🧫 Pseudomonas aeruginosa in Water
Detection and quantitative determination of Pseudomonas aeruginosa in water by membrane filtration method on CN medium (cetrimide-nalidixic acid). Key pathogen in bottled and pool water.
Overview
Pseudomonas aeruginosa is a Gram-negative, aerobic rod, one of the most important opportunistic pathogens in aquatic environment. This bacterium can multiply in water with very low nutrient content, forms biofilms on surfaces of water installations, is resistant to many disinfectants and antibiotics. It can cause wound infections, external ear infections (swimmer's ear inflammation), urinary tract infections and serious hospital infections in immunosuppressed patients.
P. aeruginosa is not a typical indicator of fecal contamination — the bacterium originates mainly from the environment (soil, vegetation, biofilms in installations). Its presence in bottled or pool water indicates hygiene problems in the production, storage or distribution process. It is a mandatory parameter for bottled water, water in swimming pools and water in healthcare facilities.
The method according to PN-EN ISO 16266 uses membrane filtration and culture on selective medium with cetrimide (cetyltrimethylammonium bromide — cationic detergent inhibiting growth of most bacteria except Pseudomonas) and nalidixic acid (inhibitor of Gram-negative bacteria sensitive to quinolones). P. aeruginosa colonies produce characteristic pigments: pyocyanin (blue-green) or fluorescein (yellow-green fluorescent).
Confirmation includes ammonia production test from acetamide (Nessler's reagent) — positive for P. aeruginosa — and optionally growth at 42°C and oxidase test.
Method principle
The water sample is filtered through a 0.45 µm membrane and placed on CN medium (Pseudomonas CN agar) containing cetrimide (0.2 g/L) and nalidixic acid (15 mg/L). Incubation at 36±2°C for 40–48 h. Cetrimide inhibits most bacteria except Pseudomonas spp. — acts as a cationic detergent damaging cell membranes. P. aeruginosa produces soluble pigments: pyocyanin (blue-green, visible in daylight) and/or fluorescein (yellow-green, fluorescent in UV 365 nm). Colonies producing pyocyanin do not require confirmation. Fluorescent or brown-red colonies require acetamide test.
Applications
- Testing of bottled and spring water (mandatory parameter)
- Monitoring of water in swimming pools and water parks
- Control of water in hospitals and healthcare facilities
- Testing of drinking water in public buildings
- Assessment of water quality in cooling installations (cooling towers)
- Control of biofilms in water distribution systems
- Testing of water for dialysis and pharmaceutical purposes
Key parameters
| Parameter | Value |
|---|---|
| Result | cfu/250 mL (bottled water) or cfu/100 mL |
| Permissible value | 0 cfu/250 mL (bottled water) |
| Incubation temperature | 36 ± 2°C |
| Incubation time | 40–48 hours |
| UV lamp | 365 nm for fluorescence observation |
| Sample volume | 250 mL (bottled water), 100 mL (other) |
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
1. Workstation preparation
Sterilize filtration set. Prepare Pseudomonas CN Agar plates (remove from refrigerator 30 min before).
2. Membrane filtration
Filter 250 mL sample (bottled water) or 100 mL (other waters) through 0.45 µm filter. Rinse funnel with sterile peptone water.
3. Transfer to medium
With sterile forceps transfer filter to Pseudomonas CN Agar surface. Avoid air bubbles.
4. Incubation
Incubate inverted plates at 36±2°C for 40–48 hours. Preliminary assessment possible after 24 h.
5. Reading — pyocyanin production
Check colonies in daylight. Blue-green colonies (pyocyanin) = confirmed P. aeruginosa without further tests.
6. Reading — UV fluorescence
Examine plates under UV lamp 365 nm. Yellow-green fluorescent colonies but without pyocyanin → require confirmation.
7. Confirmatory test — acetamide
Transfer colonies requiring confirmation to tubes with acetamide medium. Incubate 36°C/24 h. Add Nessler's reagent — yellow coloration = NH₃ = P. aeruginosa (+).
8. Growth test at 42°C (optional)
Transfer colonies to nutrient agar and incubate at 42°C/24 h. Growth confirms P. aeruginosa (other Pseudomonas do not grow at 42°C).
9. Result calculation
Result = number of confirmed colonies / sample volume. Report in cfu/250 mL (bottled water) or cfu/100 mL.
10. Quality control
Positive control: P. aeruginosa ATCC 27853. Negative control: sterile water. Selectivity control: E. coli ATCC 25922 (no growth).
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Membrane filtration system | Sartorius Combisart, Merck EZ-Fit | 3,000–12,000 PLN |
| Vacuum pump | KNF Laboport N 810 | 3,000–8,000 PLN |
| Incubator 36°C | Binder BD 56, Memmert IN30 | 5,000–15,000 PLN |
| UV lamp 365 nm (Wood) | Vilber Lourmat VL-6.L, Analytik Jena UVhand | 500–3,000 PLN |
| Autoclave | Tuttnauer ELV 2540 | 20,000–45,000 PLN |
| Incubator 42°C (confirmation) | Binder BD 23 | 5,000–15,000 PLN |
| Laminar flow hood | Esco Airstream | 15,000–40,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Pseudomonas CN Agar | — | Selective medium with cetrimide and nalidixic acid + glycerol. Manufacturers: Oxoid, Merck, Biokar |
| Cetrimide (cetyltrimethylammonium bromide) | 57-09-0 | Cationic detergent, selective inhibitor, concentration 0.2 g/L in medium |
| Nalidixic acid | 389-08-2 | Inhibitor of sensitive Gram-negative bacteria, concentration 15 mg/L in medium |
| Nessler's reagent (K₂HgI₄) | 7783-33-7 | For detecting ammonia from acetamide — yellow/brown coloration = positive result. WARNING: contains mercury! |
| Acetamide (CH₃CONH₂) | 60-35-5 | Medium for confirmatory test — P. aeruginosa deaminates acetamide to ammonia |
| Membrane filters 0.45 µm | — | MCE, 47 mm, sterile. Merck Millipore, Sartorius |
| Sodium thiosulfate (Na₂S₂O₃) | 7772-98-7 | Neutralization of chlorine in samples |
Health and safety (OHS)
- P. aeruginosa — opportunistic pathogen, BSL-2. Gloves, lab coat, safety goggles. Work in laminar flow hood
- Nessler's reagent — contains mercury (Hg), highly toxic! Use only under fume hood, nitrile gloves, disposal as hazardous waste
- Cetrimide — irritating to skin and eyes. Use protective gloves
- Nalidixic acid — avoid skin contact, possible allergic reactions
- Infectious material — autoclave 121°C/30 min before disposal