🦠 Spores of sulfite-reducing anaerobes (clostridia) in water — membrane filtration
In short
The water sample is heated for 15 min at (75 ± 5) °C in order to kill the vegetative forms, filtered through a 0. The test is performed according to PN-EN 26461-2:2001 (EN 26461-2:1993, ISO 6461-2:1986); Selection of the spores: heating at (75 ± 5) °C for 15 min from the moment the temperature is reached; control with a bottle fitted with a thermometer. The procedure comprises 6 steps; it is used for: Testing of natural mineral, spring and table waters at the point of abstraction and in packages (the requirement of 0 in 50 ml under the regulation of the Minister of Health of 2011), Drinking water, water from wells and springs, slightly contaminated surface waters — volume 100 ml, Assessment of the effectiveness of treatment and of chlorination (the spores as an indicator of remote or intermittent contamination).
At a glance
- Standard: PN-EN 26461-2:2001 (EN 26461-2:1993, ISO 6461-2:1986)
- Category: Microbiology
- Procedure steps: 6
- Selection of the spores: heating at (75 ± 5) °C for 15 min from the moment the temperature is reached; control with a bottle fitted with a thermometer
- Filter: sterile membrane of a nominal pore diameter of 0.2 µm; quality in accordance with ISO 7704
- Volume: 100 ml (drinking, spring, well, mineral, sea and surface waters); volumes < 10 ml to be mixed with 10–100 ml of sterile water
Overview
The spores of sulfite-reducing anaerobes (clostridia) are widely distributed in the environment: they occur in the faeces of humans and animals, in waste water and in the soil. Unlike Escherichia coli and other coliform bacteria, the spores survive in water for a long time, because they are more resistant than the vegetative forms to chemical and physical agents — they may therefore indicate remote or intermittent contamination; they are sometimes resistant even to chlorination at the doses used in treatment and are for that reason useful for control purposes. ISO 6461/2-1986 (in Poland PN-EN 26461-2:2001) specifies a method for the detection and enumeration of the spores of clostridia by membrane filtration; it is applied to all kinds of water, except samples in which a large amount of particles would be retained on the membrane. Clostridia are defined as sulfite-reducing, spore-forming, anaerobic micro-organisms of the family Bacillaceae and of the genus Clostridium. In Poland the regulation of the Minister of Health of 31 March 2011 on natural mineral waters, spring waters and table waters (Dz.U. 2011 nr 85 poz. 466 [Journal of Laws 2011, No. 85, item 466]) requires the absence of sulfite-reducing clostridia (including the spores) in 50 ml of water at the point of abstraction; laboratories apply this standard in the testing of bottled waters (lists of media: sulfite agar in accordance with PN-EN 26461-2:2001).
Method principle
The detection of the spores in a given volume of water requires three stages: selection of the spores by heating the sample for a time sufficient to destroy the vegetative bacteria; filtration through a membrane with pores retaining the spores (0.2 µm); placing of the filter on a selective medium (sulfite-iron agar) and incubation at (37 ± 1) °C for (20 ± 4) h and (44 ± 4) h with counting of the black colonies. The basic medium is nutrient agar (meat extract, peptone 10 g, NaCl 5 g, agar, water 1000 ml; pH 7.6 ± 0.1 adjusted with 1 mol/l NaOH; sterilisation for 20 min at (121 ± 1) °C; distributed in 18 ml portions into tubes, stored at 4–5 °C and discarded after 2 weeks); immediately before use 1 ml of a sodium sulfite solution (10 g/100 ml, freshly prepared every 2 weeks) and 5 drops of an iron(II) sulfate solution (8 g/100 ml) are added to 18 ml of the melted medium. An alternative is tryptose sulfite agar (tryptose 15 g, soytone 5 g, yeast extract 5 g, sodium metabisulfite, ammonium iron(III) citrate 1 g). The pH of the media is measured with a pH meter with reference to 25 °C, and the quality of the filters is checked in accordance with ISO 7704, because it may differ between brands and batches.
Applications
- Testing of natural mineral, spring and table waters at the point of abstraction and in packages (the requirement of 0 in 50 ml under the regulation of the Minister of Health of 2011)
- Drinking water, water from wells and springs, slightly contaminated surface waters — volume 100 ml
- Assessment of the effectiveness of treatment and of chlorination (the spores as an indicator of remote or intermittent contamination)
- Waste water and heavily contaminated waters — smaller volumes filtered after mixing with sterile water
- Indicator accompanying Clostridium perfringens (PN-EN ISO 14189) in the monitoring of waters
Key parameters
| Parameter | Value |
|---|---|
| Selection of the spores | heating at (75 ± 5) °C for 15 min from the moment the temperature is reached; control with a bottle fitted with a thermometer |
| Filter | sterile membrane of a nominal pore diameter of 0.2 µm; quality in accordance with ISO 7704 |
| Volume | 100 ml (drinking, spring, well, mineral, sea and surface waters); volumes < 10 ml to be mixed with 10–100 ml of sterile water |
| Medium | sulfite-iron agar (18 ml of medium + 1 ml of Na₂SO₃ 10 % + 5 drops of FeSO₄ 8 %), pH 7.6 ± 0.1; alternatively tryptose sulfite agar |
| Incubation | (37 ± 1) °C anaerobically; reading after (20 ± 4) h and (44 ± 4) h |
| Result | black colonies per volume of sample; the reading after 44 h is reported (the one after 20 h only as approximate) |
| Requirement (mineral waters, PL) | absence of sulfite-reducing clostridia (including the spores) in 50 ml |
Standard
- Standard number
- PN-EN 26461-2:2001 (EN 26461-2:1993, ISO 6461-2:1986)
- Title (PL)
- Jakość wody — Wykrywanie i oznaczanie ilościowe przetrwalników beztlenowców redukujących siarczyny (clostridia) — Część 2: Metoda filtracji membranowej
- Title (EN)
- Water quality — Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) — Part 2: Method by membrane filtration
Step-by-step procedure
-
Sampling and preparation
Take the samples in accordance with ISO 5667-2 and ISO 8199; store them in accordance with ISO 5667-3.
-
Selection of the spores
Heat the sample in a water bath at (75 ± 5) °C for 15 min counted from the moment that temperature is reached; from time to time use a control bottle with the same volume of water and a thermometer to check the heating time.
-
Filtration
In accordance with ISO 7704 filter a suitable volume: 100 ml for drinking, spring, well, mineral and sea water and for slightly contaminated surface water; less for heavily contaminated waters and waste water (mix volumes < 10 ml with 10–100 ml of sterile water). Choose the dilutions so that the colonies are well separated.
-
Covering the membrane with the medium
With sterile forceps transfer the membrane, filtering side downwards, onto the bottom of a Petri dish without air bubbles; holding it down, carefully pour over it 18 ml of the melted complete medium (with the sulfite and the iron sulfate) cooled to about 50 °C and leave it to solidify.
-
Incubation
Incubate anaerobically (or under other conditions ensuring anaerobiosis) at (37 ± 1) °C for (20 ± 4) h and (44 ± 4) h; in a jar or an anaerobic incubator the membrane may be placed on the surface of the agar with the filtering side upwards.
-
Reading and test report
Count all the black colonies after (20 ± 4) h and (44 ± 4) h; express the result in accordance with ISO 8199 as the number of sulfite-reducing anaerobes (clostridia) per volume of sample — usually the reading after 44 h, the reading after 20 h only as approximate; describe any deviations and events that could affect the result.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Membrane filtration set | funnel, base, vacuum pump | — |
| Water bath (75 ± 5) °C with a thermometer | a control bottle of the same volume to check the heating time | — |
| Incubator (37 ± 1) °C and an anaerobic system | anaerobic jar or anaerobic incubator (the filter may then lie on the surface of the agar facing upwards) | — |
| Autoclave, steamer, pH meter | sterilisation of the media at (121 ± 1) °C; pH at 25 °C | — |
| Tubes 160 × 16 mm, pipettes 10 ml (graduated in 0.1 ml), flasks of 2 l, jars of 1 l, Petri dishes, sterile forceps | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Nutrient agar (meat extract, peptone 10 g, NaCl 5 g, agar, water to 1 l), pH 7.6 ± 0.1 | — | basic medium, distributed in 18 ml portions |
| Sodium sulfite Na₂SO₃ — 10 g in 100 ml of water (freshly prepared every 2 weeks) | — | selective-differential component (1 ml per 18 ml of medium) |
| Iron(II) sulfate FeSO₄ — 8 g of the crystalline salt in 100 ml of water | — | indicator — black iron sulfide around the colonies (5 drops per 18 ml) |
| Tryptose sulfite agar (tryptose 15 g, soytone 5 g, yeast extract 5 g, sodium metabisulfite, ammonium iron(III) citrate 1 g/l) | — | alternative medium |
| Sodium hydroxide 1 mol/l | — | adjustment of the pH of the medium |
| Distilled or deionised water free from inhibiting substances (ISO 3696) | — | media and dilution of small volumes of sample |
Health and safety (OHS)
- Cultures of anaerobes of the genus Clostridium (including potentially C. perfringens) — work in a microbiological laboratory, autoclaving of the waste
- Hot water bath and melted agar at about 50 °C — take care when pouring it over the membrane
- Anaerobic jars with gas generators — follow the manufacturer's instructions
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN 26461-2:2001 (EN 26461-2:1993, ISO 6461-2:1986) — “Water quality — Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) — Part 2: Method by membrane filtration”.
How does this method work?
The detection of the spores in a given volume of water requires three stages: selection of the spores by heating the sample for a time sufficient to destroy the vegetative bacteria; filtration through a membrane with pores retaining the spores (0.2 µm); placing of the filter on a selective medium (sulfite-iron agar) and incubation at (37 ± 1) °C for (20 ± 4) h and (44 ± 4) h with counting of the black colonies.
What is the measuring range and accuracy?
Selection of the spores: heating at (75 ± 5) °C for 15 min from the moment the temperature is reached; control with a bottle fitted with a thermometer; Filter: sterile membrane of a nominal pore diameter of 0.2 µm; quality in accordance with ISO 7704; Volume: 100 ml (drinking, spring, well, mineral, sea and surface waters); volumes < 10 ml to be mixed with 10–100 ml of sterile water; Medium: sulfite-iron agar (18 ml of medium + 1 ml of Na₂SO₃ 10 % + 5 drops of FeSO₄ 8 %), pH 7.6 ± 0.1; alternatively tryptose sulfite agar.
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?
Membrane filtration set, Water bath (75 ± 5) °C with a thermometer, Incubator (37 ± 1) °C and an anaerobic system, Autoclave, steamer, pH meter, Tubes 160 × 16 mm, pipettes 10 ml (graduated in 0.1 ml), flasks of 2 l, jars of 1 l, Petri dishes, sterile forceps.
Where is this test used?
Testing of natural mineral, spring and table waters at the point of abstraction and in packages (the requirement of 0 in 50 ml under the regulation of the Minister of Health of 2011); Drinking water, water from wells and springs, slightly contaminated surface waters — volume 100 ml; Assessment of the effectiveness of treatment and of chlorination (the spores as an indicator of remote or intermittent contamination); Waste water and heavily contaminated waters — smaller volumes filtered after mixing with sterile water; Indicator accompanying Clostridium perfringens (PN-EN ISO 14189) in the monitoring of waters.
What safety precautions apply?
Cultures of anaerobes of the genus Clostridium (including potentially C. perfringens) — work in a microbiological laboratory, autoclaving of the waste; Hot water bath and melted agar at about 50 °C — take care when pouring it over the membrane; Anaerobic jars with gas generators — follow the manufacturer's instructions.
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 26461-2.