🦠 Fecal Enterococci in Water
In short
Detection and quantitative determination of intestinal enterococci in water by membrane filtration method on Slanetz-Bartley medium with confirmation on bile-esculin-azide agar. The test is performed according to PN-EN ISO 7899-2:2004; Result: cfu/100 mL (colony forming units). The procedure comprises 10 steps (about 1 days 19 h 37 min–2 days 3 h 37 min in total); it is used for: Control of drinking water quality (Journal of Laws 2017 — enterococci: 0 cfu/100 mL), Monitoring of bathing waters (Directive 2006/7/EC), Testing of bottled and spring water.
At a glance
- Standard: PN-EN ISO 7899-2:2004
- Category: Microbiology
- Procedure steps: 10
- Total time of stages: 1 days 19 h 37 min–2 days 3 h 37 min
- Result: cfu/100 mL (colony forming units)
- Permissible value (drinking water): 0 cfu/100 mL
- Isolation temperature: 36 ± 1°C (presumptive stage)
Overview
Intestinal enterococci are a group of bacteria from the genus Enterococcus — primarily E. faecalis and E. faecium — which naturally inhabit the gastrointestinal tract of humans and warm-blooded animals. They are a recognized indicator of fecal contamination of water, complementary to E. coli, but characterized by much greater resistance to environmental conditions, chlorination and desiccation.
Enterococci are a particularly valuable indicator in situations where E. coli may undergo rapid inactivation — in seawater, water after intensive disinfection, and when assessing old fecal contamination. The survival of enterococci in aquatic environment is longer than E. coli, making them a better marker of treatment process effectiveness.
The method according to PN-EN ISO 7899-2 includes two stages: presumptive isolation on Slanetz-Bartley medium (agar with sodium azide and triphenyltetrazolium chloride — TTC) and confirmation on bile-esculin-azide agar (BEA). Enterococci reduce TTC to red formazan, forming characteristic red to burgundy colonies. On BEA they hydrolyze esculin, giving dark brown to black coloration around colonies.
Enterococci are a mandatory parameter in drinking water testing (MoH Regulation) with a permissible value of 0 cfu/100 mL. They are also required in bathing water monitoring (Directive 2006/7/EC) and bottled water.
Method principle
The water sample is filtered through a 0.45 µm membrane, which retains bacteria. The membrane is placed on Slanetz-Bartley medium and incubated at 36±1°C for 40–48 h. The medium contains sodium azide (inhibitor of Gram-negative bacteria) and 2,3,5-triphenyltetrazolium chloride (TTC), which enterococci reduce to insoluble red formazan — presumptive colonies are red, burgundy or pink. Then the membrane is transferred to bile-esculin-azide agar (BEA) and incubated at 44±0.5°C for 2 h. Enterococci hydrolyze the glycoside esculin to esculetin, which reacts with iron ions in the medium, forming a dark brown to black complex — result confirmation.
Applications
- Control of drinking water quality (Journal of Laws 2017 — enterococci: 0 cfu/100 mL)
- Monitoring of bathing waters (Directive 2006/7/EC)
- Testing of bottled and spring water
- Assessment of water disinfection effectiveness (chlorination, UV, ozonation)
- Monitoring of seawater and brackish water — better survival than E. coli
- Testing of groundwater and infiltration intakes
- Control of pool water quality
Key parameters
| Parameter | Value |
|---|---|
| Result | cfu/100 mL (colony forming units) |
| Permissible value (drinking water) | 0 cfu/100 mL |
| Isolation temperature | 36 ± 1°C (presumptive stage) |
| Confirmation temperature | 44 ± 0.5°C (confirmatory stage) |
| Incubation time | 40–48 h (isolation) + 2 h (confirmation) |
| Sample volume | 100 mL (standard) |
Standard
- Standard number
- PN-EN ISO 7899-2:2004
- Title (PL)
- Jakość wody — Wykrywanie i oznaczanie ilościowe enterokoków kałowych — Część 2: Metoda filtracji membranowej
- Title (EN)
- Water quality — Detection and enumeration of intestinal enterococci — Part 2: Membrane filtration method
Step-by-step procedure
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Workstation preparation
Sterilize filtration system. Prepare Slanetz-Bartley and BEA plates. Check incubator temperatures (36°C and 44°C).
-
Chlorine neutralization
Add sodium thiosulfate to sample containing residual chlorine (1.8% tablet). Mix gently.
-
Membrane filtration
Place sterile 0.45 µm filter on funnel. Filter 100 mL sample. Rinse funnel with 20 mL sterile peptone water.
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Presumptive stage — Slanetz-Bartley
Transfer filter to Slanetz-Bartley medium. Incubate at 36±1°C for 40–48 hours.
-
Presumptive colony reading
Count red, burgundy or pink colonies (TTC reduction to formazan). Note number and distribution of colonies.
-
Confirmatory stage — BEA
Transfer membrane with colonies to BEA agar surface (without inverting). Incubate at 44±0.5°C for 2 hours. Read immediately after incubation.
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Confirmed colony reading
Count colonies surrounded by brown-black zone (esculin hydrolysis). These colonies = confirmed intestinal enterococci.
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Result calculation
Result = number of confirmed colonies / sample volume × 100. Report result in cfu/100 mL.
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Quality control
Positive control: E. faecalis ATCC 29212 strain. Negative control: sterile water. Media control: sterility and purity.
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Disposal
Autoclave all plates with colonies (121°C/30 min) before disposal as infectious waste.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Membrane filtration system | Sartorius Combisart, Merck EZ-Fit, Pall MicroFunnel | 3,000–12,000 PLN |
| Vacuum pump | KNF Laboport N 810, Sartorius 16612 | 3,000–8,000 PLN |
| Incubator 36°C | Binder BD 56, Memmert IN30, Pol-Eko CLN 32 | 5,000–15,000 PLN |
| Incubator 44°C (precision) | Binder BD 23 with ±0.5°C control, Memmert IN30plus | 6,000–18,000 PLN |
| Autoclave for sterilization | Tuttnauer ELV 2540, Systec VX-40 | 20,000–45,000 PLN |
| Magnifier / colony counter | Stuart SC6+, Interscience Scan 500 | 1,500–25,000 PLN |
| Laminar flow hood | Esco Airstream, Telstar Bio II Advance | 15,000–40,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Slanetz-Bartley Agar | — | Selective medium with sodium azide and TTC for presumptive isolation of enterococci. Manufacturers: Merck, Oxoid, Biokar |
| Bile-esculin-azide agar (BEA) | — | Confirmatory medium with ox bile, esculin, ferric ammonium citrate and sodium azide |
| Membrane filters 0.45 µm | — | Mixed cellulose ester (MCE), 47 mm diameter, sterile. Merck Millipore, Sartorius |
| 2,3,5-Triphenyltetrazolium chloride (TTC) | 298-96-4 | Supplement to Slanetz-Bartley medium, concentration 0.1 g/L |
| Sodium azide (NaN₃) | 26628-22-8 | Inhibitor in medium, inhibits Gram-negative bacteria. WARNING: highly toxic! |
| Sodium thiosulfate (Na₂S₂O₃) | 7772-98-7 | For neutralizing chlorine in tap water samples |
| Ringer solution (1/4 strength) | — | For diluting samples, sterile 9 mL tubes |
Health and safety (OHS)
- Sodium azide (NaN₃) — highly toxic (H300, H310, H330)! Lethal doses: 10 mg/kg. Work in fume hood, gloves, goggles
- Enterococci may cause hospital infections (VRE) — full personal protective equipment
- Biological material after incubation — autoclave 121°C/30 min before disposal
- Work with Bunsen burner — risk of burns
- Seawater/bathing water — possible presence of other pathogens, exercise special caution
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 7899-2:2004 — “Water quality — Detection and enumeration of intestinal enterococci — Part 2: Membrane filtration method”.
How does this method work?
The water sample is filtered through a 0.45 µm membrane, which retains bacteria.
What is the measuring range and accuracy?
Result: cfu/100 mL (colony forming units); Permissible value (drinking water): 0 cfu/100 mL; Isolation temperature: 36 ± 1°C (presumptive stage); Confirmation temperature: 44 ± 0.5°C (confirmatory stage).
How long does the test take?
The times given for the individual stages add up to approximately 1 days 19 h 37 min–2 days 3 h 37 min. The procedure comprises 10 steps.
What equipment is required?
Membrane filtration system, Vacuum pump, Incubator 36°C, Incubator 44°C (precision), Autoclave for sterilization, Magnifier / colony counter, Laminar flow hood. Indicative cost of the core instrument (Membrane filtration system): 3,000–12,000 PLN.
Where is this test used?
Control of drinking water quality (Journal of Laws 2017 — enterococci: 0 cfu/100 mL); Monitoring of bathing waters (Directive 2006/7/EC); Testing of bottled and spring water; Assessment of water disinfection effectiveness (chlorination, UV, ozonation); Monitoring of seawater and brackish water — better survival than E. coli; Testing of groundwater and infiltration intakes; Control of pool water quality.
What safety precautions apply?
Sodium azide (NaN₃) — highly toxic (H300, H310, H330)! Lethal doses: 10 mg/kg. Work in fume hood, gloves, goggles; Enterococci may cause hospital infections (VRE) — full personal protective equipment; Biological material after incubation — autoclave 121°C/30 min before disposal; Work with Bunsen burner — risk of burns; Seawater/bathing water — possible presence of other pathogens, exercise special caution.
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 7899-2.