ustrezno analizo tveganja (npr. izdelkov z nizko aktivnostjo vode ali s skrajnimi vrednostmi pH, hidro-alkoholnih izdelkov).
Zaradi velike raznolikosti izdelkov in možnosti njihove uporabe na tem področju uporabe ta navodila morda niso v celoti primerna za nekatere izdelke (npr. tiste, ki se ne mešajo z vodo).

General Information

• Status: Published
• Publication Date: 20-Jun-2017
• Withdrawal Date: 30-Dec-2017
• ICS: 07.100.40 - Cosmetics microbiology
• ICS: 07.100.99 - Other standards related to microbiology
• ICS: 71.100.70 - Cosmetics. Toiletries
• Technical Committee: CEN/TC 392 - Cosmetics
• Drafting Committee: CEN/TC 392 - Cosmetics
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 21-Jun-2017
• Completion Date: 21-Jun-2017
• Mandate: M/426 - Standardisation mandate assigned to CEN concerning the microbiological analysis of cosmetic products
• Ref Project: SIST EN ISO 21148:2017 - Cosmetics - Microbiology - General instructions for microbiological examination (ISO 21148:2017)

Relations

• Replaces: EN ISO 21148:2009 - Cosmetics - Microbiology - General instructions for microbiological examination (ISO 21148:2005)
• Effective Date: 31-Aug-2016

Overview - EN ISO 21148:2017 (Cosmetics microbiology)

EN ISO 21148:2017 provides general, risk‑based instructions for carrying out microbiological examinations of cosmetic products. Published in 2017 and endorsed by CEN (as EN ISO 21148:2017), it supersedes the 2009 edition. The standard sets out laboratory premises, equipment, personnel, sample handling and test‑preparation practices needed to ensure reliable cosmetics microbiology testing across diverse product types (including low water activity, hydro‑alcoholic and extreme pH formulations). It also notes that some details may not apply to all product types (for example certain water‑immiscible products).

Key topics and technical requirements

EN ISO 21148:2017 covers procedural and technical areas to support consistent microbiological examination of cosmetics:

• Premises and test areas (Clause 4): layout, location, equipping and maintenance of laboratory spaces used for microbiological testing.
• Equipment (Clause 5): requirements for microbiological cabinets, balances, homogenizers, pH meters, autoclaves, incubators, refrigerators/freezers and colony‑counting devices.
• Strains and reference organisms (Clause 6): management of microbial strains used for controls and method validation.
• Personnel (Clause 7): competence, training and hygiene requirements for laboratory staff performing tests.
• Apparatus and glassware (Clause 8): preparation, sterilization (dry heat and moist heat), handling of disposable items and contaminated material treatment.
• Culture media and reagents (Clause 9): preparation, sterilization (including filtration), storage and handling of media and reagents.
• Sampling and sample handling (Clause 10): sampling procedures from products, transport, receipt, storage, conservation and destruction.
• Operating practices (Clause 11): hygienic precautions, preparation of initial suspensions and sample dilutions, and guidance for water‑miscible vs water‑immiscible products.
• Emphasis on a risk analysis approach to determine appropriate test methods for different cosmetic matrices.

Practical applications - who uses ISO 21148:2017

This standard is used by:

• Cosmetic manufacturers for routine microbiological quality control and batch release.
• Contract testing laboratories and QC units to standardize methods and ensure reproducibility.
• Product development teams to design preservative efficacy and stability studies.
• Regulatory and compliance personnel as a reference for best practices in microbiological testing procedures.
• Auditors and certification bodies assessing laboratory competency and test environment suitability.

Related standards and implementation notes

EN ISO 21148:2017 is part of the broader set of cosmetic microbiology guidance documents developed by ISO/TC 217 and CEN/TC 392. Laboratories should implement this standard alongside product‑specific test methods, preservative efficacy tests and national regulatory requirements. Where a cosmetic matrix has atypical properties (e.g., strongly water‑immiscible systems), the standard’s general instructions should be adapted based on documented risk analysis.

Keywords: ISO 21148:2017, cosmetics microbiology, microbiological examination, cosmetic product testing, risk analysis, sampling, culture media, laboratory equipment.

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2017
1DGRPHãþD
SIST EN ISO 21148:2009
Kozmetika - Mikrobiologija - Splošna navodila za mikrobiološko preskušanje (ISO
21148:2017)
Cosmetics - Microbiology - General instructions for microbiological examination (ISO
21148:2017)
Kosmetische Mittel - Mikrobiologie - Allgemeine Anleitungen zur mikrobiologischen
Untersuchung (ISO 21148:2017)
Cosmétiques - Microbiologie - Instructions générales pour les examens microbiologiques
(ISO 21148:2017)
Ta slovenski standard je istoveten z: EN ISO 21148:2017
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 21148
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2017
EUROPÄISCHE NORM
ICS 07.100.99; 71.100.70 Supersedes EN ISO 21148:2009
English Version
Cosmetics - Microbiology - General instructions for
microbiological examination (ISO 21148:2017)
Cosmétiques - Microbiologie - Instructions générales Kosmetische Mittel - Mikrobiologie - Allgemeine
pour les examens microbiologiques (ISO 21148:2017) Anleitungen zur mikrobiologischen Untersuchung (ISO
21148:2017)
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 21148:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 21148:2017) 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 21148:2009.
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 21148:2017 has been approved by CEN as EN ISO 21148:2017 without any modification.

INTERNATIONAL ISO
STANDARD 21148
Second edition
2017-06
Cosmetics — Microbiology — General
instructions for microbiological
examination
Cosmétiques — Microbiologie — Instructions générales pour les
examens microbiologiques
Reference number
ISO 21148:2017(E)
©
ISO 2017
ISO 21148:2017(E)
© 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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2017 – All rights reserved

ISO 21148:2017(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Premises . 1
4.1 Test areas . 1
4.2 Additional areas . 2
4.3 Location of the premises . 2
4.4 Equipping the premises . 2
4.5 Maintenance . 3
5 Equipment . 3
5.1 General . 3
5.2 Microbiological cabinets . 3
5.3 Balances . 3
5.4 Homogenizer . 4
5.5 pH–meter . 4
5.6 Autoclave . 4
5.7 Incubator . 4
5.8 Water baths . 4
5.9 Refrigerator or cold-storage room . 4
5.10 Freezer . 4
5.11 Sterilizing oven . 5
5.12 Colony-counting device . 5
5.13 Other equipment . 5
6 Strains of microorganisms . 5
7 Personnel . 6
7.1 Competence . 6
7.2 Hygiene . 6
8 Preparation of the apparatus and glassware . 6
8.1 Preparation . 6
8.2 Sterilization . 6
8.2.1 Sterilization by dry heat . 6
8.2.2 Sterilization by moist heat . 7
8.3 Disposable apparatus . 7
8.4 Management of clean apparatus and glassware . 7
8.5 Management of sterile apparatus and glassware . 7
8.6 Treatment of contaminated material . 7
8.7 Washing . 7
9 Preparation and sterilization of culture media and reagents . 8
9.1 General . 8
9.2 Water . 8
9.3 Preparation of culture media . 8
9.3.1 General. 8
9.3.2 Rehydration . 8
9.3.3 Measurement of pH . 8
9.3.4 Dispensing . 8
9.4 Sterilization . 9
9.4.1 General. 9
9.4.2 Sterilization by moist heat . 9
9.4.3 Sterilization by filtration . 9
ISO 21148:2017(E)
9.5 Storage . 9
9.5.1 General. 9
9.5.2 Laboratory-prepared culture media and reagents . 9
9.5.3 Ready-to-use culture media and reagents .10
9.6 Melting of agar culture media .10
9.7 Preparation of Petri dishes .10
10 Laboratory samples .10
10.1 General .10
10.2 Sampling the cosmetic product .10
10.3 Transport .10
10.4 Receipt and storage .11
10.5 Handling products and samples.11
10.6 Conservation and destruction of products .11
11 Operating practices .11
11.1 Hygienic precautions during the testing .11
11.2 Preparation of the initial suspension and of sample dilutions .12
11.2.1 General.12
11.2.2 Water-miscible product .12
11.2.3 Water-immiscible products .13
11.3 Counting methods .13
11.4 Detection methods .13
12 Expression of results .13
13 Neutralization of the antimicrobial properties of the product .13
Annex A (informative) Basic identification techniques .14
Annex B (informative) Basic techniques for counting and plating .19
Annex C (informative) Preparation and calibration of inoculums .20
Bibliography .21
iv © ISO 2017 – All rights reserved

ISO 21148:2017(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 21148:2005), of which it constitutes a
minor revision.
It also incorporates the Technical Corrigendum ISO 21148:2005/Cor 1:2006.
The following changes have been made:
a) in the Introduction, “validated” was changed to “demonstrated to be suitable”;
b) in Clause 6, “validation of the methodology” was changed to “verification of the methods’
suitability”;
c) in 8.2.1, “validated” was changed to “demonstrated to be suitable”;
d) in Clause 13, “validated” was changed to “demonstrated”;
e) in A.5, “validated” was changed to “demonstrated to be suitable”;
f) in B.3, editorial changes were applied.
ISO 21148:2017(E)
Introduction
The purpose of this document 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 document, 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.
A large number of manipulations can, for example, unintentionally lead to cross-contamination and the
analyst 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
document at least provides the main measures to be taken when preparing, sterilizing and storing the
media and the equipment.
The given recommendations will allow 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
demonstrated to be suitable. 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.
vi © ISO 2017 – All rights reserved

INTERNATIONAL STANDARD ISO 21148:2017(E)
Cosmetics — Microbiology — General instructions for
microbiological examination
1 Scope
This document 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 Normative references
There are no normative references in this document.
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
product
portion of an identified cosmetic product received in the laboratory for testing
3.2
sample
portion of the product (3.1) (at least 1 g or 1 ml) that is used in the test to prepare the initial
suspension (3.3)
3.3
initial suspension
suspension (or solution) of the sample (3.2) in a defined volume of an appropriate enrichment broth
3.4
sample dilution
dilution of the initial suspension (3.3)
4 Premises
4.1 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;
ISO 21148:2017(E)
— 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.
4.2 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.
4.3 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.
The surface area shall be sufficiently large to keep the work areas clean and orderly.
During the course of the tests, care shall be taken to limit access to the test areas to only those persons
required to conduct the tests.
Separate rooms and/or separate areas and/or specific enclosures should be provided for the following:
— 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.
4.4 Equipping the premises
4.4.1 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;
2 © ISO 2017 – All rights reserved

ISO 21148:2017(E)
— 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.
4.4.2 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.
4.4.3 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.
4.5 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.
5 Equipment
5.1 General
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.
5.2 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.
5.3 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
ISO 21148:2017(E)
for weighing the samples to be analysed and some components of the culture media and reagents is
±0,01 g.
5.4 Homogenizer
This equipment (e.g. blender, stomacher, etc.) may be used to prepare the initial suspension from the
test samples of non-liquid products.
5.5 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.
5.6 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.
5.7 Incubator
Incubators shall be equipped with a regulation system which allows the temperature to be kept even
and stable over their entire working volume.
If the ambient temperature is close to, or higher than, that of the incubator, use an incubator with a
cooling system.
Incubators should be protected from direct sunlight.
If possible, incubators should not be completely filled in one single operation because the culture media
will take a long time to equilibrate to temperature, whatever type of incubator is used (forced-air
convection or otherwise).
The temperature shall be checked and recorded at least every working day.
5.8 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.
5.9 Refrigerator or cold-storage room
The temperature, unless otherwise specified, shall be 5 °C ± 3 °C.
5.10 Freezer
The temperature, unless otherwise specified, shall be below −18 °C.
4 © ISO 2017 – All rights reserved

ISO 21148:2017(E)
5.11 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.
5.12 Colony-counting device
A colony-counting device may be used.
5.13 Other equipment
WARNING — Volumetric glassware shall not be sterilized in a sterilizing oven.
Other equipment and apparatus for everyday use include the following:
a) filtration apparatus (see below);
b) glass or plastic containers (test tubes, flasks, bottles);
c) glass or plastic Petri dishes (most commonly between 85 mm and 100 mm in diameter);
d) glass or plastic pipettes (10 ml, 2 ml, 1 ml), automatic pipettes;
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).
6 Strains of microorganisms
The strains needed for the verification of the methods’ suitability are indicated in each method of
application.
ISO 21148:2017(E)
7 Personnel
7.1 Competence
All personnel working in a microbiology laboratory shall have received adequate training to enable
them to conduct properly the operations entrusted to them.
The personnel who perform the tests shall have a good knowledge and sufficient practical experience
with microbiological techniques and the microorganisms sought. The person in charge shall be able to
interpret the accuracy and precision required to yield acceptable results.
7.2 Hygiene
In the field of personal hygiene, the following precautions shall be taken not only in order to avoid
contaminating the samples and culture media, but also in order to avoid risk of personnel infection:
— wear laboratory clothing that is light-coloured, clean and in good condition, manufactured from a
fabric which limits the risks of flammability; this clothing shall not be worn outside the work areas;
— keep nails perfectly clean and well groomed, and preferably short;
— wash hands, before and after microbiological examinations and immediately after visiting the
toilets or eating; for drying hands, use disposable paper or disposable cloth towels;
— when inoculating, avoid speaking, coughing, etc.;
— do not smoke, drink or eat in the test areas;
— do not put food for personal consumption in the laboratory refrigerators;
— special precautions shall be taken by persons having infections or illnesses that are likely to
contaminate samples with microorganisms and may invalidate results.
8 Preparation of the apparatus and glassware
8.1 Preparation
The apparatus and glassware used in microbiology shall be prepared in such a manner as to guarantee
its cleanliness and/or sterility up until the time of use.
Stopper tubes and cap bottles by appropriate material prior to sterilization.
Stopper the pipettes with cotton or any other appropriate material.
If necessary, the apparatus and glassware to be sterilized should be placed in special containers or
wrapped in an appropriate material (e.g. special paper, aluminium, etc.).
8.2 Sterilization
8.2.1 Sterilization by dry heat
Heat in sterilizing oven for at least 1 h at 170 °C to 180 °C or use any combination of time and temperature
if they are demonstrated to be suitable.
Indicators can be used in order to make certain that the sterilization has been achieved.
6 © ISO 2017 – All rights reserved

ISO 21148:2017(E)
8.2.2 Sterilization by moist heat
Heat for at least 15 min at minimum of 121 °C in an autoclave. Indicators can be used in order to make
certain that the sterilization has been achieved.
8.3 Disposable apparatus
Disposable apparatus may be used in the same way as the re-usable glassware (Petri dishes, pipettes,
tubes, etc.) if the specifications are similar.
In this case, contact the manufacturer to determine that the proposed apparatus and glassware is
suitable for use in microbiology (in particular sterility) and that the material contains no substances
that inhibit the growth of microorganisms.
Disposable apparatus shall be decontaminated prior to its disposal. Other than the methods described
in 8.6, and depending upon national regulations, incineration may be used. If there is an incinerator on
the premises, decontamination and disposal may be achieved in a single operation.
8.4 Management of clean apparatus and glassware
Clean apparatus and glassware shall be protected against external contamination during storage,
under conditions which maintain their cleanliness.
8.5 Management of sterile apparatus and glassware
Prior to use, the apparatus and glassware shall be stored under conditions which allow them to remain
sterile. Disposable apparatus and glassware shall be stored in accordance to the manufacturer’s
specifications, without any damage to the packaging; laboratory-prepared apparatus and glassware
shall be stored in clean containers.
When sterilizing apparatus and glassware intended for microbiology, an expiry date (or a manufacturing
date) shall be put on each packaging. Hermetically sealed apparatus and glassware can be stored for up
to 3 months prior to use unless otherwise specified.
8.6 Treatment of contaminated material
After use (culture of microorganisms or material in contact with microorganisms), the apparatus and
glassware and their contents shall be treated for destruction of microorganisms, prior to cleaning or
disposal, whatever the microorganism involved.
According to the nature of the materials, disinfection (see 11.1), sterilization (see 8.2) or incineration of
disposable material (see 8.3) may be used.
8.7 Washing
Wash apparatus and glassware after they have been treated (see 8.6).
Empty the containers of their contents.
Prior to washing, separate seals from stoppers or caps, as appropriate.
Rinse detergent residue from equipment with tap water. Rinse clean equipment with water (see 9.2).
In the absence of any commercial product, a sodium carbonate solution of 0,125 % (mass fraction) can
[2]
be used, followed by immersion in diluted acid [e.g. hydrochloric acid c(HCl) = 0,1 mol/l].
Specialized apparatus and glassware may be used in order to facilitate cleaning operations (e.g. pipette
washers, dishwashers, ultrasonic baths, etc.).
ISO 21148:2017(E)
9 Preparation and sterilization of culture media and reagents
9.1 General
The accurate preparation of culture media is one of the fundamental steps in microbiological analysis
and it shall be given special care.
9.2 Water
WARNING — Water processed through an ion exchanger (deionized) may have a high
microorganism content; it is therefore advisable not to use such water without verifying that
the microorganism content of the water is low. Consult the manufacturer for the best way to
minimize microbial contamination. Heavily contaminated deionized water that has been filter
sterilized may still contain substances inhibitory to the growth of some microorganisms.
[7][9][11] [3]
Use distilled water or water of equivalent quality, i.e. purified water or deionized water . If the
distilled water is prepared from chlorinated water, neutralize the chlorine prior to the distillation.
The water shall be stored in containers manufactured of inert materials (e.g. neutral glass,
polyethylene, etc.).
9.3 Preparation of culture media
9.3.1 General
Two types of culture media preparation exist:
— from basic ingredients, dehydrated or not; or
— from dehydrated complete media.
Follow the manufacturer’s specified storage conditions and expiration date.
Do not use the culture media beyond the stated shelf life.
Protect laboratory culture media that is in dehydrate from absorbing additional moisture from the
environment during storage and use.
9.3.2 Rehydration
Follow the manufacturer’s recommendation for rehydration.
9.3.3 Measurement of pH
Measure the pH using a pH-meter (5.5) and adjust it, if necessary, so that, after sterilization and cooling
down to room temperature, the medium is at the required pH ±0,2 units, unless otherwise stated.
NOTE The adjustment is normally carried out using a solution of approximately 40 g/l (about 1 mol/l) of
[2]
sodium
...