🦠 E. coli in surface water and waste water — miniaturised MPN method in microtitre plates (MUG)
In short
Dilutions of the sample (1/2, 1/20, 1/200 and so on) in a special diluent with sea salt are inoculated in 200 µl portions into the wells of 96-well plates containing dehydrated MUG/EC medium, incubated for 36–72 h at (44 ± 0. The test is performed according to PN-EN ISO 9308-3:2002 (ISO 9308-3:1998 + Cor 1:2000); Range: bathing water 15–3.5·10⁴; surface waters 40–3.2·10⁶; waste water 60–6.7·10⁸ E. coli/100 ml. The procedure comprises 7 steps; it is used for: Monitoring of bathing water quality — the reference method for E. coli under Directive 2006/7/EC (inland, coastal and transitional waters), Surface waters (rivers, lakes) rich in suspended matter, where membrane filtration is of no use, Raw and treated waste water, effluents from treatment plants (range up to 6.7·10⁸/100 ml).
At a glance
- Standard: PN-EN ISO 9308-3:2002 (ISO 9308-3:1998 + Cor 1:2000)
- Category: Microbiology
- Procedure steps: 7
- Range: bathing water 15–3.5·10⁴; surface waters 40–3.2·10⁶; waste water 60–6.7·10⁸ E. coli/100 ml
- Limitation: not for drinking water or for waters with a guideline value < 15/100 ml; it does not enumerate other coliform bacteria
- Incubation: (44 ± 0.5) °C, min. 36 h, max. 72 h
Overview
ISO 9308-3:1998 specifies a miniaturised method for the detection and enumeration of Escherichia coli in surface water and waste water by inoculation in a liquid medium; it is applied to all kinds of surface water and waste water, in particular those rich in suspended matter. It is not suitable for drinking water or for other waters for which the guideline value is lower than 15 bacteria/100 ml, and it does not serve to detect coliform bacteria other than E. coli. E. coli is defined as a β-D-glucuronidase-positive micro-organism growing at 44 °C in a liquid medium with 4-methylumbelliferyl-β-D-glucuronide (MUG). The method has been validated in interlaboratory studies (annex D). In the European Union, Directive 2006/7/EC concerning the management of bathing water quality names ISO 9308-3 or ISO 9308-1 as the reference methods for the determination of E. coli (cfu/100 ml) in inland waters and in coastal and transitional waters. In Poland it is in force as PN-EN ISO 9308-3:2002.
Method principle
The diluted sample is inoculated into a row of wells of a microtitre plate containing dehydrated culture medium. After incubation for at least 36 h and at most 72 h at (44 ± 0.5) °C the plates are examined in the dark under UV light at 366 nm — the presence of E. coli is shown by the blue fluorescence arising from the hydrolysis of MUG; the result is given as the most probable number (MPN) per 100 ml. The MUG/EC medium contains tryptose 40 g, salicin 1 g, Triton X-100 1 g and MUG 100 mg per 1000 ml (the MUG dissolved in 2 ml of N,N-dimethylformamide), pH 6.9 ± 0.2, sterilised by filtration at 0.2 µm, distributed in 100 µl portions into the wells of 96-well plates (capacity ≥ 350 µl) and immediately dehydrated in a tunnel dryer or in a laminar-flow cabinet; its manufacture must meet the quality criteria of annex E. The special diluent (SD) is 22.5 g of synthetic sea salt per 1000 ml of water (optionally with bromophenol blue to distinguish it from water), sterilised for 15–20 min at (121 ± 3) °C; for sea water (salinity > 30 g/kg) the first dilution is made in demineralised water. The number of dilutions and of wells depends on the expected contamination: bathing water — 2 dilutions (64 wells of 1/2 and 32 wells of 1/20; range 15–3.5·10⁴/100 ml), fresh surface waters — 4 dilutions of 24 wells each (40–3.2·10⁶), waste water and treatment plants — 6 dilutions of 16 wells each (60–6.7·10⁸). From the number of positive wells in each dilution a characteristic number is formed, and the MPN and the 95 % confidence interval are calculated by the program of annex A or B (Poisson distribution); where there are no positive wells the result is given as "< n/100 ml".
Applications
- Monitoring of bathing water quality — the reference method for E. coli under Directive 2006/7/EC (inland, coastal and transitional waters)
- Surface waters (rivers, lakes) rich in suspended matter, where membrane filtration is of no use
- Raw and treated waste water, effluents from treatment plants (range up to 6.7·10⁸/100 ml)
- Sea water and brackish water — thanks to the diluent with sea salt
- Large series of tests — the 96-well plate format and multichannel pipettes
Key parameters
| Parameter | Value |
|---|---|
| Range | bathing water 15–3.5·10⁴; surface waters 40–3.2·10⁶; waste water 60–6.7·10⁸ E. coli/100 ml |
| Limitation | not for drinking water or for waters with a guideline value < 15/100 ml; it does not enumerate other coliform bacteria |
| Incubation | (44 ± 0.5) °C, min. 36 h, max. 72 h |
| Reading | blue fluorescence under UV at 366 nm (Wood's lamp), in the dark; the fluorescence does not change with time |
| Inoculum | 200 µl per well; 96-well plate, 350 µl, flat-bottomed, non-fluorescing |
| Diluent SD | 22.5 g of synthetic sea salt per litre; sterilisation at (121 ± 3) °C for 15–20 min |
| MUG/EC medium | tryptose 40 g, salicin 1 g, Triton X-100 1 g, MUG 100 mg/l; pH 6.9 ± 0.2; filtration at 0.2 µm; 100 µl/well, dehydrated |
Standard
- Standard number
- PN-EN ISO 9308-3:2002 (ISO 9308-3:1998 + Cor 1:2000)
- Title (PL)
- Jakość wody — Wykrywanie i oznaczanie ilościowe Escherichia coli i bakterii grupy coli — Część 3: Zminiaturyzowana metoda wykrywania i oznaczania E. coli w wodach powierzchniowych i w ściekach (najbardziej prawdopodobna liczba bakterii)
- Title (EN)
- Water quality — Detection and enumeration of Escherichia coli and coliform bacteria in surface and waste water — Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium
Step-by-step procedure
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Sampling
Take and deliver the samples in accordance with ISO 8199 and ISO 5667-1, -2 and -3.
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Choice of the dilutions
Match the number of dilutions and of wells to the expected contamination (table 1 of the standard): bathing water — 64 wells of 1/2 and 32 wells of 1/20; surface waters — 4 dilutions of 24 wells each; waste water — up to 6 dilutions of 16 wells each.
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Dilutions
Add 9 ml portions of the diluent SD to the tubes (for sea water of a salinity > 30 g/kg — the first tube with 9 ml of demineralised water). Mix the sample vigorously and transfer 9 ml into the first tube (1/2), then 1 ml into the next one (1/20), and further 1 ml portions (1/200 and so on), changing the pipettes. Work in a biological safety cabinet (aerosols).
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Inoculation of the plates
Pour the contents of the tube into a sterile Petri dish 90 mm and, with an 8-channel pipette, distribute 200 µl portions into the wells corresponding to the given dilution; for the successive dilutions change the Petri dish and the tips. Take care that there is no overflow between the wells.
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Incubation
Seal the plate with a sterile self-adhesive film and incubate it at (44 ± 0.5) °C for 36–72 h, without tilting the plates.
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Reading
Place the plate with its film in the UV cabinet; consider as positive all the wells with a blue fluorescence (the reading is possible at any time after 36 h).
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Calculations
For each dilution record the number of positive wells as the characteristic number (with ≥ 3 dilutions — a three-digit one, the last digit being 0 as far as possible, in accordance with ISO 8199); calculate the MPN per ml and the 95 % confidence interval with the program of annex A or B and convert it to 100 ml (e.g. 32/5 → 756/100 ml, 542–1054); where there are no positive wells give "< n/100 ml".
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Thermostatically controlled incubator (44 ± 0.5) °C | — | |
| UV viewing cabinet (Wood's lamp 366 nm) | reading in the dark; protective goggles and gloves | — |
| 8-channel pipette (200 µl) with sterile tips | or another system dispensing 200 µl per well | — |
| Sterile 96-well microtitre plates, 350 µl, flat-bottomed, non-fluorescing, with sterile self-adhesive films | with dehydrated MUG/EC medium | — |
| Tunnel dryer or laminar-flow cabinet (preferably class II) | dehydration of the medium in the plates | — |
| Membrane filtration set 0.2 µm, pH meter (± 0.1), portable refractometer (optional), tubes 16 × 160 and 20 × 200 mm, sterile Petri dishes 90 mm | sterilisation of the medium, measurement of the salinity, dilutions | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| MUG/EC medium (tryptose, salicin, Triton X-100, MUG) | — | detection of the β-D-glucuronidase of E. coli — fluorescence |
| N,N-dimethylformamide (2 ml per 1000 ml of medium) | — | dissolution of the MUG; toxic and carcinogenic — work in a fume cupboard |
| Synthetic sea salt (22.5 g/l) — the special diluent SD | — | dilutions; optionally bromophenol blue 0.04 g/100 ml of 50 % ethanol as a dye |
| Demineralised or distilled water, sterile | — | first dilution of sea water; preparation of the media |
Health and safety (OHS)
- N,N-dimethylformamide is toxic and may cause cancer; harmful by inhalation, through the skin and if swallowed — work in a chemical fume cupboard
- UV radiation irritates the eyes and the skin — protective goggles and gloves during the reading
- Diluting and pipetting waste water generates aerosols — biological safety cabinet
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 9308-3:2002 (ISO 9308-3:1998 + Cor 1:2000) — “Water quality — Detection and enumeration of Escherichia coli and coliform bacteria in surface and waste water — Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium”.
How does this method work?
The diluted sample is inoculated into a row of wells of a microtitre plate containing dehydrated culture medium. After incubation for at least 36 h and at most 72 h at (44 ± 0.
What is the measuring range and accuracy?
Range: bathing water 15–3.5·10⁴; surface waters 40–3.2·10⁶; waste water 60–6.7·10⁸ E. coli/100 ml; Limitation: not for drinking water or for waters with a guideline value < 15/100 ml; it does not enumerate other coliform bacteria; Incubation: (44 ± 0.5) °C, min. 36 h, max. 72 h; Reading: blue fluorescence under UV at 366 nm (Wood's lamp), in the dark; the fluorescence does not change with time.
How long does the test take?
The procedure comprises 7 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Thermostatically controlled incubator (44 ± 0.5) °C, UV viewing cabinet (Wood's lamp 366 nm), 8-channel pipette (200 µl) with sterile tips, Sterile 96-well microtitre plates, 350 µl, flat-bottomed, non-fluorescing, with sterile self-adhesive films, Tunnel dryer or laminar-flow cabinet (preferably class II), Membrane filtration set 0.2 µm, pH meter (± 0.1), portable refractometer (optional), tubes 16 × 160 and 20 × 200 mm, sterile Petri dishes 90 mm.
Where is this test used?
Monitoring of bathing water quality — the reference method for E. coli under Directive 2006/7/EC (inland, coastal and transitional waters); Surface waters (rivers, lakes) rich in suspended matter, where membrane filtration is of no use; Raw and treated waste water, effluents from treatment plants (range up to 6.7·10⁸/100 ml); Sea water and brackish water — thanks to the diluent with sea salt; Large series of tests — the 96-well plate format and multichannel pipettes.
What safety precautions apply?
N,N-dimethylformamide is toxic and may cause cancer; harmful by inhalation, through the skin and if swallowed — work in a chemical fume cupboard; UV radiation irritates the eyes and the skin — protective goggles and gloves during the reading; Diluting and pipetting waste water generates aerosols — biological safety cabinet.
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 9308-3.