🔴 Legionella in Water
Detection and quantitative determination of Legionella bacteria in water by culture method on BCYE/GVPC medium with heat or acid treatment. The only method recognized by Sanepid.
Overview
Legionella is a genus of Gram-negative rods, of which the most dangerous to humans is Legionella pneumophila — the causative agent of legionellosis (Legionnaires' disease), severe pneumonia with 5–30% mortality. Legionella bacteria naturally inhabit aquatic environment, but pose a health risk when they multiply in artificial water systems: hot water installations, cooling towers, fountains, jacuzzis, humidifiers and air conditioners.
Infection occurs by inhalation — through inhaling water aerosol containing bacteria. Legionella is not transmitted from person to person. Optimal multiplication temperature: 25–45°C, with maximum at 35–37°C. Below 20°C bacteria do not grow, above 60°C they die.
The culture method according to PN-EN ISO 11731:2017 is the only method recognized by the State Sanitary Inspectorate in Poland. It includes membrane filtration or direct plating, preliminary sample treatment (heat 50°C/30 min or acid pH 2.2/5 min) to eliminate accompanying flora, culture on GVPC medium (selective) and BCYE (non-selective) at 36±1°C in moist atmosphere for 7–10 days.
Testing is required by the Regulation of the Minister of Health on water quality in internal installations (≤100 cfu/100 mL — no action, >100 — cleaning and disinfection, >1000 — immediate corrective actions). Waiting period for results: 9–15 days, which is the main disadvantage of the culture method.
Method principle
The water sample is concentrated by membrane filtration (0.2 µm) or centrifugation. The concentrate is subjected to treatment eliminating accompanying flora: heat treatment (50±1°C/30 min) or acid treatment (HCl/KCl buffer pH 2.2 for 5 min). The sample is plated on GVPC medium (BCYE agar with supplements: glycine, polymyxin B, vancomycin, cycloheximide — inhibiting bacteria and fungi) and on BCYE medium without supplements (control). Incubation: 36±1°C in moist atmosphere (>80% RH) for 7–10 days. Legionella colonies: grayish, glassy, with "cut-glass" structure. Confirmation: no growth on BCYE without L-cysteine (Legionella requires L-cysteine for growth).
Applications
- Monitoring of hot water installations (hospitals, hotels, multi-family buildings)
- Control of cooling towers and air conditioning systems
- Testing of water in pools, jacuzzis, SPAs, fountains
- Epidemiological investigation — legionellosis outbreaks
- Control of thermal disinfection effectiveness (>70°C) and chemical disinfection
- Monitoring of water in healthcare facilities (mandatory)
- Testing of water commissioned by property managers
Key parameters
| Parameter | Value |
|---|---|
| Result | cfu/100 mL (colony forming units) |
| Permissible value | ≤100 cfu/100 mL (no action), >1000 — alarm |
| Incubation temperature | 36 ± 1°C |
| Incubation time | 7–10 days (reading after 3, 5, 7 and 10 days) |
| Atmosphere | Aerobic, humidity >80% RH |
| Waiting time for result | 9–15 days (with treatment and confirmation) |
Standard
- Standard number
- PN-EN ISO 11731:2017
- Title (PL)
- Jakość wody — Oznaczanie ilościowe Legionella
- Title (EN)
- Water quality — Enumeration of Legionella
Step-by-step procedure
1. Sample collection
Collect min. 1 L water. DO NOT rinse sampling point (no flushing). Add sodium thiosulfate (if chlorinated water). Transport at 5–20°C, analysis within 24 h.
2. Membrane filtration — concentration
Filter 1 L sample through 0.2 µm filter (polycarbonate). Aseptically cut membrane, place in sterile container with 10 mL of reserved sample. Vortex vigorously 2 min.
3. Heat treatment
Divide concentrated sample into 2 portions. Heat one in water bath 50±1°C for 30 min (elimination of heat-sensitive flora). Leave the other without treatment.
4. Acid treatment (alternative)
Instead of heat treatment: add HCl/KCl buffer (pH 2.2) in 1:1 ratio. Contact 5 min. Then neutralize with KOH buffer.
5. Plating on media
Plate 0.1 mL sample (treated and untreated) on GVPC and BCYE plates. Spread with glass spatula. Plate in duplicate.
6. Incubation
Incubate plates at 36±1°C in moist atmosphere (>80% RH). Use tightly sealed bags or incubator with water tray. DO NOT invert plates.
7. Reading — day 3 and 5
Check plates under stereomicroscope. If accompanying flora overgrows GVPC plates — perform additional plating with another treatment.
8. Reading — day 7 and 10
Count colonies with typical morphology: grayish, glassy, shiny, with "cut-glass" structure. Diameter 1–3 mm. Mark suspect colonies.
9. Confirmation — subculture
Transfer suspect colonies to BCYE with L-cysteine and BCYE without L-cysteine. Incubate 36°C/48 h. Growth on BCYE+cysteine and no growth on BCYE−cysteine = Legionella spp.
10. Serogroup identification
Test confirmed Legionella colonies with latex test (agglutination) to identify L. pneumophila sg 1 (most commonly pathogenic) and sg 2–14.
11. Result calculation
Result = (number of confirmed colonies × dilution factor) / sample volume. Report in cfu/100 mL or cfu/L.
12. Quality control
Positive control: L. pneumophila ATCC 33152. Negative control: sterile water. Check incubator humidity and media sterility.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Membrane filtration system (0.2 µm) | Sartorius Combisart, Merck EZ-Fit with 0.2 µm filters | 3,000–12,000 PLN |
| Incubator with humidity control | Binder KBF 115, Memmert HPP110 with water tray | 8,000–25,000 PLN |
| Thermostatic water bath | Memmert WNB 14, Julabo CORIO C-B5 (50°C for heat treatment) | 5,000–15,000 PLN |
| Laboratory vortex | IKA Vortex 3, Scientific Industries Vortex-Genie 2 | 1,500–4,000 PLN |
| Autoclave | Tuttnauer ELV 2540 | 20,000–45,000 PLN |
| Class II laminar flow hood | Esco Airstream Class II, Telstar Bio II Advance | 20,000–50,000 PLN |
| Stereomicroscope | Zeiss Stemi 305, Olympus SZ61 | 5,000–15,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| BCYE Agar (Buffered Charcoal Yeast Extract) | — | Base medium with L-cysteine, ferric pyrophosphate, activated carbon, yeast extract, ACES buffer. Manufacturers: Oxoid, Merck, bioMérieux |
| GVPC Agar (selective supplement) | — | BCYE + glycine + polymyxin B + vancomycin + cycloheximide. Inhibits accompanying flora |
| BCYE without L-cysteine | — | Control medium — Legionella does not grow without L-cysteine. For confirmation |
| L-Cysteine hydrochloride | 52-89-1 | BCYE growth supplement, concentration 0.4 g/L. Legionella requires L-cysteine |
| Acid buffer HCl/KCl pH 2.2 | — | For acid treatment of sample — elimination of accompanying flora. Contact 5 min |
| Membrane filters 0.2 µm (polycarbonate) | — | Polycarbonate (PC), 47 mm, black or white. For filtration of Legionella samples |
| Latex test reagents | — | Latex agglutination test for identification of L. pneumophila serogroups (sg 1–14). Oxoid DR0800 |
Health and safety (OHS)
- Legionella pneumophila — BSL-2 pathogen! All work in class II laminar flow hood. FFP2/FFP3 mask when working with aerosol
- DO NOT vortex open tubes with sample — risk of creating infectious aerosol
- Acid buffer pH 2.2 — corrosive, use gloves and safety goggles
- Cycloheximide (in GVPC) — toxic, mutagenic. Avoid contact, work in fume hood
- Infectious material — autoclave 121°C/30 min. Pack plates in autoclavable bags
- Water samples from installations >40°C — risk of burns during collection