Iso 9308 2 2012

ISO
INTERNATIONAL
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
iTeh STANDARD PREVIEWorganismes coliformes

—
Partie 2: Méthode du nombre le plus probable
(standards.iteh.ai )
ISO 9308-2:2012
https://standards.iteh.ai/catalog/standards/sist/a05b6929-1d9c-478b-
86fe3548911709b6/iso-9308-2-2012
ISO 9308-2:2012(E)
iTeh STANDARD PREVIEW

(standards.iteh.ai)
ISO 9308-2:2012
86fe3548911709b6/iso-9308-2-2012
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 [email protected]
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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
© ISO 2012 – All rights reserved

10 Test report ..............................................................................................................................................
4
11 Quality assurance ..................................................................................................................................
4iTeh STANDARD PREVIEW

Annex A (informative) Further microbiological information on coliform bacteria .......................................
5(standards.iteh.ai)
5)
Annex B (normative) The Quanti-Tray Sealer and calculation of results ..................................................
6
ISO 9308-2:2012
Annex C (informative) https://standards.iteh.ai/catalog/standards/sist/a05b6929-1d9c-478b-86fe- Composition of
the Colilert-18 medium ................................................................... 42
Annex D (informative) Validation of Colilert-18/Quanti-Tray3548911709b6/iso-9308-2-20128) for the
enumeration of E.coli and coliform bacteria from
water .............................................................................................................. 44
Bibliography ......................................................................................................................................................
46
iii
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.iTeh STANDARD PREVIEW
ISO 9308 consists of the following parts, under the general title Water quality — Enumeration of Escherichia coli
and coliform bacteria: (standards.iteh.ai)

 Part 1: Membrane filtration method for waters with low bacterial background flora
ISO 9308-2:2012
86fe-
 Part 2: Most probable number method
3548911709b6/iso-9308-2-2012
 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

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: iTeh STANDARD PREVIEW

IDEXX Laboratories, Inc.
One IDEXX Drive

(standards.iteh.ai)
Westbrook, Maine 04092 USA
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
ISO 9308-2:2012
rights other than those identified above. ISO shall not be held responsible for identifying any
or all such patent https://standards.iteh.ai/catalog/standards/sist/a05b6929-1d9c-478b-86fe-
rights. 3548911709b6/iso-9308-2-2012
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.
v

(standards.iteh.ai)
ISO 9308-2:2012
86fe3548911709b6/iso-9308-2-2012

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
(standards.iteh.ai)
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
ISO 9308-2:2012
heterotrophic bacteria. However, it must not be used for the enumeration of coliform bacteria in marine water.
https://standards.iteh.ai/catalog/standards/sist/a05b6929-1d9c-478b-86fe-
When using for the enumeration of E. coli in marine waters, a 1→10 dilution in sterile water is typically
3548911709b6/iso-9308-2-2012
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.
© ISO 2012 – All rights reserved 1

ISO 9308-2:2012(E)
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 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
medium. The sample plus medium is then aseptically poured into a Quanti-Tray 1) or Quanti-Tray/20001) to
enumerate up to 201 organisms or 2 419 organisms per 100 ml, respectively. Trays are sealed with a Quanti-
Tray1) 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 iTeh
STANDARD PREVIEW comparator wells are considered positive for coliform
bacteria. Yellow wells that also exhibit any degree of fluorescence are considered positive for E.
coli(standards.iteh.ai. )
By means of statistical tables, or a simple computer program, the most probable number (MPN) of coliform
ISO 9308-2:2012
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
3548911709b6/iso-9308-2-2012 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:
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

ISO 9308-2:2012(E)
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.
5.4 Quanti-Tray1) sealer.
5.5 Sterile wide mouthed vessels of at least 110 ml.

5.6 Quanti-Tray2) comparator.
5.7 Ultraviolet lamp, 365 nm.
5.8 Quanti-Tray2) or Quanti-Tray/20002), see Annex B.
6 Culture media and reagents
6.1 Basic materials
The method utilises Colilert3)-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
For dilutions to be used with Colilert3)-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 iTeh STANDARD PREVIEW

Antifoam B is a 10 % active, water soluble suspension of silicone. (standards.iteh.ai)
ISO 9308-2:2012
7 Sampling
3548911709b6/iso-9308-2-2012
Take the samples and deliver them to the laboratory in accordance with ISO 19458.
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.

ISO 9308-2:2012(E)
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
Aseptically add a single snap pack of Colilert 4)-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
Quanti-Tray4) or Quanti-Tray4)/2000 and then sealed with the Quanti-Tray4) 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
which the Colilert4)-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
Incubate the inoculated Quanti-Trays4) for 18 h to 22 h at (36  2) °C for coliform bacteria and E. coli.
8.4 Examination of results
Examine the Quanti-Tray4) or Quanti-Tray4)/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
iTeh STANDARD PREVIEW 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 (standards.iteh.ai) 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.
ISO 9308-2:2012
3548911709b6/iso-9308-2-2012
9 Expression of results
From the number of wells on a Quanti-Tray4) 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.
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 9308-2:2012(E)
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.
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.

(standards.iteh.ai)
ISO 9308-2:2012
86fe3548911709b6/iso-9308-2-2012
Annex B
(normative)

ISO 9308-2:2012(E)
The Quanti-Tray5) Sealer and calculation of results
B.1 General
The Quanti-Tray5) 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
B.2.1 Calculation of MPN for IDEXX Quanti-Tray5 and Quanti-Tray/2000
B.2.1.1 Quanti-Tray (51-well) iTeh STANDARD PREVIEW

Quanti-Tray MPN was originally developed at Yale University; an additional, good example of this serial
(standards.iteh.ai) 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).
ISO 9308-2:2012
https://standards.iteh.ai/catalog/standards/sist/a05b6929-1d9c-478b-86feEach sample well has an
approximate volume of 1,96 ml.
3548911709b6/iso-9308-2-
2012 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).
NMPN = N · ln [N/(N  X)] (B.1)
where
NMPN is the 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.
B.2.1.2 Quanti-Tray5)/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).
Overflow well will hold approximately 11 ml.

5 Quanti-Tray is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States

ISO 9308-2:2012(E)
For the calculation of the Quanti-Tray6) /2000 MPN (Table B.2), see Equation (B.2): K
VdP
 Vdn (B.2)
 i ii i i mpn  i i i
V d N
i1 1 e i1
where
di is the dilution factor at level i (e.g. 0,1 for 1→10 dilution);
K is the number of dilution levels;
ni is the number of wells at level i;
N is the MPN;
mpn
Pi is the number of positive wells at level i;
Vi is the volume of the wells at level i.
The 95 % confidence intervals can be found at : iTeh STANDARD

PREVIEW
T0  (ln Nmpn  1,96)  (ln Nmpn (standards.iteh.ai) )
T1  (ln Nmpn  1,96)  (ln Nmpn) ISO 9308-2:2012
where 3548911709b6/iso-9308-2-2012
T0 is the lower confidence interval;
T1 is the upper confidence interval;
 is the standard error; and

K 222
Vdn
ln Nmpn  Nmpn2 V d Nii i impni (B.3) i1 e 1
Table B.1 — 51-Well Quanti-Tray MPN

No. of wells giving positive Most probable number 95 % Confidence limits
reaction (MPN) per 100 ml sample
Lower Upper
0 < 1a 0,0 3,7

ISO 9308-2:2012(E)
1 1 0,3 5,6
2 2 0,6 7,3
3 3,1 1,1 9
4 4,2 1,7 10,7
5 5,3 2,3 12,3
6 6,4 3 13,9
7 7,5 3,7 15,5
8 8,7 4,5 17,1
9 9,9 5,3 18,8
10 11,1 6,1 20,5
11 12,4 7 22,1
12 23,9
VIEW7,
iTeh STANDARD P13,7 RE9
13 15 8,8 25,7
9,8 27,5
(standards.iteh 16,4 .ai)
14
15 17,8 10,8 29,4
ISO 9308-2:2012
16https://standards.iteh.ai/catalog/standards/sist/ 929-d9c-478b- 6fe- 31,3
a05b619,2 1 811,9
17 3548911709b620/iso,7 -9308-2-20 12 13 33,3
18 22,2 14,1 35,2
19 23,8 15,3 37,3
20 25,4 16,5 39,4
21 27,1 17,7 41,6
22 28,8 19 43,9
23 30,6 20,4 46,3
24 32,4 21,8 48,7
25 34,4 23,3 51,2
26 36,4 24,7 53,9
27 38,4 26,4 56,6
28 40,6 28 59,5
29 42,9 29,7 62,5
30 45,3 31,5 65,6
31 47,8 33,4 69

ISO 9308-2:2012(E)
32 50,4 35,4 72,5
33 53,1 37,5 76,2
34 56 39,7 80,1
Table B.1 (continued)

No. of wells giving positive reaction Most probable number 95 % Confidence limits
(MPN) per 100 ml
sample
Lower Upper
35 59,1 42 84,4
36 62,4 44,6 88,8
37 65,9 47,2 93,7
38 69,7 50 99
39 73,8 53,1 104,8
40 78,2 56,4 111,2
41 83,1 59,9 118,3
42 88,5 63,9 126,2
43 94,5 68,2 135,4
44 101,3 73,1 146
45 109,1 78,6 158,7
46 118,4 85 174,5
47 129,8 92,7 195
224,1
iTeh STAN DARD 48 IEW102,3
PREV144,5
115,2 272,2
49 (standards.iteh.ai165,
2 )
50 200,5 O
135,8 387,6
IS 9308-2:2012
https://standards.iteh.ai/catalog51/standards/sist/a05b6929-c-478b-86fe- infinite
1d9> 200,5 146,1
a
A value of < 1 means that there were no target bacteria detected in the test. For undiluted samples this can
3548911709b6/iso-9308-2-2012
be reported as zero. However, if dilutions of the sample have been made,
then the appropriate multiplication factor must be used. For example, if a 1→10 dilution has been made and no
bacteria are detected then the result should be reported as < 10.
Table B.2 — 97-Well Quanti Tray (Quanti-Tray/2000) MPN with 95 % confidence limits
Positive Positive MPN 95% Confidence Positive Positive MPN 95% Confidence
large wells small wells large wells small wells
Lower Upper Lower Upper limit
limit limit limit
0 0 a 0,0 3,7 25 0 33,6 22,0 48,9

<1

ISO 9308-2:2012(E)
0 1 1,0 0,0 3,7 25 1 35,0 22,9 51,2
0 2 2,0 0,3 5,6 25 2 36,4 23,8 52,6
0 3 3,0 0,6 7,3 25 3 37,9 25,5 54,0
0 4 4,0 1,1 8,9 25 4 39,3 26,5 55,9
0 5 5,0 1,7 10,5 25 5 40,8 28,3 57,3
0 6 6,0 2,3 12,1 25 6 42,2 29,3 59,0
0 7 7,0 2,9 13,7 25 7 43,7 30,3 60,7
0 8 8,0 3,7 15,3 25 8 45,2 31,3 62,5
0 9 9,0 4,5 15,8 25 9 46,7 33,3 64,2
0 10 10,0 5,2 16,9 25 10 48,2 34,4 66,0

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ISO

Water quality — Enumeration of

iTeh STANDARD PREVIEWorganismes coliformes

iTeh STANDARD PREVIEW

ii © ISO 2012 – All rights reserved

© ISO 2012 – All rights reserved

11 Quality assurance ..................................................................................................................................

4iTeh STANDARD PREVIEW

and coliform bacteria: (standards.iteh.ai)

iv © ISO 2012 – All rights reserved

© ISO 2012 – All rights reserved

be obtained from: iTeh STANDARD PREVIEW

One IDEXX Drive

iTeh STANDARD PREVIEW

© ISO 2012 – All rights reserved

Water quality — Enumeration of Escherichia coli and coliform

© ISO 2012 – All rights reserved 1

ISO/IEC Guide 2:2004, Standardization and related activities — General vocabulary

3 Terms and definitions

5 Apparatus and glassware

2 © ISO 2012 – All rights reserved

5.1 Apparatus for sterilization by steam (autoclave)

5.2 Hot air oven, for dry heat sterilization.

5.3 Incubator, thermostatically controlled at (36 ± 2) °C.

5.4 Quanti-Tray1) sealer.

5.5 Sterile wide mouthed vessels of at least 110 ml.

5.7 Ultraviolet lamp, 365 nm.

5.8 Quanti-Tray2) or Quanti-Tray/20002), see Annex B.

6 Culture media and reagents

6.1 Basic materials

6.3 Antifoam B iTeh STANDARD PREVIEW

© ISO 2012 – All rights reserved 3

8.1 Preparation of the sample

8.2 Inoculation of media

8.3 Incubation and differentiation

8.4 Examination of results

4 © ISO 2012 – All rights reserved

b) all information required for the complete identification of the sample;

c) the results expressed in accordance with Clause 9;

Further microbiological information on coliform bacteria

In addition to expressing -D-galactosidase, coliform bacteria are typically Gram-negative non-sporeforming,

iTeh STANDARD PREVIEW

© ISO 2012 – All rights reserved 5

The Quanti-Tray5) Sealer and calculation of results

B.2 Calculation of the most probable number

B.2.1 Calculation of MPN for IDEXX Quanti-Tray5 and Quanti-Tray/2000

B.2.1.1 Quanti-Tray (51-well) iTeh STANDARD PREVIEW

NMPN = N · ln [N/(N  X)] (B.1)

NMPN is the 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.

B.2.1.2 Quanti-Tray5)/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.

Overflow well will hold approximately 11 ml.

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di is the dilution factor at level i (e.g. 0,1 for 1→10 dilution);

K is the number of dilution levels;

ni is the number of wells at level i;