🦠 E. coli and Coliform Bacteria in Water
Quantitative determination of E. coli and coliform bacteria in water by membrane filtration method on chromogenic CCA medium. Basic indicator of fecal contamination of drinking water.
Overview
Escherichia coli is the most important indicator of fecal contamination of water. The presence of E. coli in drinking water indicates contamination with human or animal feces and potential risk from pathogenic bacteria, viruses and intestinal protozoa. Coliform bacteria (coliforms) comprise a broader group — in addition to E. coli also Klebsiella, Enterobacter, Citrobacter — and serve as a general indicator of treatment process hygiene.
The method according to PN-EN ISO 9308-1 is based on membrane filtration of water sample through a 0.45 µm filter, which retains bacteria. The filter is placed on selective chromogenic CCA medium (Chromogenic Coliform Agar), containing enzymatic substrates: Salmon-GAL (6-chloro-3-indolyl-β-D-galactopyranoside) for coliform β-galactosidase and X-Glucuronide (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) for E. coli β-glucuronidase.
After incubation at 36±2°C for 18–24 hours, coliform colonies color pink to red (β-galactosidase activity), and E. coli colonies color dark blue-violet (activity of both enzymes). The result is read directly — without need for biochemical confirmation, which significantly speeds up analysis.
The method is intended for waters with low background flora — drinking water, treated water, groundwater. For surface waters and wastewater (high background flora), PN-EN ISO 9308-2 is used (MPN method with Colilert medium).
Method principle
The water sample is filtered through a 0.45 µm membrane (47 mm diameter), which retains bacteria. The membrane is transferred to the surface of chromogenic CCA medium. The medium contains two chromogenic substrates: Salmon-GAL cleaved by β-galactosidase (coliform enzyme) giving a salmon-pink product, and X-Glucuronide cleaved by β-glucuronidase (E. coli specific enzyme) giving a dark blue product. Selectivity of the medium is provided by Tergitol-7, which inhibits growth of Gram-positive bacteria and most non-coliforms.
Applications
- Control of drinking water quality (Journal of Laws 2017 — E. coli: 0 cfu/100 mL)
- Monitoring of water supply network — sampling points at consumers
- Testing of groundwater and water intakes
- Control of water after treatment and disinfection
- Testing of bottled and spring water
- Monitoring of water in public buildings (hospitals, schools)
- Water for production purposes in food industry
- Emergency testing — after water supply network failures
Key parameters
| Parameter | Value |
|---|---|
| Result | cfu/100 mL (colony forming units) |
| Permissible value (drinking water) | E. coli: 0 cfu/100 mL; Coliforms: 0 cfu/100 mL |
| Incubation temperature | 36 ± 2°C |
| Incubation time | 18–24 hours |
| Sample volume | 100 mL (standard) or 250 mL |
| Linear range | 10–100 colonies on filter (optimal) |
Standard
- Standard number
- PN-EN ISO 9308-1:2014+A1:2017
- Title (PL)
- Jakość wody — Oznaczanie ilościowe Escherichia coli i bakterii grupy coli — Część 1: Metoda filtracji membranowej do wód o małej ilości flory towarzyszącej
- Title (EN)
- Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 1: Membrane filtration method for waters with low bacterial background flora
Step-by-step procedure
1. Workstation preparation
Sterilize filtration system (autoclave 121°C/15 min or flame). Prepare CCA plates — remove from refrigerator 30 min before.
2. Chlorine neutralization
If sample contains chlorine (tap water), add sodium thiosulfate (1.8% tablet — 0.1 mL per 100 mL sample). Mix gently.
3. Membrane filtration
Place sterile 0.45 µm filter on filtration funnel. Pour 100 mL sample. Turn on vacuum pump. After filtration rinse funnel with 20 mL sterile peptone water.
4. Transfer filter to medium
With sterile forceps transfer filter to CCA plate. Ensure filter adheres evenly, without air bubbles underneath.
5. Incubation
Incubate inverted plates (bottom up) in incubator 36±2°C for 18–24 hours. Do not exceed 24 hours.
6. Result reading — coliforms
Count pink to red colonies (β-galactosidase-positive) = coliform bacteria. Optimal count: 10–100 colonies on filter.
7. Result reading — E. coli
Count dark blue to violet colonies (β-galactosidase- and β-glucuronidase-positive) = E. coli. E. coli is included in total coliform count.
8. Oxidase test (confirmation)
Transfer suspect colonies to oxidase strip. E. coli is oxidase-negative (no color change). Discard oxidase-positive colonies.
9. Result calculation
Result = number of confirmed colonies per 100 mL sample. For drinking water required result: 0 cfu/100 mL for both E. coli and coliforms.
10. Quality control
Daily: negative control (sterile water), duplicate every 10 samples, CRM control sample. Recovery 90–110%.
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 |
| Bacteriological incubator 36°C | Binder BD 56, Memmert IN30, Pol-Eko CLN 32 | 5,000–15,000 PLN |
| Autoclave for sterilization | Tuttnauer ELV 2540, Systec VX-40 | 20,000–45,000 PLN |
| UV lamp / Bunsen burner | UVC bactericidal lamp for chamber, Teclu burner | 200–1,500 PLN |
| Laminar flow hood (optional) | Esco Airstream, Telstar Bio II Advance | 15,000–40,000 PLN |
| Magnifier / colony counter | Stuart SC6+, Interscience Scan 500 | 1,500–25,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Chromogenic Coliform Agar (CCA) | — | Chromogenic medium per ISO 9308-1, contains Salmon-GAL + X-Glucuronide + Tergitol-7. Manufacturers: Merck, Biokar, Oxoid |
| Membrane filters 0.45 µm | — | Mixed cellulose ester (MCE), 47 mm diameter, sterile, gridded. Merck Millipore, Sartorius, Pall |
| Sodium thiosulfate (Na₂S₂O₃) | 7772-98-7 | Tablets for neutralizing chlorine in samples, 18 mg/L per 500 mL sample |
| Ringer solution (1/4 strength) | — | For diluting samples, sterile, 9 mL tubes |
| Buffered peptone water | — | Alternative for dilutions, 0.1% peptone + 0.85% NaCl, pH 7.0 |
| Oxidase reagent (Kovacs) | 2436-85-3 | Oxidase strips for E. coli confirmation (oxidase-negative). Tetramethyl-p-phenylenediamine |
Health and safety (OHS)
- Water samples may contain pathogens — work with nitrile gloves and laboratory coat
- Media with colonies after incubation treat as infectious material — autoclave before disposal
- Work with Bunsen burner — risk of burns, do not leave unattended
- Sodium thiosulfate — low risk, avoid eye contact
- UV lamp — do not look directly at radiation source, risk of eye damage