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BRITISH STANDARD BS EN

12322:1999
Incorporating
Amendment No. 1

In vitro diagnostic
medical devices —
Culture media for
microbiology —
Performance criteria
for culture media

The European Standard EN 12322:1999, with the incorporation of


amendment A1, has the status of a British Standard

ICS 07.100.10

NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW


BS EN 12322:1999

National foreword
This British Standard is the English language version of EN 12322:1999,
including amendment A1:2001.
The UK participation in its preparation was entrusted to Technical Committee
CH/69, In vitro diagnostic systems, which has the responsibility to:
— aid enquirers to understand the text;
— present to the responsible European committee any enquiries on the
interpretation, or proposals for change, and keep the UK interests informed;
— monitor related international and European developments and
promulgate them in the UK.
A list of organizations represented on this committee can be obtained on
request to its secretary.
The UK submitted a negative vote to prEN 12322 but the standard received an
overall positive vote from CEN Member Bodies and the UK comments were not
accepted. The reasons for UK disapproval were as follows.
Clause 4, Performance evaluation. It is not clear how the requirements are to
be verified, contrary to the ISO/IEC Directives Part 3:1997.
Clause 4.1, General quality criteria. It is expected that manufacturers will use
appropriate European and international quality systems standards in
conjunction with EN 12322, so inclusion by cross reference is unnecessary. The
verbal form “shall”, used to describe the requirements of a quality assurance
scheme, cannot be taken as normative as the standard relates to culture media
and not to quality systems, which are outside its scope.
Cross-references
The British Standards which implement international or European
publications referred to in this document may be found in the BSI Standards
Catalogue under the section entitled “International Standards Correspondence
Index”, or by using the “Find” facility of the BSI Standards Electronic
Catalogue.
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a contract. Users of British Standards are responsible for their correct
application.
Compliance with a British Standard does not of itself confer immunity
from legal obligations.
Summary of pages
This British Standard, having This document comprises a front cover, an inside front cover, the EN title page,
been prepared under the pages 2 to 10, an inside back cover and a back cover.
direction of the Health and The BSI copyright notice displayed throughout this document indicates when
Environment Sector
Committee, was published the document was last issued.
under the authority of the
Standards Committee and
comes into effect on
15 August 1999
Amendments issued since publication
Amd. No. Date Comments
13482 24 December 2001 Addition of Annex ZA
© BSI 24 December 2001

ISBN 0 580 32698 5


EUROPEAN STANDARD EN 12322
NORME EUROPÉENNE April 1999
EUROPÄISCHE NORM + A1
October 2001

ICS 07.100.10

English version

In vitro diagnostic medical devices — Culture media for


microbiology — Performance criteria for culture media
(includes amendment A1:2001)

Dispositifs médicaux de diagnostic in vitro — In-vitro-Diagnostika — Kulturmedien für die


Milleux de culture de microbiologie — Critères Mikrobiologie — Leistungskriterien für
de performance des milieux de culture Kulturmedien
(inclut l’amendement A1:2001) (enthält Änderung A1:2001)

This European Standard was approved by CEN on 16 March 1999. Amendment


A1:2001 was approved by CEN on 30 September 2001. 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 biographical references concerning
such national standards may be obtained on application to the Central
Secretariat 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
Central Secretariat has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Czech
Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and
United Kingdom.

CEN
European Committee for Standardization
Comité Européen de Normalisation
Europäisches Komitee für Normung

Central Secretariat: rue de Stassart 36, B-1050 Brussels


© 1999 CEN — All rights of exploitation in any form and by any means reserved worldwide for CEN
national Members.
Ref. No. EN 12322:1999 + A1:2001 E
EN 12322:1999

Foreword Contents
This European Standard has been prepared by Page
Technical Committee CEN/TC 140, In vitro Foreword 2
diagnostic medical devices, the Secretariat of which Introduction 3
is held by DIN.
1 Scope 3
This European Standard shall be given the status of
a national standard, either by publication of an 2 Normative references 3
identical text or by endorsement, at the latest by 3 Definitions 3
October 1999, and conflicting national standards 4 Performance evaluation 4
shall be withdrawn at the latest by October 1999. 4.1 General quality criteria 4
This European Standard has been prepared under a 4.2 Control strains 4
mandate given to CEN by the European 4.3 Microbiological quality criteria 5
Commission and the European Free Trade
Association, and supports essential requirements of 4.4 Performance evaluation and interpretation
EU Directive(s). of results 6
Annex A and Annex B are for information only. 5 Information to be supplied by the
manufacturer 6
According to the CEN/CENELEC Internal 6 Documentation 6
Regulations, the national standards organizations
of the following countries are bound to implement Annex A (informative) Guidance on preservation
this European Standard: Austria, Belgium, Czech and maintenance of control strains 7
Republic, Denmark, Finland, France, Germany, Annex B (informative) Bibliography 8
Greece, Iceland, Ireland, Italy, Luxembourg, Annex ZA (informative) Clauses of this European
Netherlands, Norway, Portugal, Spain, Sweden, Standard addressing essential requirements or
Switzerland and the United Kingdom. other provisions of EU Directives 10
Foreword to amendment A1
This amendment EN 12322:1999/A1:2001 to the
EN 12322:1999 has been prepared by Technical
Committee CEN/TC 140, In vitro diagnostic medical
devices, the Secretariat of which is held by DIN.
This amendment to the European Standard
EN 12322:1999 shall be given the status of a
national standard, either by publication of an
identical text or by endorsement, at the latest by
April 2002, and conflicting national standards shall
be withdrawn at the latest by April 2002.
This amendment to the European Standard
EN 12322:1999 has been prepared under a mandate
given to CEN by the European Commission and the
European Free Trade Association, and supports
essential requirements of EU Directive(s).
For relationship with EU Directive(s), see
informative Annex ZA, which is an integral part of
this standard.
According to the CEN/CENELEC Internal
Regulations, the national standards organizations
of the following countries are bound to implement
this European Standard: Austria, Belgium, Czech
Republic, Denmark, Finland, France, Germany,
Greece, Iceland, Ireland, Italy, Luxembourg,
Netherlands, Norway, Portugal, Spain, Sweden,
Switzerland and the United Kingdom.

2 © BSI 24 December 2001


EN 12322:1 999

Introduction
In the microbiology laboratory many tests and procedures depend on culture media being consistent and
providing reproducible results. Several hundreds of formulae of dehydrated culture media are
commercially available, and many more designed for specific growth responses or purposes are described
in the literature. In addition, in clinical and industrial laboratories, the main objectives are growth and
rapid and sensitive detection of micro-organisms. The requirements for media are specific to both the
sample and the organism(s) to be detected. Standardized culture media are therefore a prerequisite for any
reliable microbiological work (see Annex B [1]), and it is of primary importance to define the major
objectives and criteria related to how a culture medium is supposed to perform. Determining the
performance characteristics of culture media is necessary for commercially prepared culture media. For
in-house prepared culture media the internal quality control is carried out by the user.
Performance criteria for culture media are necessary for obtaining comparable products of the same
medium type, regardless of source. In addition these criteria may be used by all microbiology laboratories
for the evaluation of nutritive and/or selective properties of (new) culture media.
Uniform performance criteria should therefore result in the manufacture of “standardized” products, and
limit the amount of testing of commercial culture media in microbiology laboratories.
1 Scope
This European Standard specifies requirements for the performance of culture media. It is concerned with
the traceability, comparability, reproducibility and suitability of culture media used in microbiological
laboratories. These characteristics are achieved by applying the quality criteria outlined in this standard.
This European Standard is applicable to:
a) commercial organizations distributing media to microbiology laboratories in ready-to-use form, as
dehydrated media or as semi-finished media (see 2.5 in EN 1659:1996);
b) non-commercial organizations that distribute media to satellite locations;
c) laboratories that prepare culture media for their own use.
Cell culture media are not covered by this standard.
2 Normative references
This European Standard incorporates by dated or undated reference, provisions from other publications.
These normative references are cited at the appropriate places in the text and the publications are listed
hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply
to this European Standard only when incorporated in it by amendment or revision. For undated references
the latest edition of the publication referred to applies.
EN 1659:1996, In vitro diagnostic systems — Culture media for microbiology — Terms and definitions.
EN ISO 8402:1995 , Quality management and quality assurance — Vocabulary. (ISO 8402:1994)
3 Definitions
For the purposes of this standard, the definitions given in EN 1659:1996 and in EN ISO 8402:1995 apply,
together with the following.
3.1
batch of culture media; lot of culture media
fully traceable unit referring to a defined amount of bulk, semi-finished product or end product, which is
consistent in type and quality and which has passed the requirements of production (in-process control)
and quality assurance testing, and which has been produced within one defined production period having
been assigned the same lot number
3.2
control strain
micro-organism used for microbial performance evaluation of culture media

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EN 12322:1999

3.3
reference strain
micro-organism defined to at least the genus and species level, catalogued and described according to its
characteristics
3.4
reference stock
lot of containers obtained in the laboratory by a single propagation from a reference strain or multiple
containers from the same lot of a reference strain from a supplier
3.5
stock culture
subculture(s) of a reference stock
3.6
working culture
subculture of a stock culture
4 Performance evaluation
4.1 General quality criteria
NOTE This standard is not intended to give a detailed description of quality management practices for manufacturing of culture
media, which are described elsewhere (see Annex B, [l] to [7], [11] and [13]).
It should be noted that, according to some quality systems in use by manufacturers (e.g. according to
EN ISO 9001 and EN 46001 or EN ISO 9002 and EN 46002), besides microbiological performance
evaluation of culture media, performance testing shall also include testing of physical and chemical
properties (see Annex B, [13]), such as:
a) quantity filled;
b) layer thickness and filling format;
c) colour;
d) clarity and/or optical artifacts;
e) homogeneity;
f) gel stability and consistency;
g) moisture content or homogeneity of dehydrated media;
h) appearance of specific media (e.g. blood agar);
i) pH value.
The quality of culture media depends on the quality of the basic ingredients, correct formulation and
accuracy in preparation, adequate removal of microbial contaminants, proper packaging and storage.
Raw materials, nutritive or inhibitory supplements or miniaturized test systems for identification of
micro-organisms should be supplied according to the same appropriate quality procedures.
4.2 Control strains
The set of control strains shall include reference strains.
NOTE 1 Reference strains can be obtained from service or reference collections affiliated to ECCO or WFCC (see Annex B, [9]
and [15]). The set of control strains can include well characterized positive (sensitive, resp. well-growing), weakly positive (weakly
sensitive, resp. poor-growing), and negative (insensitive, resp. suppressed or inhibited) control strains (see Annex B, [5] , as well as
DIN 58959-6 and DIN 58959-7). Control strains for commonly used culture media are referenced by a number of sources
(see Annex B [2] to [6] , [9], [11] and [12] , and DIN 58959-6, DIN 58959-6 suppl. 1, DIN 58959-7 and DIN 59959-9) and may be
selected as appropriate. For traceability, all control strains should be available from a reference collection.
Working cultures shall be used once only. Subcultures from working cultures shall not be used for
performance testing purposes or production of stock cultures.
NOTE 2 For information on preservation, maintenance techniques and service culture collections, see Annex A and Annex B, [8].

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EN 12322:1999

4.3 Microbiological quality criteria


4.3.1 Routine quality control
4.3.1.1 Growth
4.3.1.1.1 General
For the lot-control of culture media and nutritive ingredients for culture media, as appropriate, growth
shall be assessed:
— either semi-quantitatively; or
— quantitatively.
Quantitative and semi-quantitative evaluations shall be performed by a validated technique
(see Annex B, [16]).
NOTE Semi-quantitative evaluations can be performed by assigned growth-scores, e.g. 0 to 3+. When reading solid media, 0
corresponds to no growth, 1+ to very poor growth (< 10 colonies), 2+ to heavy growth (single colonies) and 3+ to very heavy growth
(confluent colonies). When reading fluid or semi-solid media visually, 0 represents no turbidity, 1+ faint turbidity and 2+ heavy
turbidity.
4.3.1.1.2 Growth-promoting properties
With an appropriate device and a defined volume of appropriate control strains, solid, semi-solid or liquid
culture media shall be inoculated with an appropriate inoculum of the control strains.
NOTE For light inocula, 100 to 1 000 CFU (colony forming unit) for each control strain or a sufficient amount of CFU to provide
isolated colonies by an appropriate streaking plate technique can be appropriate.
4.3.1.1.3 Growth-inhibiting properties
For the assessment of growth-inhibiting properties of selective culture media, with an appropriate device
and a defined volume, solid, semi-solid or liquid media shall be inoculated with an appropriate inoculum of
the control strains.
NOTE About 10 4 or more cells of each appropriate control strain can be used as inoculum of each unit to be tested.
4.3.1.2 Microbial contamination
4.3.1.2.1 General
Culture media may be classified into culture media sterilized in the final container (sterilized culture
media, see 4.3.1.2.2 ) and ready-for-use culture media produced in a manner where contamination is
eliminated or reduced to an accepted low level (see 4.3.1.2.3 ). Manufacturers shall set specifications based
on media components, process limitations and package configuration. Each lot shall meet established
target limits for the level of microbial contamination (see 4.4 ).
4.3.1.2.2 Culture media sterilized in the final container
Appropriate sterility testing procedures, e.g. as given in Annex B [10], shall be followed, or sterility shall
be guaranteed by a validated sterilization process.
4.3.1.2.3 Other culture media
To test for microbial contamination, depending on the size of the lot, an appropriate amount of each lot of
culture media shall be incubated for at least three days (? 72 h) at appropriate temperatures.
4.3.1.3 Biochemical and physiological characteristics
Where appropriate (e.g. for differentiation media), the presence, absence and/or grade of expression of
biochemical or physiological reaction(s) shall be tested with an appropriate set of control strains.
4.3.1.4 Antimicrobial susceptibility testing
For antibiotic susceptibility testing media, both the reference method and reference values for each control
strain-antimicrobial agent combination shall be used and specified.

© BSI 24 December 2001 5


EN 12322:1 999

4.3.2 Extended quality control


As appropriate, extended quality control shall be performed for:
— culture media development and validation;
— lot out-of-specification result;
— lot rejection analysis;
using an extended range of appropriate cultures and more precise methods of enumeration
(see Annex B, [16]).
4.4 Performance evaluation and interpretation of results
A culture medium performs satisfactorily if all control strains used perform according to the specifications
set. A lot shall be accepted if both general and microbiological quality criteria are met.
NOTE For the assessment of growth, the appropriate control strains should achieve growth within the specified time limit with
typical colony morphology and size, and/or expected colony counts.
Media according to 4.3.1.2.2 shall not show growth in any of the final containers within the criteria and
incubation period specified by the test method applied. Other culture media shall be rejected if the
contamination level is ? 5 %.
5 Information to be supplied by the manufacturer
The specific microbiological characteristics of the culture media shall be indicated by the manufacturer or
the supplier:
a) classification according to the type of product (dehydrated medium, partially-completed medium,
ready-to-use medium);
b) evaluated performance criteria;
c) control strains used;
d) expected results of general and microbiological testing;
e) pH value (where appropriate);
f) information on storage and expiry date.
NOTE The certificates of individual lots should be made available on request.
6 Documentation
All the documentation for each lot tested shall be recorded, documented and retained for a sufficient period
of time according to the appropriate local legal requirements.

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EN 12322:1999

Annex A (informative)
Guidance on preservation and maintenance of control strains
A.1 General
There is no universal method available for the successful preservation and maintenance of all
micro-organisms. Even strains within a species exhibit significant differences with regard to culture
preservation and resuscitation.
NOTE For the maintenance of a wide range of micro-organisms, technology is still experimental and precise methodology cannot
yet be recommended (see Annex B, [8]).
A.2 Reference strains from commercial sources
If control cultures from service collections or commercial suppliers are purchased and maintained in their
original containers (reference stock), the directions of the manufacturers should be followed.
A.3 In house prepared control strains (stock cultures)
A.3.1 Maintenance
Stock cultures of control strains for performance evaluation should be maintained and handled in a
standardized manner that should minimize the opportunity for contamination, changes in genetic
properties, or alteration of characteristics (see Annex B, [8] and DIN 58959-6). Control strain suspensions
should be stored in aliquots, either deep-frozen (? 70 ? C or below) or as lyophilized or ready-to-use cultures
from a service collection (see Annex B, [6], [8] and [9]).
NOTE For certain micro-organisms these methods may be applicable only in modified form or are not to be recommended. In this
case other suitable maintenance procedures may be considered.
For control strains, a sufficient number of stock cultures of the reference strains should be prepared,
preserved and maintained according to a written protocol with an appropriate method as recommended,
e.g. Annex B [8] and DIN 58959-6 suppl. 1.
A.3.2 Storage of control cultures
If frozen control cultures are used they should be stored by a method suitable for that control strain.
NOTE Storage on glass/ceramic beads (see Annex B, [8]) or freezing over liquid nitrogen (cryopreservation) (see Annex B, [9]) are
suitable for most strains.
A.4 Working cultures
Sub-cultures of control strains (stock cultures) are the source for working cultures. For retrieval of frozen
stock cultures, the appropriate culture vials are removed from the freezer. A portion [e.g. one or two glass
or ceramic bead(s)] is dislodged in the frozen state using a sterile instrument (e.g. forceps). During
retrieval, care should be taken to avoid de-freezing of the cultures, and the vials should be returned to the
freezer immediately.
For the preparation of an inoculum e.g. for performance testing of culture media, the working culture of the
control strain should be adjusted or colonies suspended within a suitable sterile diluent to the desired cell
density.

© BSI 24 December 2001 7


EN 12322:1999

Annex B (informative)
Bibliography
EN 46001, Quality systems — Medical devices — Particular requirements for the application of
EN ISO 9001.
EN 46002, Quality systems — Medical devices — Particular requirements for the application of
EN ISO 9002.
EN ISO 9001, Quality systems — Model for quality assurance in design/development, production,
installation and servicing. (ISO 9001:1994)
EN ISO 9002, Quality systems — Model for quality assurance in production, installation and servicing.
(ISO 9002:1994)
DIN 58959-6, Quality management in medical microbiology — Part 6: Requirements for control strains —
Examples for production and preservation of bacteria used as stock cultures and working cultures.
DIN 58959-6, Supplement 1, Quality management in medical microbiology — Part 6: Requirements for
control strains — Examples for production and preservation of bacteria used as stock cultures and working
cultures.
DIN 58959-7, Quality management in medical microbiology — Part 7: General requirements for the use of
control strains.
DIN 58959-9, Supplement 1, Quality management in medical microbiology — Part 9: Requirements for the
use of control strains for testing culture media.
DIN 59959-9, Quality management in medical microbiology — Part 9: Requirements for the use of control
strains for testing culture media.

[1] Power, D. A. Quality Control of Commercially Prepared Bacteriological Media. In: Prier, J. E.,
Bartola, J.T., Friedmann, H (eds.). Quality Control in Microbiology, University Park Press,
Baltimore, 1975, pp 47–53.
[2] Miller, J. M. Media and Reagents. In: Miller, J. M., Wentworth, B. B. (eds.). Methods for
Quality Control in Diagnostic Microbiology, American Public Health Association,
Washington D. C., 1985, pp 59–87.
[3] MacFaddin, J. F., ed. Media for Isolation — Cultivation — Identification — Maintenance of
Medical Bacteria. Vol 1. Williams & Wilkins, Baltimore-London, 1985, pp 928.
[4] Campbell, M.C., J. L. Stewart (eds.). Media Quality Control Tests. In: The Medical Mycology
Handbook, J. Wiley & Sons, New York, 1980, pp 393–397.
[5] Quality Assurance for Commercially Prepared Microbiological Culture Media — Second
Edition; Approved Standard. NCCLS publication M22-A. Villanova, PA., USA, 1990, pp 24.
[6] Burkhardt, E. (Publisher): Qualitätssicherung in der medizinischen Mikrobiologie.
DGHM-Verfahrensrichtlinien für die mikrobiologische Diagnostik. Teil 4.1.
Qualitätssicherung. Gustav Fischer Verlag, Stuttgart — New York, 1985.
[7] Costin, J. D., M. Kappner. Qualitätssicherung von Trockenmaterialien für die Mikrobiologie.
Abstr. 28. DGHM-Tagung, Göttingen, FRG, 1981.
[8] Kirsop, B. E., Doyie, A. eds. Maintenance of Micro-organisms and Cultured Cells — A Manual
of Laboratory Methods, 2nd. ed. Academic Press, London-New York, 1991.
[9] European Culture Collection’s Organization (ECCO). ICECC Publication No. 4 — Information
on holdings and services. Information Center for European Culture Collections (ICECC),
Mascheroder Weg 1b, D-38124 Braunschweig, FRG, 1992.
[10] European Pharmacopoeia. 2.6 — Biological tests. 2.6.1 Sterility — culture media — sterility,
1999.
[11] Sewell, D., L. Quality Assurance, Quality Control, Laboratory Records, and Water Quality. In:
Clinical Microbiology Procedures Handbook, Volume 2, Section 13. H. D. Isenberg, ed.,
American Society for Microbiology, Washington, D.C., 1992.
[12] Isenberg, H. D. (ed.). Clinical Microbiology Procedures Handbook. Volume 2, Chapters 13.1
and 13.2.4 to 13.2.22, American Society for Microbiology, Washington, D. C., USA, 1992

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EN 12322:1999

[13] Costin, I. D. Quality Assurance and Quality Control of Ingredients for Microbiological Culture
Media. In: Quality Assurance and Quality Control of Microbiological Culture Media,
J.E.L. Corry (ed.). G-I-T Verlag, Darmstadt, FRG, 1982, pp 65–87.
[14] Woolsen, R. F. (ed.). Statistical Methods for the Analysis of Biomedical Data.
John Wiley & Sons, Inc., New York, USA, 1987.
[15] World Directory of Collections of Cultures of Micro-organisms, 4th Edition, Eds. H. Sugawara,
M. Juncai, M. Saturo, T. Youko. Published by the WFCC’s World Data Center on
Micro-organisms, 1993.
[16] Weenk G. H. Microbiological assessment of culture media: Comparison and statistical
evaluation of methods. In: Culture media for food microbiology, J.E.L. Corry et al. (ed.).
Elsevier, Amsterdam, 1995, pp 1–23.

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EN 12322:1999

Annex ZA (informative)
Clauses of this European Standard addressing essential requirements or other
provisions of EU Directives
This European Standard has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association and supports essential requirements of EU Directive 98/79/EC.
WARNING: Other requirements and other EU Directives may be applicable to the product(s) falling
within the scope of this standard.
The following clauses of this standard, as detailed in Table ZA.1, are likely to support requirements of
Directive 98/79/EC.
Compliance with these clauses of this standard provides one means of conforming with the specific
essential requirements of the Directive concerned and associated EFTA regulations.
Table ZA.1 — Correspondence between this European Standard and Directive 98/79/EC
Clause/subclause of this Corresponding essential Qualifying remarks/Notes
European Standard requirement of Directive 98/79/EC
4.1 A.3, B.2.5
4.2 A.3
4.3 A.3, B.2.2
4.4 A.3
5 B.8.4, B.8.7

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BS EN
12322:1999
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