INTERNATIONAL ISO

STANDARD 16266

First edition
2006-04-15

Water quality — Detection and

enumeration of Pseudomonas
aeruginosa — Method by membrane
filtration
Qualité de l'eau — Recherche et dénombrement de Pseudomonas
aeruginosa — Méthode par filtration sur membrane
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ISO 16266:2006
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Reference number
ISO 16266:2006(E)

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ISO 16266:2006(E)

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Contents Page

Foreword............................................................................................................................................................ iv
Introduction ........................................................................................................................................................ v
1 Scope ..................................................................................................................................................... 1
2 Normative references ........................................................................................................................... 1
3 Terms and definitions........................................................................................................................... 2
4 Principle ................................................................................................................................................. 2
5 Diluents, culture media and reagents................................................................................................. 2
6 Apparatus and glassware .................................................................................................................... 5
7 Sampling ................................................................................................................................................ 5
8 Procedure .............................................................................................................................................. 6
9 Expression of results ........................................................................................................................... 7
10 Test report ............................................................................................................................................. 8
11 iTeh STANDARD PREVIEW
Performance data.................................................................................................................................. 8
12 (standards.iteh.ai)
Interferences ......................................................................................................................................... 9
13 Quality assurance ................................................................................................................................. 9
ISO 16266:2006
Annex A (informative) Further information about Pseudomonas aeruginosa........................................... 10
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Annex B (informative) Alternative media ....................................................................................................... 11
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Bibliography ..................................................................................................................................................... 12

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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.

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.

ISO 16266 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
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This International Standard is the equivalent of EN 12780:2002.
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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, e.g. Council Directive 80/777/EEC[1] and
Council Directive 96/70/EC[2]). 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[3]). Other waters, including pool waters
and water for human consumption, may sometimes be tested for Pseudomonas aeruginosa for reasons of public
health. In these cases, it is typical to examine 100 ml volumes.

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INTERNATIONAL STANDARD ISO 16266:2006(E)

Water quality — Detection and enumeration of Pseudomonas

aeruginosa — Method by membrane filtration

WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This 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 according to this International Standard
be carried out by suitably trained staff.

1 Scope
This International Standard specifies a method for the isolation and enumeration of Pseudomonas aeruginosa in
samples of bottled water by a membrane filtration technique. This method can also be applied to other types of
water with a low background flora, for example, pool waters and waters intended for human consumption.
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2 Normative references (standards.iteh.ai)
The following referenced documents are ISO indispensable
16266:2006 for the application of this document. For dated
references, only thehttps://standards.iteh.ai/catalog/standards/sist/8ec08368-c079-4ac4-a239-
edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
702d393827bc/iso-16266-2006
ISO 3696, Water for analytical laboratory use — Specification and test methods

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

ISO 5667-21), Water quality — Sampling — Part 2: Guidance on sampling techniques

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

ISO 6887-1, Microbiology of food and feeding stuffs — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions

ISO 7704, Water quality — Evaluation of membrane filters used for microbiological analyses

ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture

ISO 194582), Water quality — Sampling for microbiological analysis

1) ISO 5667-1 and ISO 5667-2 are currently undergoing joint revision, which will be published as ISO 5667-1.
2) To be published.

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ISO 16266:2006(E)

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1
Pseudomonas aeruginosa
micro-organisms that grow on selective media containing cetrimide and produce pyocyanin, or
micro-organisms that grow on selective media containing cetrimide, are oxidase positive, fluoresce under UV
radiation (360 ± 20) nm, and are able to produce ammonia from acetamide

4 Principle

4.1 Filtration

A measured volume of the water sample, or a dilution of the sample, is filtered through a membrane filter of
0,45 µm. The membrane filter is placed on the selective medium and incubated under the conditions specified
for the medium.

4.2 Enumeration

The numbers of presumptive Pseudomonas aeruginosa are obtained by counting the number of characteristic
colonies on the membrane filter after incubation. Pyocyanin-producing colonies are considered as confirmed
Pseudomonas aeruginosa but other fluorescing or reddish brown colonies require confirmation.
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4.3 Confirmation
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Subcultures of colonies requiring confirmation are made from the membrane filter onto plates of nutrient agar
(but see Annex B). After incubation, cultures that ISO were 16266:2006
not initially fluorescent are tested for the oxidase
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reaction, and oxidase-positive cultures are tested for the production of fluorescein and the ability to produce
ammonia from acetamide. Cultures that were702d393827bc/iso-16266-2006
fluorescent initially are tested for the ability to produce ammonia
from acetamide.

5 Diluents, culture media and reagents

Use reagents of analytical reagent quality in the preparation of culture media and diluents, unless otherwise
specified. Prepare the medium as follows and add the selective agents as supplements at the given
concentrations or use commercially available media and reagents prepared according to the manufacturer’s
instructions. Prepare media and reagents using water grade 3 as specified in ISO 3696, or water of equivalent
purity and free from substances which might inhibit growth under the conditions of the test.

5.1 Culture medium

Use the following medium for the determination of Pseudomonas aeruginosa.

5.1.1 Pseudomonas agar base/CN-agar

5.1.1.1 Composition

Gelatin peptone 16,0 g

Casein hydrolysate 10,0 g

Potassium sulfate (anhydrous) (K2SO4) 10,0 g

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Magnesium chloride (anhydrous) (MgCl2) 1,4 g

Glycerol 10 ml

Agar 11,0 g to 18,0 g

Water (distilled or equivalent) 1 000 ml

NOTE The amount of agar required depends on the gel strength. Follow the manufacturer’s instructions for the agar
used.

CN supplement

Hexadecyltrimethyl ammonium bromide (cetrimide) 0,2 g

Nalidixic acid 0,015 g

5.1.1.2 Preparation

Suspend the peptone, casein hydrolysate, potassium sulfate, magnesium chloride and agar in 1 000 ml of
distilled water (or equivalent). Add 10 ml of glycerol. Heat to boiling in order to dissolve completely and
sterilize by autoclaving at (121 ± 3) °C for 15 min. Allow the medium to cool to (45 to 50) °C. Add the CN
supplement rehydrated in 2 ml of sterile distilled water, mix well and add to the sterile molten basal medium.
Mix well and pour into sterile Petri dishes to give a depth of at least 5 mm of agar. The final pH of the solidified
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medium should correspond to 7,1 ± 0,2 at 25 °C. Store prepared plates in the dark protected from desiccation
at (5 ± 3) °C and use within 1 month. Do not keep the agar molten for more than 4 h. Do not remelt the
medium. (standards.iteh.ai)
5.2 Confirmatory media and reagents ISO 16266:2006
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5.2.1 King's B medium 702d393827bc/iso-16266-2006
5.2.1.1 Composition

Peptone 20,0 g

Glycerol 10 ml

Di-potassium hydrogen phosphate (K2HPO4) 1,5 g

Magnesium sulfate heptahydrate (MgSO4·7H2O) 1,5 g

Agar 15,0 g

Water (distilled or equivalent) 1 000 ml

5.2.1.2 Preparation

Dissolve the ingredients in the water by heating. Cool down to (45 to 50) °C and adjust the pH corresponding
to 7,2 ± 0,2 at 25 °C, using either hydrochloric acid or sodium hydroxide. Dispense the medium in 5 ml
aliquots into culture tubes which are capped and autoclaved at (121 ± 3) °C for 15 min. Allow the tubes to cool
and solidify in slants.

Store in the dark at (5 ± 3) °C and use within 3 months.

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5.2.2 Acetamide broth

5.2.2.1 Composition

Solution A

Potassium di-hydrogenphosphate (KH2PO4) 1,0 g

Magnesium sulfate (anhydrous) (MgSO4) 0,2 g

Acetamide 2,0 g

Sodium chloride (NaCl) 0,2 g

Water (distilled or equivalent, ammonia free) 900 ml

Dissolve the ingredients in water and then adjust the pH to correspond to 7,0 ± 0,5 at 25 °C with either
hydrochloric acid or sodium hydroxide.

CAUTION — Acetamide is carcinogenic and irritant — appropriate precautions shall be taken when
weighing out, preparing and discarding the medium.

Solution B

Sodium molybdate (Na2MoO4·2H2O) 0,5 g

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Iron sulfate heptahydrate (FeSO ·7H O) 0,05 g
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Water 100 ml
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To prepare the acetamide broth, add 1 ml of solution B to 900 ml of a freshly prepared solution A (5.2.2.1).
Add water with constant stirring to a total volume of 1 l. Dispense this mixture in 5 ml aliquots to culture tubes
which are then capped and sterilized in an autoclave at (121 ± 3) °C for 15 min. Store in the dark at (5 ± 3) °C
and use within 3 months.

5.2.3 Nutrient agar

5.2.3.1 Composition

Peptone 5,0 g

Meat extract 1,0 g

Yeast extract 2,0 g

Sodium chloride (NaCl) 5,0 g

Agar 15,0 g

Water 1 000 ml

5.2.3.2 Preparation

Dissolve the ingredients in the water by heating. Sterilize by autoclaving at (121 ± 3) °C for 15 min. The pH of
the solidified prepared medium should correspond to 7,4 ± 0,2 at 25 °C. Dry the plates to remove excess
surface moisture before use. Store prepared plates in the dark protected from desiccation at (5 ± 3) °C and
use within 1 month.

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5.2.4 Oxidase reagent

5.2.4.1 Composition

Tetramethyl-p-phenylenediamine dihydrochloride 0,1 g

Water 10 ml

5.2.4.2 Preparation

Dissolve the tetramethyl-p-phenylenediamine dihydrochloride in the water immediately before use and protect
from light. This reagent is not stable. Prepare in small amounts freshly before use.

Alternatively, use commercially available oxidase tests.

5.2.5 Nessler reagent

5.2.5.1 Composition

Mercuric chloride (HgCI2) 10 g

Potassium iodide (KI) 7g

Sodium hydroxide (NaOH) 16 g

Water (ammonia free) iTeh STANDARDto 100 PREVIEW

ml

Dissolve 10 g of HgCI2 and 7 g of (standards.iteh.ai)

KI in a small quantity of water and add this mixture slowly, with stirring, to a
cooled solution of 16 g of NaOH dissolved in 50 ml of water. Dilute to 100 ml. Store in rubber-stoppered
borosilicate glassware out of sunlight for a maximum of 1 year.
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ingestion.

6 Apparatus and glassware

Use usual microbiological laboratory equipment.

6.1 Glassware

Sterilize all glassware at (170 ± 5) °C for 1 h in a dry oven or at (121 ± 3) °C for 15 min in an autoclave before
use.

6.2 Incubator, capable of being maintained at (36 ± 2) °C.

6.3 Ultra violet lamp, capable of emitting radiation of wavelength (360 ± 20) nm.

6.4 Sterile membrane filters, with nominal pore size of 0,45 µm.

Check filters on a regular basis as specified in ISO 7704.

7 Sampling
Carry out the collection, preservation and handling of samples as specified in ISO 5667-1, ISO 5667-2,
ISO 5667-3 and ISO 19458.

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