🦠 E. coli and coliform bacteria in water — MPN method with an enzymatic medium (Colilert-18 / Quanti-Tray)
In short
Dehydrated Colilert-18 medium (defined substrate technology, ONPG/MUG) is added to 100 ml of water, the mixture is poured into a Quanti-Tray or a Quanti-Tray/2000, sealed and incubated for 18–22 h at (36 ± 2) °C; yellow wells = coliform bacteria, wells that are yellow and fluoresce under UV at 365 nm = E. The test is performed according to PN-EN ISO 9308-2:2014-06 (ISO 9308-2:2012); MPN range: Quanti-Tray: < 1 to > 200.5/100 ml; Quanti-Tray/2000: < 1 to 2419/100 ml. The procedure comprises 6 steps; it is used for: Monitoring of drinking water (the reference method under the regulation of the Minister of Health of 22 May 2026, Dz.U. 2026 poz. 748 [Journal of Laws 2026, item 748], annex No 4 part A point 1) and of water in distribution networks — a rapid result after 18 h, Waters with a high content of suspended matter and a high background of heterotrophs, where membrane filtration fails (surface waters, raw waters, wells), Sea water and brackish water — determination of E. coli after a dilution of 1→10 (coliform bacteria in sea water excluded).
At a glance
- Standard: PN-EN ISO 9308-2:2014-06 (ISO 9308-2:2012)
- Category: Microbiology
- Procedure steps: 6
- Volume of sample: 100 ml (or a dilution made up to 100 ml); sea water 1→10
- Medium: Colilert-18, 2.8 g per test; storage at 2–25 °C away from sunlight
- Incubation: (36 ± 2) °C, 18–22 h
Overview
The presence and the scale of faecal contamination is an important factor in the assessment of the quality of water and of the risk of infection; testing for the presence of Escherichia coli, which lives in the intestines of humans and of warm-blooded animals, indicates such contamination. The result for coliform bacteria is more difficult to interpret, because some of them live in the soil and in surface water — their presence does not prove faecal contamination, but it may point to a failure of the treatment or to an ingress of water into the network. ISO 9308-2:2012 (the second edition, replacing ISO 9308-2:1990) specifies a method for the enumeration of E. coli and coliform bacteria in water based on growth in a liquid medium and on the calculation of the most probable number (MPN) from tables. It is applied to all kinds of water, including water with a large amount of suspended matter and a high background of heterotrophic bacteria; it must not be used for the enumeration of coliform bacteria in sea water, and for E. coli in sea water a dilution of 1→10 in sterile water is usually required. The detection of E. coli is based on the expression of β-D-glucuronidase, so the method does not detect many enterohaemorrhagic strains (e.g. O157), which usually do not produce that enzyme — they appear as coliform bacteria. The result is confirmed without any further confirmation of the positive wells. ISO draws attention to the fact that the use of the standard may involve IDEXX patents (Colilert-18, Quanti-Tray). In Poland the regulation of the Minister of Health of 22 May 2026 on the quality of water intended for human consumption (Dz.U. 2026 poz. 748 [Journal of Laws 2026, item 748]), annex No 4 part A point 1, indicates for E. coli and coliform bacteria the reference method: the standard PN-EN ISO 9308-1 OR the standard PN-EN ISO 9308-2 — both on an equal footing. The regulation of 7 December 2017 (Dz.U. 2017 poz. 2294 [Journal of Laws 2017, item 2294]), repealed on 24-06-2026, provided the same, so the change of the act does not change the choice of the standard.
Method principle
A single-use snap pack of dehydrated Colilert-18 medium (2.8 g) is added to the water sample (100 ml) or to a dilution made up to 100 ml, mixed until it has dissolved and aseptically poured into a Quanti-Tray (up to 201 organisms/100 ml, 51 wells) or a Quanti-Tray/2000 (up to 2419 organisms/100 ml, 97 wells); the trays are sealed in a Quanti-Tray sealer and incubated at (36 ± 2) °C for 18–22 h. Wells of a yellow colour equal to or more intense than the comparator are considered positive for coliform bacteria; yellow wells showing any fluorescence under UV (365 nm) — positive for E. coli. The yellow colour comes from the breakdown of ortho-nitrophenyl galactoside (ONPG) by β-D-galactosidase, and the fluorescence from the breakdown of 4-methylumbelliferyl glucuronide (MUG) by β-D-glucuronidase into fluorescing methylumbelliferone. A coliform bacterium is a member of the Enterobacteriaceae producing β-D-galactosidase, E. coli — one producing both of these enzymes. The MPN per 100 ml is read from statistical tables or from a program; for the 51-well tray MPN = N·ln[N/(N − X)]. The medium can also be used in the classical arrangement of MPN tubes (e.g. 1 × 50 ml and 5 × 10 ml) or as a presence/absence test in 100 ml — the tubes are then pre-warmed for 20 min at (36 ± 2) °C. Only sterile, non-inhibitory water free from oxidants (deionised or tap water) is used for the dilutions; buffered, saline and peptone diluents interfere with the test.
Applications
- Monitoring of drinking water (the reference method under the regulation of the Minister of Health of 22 May 2026, Dz.U. 2026 poz. 748 [Journal of Laws 2026, item 748], annex No 4 part A point 1) and of water in distribution networks — a rapid result after 18 h
- Waters with a high content of suspended matter and a high background of heterotrophs, where membrane filtration fails (surface waters, raw waters, wells)
- Sea water and brackish water — determination of E. coli after a dilution of 1→10 (coliform bacteria in sea water excluded)
- Presence/absence test in 100 ml and the classical arrangement of MPN tubes with the same medium
- Laboratories of sanitary-epidemiological stations and of waterworks — no need for confirmation and for counting colonies
Key parameters
| Parameter | Value |
|---|---|
| Volume of sample | 100 ml (or a dilution made up to 100 ml); sea water 1→10 |
| Medium | Colilert-18, 2.8 g per test; storage at 2–25 °C away from sunlight |
| Incubation | (36 ± 2) °C, 18–22 h |
| Reading | yellow ≥ comparator = coliforms; yellow + fluorescence under UV at 365 nm = E. coli |
| MPN range | Quanti-Tray: < 1 to > 200.5/100 ml; Quanti-Tray/2000: < 1 to 2419/100 ml |
| Transport and storage | (5 ± 3) °C in accordance with ISO 19458; analysis on the day of sampling or within 18 h, exceptionally within 24 h |
| Volume of the wells | Quanti-Tray about 1.96 ml; Quanti-Tray/2000: small 0.186 ml, large about 1.86 ml |
Standard
- Standard number
- PN-EN ISO 9308-2:2014-06 (ISO 9308-2:2012)
- Title (PL)
- Jakość wody — Oznaczanie ilościowe Escherichia coli i bakterii grupy coli — Część 2: Metoda najbardziej prawdopodobnej liczby
- Title (EN)
- Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 2: Most probable number method
Step-by-step procedure
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Sampling and transport
Take the samples in accordance with ISO 19458, transport and store them at (5 ± 3) °C; begin the analysis on the day of sampling or within 18 h (exceptionally within 24 h).
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Addition of the medium
To 100 ml of the sample (or of a dilution; sea water usually 1→10 in sterile water) aseptically add one pack of Colilert-18 (2.8 g) and shake gently until it has dissolved completely.
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Pouring and sealing
Pour the sample with the medium into a Quanti-Tray or a Quanti-Tray/2000 and seal it in the sealer; alternatively distribute it into sterile tubes in an MPN arrangement (e.g. 1 × 50 ml and 5 × 10 ml) or incubate the whole 100 ml as a presence/absence test — then pre-warm it for 20 min at (36 ± 2) °C.
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Incubation
Incubate the trays for 18–22 h at (36 ± 2) °C.
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Reading
Consider as positive for coliform bacteria the wells of a yellow colour at least equal to that of the comparator; examine the trays under a UV lamp at 365 nm in a dark room — yellow wells with any fluorescence are positive for E. coli. Where the colour is doubtful after 18 h, extend the incubation to 22 h; positive results before 18 h and negative results after 22 h are valid.
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Calculation and test report
From the number of positive wells read the MPN/100 ml for coliform bacteria and for E. coli from tables B.1/B.2 or from a program; the result is "< 1" when there are no positive wells (with a dilution of 1→10 — "< 10"). State the method, the identification of the sample, the result and any deviations; use positive, negative and blank controls.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator (36 ± 2) °C | thermostatically controlled | — |
| Quanti-Tray sealer | seals the tray and automatically distributes the liquid into the wells | — |
| Quanti-Tray trays (51 wells) or Quanti-Tray/2000 (97 wells) | the choice depends on the expected number (< 200 or up to 2419/100 ml) | — |
| Quanti-Tray comparator and UV lamp 365 nm | reading in a dark room or in a darkened cabinet | — |
| Sterile wide-mouth vessels of ≥ 110 ml | bottles with antifoam B (optional) | — |
| Autoclave and drying oven for sterilisation | sterilisation in accordance with ISO 8199 | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Colilert-18 (dehydrated medium in a single-use pack, 2.8 g) | — | medium with the defined substrates ONPG and MUG |
| Sterile deionised or tap water free from oxidants | — | diluent (without buffer, salt or peptone) |
| Antifoam B (10 % silicone suspension) | — | optional — limits air bubbles in the wells |
Health and safety (OHS)
- Work with material potentially containing intestinal pathogens — the rules of good microbiological practice, disinfection and autoclaving of the used trays
- UV lamp 365 nm — protection of the eyes and the skin during the reading
- The sealer becomes hot — do not touch the heating elements
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 9308-2:2014-06 (ISO 9308-2:2012) — “Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 2: Most probable number method”.
How does this method work?
A single-use snap pack of dehydrated Colilert-18 medium (2.8 g) is added to the water sample (100 ml) or to a dilution made up to 100 ml, mixed until it has dissolved and aseptically poured into a Quanti-Tray (up to 201 organisms/100 ml, 51 wells) or a Quanti-Tray/2000 (up to 2419 organisms/100 ml, 97 wells); the trays are sealed in a Quanti-Tray sealer and incubated at (36 ± 2) °C for 18–22 h.
What is the measuring range and accuracy?
Volume of sample: 100 ml (or a dilution made up to 100 ml); sea water 1→10; Medium: Colilert-18, 2.8 g per test; storage at 2–25 °C away from sunlight; Incubation: (36 ± 2) °C, 18–22 h; Reading: yellow ≥ comparator = coliforms; yellow + fluorescence under UV at 365 nm = E. coli.
How long does the test take?
The procedure comprises 6 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Incubator (36 ± 2) °C, Quanti-Tray sealer, Quanti-Tray trays (51 wells) or Quanti-Tray/2000 (97 wells), Quanti-Tray comparator and UV lamp 365 nm, Sterile wide-mouth vessels of ≥ 110 ml, Autoclave and drying oven for sterilisation.
Where is this test used?
Monitoring of drinking water (the reference method under the regulation of the Minister of Health of 22 May 2026, Dz.U. 2026 poz. 748 [Journal of Laws 2026, item 748], annex No 4 part A point 1) and of water in distribution networks — a rapid result after 18 h; Waters with a high content of suspended matter and a high background of heterotrophs, where membrane filtration fails (surface waters, raw waters, wells); Sea water and brackish water — determination of E. coli after a dilution of 1→10 (coliform bacteria in sea water excluded); Presence/absence test in 100 ml and the classical arrangement of MPN tubes with the same medium; Laboratories of sanitary-epidemiological stations and of waterworks — no need for confirmation and for counting colonies.
What safety precautions apply?
Work with material potentially containing intestinal pathogens — the rules of good microbiological practice, disinfection and autoclaving of the used trays; UV lamp 365 nm — protection of the eyes and the skin during the reading; The sealer becomes hot — do not touch the heating elements.
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 9308-2.