🦠 E. coli and Coliform Bacteria in Water

Microbiology PN-EN ISO 9308-1

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

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.

   ⏱ Time: 30 min

2. Chlorine neutralization

   If sample contains chlorine (tap water), add sodium thiosulfate (1.8% tablet — 0.1 mL per 100 mL sample). Mix gently.

   ⏱ Time: 2 min

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.

   ⏱ Time: 5 min

4. Transfer filter to medium

   With sterile forceps transfer filter to CCA plate. Ensure filter adheres evenly, without air bubbles underneath.

   ⏱ Time: 2 min

5. Incubation

   Incubate inverted plates (bottom up) in incubator 36±2°C for 18–24 hours. Do not exceed 24 hours.

   ⏱ Time: 18–24 h • 🌡 Temperature: 36 ± 2°C

6. Result reading — coliforms

   Count pink to red colonies (β-galactosidase-positive) = coliform bacteria. Optimal count: 10–100 colonies on filter.

   ⏱ Time: 10 min

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.

   ⏱ Time: 10 min

8. Oxidase test (confirmation)

   Transfer suspect colonies to oxidase strip. E. coli is oxidase-negative (no color change). Discard oxidase-positive colonies.

   ⏱ Time: 5 min

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%.

    ⏱ Time: 15 min

Required equipment and apparatus

Reagents, media and consumables

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

Frequently asked questions