🦠 E. coli and Coliform Bacteria in Water
In short
Quantitative determination of E. coli and coliform bacteria in water by membrane filtration method on chromogenic CCA medium. The test is performed according to PN-EN ISO 9308-1:2014-12 (wersja polska) wraz ze ZMIANĄ A1:2017-04 (wersja angielska); Linear range: 10–100 colonies on filter (optimal). The procedure comprises 10 steps (about 19 h 19 min–1 days 1 h 19 min in total); it is used for: 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.
At a glance
- Standard: PN-EN ISO 9308-1:2014-12 (wersja polska) wraz ze ZMIANĄ A1:2017-04 (wersja angielska)
- Category: Microbiology
- Procedure steps: 10
- Total time of stages: 19 h 19 min–1 days 1 h 19 min
- PARTICULARS OF AMENDMENT A1 (status of the PKN cards as of 23.09.2026): PN-EN ISO 9308-1:2014-12/A1:2017-04 — ENGLISH version, published 04-04-2017, 12 pages, KT 120 (Water Quality — Microbiological and Biological Testing), "Implements: EN ISO 9308-1:2014/A1:2017 [IDT], ISO 9308-1:2014/Amd 1:2016 [IDT]", ICS 07.100.20. WHAT THE AMENDMENT CONCERNS — NOT GIVEN: PKN does not publish the content of "A" amendments free of charge (unlike the national "Ap" corrigenda), and we did not have the text
- The weight of this amendment relative to the standard: the base standard PN-EN ISO 9308-1:2014-12 (POLISH version, published 14-10-2015) has 18 pages, and amendment A1 alone — 12 pages, i.e. two thirds of the volume of the standard. An amendment of this size is not an erratum
- SCALE AND NOTATION OF THE AMENDMENT IN THE SCOPES (measurement of 23.09.2026): FOUR DEFINITIONS, FOUR NUMBERS — and neither of the first two is clean (measurement repeated on 23.09.2026). Matching by the BEGINNING of the string "PN-EN ISO 9308-1", i.e. the way the "Laboratories" tab searches: 458 entries at 206 laboratories. The core "9308-1" alone, anywhere: 481 / 210. The string "9308" alone, i.e. together with parts 2 and 3: 726 / 225. THE RIGHT NUMBER IS THE FOURTH: all records WITH THE EN ELEMENT, with any separator in the PN element, but WITHOUT the 1999 generation — 465 ENTRIES AT 207 LABORATORIES. This is because the difference of 23 entries between the first and the second splits into three classes: 13 entries are the PREVIOUS GENERATION OF THE STANDARD ("PN-ISO 9308-1:1999" 11 times, "PN ISO 9308-1:1999" 2) — a separate document from before the EN version was implemented, which the core wrongly adds to the count; 7 entries (9 records) are INCONSISTENTLY WRITTEN RECORDS OF THE SAME STANDARD, which prefix matching misses: "PN–EN" with an en dash (3), "PN -EN" with a space before the hyphen (4), "PN EN" without a hyphen (2); 3 entries are records "ISO 9308-1" without the PN element — a direct citation of the international document, which the fourth definition deliberately does not cover. The right number is therefore seven higher than the one from the tab and sixteen lower than the core. All four numbers were confirmed to the unit on a second, independent copy of the scope database. The standard remains one of the most frequently cited in the whole database. The A1 element is given by 200 laboratories; 23 entries at 6 laboratories cite it EXCLUSIVELY without the A1 element
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).
MIND THE NOTATION OF AMENDMENT A1 (23.09.2026). We previously gave "PN-EN ISO 9308-1:2014+A1:2017" here, i.e. with a plus in the number — and in the PKN catalogue a plus in the number denotes a consolidated edition (a whole standard adopting the amended EN), whereas what is meant here is a separate amendment sheet attached to the standard: PN-EN ISO 9308-1:2014-12/A1:2017-04. The base standard: Polish version, published 14-10-2015, 18 pages, KT 120 for Water Quality — Microbiological and Biological Testing (PKN card, read on 23.09.2026).
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 |
|---|---|
| PARTICULARS OF AMENDMENT A1 (status of the PKN cards as of 23.09.2026) | PN-EN ISO 9308-1:2014-12/A1:2017-04 — ENGLISH version, published 04-04-2017, 12 pages, KT 120 (Water Quality — Microbiological and Biological Testing), "Implements: EN ISO 9308-1:2014/A1:2017 [IDT], ISO 9308-1:2014/Amd 1:2016 [IDT]", ICS 07.100.20. WHAT THE AMENDMENT CONCERNS — NOT GIVEN: PKN does not publish the content of "A" amendments free of charge (unlike the national "Ap" corrigenda), and we did not have the text |
| The weight of this amendment relative to the standard | the base standard PN-EN ISO 9308-1:2014-12 (POLISH version, published 14-10-2015) has 18 pages, and amendment A1 alone — 12 pages, i.e. two thirds of the volume of the standard. An amendment of this size is not an erratum |
| SCALE AND NOTATION OF THE AMENDMENT IN THE SCOPES (measurement of 23.09.2026) | FOUR DEFINITIONS, FOUR NUMBERS — and neither of the first two is clean (measurement repeated on 23.09.2026). Matching by the BEGINNING of the string "PN-EN ISO 9308-1", i.e. the way the "Laboratories" tab searches: 458 entries at 206 laboratories. The core "9308-1" alone, anywhere: 481 / 210. The string "9308" alone, i.e. together with parts 2 and 3: 726 / 225. THE RIGHT NUMBER IS THE FOURTH: all records WITH THE EN ELEMENT, with any separator in the PN element, but WITHOUT the 1999 generation — 465 ENTRIES AT 207 LABORATORIES. This is because the difference of 23 entries between the first and the second splits into three classes: 13 entries are the PREVIOUS GENERATION OF THE STANDARD ("PN-ISO 9308-1:1999" 11 times, "PN ISO 9308-1:1999" 2) — a separate document from before the EN version was implemented, which the core wrongly adds to the count; 7 entries (9 records) are INCONSISTENTLY WRITTEN RECORDS OF THE SAME STANDARD, which prefix matching misses: "PN–EN" with an en dash (3), "PN -EN" with a space before the hyphen (4), "PN EN" without a hyphen (2); 3 entries are records "ISO 9308-1" without the PN element — a direct citation of the international document, which the fourth definition deliberately does not cover. The right number is therefore seven higher than the one from the tab and sixteen lower than the core. All four numbers were confirmed to the unit on a second, independent copy of the scope database. The standard remains one of the most frequently cited in the whole database. The A1 element is given by 200 laboratories; 23 entries at 6 laboratories cite it EXCLUSIVELY without the A1 element |
| SEVEN FORMS OF APPENDING THE A1 ELEMENT | ":2014-12+A1", ":2014-12 +A1", ":2014-12+ A1", ":2014-12 + A1", ":2014-12/A1", ":2014-12/ A1", ":2014-12 /A1". The element that is meant to serve precision is written the least uniformly of anything in this citation |
| Language versions diverge | the base standard is available in POLISH, and amendment A1 only in ENGLISH — a laboratory working on the Polish text of the standard reads the amendment in the original or in its own translation |
| 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 9308-1:2014-12 (wersja polska) wraz ze ZMIANĄ A1:2017-04 (wersja angielska)
- 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
-
Workstation preparation
Sterilize filtration system (autoclave 121°C/15 min or flame). Prepare CCA plates — remove from refrigerator 30 min before.
-
Chlorine neutralization
If sample contains chlorine (tap water), add sodium thiosulfate (1.8% tablet — 0.1 mL per 100 mL sample). Mix gently.
-
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.
-
Transfer filter to medium
With sterile forceps transfer filter to CCA plate. Ensure filter adheres evenly, without air bubbles underneath.
-
Incubation
Incubate inverted plates (bottom up) in incubator 36±2°C for 18–24 hours. Do not exceed 24 hours.
-
Result reading — coliforms
Count pink to red colonies (β-galactosidase-positive) = coliform bacteria. Optimal count: 10–100 colonies on filter.
-
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.
-
Oxidase test (confirmation)
Transfer suspect colonies to oxidase strip. E. coli is oxidase-negative (no color change). Discard oxidase-positive colonies.
-
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.
-
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
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 9308-1:2014-12 (wersja polska) wraz ze ZMIANĄ A1:2017-04 (wersja angielska) — “Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 1: Membrane filtration method for waters with low bacterial background flora”.
How does this method work?
The water sample is filtered through a 0.45 µm membrane (47 mm diameter), which retains bacteria.
What is the measuring range and accuracy?
PARTICULARS OF AMENDMENT A1 (status of the PKN cards as of 23.09.2026): PN-EN ISO 9308-1:2014-12/A1:2017-04 — ENGLISH version, published 04-04-2017, 12 pages, KT 120 (Water Quality — Microbiological and Biological Testing), "Implements: EN ISO 9308-1:2014/A1:2017 [IDT], ISO 9308-1:2014/Amd 1:2016 [IDT]", ICS 07.100.20. WHAT THE AMENDMENT CONCERNS — NOT GIVEN: PKN does not publish the content of "A" amendments free of charge (unlike the national "Ap" corrigenda), and we did not have the text; The weight of this amendment relative to the standard: the base standard PN-EN ISO 9308-1:2014-12 (POLISH version, published 14-10-2015) has 18 pages, and amendment A1 alone — 12 pages, i.e. two thirds of the volume of the standard. An amendment of this size is not an erratum; SCALE AND NOTATION OF THE AMENDMENT IN THE SCOPES (measurement of 23.09.2026): FOUR DEFINITIONS, FOUR NUMBERS — and neither of the first two is clean (measurement repeated on 23.09.2026). Matching by the BEGINNING of the string "PN-EN ISO 9308-1", i.e. the way the "Laboratories" tab searches: 458 entries at 206 laboratories. The core "9308-1" alone, anywhere: 481 / 210. The string "9308" alone, i.e. together with parts 2 and 3: 726 / 225. THE RIGHT NUMBER IS THE FOURTH: all records WITH THE EN ELEMENT, with any separator in the PN element, but WITHOUT the 1999 generation — 465 ENTRIES AT 207 LABORATORIES. This is because the difference of 23 entries between the first and the second splits into three classes: 13 entries are the PREVIOUS GENERATION OF THE STANDARD ("PN-ISO 9308-1:1999" 11 times, "PN ISO 9308-1:1999" 2) — a separate document from before the EN version was implemented, which the core wrongly adds to the count; 7 entries (9 records) are INCONSISTENTLY WRITTEN RECORDS OF THE SAME STANDARD, which prefix matching misses: "PN–EN" with an en dash (3), "PN -EN" with a space before the hyphen (4), "PN EN" without a hyphen (2); 3 entries are records "ISO 9308-1" without the PN element — a direct citation of the international document, which the fourth definition deliberately does not cover. The right number is therefore seven higher than the one from the tab and sixteen lower than the core. All four numbers were confirmed to the unit on a second, independent copy of the scope database. The standard remains one of the most frequently cited in the whole database. The A1 element is given by 200 laboratories; 23 entries at 6 laboratories cite it EXCLUSIVELY without the A1 element; SEVEN FORMS OF APPENDING THE A1 ELEMENT: ":2014-12+A1", ":2014-12 +A1", ":2014-12+ A1", ":2014-12 + A1", ":2014-12/A1", ":2014-12/ A1", ":2014-12 /A1". The element that is meant to serve precision is written the least uniformly of anything in this citation.
How long does the test take?
The times given for the individual stages add up to approximately 19 h 19 min–1 days 1 h 19 min. The procedure comprises 10 steps.
What equipment is required?
Membrane filtration system, Vacuum pump, Bacteriological incubator 36°C, Autoclave for sterilization, UV lamp / Bunsen burner, Laminar flow hood (optional), 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 (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.
What safety precautions apply?
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.
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 9308-1.