EN ISO 9308-3:1998

Water quality - Detection and enumeration of Escherichia coli and coliform bacteria in surface and wastewater - Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO 9308-3:1998)

Migrated from Progress Sheet (TC Comment) (2000-07-10): N315: New TD (TA/980915)

Wasserbeschaffenheit - Nachweis und Zählung von Escherichia coli und coliformen Bakterien in Oberflächenwasser und Abwasser - Teil 3: Miniaturisiertes Verfahren durch Animpfen in Flüssigmedium (MPN-Verfahren) (ISO 9308-3:1998)

Dieser Teil von ISO 9308 legt ein miniaturisiertes Verfahren zum Nachweis und Zählung von E. colin in Oberflächenwasser und Abwasser durch Kultivierung in Flüssigmedien fest. Das Verfahren kann für alle Oberflächen- und Abwasserarten angewandt werden, besonders für solche, die reich an Schwebstoffen sind. Das Verfahren ist nicht geeignet für Trinkwasser und sonstige Wasserarten, für die der Leitwert weniger als 15 pro 100 ml beträgt.

Qualité de l'eau - Recherche et dénombrement des Escherichia coli et des bactéries coliformes dans les eaux de surface et résiduaires - Partie 3: Méthode miniaturisée (nombre le plus probable) pour ensemencement en milieu liquide (ISO 9308-3:1998)

La présente partie de l'ISO 9308 spécifie une méthode miniaturisée pour la recherche et le dénombrement d'Escherichia coli (E. coli) dans les eaux de surface et résiduaires par ensemencement en milieu liquide. La présente méthode est applicable à tous les types d'eaux de surface et résiduaires, plus particulièrement celles riches en matières en suspension. La présente méthode n'est pas applicable à l'eau potable ou tout autre type d'eau dont la valeur guide est inférieure à 15 pour 100 ml. La présente méthode ne convient pas à la recherche et au dénombrement de bactéries coliformes autres que les E. coli.

Kakovost vode – Ugotavljanje prisotnosti in števila Escherichia coli in koliformnih bakterij v površinski in odpadnih vodah – 3. del: Metoda z miniaturizacijo (najbolj verjetno število) z nasajanjem v tekočem gojišču (ISO 9308-3:1998)

Standard SIST EN ISO 9308-3:1999 opisuje metodo najverjetnejšega števila za odkrivanje in določanje števila bakterije Escherichia coli (E. coli) v površinskih in odpadnih vodah z nasajanjem v tekočem gojišču. Uporaben je za vse vrste površinskih in odpadnih voda, predvsem tistih, ki vsebujejo veliko lebdečih delcev.

General Information

• Status: Published
• Publication Date: 27-Feb-1999
• ICS: 07.100.20 - Microbiology of water
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230 - Water analysis
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 21-May-2008
• Completion Date: 21-May-2008
• Ref Project: SIST EN ISO 9308-3:1999 - Water quality - Detection and enumeration of Escherichia coli and coliform bacteria in surface and wastewater - Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO 9308-3:1998)

Relations

• Corrected By: EN ISO 9308-3:1998/AC:2000 - Water quality - Detection and enumeration of Escherichia coli and coliform bacteria in surface and wastewater - Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO 9308-3:1998)
• Effective Date: 22-Dec-2008

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 9308-3:1999
01-november-1999

.DNRYRVWYRGH±8JRWDYOMDQMHSULVRWQRVWLLQãWHYLOD(VFKHULFKLDFROLLQNROLIRUPQLK

EDNWHULMYSRYUãLQVNLLQRGSDGQLKYRGDK±GHO0HWRGD]PLQLDWXUL]DFLMRQDMEROM

YHUMHWQRãWHYLOR]QDVDMDQMHPYWHNRþHPJRMLãþX,62

Water quality - Detection and enumeration of Escherichia coli and coliform bacteria in

surface and wastewater - Part 3: Miniaturized method (Most Probable Number) by
inoculation in liquid medium (ISO 9308-3:1998)
Wasserbeschaffenheit - Nachweis und Zählung von Escherichia coli und coliformen

Bakterien in Oberflächenwasser und Abwasser - Teil 3: Miniaturisiertes Verfahren durch

Animpfen in Flüssigmedium (MPN-Verfahren) (ISO 9308-3:1998)

Qualité de l'eau - Recherche et dénombrement des Escherichia coli et des bactéries

coliformes dans les eaux de surface et résiduaires - Partie 3: Méthode miniaturisée

(nombre le plus probable) pour ensemencement en milieu liquide (ISO 9308-3:1998)
Ta slovenski standard je istoveten z: EN ISO 9308-3:1998
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST EN ISO 9308-3:1999 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
INTERNATIONAL ISO
STANDARD 9308-3
First edition
1998-11-15
Water quality — Detection and enumeration
of Escherichia coli and coliform bacteria
in surface and waste water —
Part 3:
Miniaturized method (Most Probable Number)
by inoculation in liquid medium
Qualité de l’eau — Recherche et dénombrement des Escherichia coli
et des bactéries coliformes dans les eaux de surface et résiduaires —
Partie 3: Méthode miniaturisée (nombre le plus probable) pour
ensemencement en milieu liquide
Reference number
ISO 9308-3:1998(E)
---------------------- Page: 2 ----------------------
ISO 9308-3:1998(E)
Contents Page

1 Scope ........................................................................................................................................................................1

2 Normative references ..............................................................................................................................................1

3 Terms and definitions .............................................................................................................................................2

4 Principle....................................................................................................................................................................2

5 Apparatus .................................................................................................................................................................2

6 Sampling...................................................................................................................................................................3

7 Culture media and diluent.......................................................................................................................................3

8 Procedure .................................................................................................................................................................4

9 Expression of results ..............................................................................................................................................6

10 Test report ..............................................................................................................................................................7

11 Performance data...................................................................................................................................................7

Annex A (informative) Example of software for statistical analysis of MPNs .......................................................8

Annex B (informative) Example of software for computation of MPN .................................................................12

Annex C (informative) Synthetic sea salt................................................................................................................14

(informative)

Annex D Performance characteristics of the method.....................................................................16

Annex E (normative) Quality criteria for manufacturing of the medium in microtitre plates ............................17

Annex F (normative) Preparation of calibration microtitre plates........................................................................18

Bibliography..............................................................................................................................................................20

© ISO 1998

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic

or mechanical, including photocopying and microfilm, without permission in writing from the publisher.

International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet [e-mail]
Printed in Switzerland
---------------------- Page: 3 ----------------------
© ISO
ISO 9308-3:1998(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.

International Standard ISO 9308-3 was prepared by Technical Committee ISO/TC 147, Water quality,

Subcommittee SC 4, Microbiological methods.

ISO 9308 consists of the following parts, under the general title Water quality — Detection and enumeration of

Escherichia coli and coliform bacteria in surface and waste water:
 Part 1: Membrane filtration method
 Part 2: Liquid enrichment method

 Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid media

Annexes E and F form a normative part of this part of ISO 9308. Annexes A to D are for information only.

iii
---------------------- Page: 4 ----------------------
INTERNATIONAL STANDARD © ISO ISO 9308-3:1998(E)
Water quality — Detection and enumeration of Escherichia coli
and coliform bacteria in surface and waste water —
Part 3:
Miniaturized method (Most Probable Number) by inoculation in liquid
medium
1 Scope

This part of ISO 9308 specifies a miniaturized method for the detection and enumeration of Escherichia coli (E. coli)

in surface and waste water by inoculation in a liquid medium. The method is applicable to all types of surface and

waste waters, particularly those rich in suspended matter.

This method is not suitable for drinking water and any other type of water for which the guideline is less than 15

counts per 100 ml.

This method is not appropriate for enumeration and detection of coliform bacteria other than E. coli.

2 Normative references

The following normative documents contain provisions which, through reference in this text, constitute provisions of

this International Standard. For dated references, subsequent amendments to, or revisions of, any of these

publications do not apply. However, parties to agreements based on this International Standard 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 3951, Sampling procedures and charts for inspection by variables for percent nonconforming.

ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes.

ISO 5667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques.

ISO 5667-3, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples.

ISO 8199, Water quality — General guide to the enumeration of microorganisms by culture.

ISO/IEC Guide 2, Standardization and related activities — Vocabulary.
---------------------- Page: 5 ----------------------
© ISO
ISO 9308-3:1998(E)
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
Escherichia coli
E. coli

b-D-glucuronidase-positive microorganism growing at an incubation temperature of 44 °C in the specified liquid

medium containing 4-methylumbelliferyl-b-D-glucuronide (MUG)
4 Principle

The diluted sample is inoculated in a row of microtitre plate wells containing dehydrated culture medium.

The microtitre plates are examined under ultraviolet light at 366 nm in the dark after an incubation period of 36 h

minimum and 72 h maximum at 44 °C – 0,5 °C. The presence of E. coli is indicated by a blue fluorescence resulting

from hydrolysis of MUG. The results are given as most probable number (MPN) per 100 ml.

5 Apparatus

With the exception of equipment supplied sterile, the glassware shall be sterilized in accordance with the

instructions given in ISO 8199.
Usual microbiological laboratory equipment, and in particular:
5.1 Apparatus for sterilization by dry heat (oven) or steam (autoclave).
5.2 Thermostatic incubator regulated at 44 °C – 0,5 °C.
5.3 Tunnel drier or vertical laminar air flow cabinet (preferably class II).
5.4 UV observation chamber (Wood's Lamp, 366 nm).

WARNING: UV light causes irritation of eyes and skin. Use protective glasses and gloves.

5.5 Portable refractometer (optional).
5.6 pH meter with an accuracy of – 0,1

5.7 Test tubes of dimensions 16 mm · 160 mm and 20 mm · 200 mm, or flasks with similar capacity.

5.8 8-channel multipipette, adjustable or preset, or any other system suitable for measuring and distributing

200 μl per well.
5.9 Sterile tips for multipipette.

5.10 Equipment for membrane filtration in accordance with ISO 8199, including membrane filters with a nominal

pore size of 0,2 μm, for sterilization of liquid media.

5.11 Sterile microtitre plates, 96-well, 350 μl, flat bottomed, nonfluorescent.

Sterile adhesive covering strips for sealing microtitre plates.
5.12
, 90 mm in diameter.
5.13 Sterile Petri dishes
---------------------- Page: 6 ----------------------
© ISO
ISO 9308-3:1998(E)
6 Sampling

Take the samples and deliver them to the laboratory in accordance with ISO 8199 and ISO 5667-1, ISO 5667-2 and

ISO 5667-3.
7 Culture media and diluent
7.1 General instructions

To ensure reproducible results, prepare culture medium and diluents, using either constituents of uniform quality

and chemicals of recognized analytical grade, or a dehydrated diluent or complete medium prepared following the

manufacturer's instructions. Prepare them with demineralized or distilled water free from substances capable of

inhibiting growth under the test conditions. If the media are not used immediately, preserve them in the dark at

(5 – 3) °C for up to one month in conditions avoiding any alterations to their composition.

NOTE The use of chemicals of other grades is permissible providing they are shown to be of equivalent performance in the

test.
7.2 Diluents
7.2.1 Special diluent (SD)
Synthetic sea salt 22,5 g
Bromophenol blue solution (optional) 10 ml
Demineralized or distilled water (7.2.2) 1000 ml
Sterilize in the autoclave (5.1) at 121 °C – 3 °C for 15 min to 20 min.

The bromophenol blue solution is prepared by adding 0,04 g in 100 ml of 50 % ethanol. It is only used to colour the

SD blue and avoid confusing it with demineralized or distilled water.

7.2.2 Demineralized or distilled water, free from substances inhibiting growth under the test conditions.

Sterilize in the autoclave (5.1) at 121 °C – 3 °C for 15 min to 20 min.
7.3 Culture medium: MUG/EC medium
Composition
Tryptone 40 g
Salicin 1 g
Triton X 100® 1 g
MUG (4-methylumbelliferyl-b-D-glucuronide) 100 mg
Demineralized or distilled water (7.2.2) 1000 ml

A typical analysis of a commercially available and suitable synthetic sea salt is given in annex C. Other diluents, such as

distilled water, can be used for E. coli enumeration, unless intestinal enterococci are to be enumerated from the same dilution

tubes.
---------------------- Page: 7 ----------------------
© ISO
ISO 9308-3:1998(E)

Successively add tryptone, salicin and Triton to one litre of water, whilst maintaining a gentle heat and magnetic

stirring, then bring to the boil until completely dissolved. Allow to cool and add the fluorogenic constituent MUG,

dissolved in 2 ml of N,N-dimethylformamide.

WARNING: N,N-dimethylformamide is toxic and can cause cancer. Harmful by inhalation, in contact with

skin and if swallowed. Use in a chemicals fume hood.
Adjust the pH to 6,9 – 0,2.
Sterilize by filtration with membranes of average pore size 0,2 μm (5.10).

Distribute in 96-well microtitre plates (5.11) with a volume of 100 μl of media in each well (minimum capacity 350 μl)

and dehydrate immediately in a tunnel drier or laminar air flow cabinet (5.3).

The manufacturing of the medium shall meet the quality criteria given in annex E.

8 Procedure
8.1 Choice of dilutions

The number of dilutions to inoculate varies according to the presumed level of contamination of the water to be

tested. Table 1 gives examples.
Table 1
Measurement limits,
Origin of sample No. of dilutions No. of wells/dilution
bacteria/100 ml
Bathing water 2 64 wells to 1/2
15 to 3,5 · 10
32 wells to 1/20
Fresh surface water 4 24 wells to 1/2
24 wells to 1/20
40 to 3,2 · 10
24 wells to 1/200
24 wells to 1/2 000
Waste water and 6 16 wells to 1/2
treatment plants Up to 16 wells to 60 to 6,7 · 10
1/200 000
8.2 Preparation of dilutions

NOTE These procedures should be performed in a biological safety cabinet, as aerosols may be created by the diluting

and pipetting.

8.2.1 Fresh and brackish (waste) water (salinity , 30 g/kg, measured with a refractometer (5.5) or equivalent

method)

Prepare the relevant number of sterile tubes (5.7) in a rack, according to the number of selected dilutions; add 9 ml

of the special diluent (7.2.1) to each tube.

Vigorously stir the sample (clause 6) in order to obtain a homogeneous distribution of the microorganisms and using

a sterile pipette, immediately transfer 9 ml of this homogenized sample to the first tube containing 9 ml of diluent

(1/2 dilution).

Triton X 100 is an example of a suitable product available commercially. This information is given for the convenience of

users of this part of ISO 9308 and does not constitute an endorsement by ISO of this product. Equivalent products may be

used if they can be shown to lead to the same results.
---------------------- Page: 8 ----------------------
© ISO
ISO 9308-3:1998(E)

Using a fresh pipette, transfer 1 ml of this dilution (homogenized) to the second tube (1/20 dilution).

From the second tube (dilution 1/20 carefully homogenized) proceed, if necessary to another 1/10 dilution giving the

dilution 1/200.
Continue as above until all the dilutions have been prepared.
8.2.2 Sea water (salinity > 30 g/kg)

Prepare the relevant number of sterile tubes in a rack, according to the number of selected dilutions; add 9 ml of

demineralized or distilled water (7.2.2) to the first tube and 9 ml of the special diluent (7.2.1) to the following tubes.

Vigorously stir the sample (clause 6) in order to obtain a homogeneous distribution of the microorganisms and using

a sterile pipette, immediately transfer 9 ml of this homogenized sample to the first tube containing 9 ml of diluent

(7.2.2) (1/2 dilution).

Using a fresh sterile pipette, transfer 1 ml of this dilution (homogenized) to the second tube (1/20 dilution).

From the second tube (dilution 1/20 carefully homogenized) proceed, if necessary to another 1/10 dilution, giving

the dilution 1/200.
Continue as above until all the dilutions have been prepared.
8.3 Inoculation and incubation of microtitre plates
8.3.1 Inoculation

Transfer the contents of the first tube of dilution to an empty, sterile Petri dish of diameter 90 mm.

Using a multichannel pipette (5.8) with eight sterile tips (5.9), distribute 200 μl into each well of microtitre plate (5.11)

corresponding to this first dilution.

For subsequent dilutions (1/20, 1/200, etc.) operate in an identical manner, changing the Petri dish and the row of

eight sterile tips between each dilution.

Alternatively, any other suitable system (5.8) may be used to distribute 200 μl of each dilution per well according to

Table 1.
CAUTION: Beware of contamination via overflow from one well to another.
8.3.2 Incubation

Once the microtitre plate is inoculated, cover with the disposable sterile adhesive tape (5.12) provided for this

purpose.

Incubate the microtitre plate in an incubator (5.2) at 44 °C – 0,5 °C for a minimum of 36 h and a maximum of 72 h.

NOTE The microtitre plates should be handled with care, without tilting.
8.4 Reading of results

Place each microtitre plate, with the adhesive on, in the UV observation chamber (5.4).

Consider all wells
...