🧫 Pseudomonas aeruginosa in Water
In short
Detection and enumeration of Pseudomonas aeruginosa by membrane filtration on CN medium with cetrimide and nalidixic acid, intended for bottled water and other waters with a low number of background bacteria, such as pool water and water intended for human consumption. The test is performed according to PN-EN ISO 16266:2009; Membrane filter: sterile, nominal pore size 0.45 µm; checked according to ISO 7704 (4.1, 6.4). The procedure comprises 7 steps; it is used for: Bottled water — usually a 250 ml sample (clause 1, introduction), Pool water and water intended for human consumption — usually 100 ml (clause 1, introduction), Natural mineral waters and spring waters — exclusion in 250 ml at the source and in packaging (Journal of Laws 2011, No 85, item 466, § 4 and § 10).
At a glance
- Standard: PN-EN ISO 16266:2009
- Category: Microbiology
- Procedure steps: 7
- Membrane filter: sterile, nominal pore size 0.45 µm; checked according to ISO 7704 (4.1, 6.4)
- Medium: Pseudomonas agar base with CN supplement: cetrimide 0.2 g/l, nalidixic acid 0.015 g/l (5.1.1)
- Incubation: (36 ± 2) °C (6.2); (44 ± 4) h — according to the data sheet of a medium manufactured in accordance with the standard (clause 8 beyond the available text)
Overview
STATUS (as of 29.10.2026). PN-EN ISO 16266:2009 — Polish version, published on 09.11.2009, adopts EN ISO 16266:2008 [IDT] and ISO 16266:2006 [IDT]; it replaced the English version PN-EN ISO 16266:2008. The standard is current — no withdrawal note. ISO 16266:2006 is the equivalent of EN 12780:2002 (foreword of the standard) and has the status of a current standard in the catalogue.
SCOPE (clause 1). Isolation and enumeration of Pseudomonas aeruginosa in samples of bottled water by a membrane filtration technique. The method can also be applied to other waters with a low background flora, such as pool water and water intended for human consumption. Part 2 of this series (PN-EN ISO 16266-2) describes a different method — the most probable number method.
SIGNIFICANCE (introduction). P. aeruginosa is an opportunistic pathogen of humans that is capable of growth in water at very low nutrient concentrations. In bottled water a 250 ml sample is usually examined; other waters, including pool water and water intended for human consumption, are sometimes tested for public health reasons — then typically 100 ml.
DEFINITION (clause 3). P. aeruginosa are micro-organisms that grow on selective media containing cetrimide and produce pyocyanin, or that grow on such media, are oxidase positive, fluoresce under UV radiation of (360 ± 20) nm and are able to produce ammonia from acetamide.
REGULATIONS. Natural mineral waters and spring waters: the examination at the source is to exclude P. aeruginosa in 250 ml, and packaged water placed on the market must meet this requirement (Journal of Laws 2011, No 85, item 466, § 4(3) and (5), § 10). Swimming pools: 0 in 100 ml in water supplied to the basin and in water in the basin (Journal of Laws 2022, item 1230, Annex 1). Water intended for human consumption in tanks of land and water transport: 0 in 100 ml; in air transport: up to 10 in 100 ml at the tap furthest from the tank, after the flight — a result above 10 requires corrective action (Journal of Laws 2026, item 748, Annex 1 Part A Tables 2–3); the reference method is PN-EN ISO 16266 (Annex 4 Part A).
WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends at clause 7 (sampling). The procedure (clause 8), expression of results (clause 9), performance data (clause 11), interferences (clause 12), quality assurance (clause 13) and alternative media (Annex B) are beyond it. The incubation time of (44 ± 4) h is given according to the data sheet of a medium manufactured in accordance with the standard — the available text gives the incubator temperature of (36 ± 2) °C.
Method principle
A measured volume of the sample, or a dilution of it, is filtered through a 0.45 µm membrane filter, which is placed on the selective medium and incubated under the conditions specified for that medium (4.1). The number of presumptive P. aeruginosa is obtained by counting characteristic colonies on the filter: pyocyanin-producing colonies are regarded as confirmed, while other fluorescent or reddish brown colonies require confirmation (4.2). Colonies requiring confirmation are subcultured from the filter onto nutrient agar; after incubation, cultures that were not initially fluorescent are tested for the oxidase reaction, and oxidase-positive ones — for the production of fluorescein and of ammonia from acetamide; cultures that were initially fluorescent are tested only for the production of ammonia from acetamide (4.3).
Applications
- Bottled water — usually a 250 ml sample (clause 1, introduction)
- Pool water and water intended for human consumption — usually 100 ml (clause 1, introduction)
- Natural mineral waters and spring waters — exclusion in 250 ml at the source and in packaging (Journal of Laws 2011, No 85, item 466, § 4 and § 10)
- Swimming pools — 0 in 100 ml in water supplied to the basin and in the basin (Journal of Laws 2022, item 1230, Annex 1)
- Water intended for human consumption in tanks of means of transport — reference method (Journal of Laws 2026, item 748, Annex 1 Part A and Annex 4 Part A)
Key parameters
| Parameter | Value |
|---|---|
| Membrane filter | sterile, nominal pore size 0.45 µm; checked according to ISO 7704 (4.1, 6.4) |
| Medium | Pseudomonas agar base with CN supplement: cetrimide 0.2 g/l, nalidixic acid 0.015 g/l (5.1.1) |
| Incubation | (36 ± 2) °C (6.2); (44 ± 4) h — according to the data sheet of a medium manufactured in accordance with the standard (clause 8 beyond the available text) |
| UV lamp | radiation of wavelength (360 ± 20) nm (3.1, 6.3) |
| Sample volume | bottled water — usually 250 ml; pool water and water intended for human consumption — usually 100 ml (introduction) |
| Confirmation | pyocyanin — confirmed; fluorescent or reddish brown colonies — oxidase, fluorescein on King’s B medium, ammonia from acetamide (4.2–4.3) |
Standard
- Standard number
- PN-EN ISO 16266:2009
- Title (PL)
- Jakość wody — Wykrywanie i oznaczanie ilościowe Pseudomonas aeruginosa — Metoda filtracji membranowej
- Title (EN)
- Water quality — Detection and enumeration of Pseudomonas aeruginosa — Method by membrane filtration
Step-by-step procedure
-
Sampling
Collection, preservation and handling of samples — according to ISO 5667-1, ISO 5667-2, ISO 5667-3 and ISO 19458 (clause 7).
-
Filtration
Filter a measured volume of the sample or its dilution through a 0.45 µm membrane filter — bottled water usually 250 ml, other waters usually 100 ml (4.1, introduction).
-
Incubation on CN agar
Place the filter on CN medium and incubate at (36 ± 2) °C (4.1, 6.2) — for (44 ± 4) h according to the data sheet of a medium manufactured in accordance with the standard.
-
Counting colonies
Count characteristic colonies, examining the filter also under the UV lamp (360 ± 20) nm: pyocyanin-producing colonies are confirmed P. aeruginosa; other fluorescent or reddish brown colonies require confirmation (4.2, 6.3).
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Subculture
Subculture the colonies requiring confirmation from the filter onto nutrient agar plates (alternative media — Annex B) (4.3).
-
Confirmation
Test cultures that were not initially fluorescent for oxidase; oxidase-positive ones — for production of fluorescein (King’s B medium) and of ammonia from acetamide (acetamide broth, detection with Nessler reagent). Test initially fluorescent cultures only for production of ammonia from acetamide (3.1, 4.3, 5.2).
-
Result
Number of confirmed colonies in the volume filtered; calculation rules — clause 9 and ISO 8199, beyond the available text of the standard.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Glassware | sterilized before use — in a dry oven at (170 ± 5) °C for 1 h or in an autoclave at (121 ± 3) °C for 15 min (6.1) | — |
| Incubator | capable of being maintained at (36 ± 2) °C (6.2) | — |
| UV lamp | emitting radiation of wavelength (360 ± 20) nm (6.3) | — |
| Membrane filters | sterile, nominal pore size 0.45 µm; checked regularly according to ISO 7704 (6.4) | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Pseudomonas agar base (CN agar) | — | gelatin peptone 16.0 g, casein hydrolysate 10.0 g, anhydrous potassium sulfate 10.0 g, anhydrous magnesium chloride 1.4 g, glycerol 10 ml, agar 11.0–18.0 g, water 1000 ml; suspend, add the glycerol, bring to the boil, sterilize at (121 ± 3) °C for 15 min, cool to 45–50 °C, add the CN supplement rehydrated in 2 ml of sterile water, pour to a depth of at least 5 mm; pH 7.1 ± 0.2 at 25 °C; plates in the dark at (5 ± 3) °C for up to a month; do not keep the agar molten longer than 4 h or remelt it (5.1.1) |
| CN supplement — cetrimide (hexadecyltrimethylammonium bromide) | 57-09-0 | 0.2 g per 1000 ml of medium (5.1.1.1) |
| CN supplement — nalidixic acid | 389-08-2 | 0.015 g per 1000 ml of medium (5.1.1.1) |
| King’s B medium | — | peptone 20.0 g, glycerol 10 ml, dipotassium hydrogen phosphate 1.5 g, magnesium sulfate heptahydrate 1.5 g, agar 15.0 g, water 1000 ml; pH 7.2 ± 0.2 at 25 °C; 5 ml portions in tubes, sterilization (121 ± 3) °C, 15 min, slants; in the dark at (5 ± 3) °C for up to 3 months (5.2.1) |
| Acetamide broth | 60-35-5 | solution A: KH₂PO₄ 1.0 g, anhydrous MgSO₄ 0.2 g, acetamide 2.0 g, NaCl 0.2 g in 900 ml of ammonia-free water, pH 7.0 ± 0.5; solution B: sodium molybdate dihydrate 0.5 g and iron(II) sulfate heptahydrate 0.05 g in 100 ml of water; 1 ml of solution B to 900 ml of freshly prepared solution A, make up to 1 l, 5 ml portions in tubes, sterilization (121 ± 3) °C, 15 min; in the dark at (5 ± 3) °C for up to 3 months (5.2.2) |
| Nutrient agar | — | peptone 5.0 g, meat extract 1.0 g, yeast extract 2.0 g, NaCl 5.0 g, agar 15.0 g, water 1000 ml; sterilization (121 ± 3) °C, 15 min; pH 7.4 ± 0.2 at 25 °C; dry the plates before use, store in the dark at (5 ± 3) °C for up to a month (5.2.3) |
| Oxidase reagent | 637-01-4 | 0.1 g of tetramethyl-p-phenylenediamine dihydrochloride in 10 ml of water, dissolve immediately before use and protect from light — the reagent is not stable, prepare in small amounts; commercial oxidase tests are allowed (5.2.4) |
| Nessler reagent | 7487-94-7 | dissolve 10 g of mercury(II) chloride and 7 g of potassium iodide (CAS 7681-11-0) in a small quantity of water and add slowly, with stirring, to a cooled solution of 16 g of NaOH in 50 ml of water; dilute to 100 ml; store in rubber-stoppered borosilicate glassware, out of sunlight, for a maximum of one year (5.2.5) |
| Water | — | grade 3 according to ISO 3696 or of equivalent purity, free from growth-inhibiting substances; analytical-grade reagents or commercial media prepared according to the manufacturer’s instructions (clause 5) |
Health and safety (OHS)
- Acetamide is carcinogenic and irritant — take precautions when weighing, preparing and discarding the medium (5.2.2.1)
- Mercury(II) chloride in the Nessler reagent is toxic — avoid ingestion (5.2.5)
- Persons using the standard should be familiar with normal laboratory practice, and tests must be carried out by suitably trained staff; establishing safety and health practices is the responsibility of the user (warning of the standard)
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 16266:2009 — “Water quality — Detection and enumeration of Pseudomonas aeruginosa — Method by membrane filtration”.
How does this method work?
A measured volume of the sample, or a dilution of it, is filtered through a 0.45 µm membrane filter, which is placed on the selective medium and incubated under the conditions specified for that medium (4.
What is the measuring range and accuracy?
Membrane filter: sterile, nominal pore size 0.45 µm; checked according to ISO 7704 (4.1, 6.4); Medium: Pseudomonas agar base with CN supplement: cetrimide 0.2 g/l, nalidixic acid 0.015 g/l (5.1.1); Incubation: (36 ± 2) °C (6.2); (44 ± 4) h — according to the data sheet of a medium manufactured in accordance with the standard (clause 8 beyond the available text); UV lamp: radiation of wavelength (360 ± 20) nm (3.1, 6.3).
How long does the test take?
The procedure comprises 7 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Glassware, Incubator, UV lamp, Membrane filters.
Where is this test used?
Bottled water — usually a 250 ml sample (clause 1, introduction); Pool water and water intended for human consumption — usually 100 ml (clause 1, introduction); Natural mineral waters and spring waters — exclusion in 250 ml at the source and in packaging (Journal of Laws 2011, No 85, item 466, § 4 and § 10); Swimming pools — 0 in 100 ml in water supplied to the basin and in the basin (Journal of Laws 2022, item 1230, Annex 1); Water intended for human consumption in tanks of means of transport — reference method (Journal of Laws 2026, item 748, Annex 1 Part A and Annex 4 Part A).
What safety precautions apply?
Acetamide is carcinogenic and irritant — take precautions when weighing, preparing and discarding the medium (5.2.2.1); Mercury(II) chloride in the Nessler reagent is toxic — avoid ingestion (5.2.5); Persons using the standard should be familiar with normal laboratory practice, and tests must be carried out by suitably trained staff; establishing safety and health practices is the responsibility of the user (warning of the standard).
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 16266.