Water quality -- Detection and enumeration of Pseudomonas aeruginosa

This document specifies a method for the enumeration of Pseudomonas aeruginosa 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 document is applicable to a range of types of water. For example, hospital waters, drinking water and non‑carbonated bottled waters intended for human consumption, groundwater, swimming pool and spa pool waters including those containing high background counts of heterotrophic bacteria. This document does not apply to carbonated bottled waters, flavoured bottle waters, cooling tower waters or marine waters, for which the method has not been validated. These waters are, therefore, outside the scope of this document. Laboratories can employ the method presented in this document for these matrices by undertaking appropriate validation of performance of this method prior to use. The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa through the hydrolysis of a 7‑amino‑4‑methylcoumarin aminopeptidase substrate present in a special reagent. P. aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins and other nutrients present in the reagent. Actively growing strains of P. aeruginosa have an enzyme that cleaves the 7‑amido‑coumarin aminopeptidase substrate releasing a product which fluoresces under ultraviolet (UV) light. The test described in this document provides a confirmed result within 24 h with no requirement for further confirmation of positive wells.

Qualité de l'eau -- Recherche et dénombrement de Pseudomonas aeruginosa

Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa - 2. del: Metoda najverjetnejšega števila

Ta dokument določa metodo za ugotavljanje števila Pseudomonas aeruginosa v vodi. Metoda temelji na rasti ciljnih organizmov v tekočem mediju in izračunu najverjetnejšega števila (MPN) organizmov v skladu s preglednicami MPN.
Ta dokument se uporablja za različne vrste vod, na primer vodo v bolnišnicah, pitno vodo in ustekleničeno vodo brez dodanega ogljikovega dioksida, ki je namenjena za pitje, podtalnico, vodo v bazenih in toplicah, vključno s tistimi, ki vsebujejo veliko število heterotrofnih bakterij v ozadju. Ta dokument se ne uporablja za ustekleničeno vodo z dodanim ogljikovim dioksidom, ustekleničeno vodo z okusom, vodo v hladilnem stolpu ali morsko vodo, za katere metoda ni bila potrjena. Slednje zato ne spadajo na področje uporabe tega dokumenta. Laboratoriji lahko metodo, predstavljeno v tem dokumentu, uporabijo za te matrice, če pred uporabo ustrezno potrdijo njeno učinkovitost. Preskus temelji na tehnologiji zaznavanja bakterijskih encimov, ki signalizira prisotnost P. aeruginosa s hidrolizo substrata 7-amino-4-metilkumarin aminopeptidaze, ki je prisoten v posebnem reagentu. Celice P. aeruginosa hitro rastejo in se razmnožujejo z uporabo bogate zaloge aminokislin, vitaminov in drugih hranilnih snovi, ki so prisotne v reagentu. Aktivno rastoči sevi P. aeruginosa imajo encim, ki cepi substrat 7-amido-kumarinske aminopeptidaze, pri čemer se sprosti produkt, ki fluorescira pod ultravijolično (UV) svetlobo. Preskus, opisan v tem dokumentu, poda potrjen rezultat v 24 urah brez potrebe po nadaljnjem potrjevanju pozitivnih izvorov.

General Information

Status
Published
Publication Date
18-Jul-2018
Current Stage
6060 - International Standard published
Start Date
02-Jun-2018
Completion Date
19-Jul-2018

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INTERNATIONAL ISO
STANDARD 16266-2
First edition
2018-07
Water quality — Detection and
enumeration of Pseudomonas
aeruginosa —
Part 2:
Most probable number method
Qualité de l'eau — Recherche et dénombrement de Pseudomonas
aeruginosa —
Partie 2: Méthode du nombre le plus probable
Reference number
ISO 16266-2:2018(E)
ISO 2018
---------------------- Page: 1 ----------------------
ISO 16266-2:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 16266-2:2018(E)
Contents Page

Foreword ........................................................................................................................................................................... v

Introduction ................................................................................................................................................................... vi

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

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

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

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

5  Apparatus and gla sswa r e .............................................................................................................................. 2

6  Culture media, diluents and reagents ...................................................................................................... 3

6.1  Basic materials ................................................................................................................................................. 3

6.2  Diluent ................................................................................................................................................................. 3

6.3  Antifoam B .......................................................................................................................................................... 3

7  Sampling ............................................................................................................................................................. 4

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

8.1  Transport and storage of the samples ..................................................................................................... 4

8.2  Preparation of the sample and inoculation of media ......................................................................... 4

8.2.1  Preparation of 100 ml samples .................................................................................................................. 4

8.2.2  Preparation of 250 ml samples .................................................................................................................. 4

8.3  Incubation and differentiation ................................................................................................................... 4

8.4  Examination of results ................................................................................................................................... 5

9  Expression of results ...................................................................................................................................... 5

10  Quality assurance ............................................................................................................................................ 5

11  Test report ......................................................................................................................................................... 5

Annex A (informative) Further microbiological information about Pseudomonas

aeruginosa .......................................................................................................................................................... 7

Annex B (normative) The Quanti-Tray Sealer and calculation of results ................................................ 8

Annex C (normative) Composition of the Pseudalert medium ............................................................... 120

Annex D (informative) Performance characteristics ................................................................................. 121

Bibliography .............................................................................................................................................................. 122

© ISO 2018 – All rights reserved
iii
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ISO 16266-2:2018(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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see

www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbibiological methods.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www.iso.org/members.html.
© ISO 2018 – All rights reserved
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ISO 16266-2:2018(E)
Introduction

Pseudomonas aeruginosa is an opportunistic pathogen of man that is capable of growth in water at very

low nutrient concentrations. At source and during marketing, a natural mineral water or a spring water

is to be free from Pseudomonas aeruginosa in any 250 ml sample examined (see, for example, Council

Directive 2009/54/EC, Reference [1]). Other bottled waters offered for sale are also to be free of

Pseudomonas aeruginosa in any 250 ml sample (see e.g. Council Directive 98/83/EC, Reference [2]).

Other waters, including swimming and spa pool waters, water for human consumption and hospital

waters, may sometimes be tested for Pseudomonas aeruginosa for reasons of public health. In these

cases, it is typical to examine 100 ml volumes.

The method described in this document can be applied to a range of types of water, for example,

hospital waters, drinking water and non‐carbonated bottled waters intended for human consumption,

groundwater, swimming pool and spa pool waters including those containing high background counts

of heterotrophic bacteria (see References [3], [4], [5], [6] and [7]).
© ISO 2018 – All rights reserved
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INTERNATIONAL STANDARD ISO 16266‐2:2018(E)
Water quality — Detection and enumeration of Pseudomonas
aeruginosa — Part 2: Most probable number method

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

are carried out by suitably qualified staff.
1 Scope

This document specifies a method for the enumeration of Pseudomonas aeruginosa 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 document is applicable to a range of types of water. For example, hospital waters, drinking water

and non‐carbonated bottled waters intended for human consumption, groundwater, swimming pool

and spa pool waters including those containing high background counts of heterotrophic bacteria.

This document does not apply to carbonated bottled waters, flavoured bottle waters, cooling tower

waters or marine waters, for which the method has not been validated. These waters are, therefore,

outside the scope of this document. Laboratories can employ the method presented in this document

for these matrices by undertaking appropriate validation of performance of this method prior to use.

The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa

through the hydrolysis of a 7‐amino‐4‐methylcoumarin aminopeptidase substrate present in a special

reagent. P. aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins

and other nutrients present in the reagent. Actively growing strains of P. aeruginosa have an enzyme

that cleaves the 7‐amido‐coumarin aminopeptidase substrate releasing a product which fluoresces

under ultraviolet (UV) light. The test described in this document provides a confirmed result within

24 h with no requirement for further confirmation of positive wells.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. 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 11133, Microbiology of food, animal feeding stuffs, food production, environment and water —

Preparation, production, storage and performance testing of culture media
ISO 19458, Water quality — Sampling for microbiological analysis
© ISO 2018 – All rights reserved
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ISO 16266-2:2018(E)
ISO/IEC Guide 2, Standardization and related activities — General vocabulary
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/IEC Guide 2 and the following

apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
Pseudomonas aeruginosa

species of microorganism that is capable of growing in a selective broth and capable of hydrolyzing a

diagnostic 7‐amino‐4‐methylcoumarin aminopeptidase substrate present in the reagent

Note 1 to entry: See Annex A for further information on P. aeruginosa.
4 Principle

A snap pack of dehydrated medium is added to a sample of water (100 ml or 250 ml), or to 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. When enumeration is required, the sample plus medium (100 ml) is

1) 1)

then aseptically poured into either a Quanti‐Tray or Quanti‐Tray/2000 to enumerate up to 201

organisms or 2 419 organisms respectively per 100 ml sample. The procedure for the enumeration of

1) 1)

250 ml samples is described in 8.2. Trays are sealed with a Quanti‐Tray Sealer. Quanti‐Trays or

vessels (for presence/absence tests) are then incubated at (38 ± 0,5) °C for 24 h to 28 h. Results are

confirmed at 24 h but may be read up to 28 h.

After incubation, vessels or Quanti‐Tray sample wells that exhibit any degree of blue fluorescence

under long wavelength ultraviolet light (365 nm) are considered positive for P. aeruginosa.

By means of statistical tables, or a simple computer program, the MPN of P. aeruginosa in 100 ml or

250 ml of the sample can be determined.
This method is also suitable as a qualitative procedure.
5 Apparatus and glassware

Usual microbiological laboratory equipment, and, in particular, the following equipment.

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 document and does

not constitute an endorsement by ISO of this product.
© ISO 2018 – All rights reserved
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ISO 16266-2:2018(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 (38 ± 0,5) °C.
5.4 Quanti-Tray sealer and rubber insert.
5.5 Sterile wide mouthed vessels of at least 110 ml.
5.6 Ultraviolet lamp, 365 nm, 6 watt, long wavelength.
1) 1)
5.7 Quanti-Tray or Quanti-Tray/2000 , in accordance with Annex B.
6 Culture media, diluents and reagents
6.1 Basic materials

The method utilises Pseudalert , a medium available as a ready‐to‐use powder dispensed in snap

packs. Each snap pack contains sufficient medium (2,45 g for 100 ml samples) for a single test. For

quantitative enumeration of 250 ml samples, one snap pack of 2,45 g is added to each aliquot of divided

sample as described in 8.2. The Pseudalert reagent should be tan in colour and free flowing. The

medium contains nutrients such as amino acids and vitamins, buffer, sodium chloride, magnesium

sulfate, growth indicators, antibiotics and nitrogen sources. The medium is stored under ambient

conditions (2 °C to 30 °C) out of direct sunlight and should be used before the expiry date listed on the

snap pack. The reagent has a shelf‐life of 12 months from the date of manufacture.

The composition of the Pseudalert medium shall be in accordance with Annex C. Performance

characteristics for this method are provided in Annex D.
6.2 Diluent

For dilutions to be used with Pseudalert , use only sterile, non‐inhibitory, oxidant‐free water. The use

of buffered, saline or peptone‐containing diluents interferes with the performance of the test.

6.3 Antifoam B

Antifoam B used as a 1 % active, water soluble suspension of silicone. This reagent is added to samples

in order to minimize the formation of air bubbles during mixing.
NOTE Vessels already containing antifoam are available.

Pseudalert 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 document and does

not constitute an endorsement by ISO of this product.
© ISO 2018 – All rights reserved
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ISO 16266-2:2018(E)
7 Sampling

Carry out the collection, preservation and handling of samples as specified in ISO 19458.

8 Procedure
8.1 Transport and storage of the samples

Samples should be transported and stored in accordance with ISO 19458. Analysis should be

commenced on the day of collection or within 12 h. Under exceptional circumstances, the samples may

be kept at (5 ± 3) °C for up to 24 h prior to examination. In this case, the storage time shall be reported

in the test report.
8.2 Preparation of the sample and inoculation of media
8.2.1 Preparation of 100 ml samples

For enumeration of 100 ml samples, aseptically add a single snap pack of Pseudalert medium to each

100 ml volume of sample or dilution of sample in a sterile, transparent vessel and mix well. When the

medium is completely dissolved, the sample plus medium is aseptically poured into either a Quanti‐

1) 1) 1)

or Quanti‐Tray/2000 and then sealed with the Quanti‐Tray Sealer. In order to minimize air

Tray

bubbles within wells, samples can be prepared in pre‐sterilized vessels containing antifoam B.

Alternatively, antifoam B can be added to each vessel using a dropper bottle. An alternative MPN

method is where the water sample in which the Pseudalert has been dissolved is distributed into

sterile tubes for determination of the MPN using a more traditional MPN format (e.g. 1 × 50 ml and

5 × 10 ml).

NOTE The presence of high mineral content (especially magnesium and/or calcium) can cause the

Pseudalert reagent mixture to become cloudy but this does not affect the outcome of the test.

8.2.2 Preparation of 250 ml samples

For enumeration of 250 ml samples, divide the sample into three sterile, transparent vessels with two

samples having aliquots of 100 ml and one sample having an aliquot of 50 ml. Make the 50 ml sample up

to 100 ml by the addition of 50 ml of sterile, non‐inhibitory, oxidant‐free water. Aseptically add a single

snap pack of Pseudalert medium to each of the three 100 ml volumes of sample and mix well. When

the medium is completely dissolved, the three 100 ml volumes of sample plus medium are each

1) 1)

aseptically poured into three separate Quanti‐Tray or Quanti‐Tray/2000 and then sealed with the

Quanti‐Tray Sealer. In order to minimize air bubbles within wells, samples can be prepared in pre‐

sterilized vessels containing antifoam B. Alternatively, antifoam B can be added to each vessel using a

dropper bottle. Appropriate labelling of the three Quanti‐Tray where the tray containing the 50 ml

portion of the sample (i.e. the diluted portion) is clearly distinguishable from the two trays containing

the 100 ml undiluted portions of the sample is essential. This is important for the correct calculation of

the final count.
8.3 Incubation and differentiation

Incubate the inoculated Quanti‐Tray for 24 h to 28 h at (38 ± 0,5) °C for P. aeruginosa.

© ISO 2018 – All rights reserved
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ISO 16266-2:2018(E)
8.4 Examination of results
1) 1)

Examine the Quanti‐Tray or Quanti‐Tray/2000 after incubation under UV irradiation (365 nm) in a

dark room or in a chamber that obscures ambient light. Ensure that the UV light is facing away from

your eyes and toward the sample. The efficacy of the UV lamp should be checked regularly using a

fluorescence positive control in accordance with Clause 10. The lamp should also be replaced according

to the manufacturer’s stated lamp life (e.g. 6 000 h) or annually, whichever is sooner. Regard and count

any wells that exhibit any degree of blue fluorescence as positive for P. aeruginosa. For interpretation

purposes compare with a negative control. If there is doubt about the fluorescence for a well at 24 h,

return the tray to the incubator for further incubation without exceeding a maximum incubation time of

1) 1)

28 h. Placing the Quanti‐Tray or Quanti‐Tray/2000 rubber insert over the sample can facilitate

identification of fluorescing wells.
9 Expression of results
1) 1)

From the number of wells on a Quanti‐Tray or Quanti‐Tray/2000 that are positive, the MPN/100 ml

for P. aeruginosa can be calculated by reference to statistical tables or by using a computer MPN

generator program, see Tables B.1 and B.2. For enumeration from 250 ml samples the MPN is calculated

containing undiluted portions of the sample as
using the sum of the counts from the two Quanti‐Tray

one count and the count from the Quanti‐Tray containing the diluted portion of sample as the second

count, see Table B.3.
10 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.

For the definition of productivity and selectivity refer to ISO 11133. The performance of Pseudalert

shall be tested according to the methods and criteria described in ISO 11133 (see Table 1).

Table 1 — Performance testing of Pseudalert
Reference Method of Characteristic
Function Incubation Control strain Criteria
medium control reactions

Productivity 24 h to 28 h/ P. aeruginosa TSA Quantitative PR ≥ 0,5 Blue fluorescence

(38 ± 0,5) °C WDCM 00024 or
WDCM 00025
Selectivity 24 h to 28 h/ P. fluorescens — Qualitative Total No blue
(38 ± 0,5) °C WDCM 00115 inhibition fluorescence

Refer to the reference strain catalogue available on http://www.wfcc.info/pdf/WDCM_Reference_Strain_Catalogue.pdf

on culture collection strain numbers and contact details.
11 Test report
The test report shall contain at least the following information:

a) the test method used, together with a reference to this document, i.e. ISO 16266‐2:2018;

b) all information required for the complete identification of the sample;
© ISO 2018 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 16266-2:2018(E)
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 document that may have influenced the results.

If, under exceptional circumstances, the sample was kept at (5 ± 3) °C for up to 24 h prior to

examination, the storage time shall be reported in the test report.
© ISO 2018 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 16266-2:2018(E)
Annex A
(informative)
Further microbiological information about Pseudomonas aeruginosa

Pseudomonas aeruginosa is the type species of the genus Pseudomonas which is the type genus of the

family Pseudomonadaceae of the order Pseudomonadales.

It is a Gram negative, non‐spore forming rod which is oxidase and catalase positive. It exhibits oxidative

metabolism as indicated by the Hugh and Leifson test, generally reduces nitrate beyond the stage of

nitrite and produces ammonia from the breakdown of acetamide. Most strains (98 %) produce a water‐

soluble fluorescing pigment. The majority of strains are able to grow at 42 °C but not at 4 °C which

differentiates P. aeruginosa from P. fluorescens which grows at 4 °C but not at 42 °C.

Gelatin is liquefied, casein is hydrolysed, but starch is not hydrolysed. The pigment pyocyanin (blue‐

green) is produced by more than 90 % of strains of P. aeruginosa.
© ISO 2018 – All rights reserved
---------------------- Page: 12 ----------------------
ISO 16266-2:2018(E)
Annex B
(normative)
The Quanti-Tray Sealer and calculation of results
B.1 General
1) 1)

The Quanti‐Tray Sealer is a thermal sealing unit that forms a seal between wells in the Quanti‐Tray .

1) 1)

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 MPN/100 ml. The Quanti‐

Tray/2000 can be used to calculate MPN values up to 2 419 MPN/100 ml. When calculating MPN the

tables supplied with the trays and sealers 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 (MPN)
B.2.1 Calculation of MPN for IDEXX Quanti-Tray (51-well)

Quanti‐Tray MPN and the MPN for this dilution series can be found at the US Food and Drug

Association in the Bacteriological Analytical Manual, see Reference [8].
Each sample well has approximate volume of 1,96 ml.

The overflow well will hold approximately 8,5 ml and should be counted together with the other wells.

For the calculation of the Quanti‐Tray MPN (Table B.1), see Formula (B.1):
NNlnN/NX
MPN
(B.1)
where
N is the most probable number (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.
© ISO 2018 – All rights reserved
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ISO 16266-2:2018(E)
B.2.2 Calculation of MPN for IDEXX Quanti-Tray/2000 (97-well)
Quanti‐Tray/2000 MPN was originally derived as described by Reference [9].
Small wells have a mean volume of 0,16 ml.
Large wells have a mean volume of approximately 1,9 ml.

Overflow well will hold approximately 11 ml and should be counted as part of the large well count.

For the calculation of the Quanti‐Tray/2000 MPN (Table B.2), see Formula (B.2):
Vd P
ii i
 Vd n
ii i
Vd N
ii MPN
1exp
ii11
(B.2)
where
d is the dilution factor at level i (e.g. 0,1 for 1→10 dilution);
K is the number of dilution levels;
n is the number of wells at level i;
N is the MPN;
MPN
P is the number of positive wells at level i;
V is the volume of the wells at level i.
B.2.3 Calculation of confidence limits

Confidence limits for the MPNs calculated as indicated in B.2.1.1 and B.2.1.3 [Formulae (B.1) and (B.2)]

can be obtained using the approaches of either Reference [10] or ISO 29201 (Reference [11]). In Tables

B.1, B.2 and B.3 for all MPN values the 95 % confidence limits are indicated.
B.2.4 Calculation of the MPN and confidence limits for 250 ml samples

Calculating MPN for 250 ml samples will require the use of three Quanti‐Tray . Two trays are to be

filled with 100 ml of sample each and the remaining tray is to be filled with 50 ml of sample and 50 ml

sterile water for a 1 in 2 dilution. The number of positive wells would be summed for the two trays with

no dilution.
Each sample well has approximate volume of 1,96 ml.

Each overflow well will hold approximately 8,5 ml and should be counted together with the other wells.

Two trays will be undiluted and one tray will have a dilution of 1 in 2.

For the calculation of the MPN for 250 ml samples using three Quanti‐Tray ; see Formula (B.2).

With two dilutions, Formula (B.2) expands to Formula (B.3):
vd p vd p
121 222
vd nv d n
111 222
1exp vd N 1expvd N
 
11 MPN 22 MPN
(B.3)
© ISO 2018 – All rights reserved
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ISO 16266-2:2018(E)

For the 250 ml test with three Quanti‐Tray with two undiluted (d = 1) and one in dilution level in 1 in

2 (d = 0,50) then the formula for bacteria per ml of sample becomes Formula (B.4):

1,961pp1,960,5
1exp 1,961NN1exp 1,960,5

MPN MPN
1,961 1021,960,551
   
(B.4)
Confidence limits for the MPNs calculated as:
Upper limitexp lnNZSE
 
MPN log
Lower limit exp lnNZ SE
 
MPN logδ
where Z = the z‐score of the probability desired.
The standard error of the log of the density function is shown by Formula (B.5):
vd n
ii i
SE N
logδ 
MPN
exp vd N 1
i1 ii MPN
(B.5)
For 95 % confidence limits, the formula expands to Formula (B.6):
 2
vd n
ii i
95 % Upper limit = exp lnNN1,96
MPN 
MPN
exp vd N 1
ii MPN
i1
(B.6)
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ISO 16266-2:2018(E)

For the 250 ml test with three Quanti‐Trays where dilution level d is undiluted (= 1) and dilution level

d is 1 in 2 (= 0,50) the confidence limits formula for bacteria per ml of sample expands to

Formula (B.7):
95 % Upper limit =
 
 22 
 2
1,961 102 1,960,551
 
 
exp lnNN1,96  
 
MPN MPN
 

exp1,961NN1 exp1,960,5 1
  
MPN MPN
  
 
 
 
95 % Lower limit =
 
 22 
 2
1,961 102 1,960,551
 
 
  
exp lnNN1,96
MPN MPN
 

exp1,96
...

SLOVENSKI STANDARD
SIST ISO 16266-2:2019
01-maj-2019
Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa - 2.
del: Metoda najverjetnejšega števila

Water quality - Detection and enumeration of Pseudomonas aeruginosa - Part 2: Most

probable number method

Qualité de l'eau - Recherche et dénombrement de Pseudomonas aeruginosa - Partie 2:

Méthode du nombre le plus probable
Ta slovenski standard je istoveten z: ISO 16266-2:2018
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 16266-2:2019 en

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

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SIST ISO 16266-2:2019
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SIST ISO 16266-2:2019
INTERNATIONAL ISO
STANDARD 16266-2
First edition
2018-07
Water quality — Detection and
enumeration of Pseudomonas
aeruginosa —
Part 2:
Most probable number method
Qualité de l'eau — Recherche et dénombrement de Pseudomonas
aeruginosa —
Partie 2: Méthode du nombre le plus probable
Reference number
ISO 16266-2:2018(E)
ISO 2018
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
Contents Page

Foreword ........................................................................................................................................................................... v

Introduction ................................................................................................................................................................... vi

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

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

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

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

5  Apparatus and gla sswa r e .............................................................................................................................. 2

6  Culture media, diluents and reagents ...................................................................................................... 3

6.1  Basic materials ................................................................................................................................................. 3

6.2  Diluent ................................................................................................................................................................. 3

6.3  Antifoam B .......................................................................................................................................................... 3

7  Sampling ............................................................................................................................................................. 4

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

8.1  Transport and storage of the samples ..................................................................................................... 4

8.2  Preparation of the sample and inoculation of media ......................................................................... 4

8.2.1  Preparation of 100 ml samples .................................................................................................................. 4

8.2.2  Preparation of 250 ml samples .................................................................................................................. 4

8.3  Incubation and differentiation ................................................................................................................... 4

8.4  Examination of results ................................................................................................................................... 5

9  Expression of results ...................................................................................................................................... 5

10  Quality assurance ............................................................................................................................................ 5

11  Test report ......................................................................................................................................................... 5

Annex A (informative) Further microbiological information about Pseudomonas

aeruginosa .......................................................................................................................................................... 7

Annex B (normative) The Quanti-Tray Sealer and calculation of results ................................................ 8

Annex C (normative) Composition of the Pseudalert medium ............................................................... 120

Annex D (informative) Performance characteristics ................................................................................. 121

Bibliography .............................................................................................................................................................. 122

© ISO 2018 – All rights reserved
iii
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SIST ISO 16266-2:2019
ISO 16266-2:2018(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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see

www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,

Microbibiological methods.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www.iso.org/members.html.
© ISO 2018 – All rights reserved
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
Introduction

Pseudomonas aeruginosa is an opportunistic pathogen of man that is capable of growth in water at very

low nutrient concentrations. At source and during marketing, a natural mineral water or a spring water

is to be free from Pseudomonas aeruginosa in any 250 ml sample examined (see, for example, Council

Directive 2009/54/EC, Reference [1]). Other bottled waters offered for sale are also to be free of

Pseudomonas aeruginosa in any 250 ml sample (see e.g. Council Directive 98/83/EC, Reference [2]).

Other waters, including swimming and spa pool waters, water for human consumption and hospital

waters, may sometimes be tested for Pseudomonas aeruginosa for reasons of public health. In these

cases, it is typical to examine 100 ml volumes.

The method described in this document can be applied to a range of types of water, for example,

hospital waters, drinking water and non‐carbonated bottled waters intended for human consumption,

groundwater, swimming pool and spa pool waters including those containing high background counts

of heterotrophic bacteria (see References [3], [4], [5], [6] and [7]).
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SIST ISO 16266-2:2019
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SIST ISO 16266-2:2019
INTERNATIONAL STANDARD ISO 16266‐2:2018(E)
Water quality — Detection and enumeration of Pseudomonas
aeruginosa — Part 2: Most probable number method

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

are carried out by suitably qualified staff.
1 Scope

This document specifies a method for the enumeration of Pseudomonas aeruginosa 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 document is applicable to a range of types of water. For example, hospital waters, drinking water

and non‐carbonated bottled waters intended for human consumption, groundwater, swimming pool

and spa pool waters including those containing high background counts of heterotrophic bacteria.

This document does not apply to carbonated bottled waters, flavoured bottle waters, cooling tower

waters or marine waters, for which the method has not been validated. These waters are, therefore,

outside the scope of this document. Laboratories can employ the method presented in this document

for these matrices by undertaking appropriate validation of performance of this method prior to use.

The test is based on a bacterial enzyme detection technology that signals the presence of P. aeruginosa

through the hydrolysis of a 7‐amino‐4‐methylcoumarin aminopeptidase substrate present in a special

reagent. P. aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins

and other nutrients present in the reagent. Actively growing strains of P. aeruginosa have an enzyme

that cleaves the 7‐amido‐coumarin aminopeptidase substrate releasing a product which fluoresces

under ultraviolet (UV) light. The test described in this document provides a confirmed result within

24 h with no requirement for further confirmation of positive wells.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. 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 11133, Microbiology of food, animal feeding stuffs, food production, environment and water —

Preparation, production, storage and performance testing of culture media
ISO 19458, Water quality — Sampling for microbiological analysis
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
ISO/IEC Guide 2, Standardization and related activities — General vocabulary
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/IEC Guide 2 and the following

apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
Pseudomonas aeruginosa

species of microorganism that is capable of growing in a selective broth and capable of hydrolyzing a

diagnostic 7‐amino‐4‐methylcoumarin aminopeptidase substrate present in the reagent

Note 1 to entry: See Annex A for further information on P. aeruginosa.
4 Principle

A snap pack of dehydrated medium is added to a sample of water (100 ml or 250 ml), or to 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. When enumeration is required, the sample plus medium (100 ml) is

1) 1)

then aseptically poured into either a Quanti‐Tray or Quanti‐Tray/2000 to enumerate up to 201

organisms or 2 419 organisms respectively per 100 ml sample. The procedure for the enumeration of

1) 1)

250 ml samples is described in 8.2. Trays are sealed with a Quanti‐Tray Sealer. Quanti‐Trays or

vessels (for presence/absence tests) are then incubated at (38 ± 0,5) °C for 24 h to 28 h. Results are

confirmed at 24 h but may be read up to 28 h.

After incubation, vessels or Quanti‐Tray sample wells that exhibit any degree of blue fluorescence

under long wavelength ultraviolet light (365 nm) are considered positive for P. aeruginosa.

By means of statistical tables, or a simple computer program, the MPN of P. aeruginosa in 100 ml or

250 ml of the sample can be determined.
This method is also suitable as a qualitative procedure.
5 Apparatus and glassware

Usual microbiological laboratory equipment, and, in particular, the following equipment.

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 document and does

not constitute an endorsement by ISO of this product.
© ISO 2018 – All rights reserved
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SIST ISO 16266-2:2019
ISO 16266-2:2018(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 (38 ± 0,5) °C.
5.4 Quanti-Tray sealer and rubber insert.
5.5 Sterile wide mouthed vessels of at least 110 ml.
5.6 Ultraviolet lamp, 365 nm, 6 watt, long wavelength.
1) 1)
5.7 Quanti-Tray or Quanti-Tray/2000 , in accordance with Annex B.
6 Culture media, diluents and reagents
6.1 Basic materials

The method utilises Pseudalert , a medium available as a ready‐to‐use powder dispensed in snap

packs. Each snap pack contains sufficient medium (2,45 g for 100 ml samples) for a single test. For

quantitative enumeration of 250 ml samples, one snap pack of 2,45 g is added to each aliquot of divided

sample as described in 8.2. The Pseudalert reagent should be tan in colour and free flowing. The

medium contains nutrients such as amino acids and vitamins, buffer, sodium chloride, magnesium

sulfate, growth indicators, antibiotics and nitrogen sources. The medium is stored under ambient

conditions (2 °C to 30 °C) out of direct sunlight and should be used before the expiry date listed on the

snap pack. The reagent has a shelf‐life of 12 months from the date of manufacture.

The composition of the Pseudalert medium shall be in accordance with Annex C. Performance

characteristics for this method are provided in Annex D.
6.2 Diluent

For dilutions to be used with Pseudalert , use only sterile, non‐inhibitory, oxidant‐free water. The use

of buffered, saline or peptone‐containing diluents interferes with the performance of the test.

6.3 Antifoam B

Antifoam B used as a 1 % active, water soluble suspension of silicone. This reagent is added to samples

in order to minimize the formation of air bubbles during mixing.
NOTE Vessels already containing antifoam are available.

Pseudalert 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 document and does

not constitute an endorsement by ISO of this product.
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ISO 16266-2:2018(E)
7 Sampling

Carry out the collection, preservation and handling of samples as specified in ISO 19458.

8 Procedure
8.1 Transport and storage of the samples

Samples should be transported and stored in accordance with ISO 19458. Analysis should be

commenced on the day of collection or within 12 h. Under exceptional circumstances, the samples may

be kept at (5 ± 3) °C for up to 24 h prior to examination. In this case, the storage time shall be reported

in the test report.
8.2 Preparation of the sample and inoculation of media
8.2.1 Preparation of 100 ml samples

For enumeration of 100 ml samples, aseptically add a single snap pack of Pseudalert medium to each

100 ml volume of sample or dilution of sample in a sterile, transparent vessel and mix well. When the

medium is completely dissolved, the sample plus medium is aseptically poured into either a Quanti‐

1) 1) 1)

or Quanti‐Tray/2000 and then sealed with the Quanti‐Tray Sealer. In order to minimize air

Tray

bubbles within wells, samples can be prepared in pre‐sterilized vessels containing antifoam B.

Alternatively, antifoam B can be added to each vessel using a dropper bottle. An alternative MPN

method is where the water sample in which the Pseudalert has been dissolved is distributed into

sterile tubes for determination of the MPN using a more traditional MPN format (e.g. 1 × 50 ml and

5 × 10 ml).

NOTE The presence of high mineral content (especially magnesium and/or calcium) can cause the

Pseudalert reagent mixture to become cloudy but this does not affect the outcome of the test.

8.2.2 Preparation of 250 ml samples

For enumeration of 250 ml samples, divide the sample into three sterile, transparent vessels with two

samples having aliquots of 100 ml and one sample having an aliquot of 50 ml. Make the 50 ml sample up

to 100 ml by the addition of 50 ml of sterile, non‐inhibitory, oxidant‐free water. Aseptically add a single

snap pack of Pseudalert medium to each of the three 100 ml volumes of sample and mix well. When

the medium is completely dissolved, the three 100 ml volumes of sample plus medium are each

1) 1)

aseptically poured into three separate Quanti‐Tray or Quanti‐Tray/2000 and then sealed with the

Quanti‐Tray Sealer. In order to minimize air bubbles within wells, samples can be prepared in pre‐

sterilized vessels containing antifoam B. Alternatively, antifoam B can be added to each vessel using a

dropper bottle. Appropriate labelling of the three Quanti‐Tray where the tray containing the 50 ml

portion of the sample (i.e. the diluted portion) is clearly distinguishable from the two trays containing

the 100 ml undiluted portions of the sample is essential. This is important for the correct calculation of

the final count.
8.3 Incubation and differentiation

Incubate the inoculated Quanti‐Tray for 24 h to 28 h at (38 ± 0,5) °C for P. aeruginosa.

© ISO 2018 – All rights reserved
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ISO 16266-2:2018(E)
8.4 Examination of results
1) 1)

Examine the Quanti‐Tray or Quanti‐Tray/2000 after incubation under UV irradiation (365 nm) in a

dark room or in a chamber that obscures ambient light. Ensure that the UV light is facing away from

your eyes and toward the sample. The efficacy of the UV lamp should be checked regularly using a

fluorescence positive control in accordance with Clause 10. The lamp should also be replaced according

to the manufacturer’s stated lamp life (e.g. 6 000 h) or annually, whichever is sooner. Regard and count

any wells that exhibit any degree of blue fluorescence as positive for P. aeruginosa. For interpretation

purposes compare with a negative control. If there is doubt about the fluorescence for a well at 24 h,

return the tray to the incubator for further incubation without exceeding a maximum incubation time of

1) 1)

28 h. Placing the Quanti‐Tray or Quanti‐Tray/2000 rubber insert over the sample can facilitate

identification of fluorescing wells.
9 Expression of results
1) 1)

From the number of wells on a Quanti‐Tray or Quanti‐Tray/2000 that are positive, the MPN/100 ml

for P. aeruginosa can be calculated by reference to statistical tables or by using a computer MPN

generator program, see Tables B.1 and B.2. For enumeration from 250 ml samples the MPN is calculated

containing undiluted portions of the sample as
using the sum of the counts from the two Quanti‐Tray

one count and the count from the Quanti‐Tray containing the diluted portion of sample as the second

count, see Table B.3.
10 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.

For the definition of productivity and selectivity refer to ISO 11133. The performance of Pseudalert

shall be tested according to the methods and criteria described in ISO 11133 (see Table 1).

Table 1 — Performance testing of Pseudalert
Reference Method of Characteristic
Function Incubation Control strain Criteria
medium control reactions

Productivity 24 h to 28 h/ P. aeruginosa TSA Quantitative PR ≥ 0,5 Blue fluorescence

(38 ± 0,5) °C WDCM 00024 or
WDCM 00025
Selectivity 24 h to 28 h/ P. fluorescens — Qualitative Total No blue
(38 ± 0,5) °C WDCM 00115 inhibition fluorescence

Refer to the reference strain catalogue available on http://www.wfcc.info/pdf/WDCM_Reference_Strain_Catalogue.pdf

on culture collection strain numbers and contact details.
11 Test report
The test report shall contain at least the following information:

a) the test method used, together with a reference to this document, i.e. ISO 16266‐2:2018;

b) all information required for the complete identification of the sample;
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
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 document that may have influenced the results.

If, under exceptional circumstances, the sample was kept at (5 ± 3) °C for up to 24 h prior to

examination, the storage time shall be reported in the test report.
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
Annex A
(informative)
Further microbiological information about Pseudomonas aeruginosa

Pseudomonas aeruginosa is the type species of the genus Pseudomonas which is the type genus of the

family Pseudomonadaceae of the order Pseudomonadales.

It is a Gram negative, non‐spore forming rod which is oxidase and catalase positive. It exhibits oxidative

metabolism as indicated by the Hugh and Leifson test, generally reduces nitrate beyond the stage of

nitrite and produces ammonia from the breakdown of acetamide. Most strains (98 %) produce a water‐

soluble fluorescing pigment. The majority of strains are able to grow at 42 °C but not at 4 °C which

differentiates P. aeruginosa from P. fluorescens which grows at 4 °C but not at 42 °C.

Gelatin is liquefied, casein is hydrolysed, but starch is not hydrolysed. The pigment pyocyanin (blue‐

green) is produced by more than 90 % of strains of P. aeruginosa.
© ISO 2018 – All rights reserved
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
Annex B
(normative)
The Quanti-Tray Sealer and calculation of results
B.1 General
1) 1)

The Quanti‐Tray Sealer is a thermal sealing unit that forms a seal between wells in the Quanti‐Tray .

1) 1)

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 MPN/100 ml. The Quanti‐

Tray/2000 can be used to calculate MPN values up to 2 419 MPN/100 ml. When calculating MPN the

tables supplied with the trays and sealers 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 (MPN)
B.2.1 Calculation of MPN for IDEXX Quanti-Tray (51-well)

Quanti‐Tray MPN and the MPN for this dilution series can be found at the US Food and Drug

Association in the Bacteriological Analytical Manual, see Reference [8].
Each sample well has approximate volume of 1,96 ml.

The overflow well will hold approximately 8,5 ml and should be counted together with the other wells.

For the calculation of the Quanti‐Tray MPN (Table B.1), see Formula (B.1):
NNlnN/NX
MPN
(B.1)
where
N is the most probable number (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.
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)
B.2.2 Calculation of MPN for IDEXX Quanti-Tray/2000 (97-well)
Quanti‐Tray/2000 MPN was originally derived as described by Reference [9].
Small wells have a mean volume of 0,16 ml.
Large wells have a mean volume of approximately 1,9 ml.

Overflow well will hold approximately 11 ml and should be counted as part of the large well count.

For the calculation of the Quanti‐Tray/2000 MPN (Table B.2), see Formula (B.2):
Vd P
ii i
 Vd n
ii i
Vd N
ii MPN
1exp
ii11
(B.2)
where
d is the dilution factor at level i (e.g. 0,1 for 1→10 dilution);
K is the number of dilution levels;
n is the number of wells at level i;
N is the MPN;
MPN
P is the number of positive wells at level i;
V is the volume of the wells at level i.
B.2.3 Calculation of confidence limits

Confidence limits for the MPNs calculated as indicated in B.2.1.1 and B.2.1.3 [Formulae (B.1) and (B.2)]

can be obtained using the approaches of either Reference [10] or ISO 29201 (Reference [11]). In Tables

B.1, B.2 and B.3 for all MPN values the 95 % confidence limits are indicated.
B.2.4 Calculation of the MPN and confidence limits for 250 ml samples

Calculating MPN for 250 ml samples will require the use of three Quanti‐Tray . Two trays are to be

filled with 100 ml of sample each and the remaining tray is to be filled with 50 ml of sample and 50 ml

sterile water for a 1 in 2 dilution. The number of positive wells would be summed for the two trays with

no dilution.
Each sample well has approximate volume of 1,96 ml.

Each overflow well will hold approximately 8,5 ml and should be counted together with the other wells.

Two trays will be undiluted and one tray will have a dilution of 1 in 2.

For the calculation of the MPN for 250 ml samples using three Quanti‐Tray ; see Formula (B.2).

With two dilutions, Formula (B.2) expands to Formula (B.3):
vd p vd p
121 222
vd nv d n
111 222
1exp vd N 1expvd N
 
11 MPN 22 MPN
(B.3)
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SIST ISO 16266-2:2019
ISO 16266-2:2018(E)

For the 250 ml test with three Quanti‐Tray with two undiluted (d = 1) and one in dilution level in 1 in

2 (d = 0,50) then the formula for bacteria per ml of sample becomes Formula (B.4):

1,961pp1,960,5
1exp 1,961NN1exp 1,960,5

MPN MPN
1,9
...

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