SIST EN ISO 9308-1:2001
(Main)Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method (ISO 9308-1:2000)
Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part 1: Membrane filtration method (ISO 9308-1:2000)
Migrated from Progress Sheet (TC Comment) (2000-07-10): PQ launched on 1991.05 has failed. A new draft shall be elaborated by CEN/TC 230
Wasserbeschaffenheit - Nachweis und Zählung von Escherichia coli und coliformen Bakterien - Teil 1: Membranfiltrationsverfahren (ISO 9308-1:2000)
Diese Internationale Norm beschreibt ein Referenzverfahren (Standardtest) für den Nachweis und die Zählung von E. coli und coliformen Bakterien in Wasser für den menschlichen Gebrauch. Der Standardtest basiert auf Membranfiltration, einer anschließenden Subkultur auf einem Selektivagar und der Berechnung der Anzahl der gesuchten Organismen in der Probe. Der Standardtest hat eine geringe Selektivität und erlaubt den Nachweis geschädigter Bakterien.
Qualité de l'eau - Recherche et dénombrement des Escherichia coli et des bactéries coliformes - Partie 1: Méthode par filtration sur membrane (ISO 9308-1:2000)
La présente partie de ISO 9308 décrit une méthode de référence (essai standard) pour la recherche et le dénombrement des Escherichia coli et des bactéries coliformes présents dans l'eau destinée à la consommation humaine. L'essai standard consiste en une filtration sur membrane, suivie d'une mise en culture dans une gélose de différentiation et d'un calcul du nombre des organismes cibles présents dans l'échantillon. L'essai standard étant peu sélectif, les bactéries endommagées peuvent être détectées. En raison du caractère peu sélectif de cette méthode, la flore bactérienne générale peut perturber la fiabilité du dénombrement des bactéries coliformes et des E. coli, par exemple, dans certaines eaux potables, telles que les eaux de puits peu profonds, qui n'ont pas été désinfectées et abritent une importante flore interférente. La présente partie de ISO 9308 est donc recommandée pour les eaux désinfectées, et autres eaux potables, ne contenant qu'un petit nombre de bactéries. La présente partie de ISO 9308 inclut une méthode rapide (essai rapide) de recherche des E. coli seuls dans les eaux destinées à la consommation humaine, en moins de 24 h, et qui peut s'avérer utile dans les cas où il y a besoin d'une information rapide. L'essai rapide consiste en une filtration sur membrane, suivie d'une mise en culture dans des conditions sélectives et d'un calcul des E. coli présents dans l'échantillon. L'essai standard et l'essai rapide décrits dans la présente partie de ISO 9308 peuvent être appliqués à d'autres types d'eau, à condition que les matières en suspension ou la microflore ne créent pas d'interférence avec la filtration, la mise en culture et le comptage.
Kakovost vode - Ugotavljanje prisotnosti in števila Escherichia coli in koliformnih bakterij - 1. del: Metoda membranske filtracije (ISO 9308-1:2000)
Standard SIST EN ISO 9308-1:2001 opisuje referenčno metodo (standardni preskus) za ugotavljanje prisotnosti in števila bakterije E. Coli in koliformnih bakterij v vodi, namenjeni za prehrano ljudi. Standardna metoda temelji na membranski filtraciji, nadaljnji obdelavi na agarskem gojišču in izračunu števila ciljnih organizmov v vzorcu. Zaradi nizke selektivnosti omogoča zaznavanje/odkrivanje poškodovanih bakterij, kar pa obenem pomeni tudi, da lahko rast na izvoru/v ozadju vpliva na zanesljivost števila koliformnih bakterij in bakterije E. Coli, na primer v nedezinficiranih pitnih izvirskih vodah. Prvi del standarda SIST EN ISO 9308 je torej posebej primeren za dezinficirane vode in druge pitne vode z nizkim številom bakterij. Standard vključuje metodo za hitro odkrivanje E. Coli (v roku 24 ur) v vodi, namenjeni prehrani ljudi, kar je še posebej pomembno v nujnih primerih, ko mora biti informacija zagotovljena čim hitreje.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 9308-1:2001
01-december-2001
Kakovost vode - Ugotavljanje prisotnosti in števila Escherichia coli in koliformnih
bakterij - 1. del: Metoda membranske filtracije (ISO 9308-1:2000)
Water quality - Detection and enumeration of Escherichia coli and coliform bacteria - Part
1: Membrane filtration method (ISO 9308-1:2000)
Wasserbeschaffenheit - Nachweis und Zählung von Escherichia coli und coliformen
Bakterien - Teil 1: Membranfiltrationsverfahren (ISO 9308-1:2000)
Qualité de l'eau - Recherche et dénombrement des Escherichia coli et des bactéries
coliformes - Partie 1: Méthode par filtration sur membrane (ISO 9308-1:2000)
Ta slovenski standard je istoveten z: EN ISO 9308-1:2000
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.30 Odpadna voda Sewage water
SIST EN ISO 9308-1:2001 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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INTERNATIONAL ISO
STANDARD 9308-1
Second edition
2000-09-15
Water quality — Detection and enumeration
of Escherichia coli and coliform bacteria —
Part 1:
Membrane filtration method
Qualité de l'eau — Recherche et dénombrement des Escherichia coli et
des bactéries coliformes —
Partie 1: Méthode par filtration sur membrane
Reference number
ISO 9308-1:2000(E)
©
ISO 2000
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ISO 9308-1:2000(E)
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ii © ISO 2000 – All rights reserved
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ISO 9308-1:2000(E)
Contents Page
Foreword.iv
Introduction.v
1 Scope .1
2 Normative references .1
3 Terms and definitions .2
4 Principle.2
5 Apparatus and glassware .3
6 Culture media and reagents .3
7 Sampling.4
8 Procedure .4
9 Expression of results .5
10 Test report .5
11 Quality assurance.5
Annex A (informative) Further microbiological information on coliform bacteria .6
Annex B (normative) Culture media and reagents .7
Bibliography.10
© ISO 2000 – All rights reserved iii
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ISO 9308-1:2000(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 3.
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 part of ISO 9308 may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 9308-1 was prepared by Technical Committee ISO/TC 147, Water quality,
Subcommittee SC 4, Microbiological methods.
This second edition cancels and replaces the first edition (ISO 9308-1:1990), which has been technically revised.
ISO 9308 consists of the following parts, under the general title Water quality — Detection and enumeration of
Escherichia coli and coliform bacteria:
� Part 1: Membrane filtration method
� Part 2: Liquid enrichment method
� Part 3: Miniaturized method (Most Probable Number, MPN) for detection and enumeration of E. coli in surface
and waste water
Annex B forms a normative part of this part of ISO 9308. Annex A is for information only.
iv © ISO 2000 – All rights reserved
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ISO 9308-1:2000(E)
Introduction
The presence and extent of faecal pollution is an important factor in assessing the quality of a body of water and
the risk to human health from infection. Examination of water samples for the presence of Escherichia coli,which
normally inhabits the bowel of man and other warm-blooded animals, provides an indication of such pollution.
Examination for coliform bacteria can be more difficult to interpret because some coliform bacteria live in soil and
surface fresh water, and are not always intestinal. Therefore, the presence of coliform bacteria, although not a
proof of faecal contamination, may indicate failure in treatment or distribution. The identification of the strains
isolated can sometimes provide an indication of their origin.
© ISO 2000 – All rights reserved v
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INTERNATIONAL STANDARD ISO 9308-1:2000(E)
Water quality — Detection and enumeration of Escherichia coli
and coliform bacteria —
Part 1:
Membrane filtration method
1 Scope
This part of ISO 9308 describes a reference method (Standard Test) for the detection and enumeration of
Escherichia coli and coliform bacteria in water for human consumption. The Standard Test is based on membrane
filtration, subsequent culture on a differential agar medium and calculation of the number of target organisms in the
sample.
The Standard Test has a low selectivity, allowing the detection of injured bacteria. Due to the low selectivity,
background growth can interfere with the reliable enumeration of coliform bacteria and E. coli, for example in some
drinking waters, like shallow well waters, that have not been disinfected and yield a high background growth. This
part of ISO 9308 is therefore especially suitable for disinfected water and other drinking waters of low bacterial
numbers.
This part of ISO 9308 includes a rapid method (Rapid Test) for the detection of E. coli only within 24 h in water for
human consumption, which can be useful in special cases when information is needed quickly. The Rapid Test is
based on membrane filtration, subsequent culture under selective conditions and calculation of the number of
E. coli in the sample.
Standard and Rapid Tests described in this part of ISO 9308 are applicable to other kinds of water provided that
suspended matter or background flora does not interfere with filtration, culture and counting.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this part of ISO 9308. For dated references, subsequent amendments to, or revisions of, any of these publications
do not apply. However, parties to agreements based on this part of ISO 9308 are encouraged to investigate the
possibility of applying the most recent editions of the normative documents indicated below. For undated
references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain
registers of currently valid International Standards.
ISO/IEC Guide 2, Standardization and related activities — General vocabulary.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods.
ISO 5667-1:1980, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes.
ISO 5667-2:1991, Water quality — Sampling — Part 2: Guidance on sampling techniques.
ISO 5667-3:1994, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples.
© ISO 2000 – All rights reserved 1
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ISO 9308-1:2000(E)
ISO 6887-1:1999, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions.
ISO 8199:1988, Water quality — General guide to the enumeration of micro-organisms by culture.
3 Terms and definitions
For the purposes of this part of ISO 9308, the terms and definitions given in ISO/IEC Guide 2 and the following
apply.
3.1
lactose-positive bacteria
�Standard Test� bacteria capable of forming colonies aerobically at (36� 3) °C on a selective and differential
lactose culture medium with the production of acid within (21� 3) h
3.2
coliform bacteria
�Standard Test� lactose-positive bacteria as defined in 3.1 which are oxidase-negative
3.3
Escherichia coli
�Standard Test� coliform bacteria as defined in 3.2 which also produce indole from tryptophan at (44,0� 0,5) °C
within (21� 3) h
3.4
Escherichia coli
�Rapid Test� bile-resistant bacteria which also produce indole from tryptophan at (44,0� 0,5) °C within (21� 3) h
4Principle
4.1 General description of the method
The method is based on membrane filtration and consists of two parts, the reference Standard Test and the
optional Rapid Test, which can be performed in parallel as described below. The Standard Test includes incubation
of the membrane on a selective medium with subsequent further biochemical characterization of the typical lactose-
positive colonies, leading to the detection and enumeration of coliform bacteria and E. coli within 2 d to 3 d. The
Rapid Test consists of two incubation steps allowing the detection and enumeration of E. coli within (21� 3) h. If
both tests, Standard Test and Rapid Test, are performed in parallel, the final result for E. coli shall be the higher of
the two.
4.2 Filtration and incubation
Test portions of the sample are filtered through membranes which retain the bacteria. One membrane (Standard
Test) is placed on a selective lactose agar medium which is incubated at (36� 2) °Cfor (21� 3) h and one
membrane (Rapid Test) on a casein (tryptic digest)-containing agar medium incubated at (36� 2) °C for 4h to 5h,
followed by incubation at (44,0� 0,5) °C for 19 h to 20 h on an agar medium containing casein (tryptic digest) and
bilesalts.
4.3 Evaluation and confirmation, Standard Test
The characteristic colonies on the membrane are counted as lactose-positive bacteria. For coliform bacteria and
E. coli, subculture is carried out of randomly selected characteristic colonies for confirmatory tests: oxidase and
indole production. The numbers of lactose-positive coliform bacteria and E. coli likely to be present in 100 ml of the
sample are counted.
2 © ISO 2000 – All rights reserved
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ISO 9308-1:2000(E)
4.4 Evaluation and confirmation, Rapid Test
The colonies on the membrane which are able to form indole from the tryptophan supplied in the agar medium are
counted as E. coli. The numbers of E. coli likely to be present in 100 ml of the sample are counted.
5 Apparatus and glassware
Usual microbiological laboratory equipment, and in particular:
5.1 Apparatus for sterilization by steam (autoclave).
Apparatus and glassware not supplied sterile shall be sterilized according to the instructions given in ISO 8199.
5.2 Water bath and/or incubator, thermostatically controlled at (36� 2) °C.
5.3 Water bath and/or incubator, thermostatically controlled at (44,0� 0,5) °C.
NOTE For the Rapid Test, instead of the incubators 5.2 and 5.3 a programmable incubator with dual setting ma
...
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