🦠 Clostridium perfringens in Water
In short
Quantitative determination of vegetative cells and spores of Clostridium perfringens in water by membrane filtration method on TSC medium under anaerobic conditions. The test is performed according to PN-EN ISO 14189:2016; Result: cfu/100 mL (colony forming units). The procedure comprises 11 steps (about 1 days 4 h 17 min in total); it is used for: Control of drinking water quality — indicator parameter (MoH Regulation), Assessment of filtration process effectiveness in water treatment stations, Detection of old fecal contamination of groundwater.
At a glance
- Standard: PN-EN ISO 14189:2016
- Category: Microbiology
- Procedure steps: 11
- Total time of stages: 1 days 4 h 17 min
- Result: cfu/100 mL (colony forming units)
- Permissible value (drinking water): 0 cfu/100 mL (including spores)
- Incubation temperature: 44 ± 1°C
Overview
Clostridium perfringens is an anaerobic, spore-forming Gram-positive bacterium, whose presence in water indicates exclusively fecal contamination. C. perfringens spores are extremely resistant to chemical disinfection (chlorination, ozonation), UV radiation, pH and temperature changes — they can survive in aquatic environment for months or even years.
This exceptional resistance makes C. perfringens a valuable supplementary indicator to E. coli and enterococci. Detection of C. perfringens spores in treated water indicates insufficient effectiveness of filtration processes and may indicate old fecal contamination, even when E. coli and enterococci are no longer detectable.
The method according to PN-EN ISO 14189 includes membrane filtration of water sample, placing the membrane on TSC medium (Tryptose Sulphite Cycloserine — tryptose-sulfite-cycloserine agar) and incubation under anaerobic conditions at 44±1°C for 21±3 hours. C. perfringens reduces sulfites to sulfides, which react with iron salt in the medium, forming black colonies. Cycloserine inhibits growth of Bacillus species.
C. perfringens is an indicator parameter of drinking water according to MoH Regulation. Its detection in treated water should result in assessment of filtration process effectiveness and verification of barrier system in water treatment plant.
Method principle
The water sample (or its dilution) is filtered through a 0.45 µm membrane, which is then placed on the surface of TSC medium (Tryptose Sulphite Cycloserine agar). The medium contains sodium sulfite (Na₂SO₃) and ferric ammonium citrate — C. perfringens reduces sulfite to sulfide (S²⁻), which forms black FeS precipitate with iron ions, giving characteristic black colonies. Cycloserine (400 mg/L) selectively inhibits Bacillus spp. Incubation under anaerobic conditions (anaerobic jar with gas generator) at 44±1°C enhances selectivity towards C. perfringens.
Applications
- Control of drinking water quality — indicator parameter (MoH Regulation)
- Assessment of filtration process effectiveness in water treatment stations
- Detection of old fecal contamination of groundwater
- Monitoring of surface waters used as drinking water sources
- Testing of water after water supply network failures
- Assessment of integrity of membrane systems (UF, NF) in treatment plants
Key parameters
| Parameter | Value |
|---|---|
| Result | cfu/100 mL (colony forming units) |
| Permissible value (drinking water) | 0 cfu/100 mL (including spores) |
| Incubation temperature | 44 ± 1°C |
| Incubation time | 21 ± 3 hours |
| Incubation conditions | Anaerobic (anaerobiosis) |
| Sample volume | 100 mL (standard) |
Standard
- Standard number
- PN-EN ISO 14189:2016
- Title (PL)
- Jakość wody — Oznaczanie ilościowe Clostridium perfringens — Metoda filtracji membranowej
- Title (EN)
- Water quality — Enumeration of Clostridium perfringens — Method using membrane filtration
Step-by-step procedure
-
Workstation preparation
Sterilize filtration set. Prepare TSC plates with cycloserine supplement. Check incubator temperature (44°C).
-
Preliminary sample heating (optional)
For determination of spores only: heat sample in water bath 75°C for 15 min — kills vegetative forms. For vegetative forms + spores: skip this step.
-
Chlorine neutralization
Add sodium thiosulfate to tap water sample. Mix.
-
Membrane filtration
Filter 100 mL sample through 0.45 µm filter. Rinse funnel with sterile peptone water.
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Placement on TSC medium
Transfer filter with sterile forceps to TSC medium surface. Pour thin layer of TSC medium (overlay 5 mL) to create anaerobic conditions on surface.
-
Anaerobic incubation
Place plate in anaerobic jar. Insert AnaeroGen sachet and indicator strip. Seal jar tightly. Incubate at 44±1°C for 21±3 h.
-
Result reading
Open jar. Count black colonies within 30 minutes — color fades quickly after contact with air! Black/gray colonies = presumptive C. perfringens.
-
Confirmation — acid phosphatase test
Transfer colonies to filter paper. Apply acid phosphatase reagent (α-naphthyl phosphate). Pink coloration in 1–2 min = acid phosphatase (+) = confirmed C. perfringens.
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Subculture on blood agar (optional)
Transfer colonies to blood agar. Incubate anaerobically 37°C/24 h. C. perfringens gives double zone of hemolysis (beta-hemolysis).
-
Result calculation
Result = number of confirmed black colonies / sample volume × 100. Report in cfu/100 mL.
-
Quality control
Positive control: C. perfringens ATCC 13124. Negative control: sterile water. Check anaerobiosis indicator strip.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Membrane filtration system | Sartorius Combisart, Merck EZ-Fit | 3,000–12,000 PLN |
| Anaerobic jar 2.5 L | Oxoid AG0025A, Merck Anaerocult | 800–2,000 PLN |
| Anaerobic atmosphere generators | Oxoid AnaeroGen AN0025A, Merck Anaerocult A | 50–100 PLN/pc (sachets) |
| Incubator 44°C | Binder BD 56, Memmert IN30plus | 6,000–18,000 PLN |
| Vacuum pump | KNF Laboport N 810 | 3,000–8,000 PLN |
| Autoclave | Tuttnauer ELV 2540 | 20,000–45,000 PLN |
| Magnifier / colony counter | Stuart SC6+, Interscience Scan 500 | 1,500–25,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| TSC Agar (Tryptose Sulphite Cycloserine) | — | Selective medium without egg yolk. Composition: casein hydrolysate, yeast extract, sodium sulfite, ferric ammonium citrate, agar. Manufacturers: Oxoid, Merck, Biokar |
| Cycloserine supplement | 68-41-7 | D-Cycloserine, final concentration 400 mg/L. Inhibits growth of Bacillus spp. |
| Membrane filters 0.45 µm | — | MCE, 47 mm, sterile. Merck Millipore, Sartorius |
| AnaeroGen sachets | — | Anaerobic atmosphere generators for 2.5 L jars. O₂ reduction < 1% in 30 min |
| Anaerobiosis indicator strips | — | Oxoid Anaerobic Indicator BR0055 — control of anaerobic conditions in jar |
| Acid phosphatase test reagent | — | α-naphthyl phosphate solution + diazo reagent. For C. perfringens confirmation (acid phosphatase+) |
| Blood agar (nutrient with 5% sheep blood) | — | For subculture and confirmation of hemolytic C. perfringens |
| Sodium thiosulfate (Na₂S₂O₃) | 7772-98-7 | Neutralization of chlorine in tap water samples |
Health and safety (OHS)
- C. perfringens produces toxins (alpha, beta, epsilon, iota) — potentially dangerous pathogen. Full personal protective equipment
- Work under anaerobic conditions — jars under pressure, open carefully
- Cycloserine — antibiotic, avoid skin contact, work with gloves
- Sodium azide (in media) — highly toxic, do not acidify (releases toxic HN₃)
- Infectious material — autoclave 121°C/30 min before disposal
- Acid phosphatase reagent — irritating, use in fume hood
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 14189:2016 — “Water quality — Enumeration of Clostridium perfringens — Method using membrane filtration”.
How does this method work?
The water sample (or its dilution) is filtered through a 0.45 µm membrane, which is then placed on the surface of TSC medium (Tryptose Sulphite Cycloserine agar).
What is the measuring range and accuracy?
Result: cfu/100 mL (colony forming units); Permissible value (drinking water): 0 cfu/100 mL (including spores); Incubation temperature: 44 ± 1°C; Incubation time: 21 ± 3 hours.
How long does the test take?
The times given for the individual stages add up to approximately 1 days 4 h 17 min. The procedure comprises 11 steps.
What equipment is required?
Membrane filtration system, Anaerobic jar 2.5 L, Anaerobic atmosphere generators, Incubator 44°C, Vacuum pump, Autoclave, Magnifier / colony counter. Indicative cost of the core instrument (Membrane filtration system): 3,000–12,000 PLN.
Where is this test used?
Control of drinking water quality — indicator parameter (MoH Regulation); Assessment of filtration process effectiveness in water treatment stations; Detection of old fecal contamination of groundwater; Monitoring of surface waters used as drinking water sources; Testing of water after water supply network failures; Assessment of integrity of membrane systems (UF, NF) in treatment plants.
What safety precautions apply?
C. perfringens produces toxins (alpha, beta, epsilon, iota) — potentially dangerous pathogen. Full personal protective equipment; Work under anaerobic conditions — jars under pressure, open carefully; Cycloserine — antibiotic, avoid skin contact, work with gloves; Sodium azide (in media) — highly toxic, do not acidify (releases toxic HN₃); Infectious material — autoclave 121°C/30 min before disposal; Acid phosphatase reagent — irritating, use in fume hood.
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 14189.