🦠 Pseudomonas aeruginosa in water — the MPN method with an enzymatic medium (Pseudalert / Quanti-Tray)
In short
To 100 ml (or 250 ml divided into three portions) of water a packet of dehydrated Pseudalert medium (2. The test is performed according to PN-EN ISO 16266-2:2022-04 (ISO 16266-2:2018); MPN range: Quanti-Tray up to 201/100 ml (expected < 200); Quanti-Tray/2000 up to 2419/100 ml. The procedure comprises 7 steps; it is used for: Water in hospitals and health care facilities (P. aeruginosa as an opportunistic pathogen in the installations), Drinking water and still bottled waters — the requirement for the absence of P. aeruginosa in 250 ml, Swimming pool water and water from spa baths, groundwater — also with a high background of heterotrophic bacteria.
At a glance
- Standard: PN-EN ISO 16266-2:2022-04 (ISO 16266-2:2018)
- Category: Microbiology
- Procedure steps: 7
- Volume of the sample: 100 ml (or a dilution made up to 100 ml); bottled waters 250 ml in three trays (100 + 100 + 50 + 50 ml of water)
- Medium: Pseudalert, 2.45 g per 100 ml; storage at 2–30 °C out of direct sunlight, until the expiry date
- Incubation: (38 ± 0.5) °C, 24–28 h; the result confirmed after 24 h
Overview
ISO 16266-2:2018 specifies a method for the enumeration of Pseudomonas aeruginosa in water based on growth in a liquid medium and the calculation of the most probable number (MPN) from tables. It is used for many kinds of water: hospital water, drinking water, still bottled water, groundwater, swimming pool water and water from spa baths, including water with a high background of heterotrophic bacteria. It does not apply to carbonated and flavoured bottled waters, to water from cooling towers or to sea water, for which the method has not been validated — a laboratory may use it for those after its own validation. Bottled waters are usually examined in a volume of 250 ml, the others in 100 ml. The requirement for the absence of P. aeruginosa in 250 ml came from Directive 98/83/EC, REPEALED by Directive (EU) 2020/2184; in the Polish regulation of 22 May 2026 (Dz.U. 2026 poz. 748 [Journal of Laws 2026, item 748]) P. aeruginosa is listed in annex No 1 part A tables 2 and 3 — for water in tankers and in the tanks of means of transport, in a volume of 100 ml, and not for bottled water. The method is also suitable as a qualitative procedure (presence/absence).
The test is based on a bacterial enzyme detection technology: actively growing strains of P. aeruginosa possess an enzyme that cleaves the aminopeptidase substrate 7-amido-4-methylcoumarin present in the medium, releasing a product that fluoresces under UV; the bacteria grow quickly thanks to a rich set of amino acids, vitamins and nutrients. The result is confirmed within 24 h without any need to confirm the positive wells further. Most strains of P. aeruginosa grow at 42 °C but not at 4 °C, which distinguishes it from P. fluorescens. The standard refers to the commercial products Pseudalert and Quanti-Tray. In Poland it is in force as PN-EN ISO 16266-2:2022-04 (Polish and English versions). The manufacturer gives the same conditions for Pseudalert: incubation at (38 ± 0.5) °C for 24–28 h, reading of the fluorescence under UV, detection level 1 cfu/100 ml.
Method principle
A packet (snap pack) of the dehydrated medium — 2.45 g per 100 ml — is added to a 100 ml or 250 ml sample of water, or to a dilution made up to 100 ml, and it is shaken gently until it dissolves. For enumeration the 100 ml of sample with the medium is poured aseptically into a Quanti-Tray (up to 201 organisms/100 ml; expected numbers < 200 MPN/100 ml) or a Quanti-Tray/2000 (up to 2419 organisms/100 ml), sealed in a Quanti-Tray sealer and incubated at (38 ± 0.5) °C for 24–28 h; the result is confirmed after 24 h, but it may be read up to 28 h. For 250 ml the sample is divided among three sterile transparent vessels: two portions of 100 ml and one of 50 ml made up with 50 ml of sterile, non-inhibitory water free of oxidants; one packet of the medium is added to each and they are poured into three trays, which must be unambiguously marked (the tray with the 50 ml portion is counted differently). After the incubation the vessels or the wells showing any degree of blue fluorescence under long-wave UV (365 nm) in comparison with the negative control are regarded as positive; where there is doubt after 24 h the incubation is extended to 28 h. The MPN per 100 ml or 250 ml is read from the statistical tables (B.1, B.2) or from a program; for 250 ml formula B.2 for three trays is used. The wells of the Quanti-Tray hold about 1.96 ml and the overflow well about 8.5 ml; in the Quanti-Tray/2000 the small wells hold about 0.16 ml, the large ones about 1.9 ml and the overflow well about 11 ml (counted with the large ones). The medium may also be poured into sterile tubes in the classical MPN arrangement (e.g. 1 × 50 ml and 5 × 10 ml) or the whole sample may be incubated as a presence/absence test. Quality control: performance — P. aeruginosa WDCM 00024 (blue fluorescence, PR ≥ 0.5 relative to TSA), selectivity — P. fluorescens WDCM 00115 (complete inhibition, no fluorescence).
Applications
- Water in hospitals and health care facilities (P. aeruginosa as an opportunistic pathogen in the installations)
- Drinking water and still bottled waters — the requirement for the absence of P. aeruginosa in 250 ml
- Swimming pool water and water from spa baths, groundwater — also with a high background of heterotrophic bacteria
- The presence/absence test and the classical arrangement of MPN tubes with the same medium
- Laboratories of waterworks and of sanitary-epidemiological stations — the result confirmed after 24 h without confirmatory subcultures
Key parameters
| Parameter | Value |
|---|---|
| Volume of the sample | 100 ml (or a dilution made up to 100 ml); bottled waters 250 ml in three trays (100 + 100 + 50 + 50 ml of water) |
| Medium | Pseudalert, 2.45 g per 100 ml; storage at 2–30 °C out of direct sunlight, until the expiry date |
| Incubation | (38 ± 0.5) °C, 24–28 h; the result confirmed after 24 h |
| Reading | any blue fluorescence under UV at 365 nm relative to the negative control |
| MPN range | Quanti-Tray up to 201/100 ml (expected < 200); Quanti-Tray/2000 up to 2419/100 ml |
| Transport and storage | examination on the day of sampling or within 12 h; exceptionally at (5 ± 3) °C for up to 24 h (to be stated in the test report) |
Standard
- Standard number
- PN-EN ISO 16266-2:2022-04 (ISO 16266-2:2018)
- Title (PL)
- Jakość wody — Wykrywanie i oznaczanie ilościowe Pseudomonas aeruginosa — Część 2: Metoda najbardziej prawdopodobnej liczby
- Title (EN)
- Water quality — Detection and enumeration of Pseudomonas aeruginosa — Part 2: Most probable number method
Step-by-step procedure
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Sampling and transport
Take the sample according to the rules of ISO 19458; begin the examination on the day of sampling or within 12 h; exceptionally store it at (5 ± 3) °C for up to 24 h and record the storage time in the test report.
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Addition of the medium — 100 ml
To 100 ml of the sample (or of a dilution made up to 100 ml) in a sterile, transparent vessel add aseptically one packet of Pseudalert medium and mix until it has dissolved completely.
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Addition of the medium — 250 ml
Divide the sample among three vessels: 100 ml, 100 ml and 50 ml; make the 50 ml portion up with 50 ml of sterile, non-inhibitory water free of oxidants. Add one packet of the medium to each portion and mix; mark the trays (which one contains the 50 ml portion).
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Pouring and sealing
Pour each portion with the medium into a Quanti-Tray (expected < 200 MPN/100 ml) or a Quanti-Tray/2000 and seal it. Alternatively pour it into sterile tubes in the MPN arrangement (e.g. 1 × 50 ml and 5 × 10 ml) or incubate the whole as a presence/absence test.
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Incubation
Incubate the trays or the vessels for 24–28 h at (38 ± 0.5) °C.
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Reading
Examine them under UV at 365 nm in comparison with the negative control: every well showing any degree of blue fluorescence is positive for P. aeruginosa. Where the fluorescence is doubtful after 24 h, extend the incubation to 28 h; the rubber insert makes the reading easier.
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Calculation and test report
From the number of positive wells read the MPN/100 ml from tables B.1/B.2 or from a program; for 250 ml calculate the MPN from the three trays using formula B.2. In the test report give the method, the identification of the sample, the result and any storage at (5 ± 3) °C for up to 24 h.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Thermostatted incubator at (38 ± 0.5) °C | — | |
| Quanti-Tray sealer and Quanti-Tray trays (51 wells) / Quanti-Tray/2000 (97 wells) | the choice according to the expected number | — |
| Long-wave UV lamp 365 nm | replacement according to the declared service life (e.g. 6 000 h) or every year; reading with a negative control | — |
| Sterile transparent vessels with a wide opening of ≥ 110 ml | for 100 ml of sample with the medium | — |
| Sterile tubes (the MPN arrangement, e.g. 1 × 50 ml and 5 × 10 ml) | the classical variant without trays | — |
| Quanti-Tray / Quanti-Tray/2000 rubber insert | makes the reading of the wells easier | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Pseudalert — dehydrated medium in a single-use packet (2.45 g) | — | the aminopeptidase substrate 7-amido-4-methylcoumarin — fluorescence when P. aeruginosa grows |
| Sterile, non-inhibitory water free of oxidants | — | dilution and making up the 50 ml portion to 100 ml |
| Control strains P. aeruginosa WDCM 00024 and P. fluorescens WDCM 00115 | — | control of the performance and of the selectivity of the medium |
Health and safety (OHS)
- P. aeruginosa is an opportunistic pathogen — work under the conditions of a microbiological laboratory, autoclave the used trays
- UV lamp 365 nm — protect the eyes and the skin while reading
- The sealer becomes hot — do not touch the heating parts
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 16266-2:2022-04 (ISO 16266-2:2018) — “Water quality — Detection and enumeration of Pseudomonas aeruginosa — Part 2: Most probable number method”.
How does this method work?
A packet (snap pack) of the dehydrated medium — 2.45 g per 100 ml — is added to a 100 ml or 250 ml sample of water, or to a dilution made up to 100 ml, and it is shaken gently until it dissolves.
What is the measuring range and accuracy?
Volume of the sample: 100 ml (or a dilution made up to 100 ml); bottled waters 250 ml in three trays (100 + 100 + 50 + 50 ml of water); Medium: Pseudalert, 2.45 g per 100 ml; storage at 2–30 °C out of direct sunlight, until the expiry date; Incubation: (38 ± 0.5) °C, 24–28 h; the result confirmed after 24 h; Reading: any blue fluorescence under UV at 365 nm relative to the negative control.
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?
Thermostatted incubator at (38 ± 0.5) °C, Quanti-Tray sealer and Quanti-Tray trays (51 wells) / Quanti-Tray/2000 (97 wells), Long-wave UV lamp 365 nm, Sterile transparent vessels with a wide opening of ≥ 110 ml, Sterile tubes (the MPN arrangement, e.g. 1 × 50 ml and 5 × 10 ml), Quanti-Tray / Quanti-Tray/2000 rubber insert.
Where is this test used?
Water in hospitals and health care facilities (P. aeruginosa as an opportunistic pathogen in the installations); Drinking water and still bottled waters — the requirement for the absence of P. aeruginosa in 250 ml; Swimming pool water and water from spa baths, groundwater — also with a high background of heterotrophic bacteria; The presence/absence test and the classical arrangement of MPN tubes with the same medium; Laboratories of waterworks and of sanitary-epidemiological stations — the result confirmed after 24 h without confirmatory subcultures.
What safety precautions apply?
P. aeruginosa is an opportunistic pathogen — work under the conditions of a microbiological laboratory, autoclave the used trays; UV lamp 365 nm — protect the eyes and the skin while reading; The sealer becomes hot — do not touch the heating parts.
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 16266-2.