SIST EN ISO 7899-1:1999
(Main)Water quality - Detection and enumeration of intestinal enterococci in surface and wastewater - Part 1: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO 7899-1:1998)
Water quality - Detection and enumeration of intestinal enterococci in surface and wastewater - Part 1: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO 7899-1:1998)
Wasserbeschaffenheit - Nachweis und Zählung von intestinalen Enterokokken in Oberflächenwasser und Abwasser - Teil 1: Miniaturisiertes Verfahren durch Animpfen in Flüssigmedium (MPN-Verfahren) (ISO 7899-1:1998)
Dieser Teil von ISO 7899 legt ein miniaturisiertes Verfahren zum Nachweis und zur Zählung von Hauptvertretern der fäkalen Enterokokken 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 die Richtlinie weniger als 15 pro 100 ml beträgt.
Qualité de l'eau - Recherche et dénombrement des entérocoques intestinaux dans les eaux de surface et résiduaires - Partie 1: Méthode miniaturisée (nombre le plus probable) par ensemencement en milieu liquide (ISO 7899-1:1998)
La présente partie de ISO 7899 spécifie une méthode miniaturisée pour la recherche et le dénombrement des entérocoques intestinaux majeurs 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.
Kakovost vode – Ugotavljanje prisotnosti in števila intestinalnih enterokokov v površinskih in odpadnih vodah – 1. del: Metoda z miniaturizacijo (najbolj verjetno število) z nasajanjem v tekočem gojišču (ISO 7899-1:1998)
Standard SIST EN ISO 7899-1:1999 določa metodo najverjetnejšega števila za ugotavljanje prisotnosti in določanje števila glavnih črevesnih enterokokov v površinskih in odpadnih vodah z nasajanjem v tekoče gojišče. Ta metoda se lahko uporablja pri vseh vrstah površinskih in odpadnih voda, posebej tistih, ki so bogate z lebdečimi delci. Metoda ni primerna za pitno vodo ali katerokoli drugo vrsto vode, za katero smernica navaja vrednosti manjše od 15 na 100 ml.
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 7899-1:1999
01-november-1999
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SRYUãLQVNLKLQRGSDGQLKYRGDK±GHO 0HWRGD]PLQLDWXUL]DFLMR QDMEROMYHUMHWQR
ãWHYLOR ]QDVDMDQMHPYWHNRþHPJRMLãþX ,62
Water quality - Detection and enumeration of intestinal enterococci in surface and
wastewater - Part 1: Miniaturized method (Most Probable Number) by inoculation in
liquid medium (ISO 7899-1:1998)
Wasserbeschaffenheit - Nachweis und Zählung von intestinalen Enterokokken in
Oberflächenwasser und Abwasser - Teil 1: Miniaturisiertes Verfahren durch Animpfen in
Flüssigmedium (MPN-Verfahren) (ISO 7899-1:1998)
Qualité de l'eau - Recherche et dénombrement des entérocoques intestinaux dans les
eaux de surface et résiduaires - Partie 1: Méthode miniaturisée (nombre le plus
probable) par ensemencement en milieu liquide (ISO 7899-1:1998)
Ta slovenski standard je istoveten z: EN ISO 7899-1:1998
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST EN ISO 7899-1:1999 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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INTERNATIONAL ISO
STANDARD 7899-1
Second edition
1998-11-15
Water quality — Detection and enumeration
of intestinal enterococci in surface and
waste water —
Part 1:
Miniaturized method (Most Probable Number)
by inoculation in liquid medium
Qualité de l’eau — Recherche et dénombrement des entérocoques
intestinaux dans les eaux de surface et résiduaires —
Partie 1: Méthode miniaturisée (nombre le plus probable) par
ensemencement en milieu liquide
A
Reference number
ISO 7899-1:1998(E)
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ISO 7899-1:1998(E)
Contents
1 Scope .1
2 Normative references .1
3 Definitions .1
4 Principle.2
5 Apparatus .2
6 Sampling.2
7 Culture media and diluents.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 MPNs .11
Annex C (informative) Synthetic sea salt.13
(informative)
Annex D Performance characteristics of the method.14
Annex E (normative) Quality criteria for manufacturing of the medium in microtitre plates .15
Annex F (normative) Preparation of calibration microtitre plates.17
Annex G (informative) Bibliography .19
© 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 iso@iso.ch
Printed in Switzerland
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ISO ISO 7899-1:1998(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.
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 7899-1 was prepared by Technical Committee ISO/TC 147, Water quality,
Subcommittee SC 4, Biological methods.
This second edition cancels and replaces the first edition (ISO 7899-1:1984), which has been technically revised.
ISO 7899 consists of the following parts, under the general title Water quality — Detection and enumeration of
intestinal enterococci in surface and waste water:
� Part 1: Miniaturized method (Most Probable Number) by inoculation in liquid medium
� Part 2: Method by membrane filtration
Annexes E and F form an integral part of this part of ISO 7899. Annexes A, B, C, D and G are for information only.
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ISO 7899-1:1998(E) ISO
Introduction
The aim of this part of ISO 7899 is to enumerate the major intestinal enterococci, namely E. faecalis, E. faecium,
E. durans and E. hirae, which occur frequently in faeces of humans and homeothermic animals. Other faecal
Enterococcus species, namely E. avium, E. cecorum, E. columbae and E. gallinarum, and Streptococcus
bovis/equinus strains may occasionally be included, but they occur rarely in the environmental samples. Their
recovery tends to be low. Enterococcus casseliflavus and E. mundtii are non-faecal species which, when present in
water samples (e.g. because of influence of plant material and some industrial effluents), are enumerated as faecal
enterococci. These species and other rare non-faecal species tend to produce yellow pigment on a non-selective
medium. The possible interference of non-faecal Enterococcus species should therefore be considered in the
interpretation of results.
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INTERNATIONAL STANDARD ISO ISO 7899-1:1998(E)
Water quality — Detection and enumeration of intestinal
enterococci in surface and waste water —
Part 1:
Miniaturized method (Most Probable Number) by inoculation in liquid
medium
1 Scope
This part of ISO 7899 specifies a miniaturized method for the detection and enumeration of major intestinal
enterococci 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 count is less than
15 per 100 ml.
2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this part of
ISO 7899. At the time of publication, the editions indicated were valid. All standards are subject to revision, and
parties to agreements based on this part of ISO 7899 are encouraged to investigate the possibility of applying the
most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid
International Standards.
ISO 3951:1989, Sampling procedures and charts for inspection by variables for percent nonconforming.
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 8199:1988, Water quality — General guide to the enumeration of microorganisms by culture.
ISO/IEC Guide 2:1996, Standardization and related activities — Vocabulary.
3 Definitions
For the purposes of this part of ISO 7899, the definitions given in ISO/IEC Guide 2 and the following definition apply.
3.1
intestinal enterococci
microorganisms capable of aerobic growth at 44 °C and of hydrolysing the 4-methylumbelliferyl-b-D-glucoside
(MUD), in the presence of thallium acetate, nalidixic acid and 2,3,5-triphenyltetrazolium chloride (TTC), in the liquid
medium specified
1
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ISO 7899-1:1998(E)
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 between
36 h and 72 h at 44 °C æ 0,5 °C. The presence of enterococci is indicated by fluorescence resulting from the
hydrolysis of MUD. 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 by steam (autoclave).
5.2 Thermostatic incubator, regulated at 44 °C æ 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 can cause irritation of skin and eyes. Use protective gloves and glasses.
5.5 Portable refractometer (optional).
5.6 pH meter, with an accuracy of æ 0,1.
16 mm x 160 mm and 20 mm x 200 mm with similar capacity.
5.7 Test tubes, , or flasks
5.8 Adjustable or pre-set 8-channel multipipette, or any 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.
5.12 Sterile adhesive cover strips for sealing microtitre plates.
5.13 Sterile Petri dishes, 90 mm in diameter.
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.
2
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7 Culture media and diluents
7.1 General instructions
To ensure reproducible results, prepare culture medium and diluents, using either constituents of uniform quality
and chemicals of recognized analytical or a dehydrated diluent or complete medium prepared following the
manufacturer’s instructions. Prepare them with distilled or demineralized water, free from substances capable of
inhibiting or promoting growth under the test conditions. If the media are not used immediately, preserve them in the
dark at (5 æ 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 Diluent
7.2.1 Special Diluent (SD)
1)
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 æ 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 used only to colour the
SD blue and avoid confusing it with demineralized or distilled water.
7.2.2 Demineralized or distilled water
Water used for dilution shall be demineralized or distilled water free from substances inhibiting growth under the test
conditions.
Sterilize in the autoclave (5.1) before use at 121 °C æ 3 °C for 15 min to 20 min.
7.3 Culture medium: MUD/SF medium
7.3.1 Composition
7.3.1.1 Solution A
Tryptose 40 g
KH PO 10 g
2 4
D(+)-galactose 2 g
® 2)
Polyoxyethylenesorbitan monooleate (Tween 80 ) 1,5 ml
Demineralized or distilled water (7.2.2) 900 ml
®
Add tryptose, KH PO , galactose and Tween 80 to 900 ml of water, whilst maintaining gentle heat and magnetic
2 4
stirring, then bring to the boil until completely dissolved. Allow to cool.
1) A typical analysis of a commercially available and suitable synthetic sea salt is given in annex C. Pure NaCl solutions are
not suitable, as they lead to marked inhibition.
®
2) Tween 80 is an example of a suitable product available commercially. This information is given for the convenience of
users of this part of ISO 7899 and does not constitute an endorsement by ISO of this product.
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7.3.1.2 Solution B
NaHCO 4 g
3
Nalidixic acid 250 mg
Demineralized or distilled water (7.2.2) 50 ml
Add both chemicals to 50 ml of water, whilst maintaining gentle heat and magnetic stirring. Allow to cool.
7.3.1.3 Solution C
Thallium(I) acetate 2 g
2,3,5-triphenyltetrazolium chloride 0,1 g
Demineralized or distilled water (7.2.2) 50 ml
Add both chemicals to 50 ml of water, whilst maintaining gentle heat and magnetic stirring. Allow to cool.
7.3.1.4 Solution D
MUD (4-methylumbelliferyl-b-D-glucoside) 150 mg
N,N-dimethylformamide 2 ml
WARNING — Thallium acetate and N,N-dimethylformamide are toxic. Use in a chemicals fume hood.
7.3.2 Preparation
Mix together solutions A+B+C+D.
Adjust the pH to 7,5 æ 0,2.
Sterilize by filtration through a membrane 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 some examples.
Table 1
Measurement limits
Origin of sample No. of dilutions No. of wells /dilution
bacteria / 100 ml
64 wells to 1/2
4
Bathing water 2 15 to 3,5x10
32 wells to 1/20
24 wells to 1/2
4 24 wells to 1/20
6
Other surface water 40 to 3,2x10
24 wells to 1/200
24 wells to 1/2 000
16 wells to 1/2
8
Waste water and treatment plants 6 60 to 6,7x10
Up to 16 wells to 1/200 000
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8.2 Preparation of dilutions
NOTE These procedures should be performed in a biological safety cabinet, as aerosols may be created by diluting and
pipetting.
8.2.1 Fresh and brackish (waste) water [salinity < 30 g/kg, measured with 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 (see 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 (7.2.1) (1/2 dilution).
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 (5.7) 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 other tubes.
Vigorously stir the sample (see 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 water
(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 following 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 minimum diameter 90 mm.
Using a multichannel pipette (5.8) with 8 sterile tips (5.9), distribute 200 μl into each well of a 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 8
sterile tips between each dilution.
Alternatively, any other suitable system (5.8) may be used to distribute 200 μl of each dilution per well in
accordance with table 1.
CAUTION — Beware of contamination via overflow from one well to another.
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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 (5.2) at 44 °C æ 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
...
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