BRITISH STANDARD BS EN

26461-2:1993
BS 6068-4.9:
1993
ISO 6461-2:
1986
Water quality —
Detection and enumeration of the
spores of sulfite-reducing anaerobes
(clostridia) —

Part 2: Method by membrane filtration

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The European Standard EN 26461-2:1993 has the status of a

British Standard

UDC 628.1/.3:620.1:543.39:579.852.13
BS EN 26461-2:1993

Cooperating organizations

The European Committee for Standardization (CEN), under whose supervision

this European Standard was prepared, comprises the national standards
organizations of the following countries:

Austria Oesterreichisches Normungsinstitut

Belgium Institut belge de normalisation
Denmark Dansk Standardiseringsraad
Finland Suomen Standardisoimisliito, r.y.
France Association française de normalisation
Germany Deutsches Institut für Normung e.V.
Greece Hellenic Organization for Standardization
Iceland Technological Institute of Iceland
Ireland National Standards Authority of Ireland
Italy Ente Nazionale Italiano di Unificazione
Luxembourg Inspection du Travail et des Mines
Netherlands Nederlands Normalisatie-instituut
Norway Norges Standardiseringsforbund
Portugal Instituto Portuguès da Qualidade
Spain Asociación Española de Normalización y Certificación
Sweden Standardiseringskommissionen i Sverige

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Switzerland
United Kingdom
Association suisse de normalisation
British Standards Institution

This British Standard, having

been prepared under the
direction of the Environment
and Pollution Standards
Policy Committee, was
published under the authority
of the Standards Board and
comes into effect on
15 April 1993 Amendments issued since publication
© BSI 09-1999
Amd. No. Date Comments

The following BSI references

relate to the work on this
standard:
Committee reference EPC/44
Special announcement in
BSI News August 1991

ISBN 0 580 21206 8

 BS EN 26461-2:1993

Contents

Page
Cooperating organizations Inside front cover
National foreword ii
Foreword 2
0 Introduction 3
1 Scope 3
2 Field of application 3
3 References 3
4 Definition 3
5 Principle 3
6 Culture media and reagents 3
7 Apparatus and glassware 4
8 Sampling 4
9 Procedure 4
10 Expression of results 5
11 Test report 5
National annex NA (informative) Committees responsible 6
National annex NB (informative) Cross-references Inside back cover

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© BSI 09-1999 i
BS EN 26461-2:1993

National foreword

This British Standard has been prepared under the direction of the Environment
and Pollution Standards Policy Committee. It is the English language version of
EN 26461-2:1993 Water quality — Detection and enumeration of the spores of
sulfite-reducing anaerobes (clostridia) — Part 2: Method by membrane filtration,
published by the European Committee for Standardization (CEN), which
endorses ISO 6461-2:1986, published by the International Organization for
Standardization (ISO).
A British Standard does not purport to include all the necessary provisions of 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.

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Summary of pages
This document comprises a front cover, an inside front cover, pages i and ii,
the EN title page, pages 2 to 6, an inside back cover and a back cover.
This standard has been updated (see copyright date) and may have had
amendments incorporated. This will be indicated in the amendment table on the
inside front cover.

ii © BSI 09-1999
EUROPEAN STANDARD EN 26461-2
NORME EUROPÉENNE
January 1993
EUROPÄISCHE NORM

UDC 628.1/.3:620.1:543.39:579.852.13

Descriptors: Water, quality, water tests, microbiological analysis, micro-organisms, sulfite reducing bacteria, clostridium, filtration
analysis

English version

Water quality — Detection and enumeration of the spores

of sulfite-reducing anaerobes (clostridia) —
Part 2: Method by membrane filtration
(ISO 6461-2:1986)

Qualité de l’eau — Recherche et dénombrement Wasserbeschaffenheit — Nachweis und

des spores de micro-organismes anaérobies Zählung der Sporen sulfitreduzierender
sulfito-réducteurs (clostridia) — Anaerobier (Clostridien) —
Partie 2: Méthode par filtration sur membrane Teil 2: Membranfiltrationsverfahren
(ISO 6461-2:1986) (ISO 6461-2:1986)

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This European Standard was approved by CEN on 1993-01-20. 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 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,
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

© 1993 Copyright reserved to CEN members

Ref. No. EN 26461-2:1993 E
EN 26461-2:1993

Foreword
This European Standard is the endorsement of
ISO 6461-2. Endorsement of ISO 6461-2 was
recommended by Technical Committee CEN/TC 230
“Water analysis” under whose competence this
European Standard will henceforth fall.
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
July 1993, and conflicting national standards shall
be withdrawn at the latest by July 1993.
The standard was approved and in accordance with
the CEN/CENELEC Internal Regulations, the
following countries are bound to implement this
European Standard:
Austria, Belgium, Denmark, Finland, France,
Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, Netherlands, Norway, Portugal,
Spain, Sweden, Switzerland, United Kingdom.

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2 © BSI 09-1999
 EN 26461-2:1992

0 Introduction 5 Principle
The spores of sulfite-reducing anaerobes (clostridia) The detection of spores of sulfite-reducing
are widespread in the environment. They are anaerobes (clostridia) in a specified volume of a
present in human and animal faecal matter, in water sample requires the following steps.
waste water and in soil. Unlike Escherichia coli and 5.1 Selection of spores
other coliform organisms, the spores survive in
water for long periods as they are more resistant Selection of spores in the sample by applying heat
than vegetative forms to the action of chemical and for a period of time sufficient to destroy vegetative
physical factors. They may thus give an indication of bacteria.
remote or intermittent pollution. They may even be 5.2 Membrane filtration and culture
resistant to chlorination at levels which are Filtration of the water sample through a membrane
normally used for the treatment of water, and they filter having a pore size such that bacterial
are thus useful for control purposes. spores (0,2 4m) are retained in or on the membrane
ISO 6461 consists of the following parts: filter.
— Part 1: Method by enrichment in a liquid Placing of the filter on a specialized selective culture
medium; medium (sulfite-iron-agar), followed by incubation
— Part 2: Method by membrane filtration. at 37 ± 1 °C for 20 ± 4 h and 44 ± 4 h, and counting
of any black colonies.
1 Scope
6 Culture media and reagents
This part of ISO 6461 specifies a method for the
detection and enumeration of the spores of 6.1 Basic materials
sulfite-reducing anaerobes (clostridia) by In order to improve the reproducibility of the
membrane filtration. results, it is recommended that, for the preparation

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of the diluents and culture media, dehydrated basic
2 Field of application components or complete dehydrated media be used.
The method can be applied to all types of water, Similarly, commercially prepared reagents may also
except when a large amount of particulate material be used. The manufacturer’s instructions shall be
is liable to be retained by the membrane. rigorously followed.
The chemical products used for the preparation of
3 References the culture media and the reagents shall be of
ISO 3696, Water for laboratory use — Specifications. recognized analytical quality.
ISO 5667, Water quality — Sampling — The water used shall be distilled or deionized water,
Part 2: Guidance on sampling techniques — free from substances that might inhibit the growth
Part 3: Guidance on the preservation and handling of micro-organisms under the test conditions
of samples. (see ISO 3696).
ISO 7704, Water quality — Evaluation of membrane Measurements of pH shall be made using a pH
filters used for microbiological analyses. meter, measurements being referred to a
temperature of 25 °C.
ISO 8199, Water quality — General guidance for
microbiological examination by enumeration of If the prepared culture media are not used
micro-organisms on culture media1). immediately, they shall, unless otherwise stated, be
stored in the dark at approximately 4 °C, for no
4 Definition longer than 1 month.
6.2 Sulfite-iron-agar
For the purpose of this part of ISO 6461, the
following definition applies. 6.2.1 Basal medium (nutrient agar)
clostridia Composition
sulfite-reducing, spore-forming, anaerobic Meat extract 3g
micro-organisms which belong to the Bacillaceae Peptone 10 g
family and the genus Clostridium Sodium chloride (NaCl) 5g
Agar 15 g
Water 1 000 ml

1)
At present at the stage of draft.

© BSI 09-1999 3
EN 26461-2:1992

Preparation 7.1 Glass flasks (Erlenmeyer flask, round-bottom

Steam to dissolve, make up to 1 litre with water, flask, or conical flask), of capacity 2 litres.
and adjust the pH to 7,6 ± 0,1 with 1 mol/l sodium 7.2 Test tubes, 160 mm × 16 mm.
hydroxide solution. Sterilize at 121 ± 1 °C in the 7.3 Graduated pipettes, of capacity 10 ml, graduated
autoclave for 20 min. in divisions of 0,1 ml.
Store in the refrigerator after solidifying.
7.4 Volumetric pipettes, of capacity 10 ml.
6.2.2 Sodium sulfite (Na2SO3) solution.
7.5 Jars, of capacity 1 litre.
Dissolve 10 g of sodium sulfite in 100 ml of water.
7.6 Steamer
It is advisable to prepare a fresh solution every two
weeks. 7.7 Water bath
6.2.3 Iron(II) sulfate (FeSO4) solution. 7.8 Membrane filtration apparatus
Dissolve 8 g of crystallized iron(II) sulfate in 100 ml 7.9 Sterile membrane filters, of nominal pore
of water. size 0,2 4m.
6.2.4 Complete medium NOTE The quality of membrane filters may vary according to
brand or even from batch to batch. It is therefore advisable to
Immediately before use, melt the basal check the quality on a regular basis according to ISO 7704.
medium (6.2.1) and to each 18 ml volume add 1 ml 7.10 Incubator, capable of being maintained
of sodium sulfite solution (6.2.2) and five drops of at 37 ± 1 °C.
iron(II) sulfate solution (6.2.3).
7.11 Petri dishes
Add 1 ml of the sodium sulfite solution and 5 drops
of the iron(II) sulfate solution to the agar tubes just 8 Sampling
before the embedding procedure (see 9.3).
6.3 Tryptose-sulfite-agar (alternative medium) Refer to ISO 5667-2 and ISO 8199 for sampling
techniques.
Composition
Tryptose
Soytone
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15 g
5g
9 Procedure
9.1 Treatment of samples
Yeast extract 5g Refer to ISO 5667-3 for guidance on the
preservation and handling of samples, and to
Sodium metabisulfite 1g
ISO 8199.
Ammonium iron(III) citrate 1g
9.2 Selection of spores (technique)
Water 1 000 ml
Before the test, the sample of water should be
Preparation heated in a water bath at 75 ± 5 °C for 15 min from
the time it reaches that temperature. A similar
Steam to dissolve, and adjust the pH bottle containing the same volume of water as the
to 7,6 ± 0,1 at 2,5 °C. test sample should be used periodically as a control
Distribute into test tubes in volumes of 18 ml. in order to check the heating time required. The
Sterilize the medium for 15 min at 121 ± 1 °C. temperature of the water in the control bottle can be
Store in the refigerator at 4 to 5 °C. constantly recorded by thermometer.
Discard unused medium 2 weeks after preparation. 9.3 Inoculation and incubation
Refer to ISO 7704 for a general description of the
7 Apparatus and glassware membrane filtration technique.
Usual microbiological laboratory equipment, and According to the instructions in that document,
filter a suitable volume of water. For drinking
water, spring and well water, mineral waters,
sea-water, and surface water, less polluted with
clostridia, filter 100 ml; for highly polluted water or
sewage use smaller volumes. Volumes of water
smaller than 10 ml should be mixed
with 10 to 100 ml of sterile water or diluent.
Adjust the dilutions so that any resultant black
colonies are well separated and can be easily
counted.

4 © BSI 09-1999
 EN 26461-2:1992

After filtration, remove the membrane with sterile 10 Expression of results

forceps and place it face downwards on the bottom of
Express the results in accordance with ISO 8199.
a Petri dish, ensuring that no air bubbles are
trapped under the filter. Then carefully pour 18 ml
of the liquefied complete culture medium,
11 Test report
previously cooled to about 50 °C, over the The test report shall state the method used and
membrane while holding it still with sterile forceps. express the results as the number of
After this layer of medium has set, incubate sulfite-reducing anaerobes (clostridia) per volume of
anaerobically or under other conditions which sample. The 44 ± 4 h count should normally be
ensure anaerobiosis at a temperature of 37 ± 1 °C reported. If this is not possible, the count at 20 ± 4 h
for 20 ± 4 h and 44 ± 4 h. If an anaerobic jar or an should be reported as approximate only.
anaerobic incubator is used, the membrane filter The test report shall also mention any operating
may be placed on the surface of the agar face details not specified in this part of ISO 6461, or
upwards. regarded as optional, together with details of any
9.4 Interpretation incidents likely to have influenced the results.
Count all black colonies after incubation for 20 ± 4 h The test report shall include all the information
and 44 ± 4h. necessary for the complete identification of the
sample.

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© BSI 09-1999 5
BS EN 26461-2:1993

National annex NA (informative)

Committees responsible
The United Kingdom participation in the preparation of this European Standard was entrusted by the
Environment and Pollution Standards Policy Committee (EPC/-) to Technical Committee EPC/44 upon
which the following bodies were represented:

Association of Consulting Engineers

British Association for Chemical Specialities
British Gas plc
Chemical Industries’ Association
Department of Trade and Industry (Laboratory of the Government Chemist)
Department of the Environment for Northern Ireland
Department of the Environment (Water Directorate)
Industrial Water Society
Institute of Petroleum
Institution of Water Officers
Institution of Gas Engineers
Institution of Water and Environmental Management
National Rivers Authority
Royal Institute of Public Health and Hygiene
Royal Society of Chemistry

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Scottish Association of Directors of Water and Sewerage Services
Soap and Detergent Industry Association
Water Companies Association
Water Research Centre
Water Services Association of England and Wales

The following bodies were also represented in the drafting of the standard, through subcommittees and
panels:

Automatic Vending Association of Great Britain

British Laboratory Ware Association
British Occupational Hygiene Society
British Soft Drinks Association Ltd.
Chartered Institution of Building Services Engineers
Department of Health
Institute of Hospital Engineering
Ministry of Agriculture, Fisheries and Food
Public Health Laboratory Service
Society for Applied Bacteriology
Society for General Microbiology

6 © BSI 09-1999
 BS EN 26461-2:1993

National annex NB (informative)

Cross-references
Publication referred to Corresponding British Standard
ISO 3696:1987 BS 3978:1987 Specification for water for laboratory use
BS 6068 Water quality
Part 6 Sampling
ISO 5667-2:1991 Section 6.2:1991 Guidance on sampling techniques
ISO 5667-3:1985 Section 6.3:1986 Guidance on the preservation and handling of samples
Part 4 Microbiological methods
ISO 8199:1988 Section 4.2:1989 Guide to the enumeration of micro-organisms by culture

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© BSI 09-1999
BS EN
26461-2:1993
BS 6068-4.9: BSI — British Standards Institution
1993
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ISO 6461-2: British Standards. It presents the UK view on standards in Europe and at the
1986 international level. It is incorporated by Royal Charter.

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