SIST EN ISO 9308-2:2014

SLOVENSKI STANDARD
SIST EN ISO 9308-2:2014
01-julij-2014
1DGRPHãþD
SIST ISO 9308-2:1998
Kakovost vode - Ugotavljanje števila Escherichia coli in koliformnih bakterij - 2.
del: Metoda najverjetnejšega števila (ISO 9308-2:2012)
Water quality - Enumeration of Escherichia coli and coliform bacteria - Part 2: Most
probable number method (ISO 9308-2:2012)
Wasserbeschaffenheit - Zählung von Escherichia coli und coliformen Organismen - Teil
2: Verfahren zur Bestimmung der Anzahl mit der höchsten Wahrscheinlichkeit (ISO 9308
-2:2012)
Qualité de l'eau - Dénombrement des Escherichia coli et des bactéries coliformes -
Partie 2: Méthode du nombre le plus probable (ISO 9308-2:2012)
Ta slovenski standard je istoveten z: EN ISO 9308-2:2014
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
13.060.01 Kakovost vode na splošno Water quality in general
SIST EN ISO 9308-2:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 9308-2:2014

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SIST EN ISO 9308-2:2014

EUROPEAN STANDARD
EN ISO 9308-2

NORME EUROPÉENNE

EUROPÄISCHE NORM
April 2014
ICS 07.100.20
English Version
Water quality - Enumeration of Escherichia coli and coliform
bacteria - Part 2: Most probable number method (ISO 9308-
2:2012)
Qualité de l'eau - Dénombrement des Escherichia coli et Wasserbeschaffenheit - Zählung von Escherichia coli und
des bactéries coliformes - Partie 2: Méthode du nombre le coliformen Bakterien - Teil 2: Verfahren zur Bestimmung
plus probable (ISO 9308-2:2012) der wahrscheinlichsten Keimzahl (ISO 9308-2:2012)
This European Standard was approved by CEN on 11 April 2014.

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 CEN-CENELEC Management Centre 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 CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 9308-2:2014 E
worldwide for CEN national Members.

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SIST EN ISO 9308-2:2014
EN ISO 9308-2:2014 (E)
Contents Page
Foreword .3
2

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SIST EN ISO 9308-2:2014
EN ISO 9308-2:2014 (E)
Foreword
The text of ISO 9308-2:2012 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 9308-2:2014 by
Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN.
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 October 2014, and conflicting national standards shall be withdrawn at
the latest by October 2014.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 9308-2:2012 has been approved by CEN as EN ISO 9308-2:2014 without any modification.
3

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SIST EN ISO 9308-2:2014

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SIST EN ISO 9308-2:2014

INTERNATIONAL ISO
STANDARD 9308-2
Second edition
2012-07-01
Water quality — Enumeration of
Escherichia coli and coliform bacteria —
Part 2:
Most probable number method
Qualité de l'eau — Dénombrement des Escherichia coli et des
organismes coliformes —
Partie 2: Méthode du nombre le plus probable




Reference number
ISO 9308-2:2012(E)
©
ISO 2012

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(E)

COPYRIGHT PROTECTED DOCUMENT


©  ISO 2012
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 either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56  CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland

ii © ISO 2012 – All rights reserved

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(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 . 2
6  Culture media and reagents . 3
7  Sampling . 3
8  Procedure . 3
9  Expression of results . 4
10  Test report . 4
11  Quality assurance . 4
Annex A (informative) Further microbiological information on coliform bacteria . 5
5)
Annex B (normative) The Quanti-Tray Sealer and calculation of results . 6
Annex C (informative) Composition of the Colilert-18 medium . 42
8)
Annex D (informative) Validation of Colilert-18/Quanti-Tray for the enumeration of E.coli and
coliform bacteria from water . 44
Bibliography . 46

© ISO 2012 – All rights reserved iii

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(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 2.
The main task of technical committees is to prepare International Standards. 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.
ISO 9308-2 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 9308-2:1990), which has been technically
revised.
ISO 9308 consists of the following parts, under the general title Water quality — Enumeration of Escherichia
coli and coliform bacteria:
 Part 1: Membrane filtration method for waters with low bacterial background flora
 Part 2: Most probable number method
 Part 3: Miniaturized method (Most Probable Number) for the detection and enumeration of E. coli in
surface and waste water

iv © ISO 2012 – All rights reserved

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(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
(E. 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 a failure in treatment or ingress of water
into the distribution system.
The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that
compliance with this document may involve the use of patents concerning Colilert-18 and Quanti-Tray and
Quanti-Tray 2000 given in this document.
ISO takes no position concerning the evidence, validity and scope of these patent rights.
The holder of this patent right has assured the ISO that he/she is willing to negotiate licences either free of
charge or under reasonable and non-discriminatory terms and conditions with applicants throughout the world.
In this respect, the statement of the holder of this patent right is registered with ISO. Information may be
obtained from:
IDEXX Laboratories, Inc.
One IDEXX Drive
Westbrook, Maine 04092 USA
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent
rights.
ISO (http://www.iso.org/patents) and IEC (http://patents.iec.ch) maintain on-line databases of patents relevant
to their standards. Users are encouraged to consult the databases for the most up to date information
concerning patents.


© ISO 2012 – All rights reserved v

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SIST EN ISO 9308-2:2014

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SIST EN ISO 9308-2:2014
INTERNATIONAL STANDARD ISO 9308-2:2012(E)

Water quality — Enumeration of Escherichia coli and coliform
bacteria —
Part 2:
Most probable number method
WARNING – Persons using this part of ISO 9308 should be familiar with normal laboratory practice.
This International Standard does not purport to address all of the safety problems, if any, associated
with its use. It is the responsibility of the user to establish appropriate safety and health practices and
to ensure compliance with any national regulatory conditions.
IMPORTANT – It is absolutely essential that tests conducted in accordance with this part of ISO 9308
be carried out by suitably qualified staff.
1 Scope
This part of ISO 9308 specifies a method for the enumeration of E. coli and coliform bacteria in water. The
method is based on the growth of target organisms in a liquid medium and calculation of the “Most Probable
Number” (MPN) of organisms by reference to MPN tables. This method can be applied to all types of water,
including those containing an appreciable amount of suspended matter and high background counts of
heterotrophic bacteria. However, it must not be used for the enumeration of coliform bacteria in marine water.
When using for the enumeration of E. coli in marine waters, a 1→10 dilution in sterile water is typically
required, although the method has been shown to work well with some marine waters that have a lower than
normal concentration of salts. In the absence of data to support the use of the method without dilution, a
1→10 dilution is used.
This method relies upon the detection of E. coli based upon expression of the enzyme -D-glucuronidase and
consequently does not detect many of the enterohaemorhagic strains of E. coli, which do not typically express
this enzyme. Additionally, there are a small number of other E. coli strains that do not express
-D-glucuronidase.
The choice of tests used in the detection and confirmation of the coliform group of bacteria, including E. coli,
can be regarded as part of a continuous sequence. The extent of confirmation with a particular sample
depends partly on the nature of the water and partly on the reasons for the examination. The test described in
this part of ISO 9308 provides a confirmed result with no requirement for further confirmation of positive wells.
NOTE While this method describes the use of an enumeration device that is commercially available, the medium
described here can also be used in a standard MPN format.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 8199, Water quality — General guide to the enumeration of micro-organisms by culture
ISO/IEC Guide 2:2004, Standardization and related activities — General vocabulary
© ISO 2012 – All rights reserved 1

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(E)
ISO 19458, Water quality — Sampling for microbiological analysis
3 Terms and definitions
For the purpose of this document, the terms and definitions given in ISO/IEC Guide 2 and the following apply.
3.1
coliform bacterium
member of the Enterobacteriaceae that express the enzyme -D-galactosidase
3.2
Escherichia coli
member of the Enterobacteriaceae that expresses both -D-galactosidase and -D-glucuronidase enzymes
4 Principle
A snap pack of dehydrated medium is added to a sample of water (100 ml), or a dilution of a sample made up
to 100 ml. Sample plus medium is gently shaken to ensure adequate mixing and to afford dissolution of the
1)
1)
medium. The sample plus medium is then aseptically poured into a Quanti-Tray or Quanti-Tray/2000 to
enumerate up to 201 organisms or 2 419 organisms per 100 ml, respectively. Trays are sealed with a

1)
Quanti-Tray Sealer and then incubated at (36 ± 2) °C for 18 h to 22 h.
After incubation, sample wells that have a yellow colour of equal or greater intensity than that of the
comparator wells are considered positive for coliform bacteria. Yellow wells that also exhibit any degree of
fluorescence are considered positive for E. coli.
By means of statistical tables, or a simple computer program, the most probable number (MPN) of coliform
bacteria and E. coli in 100 ml of the sample can be determined.
NOTE The yellow colouration can be seen with the naked eye and results from the cleavage of ortho-nitrophenol
galactoside by the enzyme -D-galactosidase. The fluorescence is demonstrable under ultraviolet light (365 nm) and
originates from the cleavage of the molecule 4-methylumbelliferyl glucuronide (MUG) by the enzyme -D-glucuronidase to
produce the fluorescent compound methyl umbelliferone.
5 Apparatus and glassware
Use microbiological laboratory equipment and, in particular, the following:
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 Hot air oven, for dry heat sterilization.
5.3 Incubator, thermostatically controlled at (36 ± 2) °C.
1)
5.4 Quanti-Tray sealer.
5.5 Sterile wide mouthed vessels of at least 110 ml.

1) Quanti-Tray is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States
and/or other countries. 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.
2 © ISO 2012 – All rights reserved

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(E)
2)
5.6 Quanti-Tray comparator.
5.7 Ultraviolet lamp, 365 nm.
2) 2)
/
5.8 Quanti-Tray or Quanti-Tray 2000 , see Annex B.
6 Culture media and reagents
6.1 Basic materials
3)
The method utilises Colilert -18 a medium based on the Defined Substrate Technology available for a 100 ml
sample as a ready to use powder dispensed in snap packs. Each snap pack contains sufficient medium
(2,8 g) for a single test. Medium is stored under ambient conditions (2 °C to 25 °C) out of direct sunlight and
should be used before the expiry date listed on the snap pack.
The medium is composed of two components to give the final concentrations as shown in Annex C.
6.2 Diluent
3)
For dilutions to be used with Colilert -18, use only sterile, non-inhibitory, oxidant-free water (deionized or tap).
The use of buffered, saline or peptone-containing diluents interferes with the performance of the test.
6.3 Antifoam B
Antifoam B is a 10 % active, water soluble suspension of silicone.
7 Sampling
Take the samples and deliver them to the laboratory in accordance with ISO 19458.
8 Procedure
8.1 Preparation of the sample
Samples should be transported and stored at (5  3) °C in accordance with ISO 19458 and analysis
commenced on the day of collection or within 18 h. Under exceptional circumstances, the samples may be
kept at (5 ± 3) °C for up to 24 h prior to examination.
8.2 Inoculation of media
4)
Aseptically add a single snap pack of Colilert -18 medium (2,8 g) to each 100 ml volume of sample or dilution.
When the medium has completely dissolved, the sample plus medium is aseptically poured into either a

2) Quanti-Tray is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States
and/or other countries. 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.
3) Colilert is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or
other countries. 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.
4) Colilert and Quanti-Tray are trademarks or registered trademarks of IDEXX Laboratories, Inc. or its affiliates in the
United States and/or other countries. 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.
© ISO 2012 – All rights reserved 3

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(E)

4) 4) 4)
Quanti-Tray or Quanti-Tray /2000 and then sealed with the Quanti-Tray Sealer. Marine water samples
should generally be diluted 1→10 with sterile water. In order to minimize air bubbles within wells, samples can
be prepared in pre-sterilized bottles containing antifoam B. Alternatively, antifoam B can be added to each
bottle using a dropper bottle. The use of either form of antifoam is optional. Alternatively, the water sample in
4)
which the Colilert -18 has been dissolved can be distributed into sterile tubes for determination of the MPN
using a more traditional MPN format (e.g. 1  50 ml and 5  10 ml). If a single 100 ml volume is incubated,
then the method can be used as a presence/absence test for the detection of coliform bacteria and E. coli. If
either of these latter two approaches are used, then the tubes should be pre-warmed to (36  2) °C for 20 min
prior to the start of incubation.
While it is recommended that marine water samples be diluted 1→10 in sterile deionized water prior to
examination, it has been noted that in some geographical areas that the salt concentration of marine water is
sufficiently low to allow culture without dilution. If this procedure is to be used, then validation data should be
available. The salinity of marine water varies considerably and it is the responsibility of the laboratory to
determine if marine water samples require dilution.
8.3 Incubation and differentiation
4)
Incubate the inoculated Quanti-Trays for 18 h to 22 h at (36  2) °C for coliform bacteria and E. coli.
8.4 Examination of results
4) 4)
Examine the Quanti-Tray or Quanti-Tray /2000 after incubation for 18 h to 22 h and regard as positive
reactions for coliform bacteria those wells that have a yellow colouration equal to or greater than the
colouration of the Quanti-Tray comparator. Examine the trays under UV light (365 nm) in a dark room or in a
chamber that obscures ambient light. Regard any yellow wells that also exhibit any degree of fluorescence, as
positive for E. coli. If results are equivocal after 18 h (i.e. the yellow colouration is less than that of the
comparator), incubation should be extended up to 22 h. Positive results for both coliform bacteria and E. coli
observed before 18 h of incubation as well as negative results observed after 22 h are also valid.
9 Expression of results
4)
From the number of wells on a Quanti-Tray that are positive, the MPN/100 ml for both coliform bacteria and
E. coli can be calculated by reference to statistical tables or by using a computer MPN generator program, see
Tables B.1 and B.2.
10 Test report
This test report shall contain at least the following information:
a) the test method used, together with a reference to this part of ISO 9308;
b) all information required for the complete identification of the sample;
c) the results expressed in accordance with Clause 9;
d) any particular occurrence(s) observed during the course of the analysis and any operation(s) not
specified in this part of ISO 9308 which may have influenced the results.
11 Quality assurance
The laboratory shall have a clearly defined quality control system to ensure that the apparatus, reagents and
techniques are suitable for the test. The use of positive controls, negative controls and blanks is part of the
test.
4 © ISO 2012 – All rights reserved

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(E)
Annex A
(informative)

Further microbiological information on coliform bacteria
In addition to expressing -D-galactosidase, coliform bacteria are typically Gram-negative non-sporeforming,
oxidase-negative, rod-shaped bacteria, which are capable of aerobic and facultatively anaerobic growth in the
presence of bile-salts (or other surface-active agents with similar growth-inhibiting properties), and which are
usually able to ferment lactose with the production of acid and aldehyde within 48 h when incubated at a
temperature of (36 ± 2) °C. In addition to expressing -D-glucuronidase, E. coli are coliform bacteria that are
able to produce indole from tryptophan within (21  3) h at (44,0 ± 0,5) °C. They give a positive result in the
methyl red test and can decarboxylate l-glutamic acid but are not able to produce acetyl methyl carbinol,
utilise citrate as the sole source of carbon or grow in KCN broth.
Some strains of Escherichia coli which are -D-glucuronidase negative, such as Escherichia coli O157, will not
be detected as E. coli. As they are -D-galactosidase positive, they will appear as coliform bacteria.
© ISO 2012 – All rights reserved 5

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(E)
Annex B
(normative)

5)
The Quanti-Tray Sealer and calculation of results
B.1 General
5)
The Quanti-Tray Sealer is a thermal sealing unit that forms a seal between wells in the Quanti-Tray. The
sealer automatically distributes liquid into the wells of the Quanti-Tray or Quanti-Tray/2000. The Quanti-Tray is
used when anticipated counts are below 200 cfu/100 ml. The Quanti-Tray/2000 can be used to calculate MPN
values up to 2419 cfu/100 ml. When calculating MPN, the tables supplied with the trays are the reference for
all counts. A simple statistical program can also be used to calculate results. If required, the MPN can be
calculated manually according to the procedures given below.
B.2 Calculation of the most probable number
5
B.2.1 Calculation of MPN for IDEXX Quanti-Tray and Quanti-Tray/2000
B.2.1.1 Quanti-Tray (51-well)
Quanti-Tray MPN was originally developed at Yale University; an additional, good example of this serial
dilution MPN can be found at the U.S. Food and Drug Association in the Bacteriological Analytical Manual
(available on BAM Appendix 2: Most Probable Number from Serial Dilutions, October 2010).
Each sample well has an approximate volume of 1,96 ml.
The overflow well will hold a minimum of 8,5 ml.
For the calculation of the Quanti-Tray MPN (Table B.1), see Equation (B.1).
N = N · ln [N/(N  X)] (B.1)
MPN
where
N is the MPN;
MPN
N is the total number of wells (tubes) used in a test;
X is the number of positive wells (tubes) observed in a test.
5)
B.2.1.2 Quanti-Tray /2000 (97-well)
Quanti-Tray/2000 MPN was originally derived as described by Reference [1].
Small wells have a mean volume of 0,186 ml.
Large wells have a mean volume of approximately 1,86 ml (ten times larger than the small wells).

5
Quanti-Tray is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States
and/or other countries. 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.
6 © ISO 2012 – All rights reserved

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SIST EN ISO 9308-2:2014
ISO 9308-2:2012(E)
Overflow well will hold approximately 11 ml.
6)
For the calculation of the Quanti-Tray /2000 MPN (Table B.2), see Equation (B.2):
KK
Vd P
ii i
 Vd n (B.2)

Vd N ii i
ii mpn
1e
ii11
where
d is the dilution factor at level i (e.g. 0,1 for 1→10 dilution);
i
K is the number of dilution levels;
n is the number of wells at level i;
i
N is the MPN;
mpn
P is the number of positive wells at level i;
i
V is the volume of the wells at level i.
i

The 95 % confidence intervals can be found at :
T  (ln N  1,96)  (ln N )
0
mpn mpn
T  (ln N  1,96)  (ln N )
1
mpn mpn
where
T is the lower confidence interval;
0
T is the upper confidence interval;
1
  is the standard error; and
22 2
K
Vd n
2 ii i
 lnNN (B.3)
 
mpn mpn Vd N
ii mpn
e1
i1


6) Quanti-Tray is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States
and/or other countries
...