The hydrogen sulfide presence/absence test detects faecal contamination by blackening a growth medium when gut bacteria that make hydrogen sulfide are present in a water sample, read after 24-48 hours of warm incubation. A black vial means act at once, but a clear vial means little: a 20 milliliter sample taken from water with 1 organism per 100 milliliters contains none about 82% of the time, so a negative result never proves the water is safe.
Part of Community Water Testing
Coliform detection is the field screening of water for bacteria that live in the gut, using a presence/absence test that signals whether faeces have reached the supply.
The Test Looks For A Marker, Not For Disease
Disease germs are too many and too hard to find to test for one by one. Testing looks instead for bacteria that live in the gut and are found where faeces have been. The community water testing page uses a hydrogen sulfide test: bacteria that make hydrogen sulfide react with iron in the medium and turn it black. It is the most important field test on that page and the biological check on which the boil-water advisory decision rests.
Two points about what it detects. First, the test is a stand-in. As I recall, the main indicator organism, E. coli, usually does not make hydrogen sulfide itself; the test finds other gut bacteria that do and that are generally found alongside faecal contamination. The link to faecal contamination is statistical, not exact. Second, bacteria that make hydrogen sulfide also exist in nature, and water that already contains sulfide and iron can darken the medium, so a black vial in an unusual water needs a blank for comparison (H2S Test Kit).
What You Need
- A test kit made as described in H2S Test Kit
- Clean, sealable sample containers
- A warm place, 25-35 °C, for 24-48 hours
- A positive and a negative control for each batch
- A log (Test Log)
Method: Collect Cleanly, Test Promptly, Read Twice
Step 1: Collect the sample. Use a container washed and rinsed in boiled, cooled water. At a tap, wipe the spout and let the water run for a minute or two first. At a source, hold the container by its base and lower it below the surface, away from the edge, and fill it without letting hands or the rim touch the water.
Step 2: Test promptly. Add the sample to the vial as soon as you can, within hours, keeping it cool and out of the sun. A sample sitting in a warm bottle changes. If the water is chlorinated, residual chlorine keeps killing the bacteria in the bottle; the medium contains a large excess of thiosulfate, which neutralises it, so move the sample into the medium without delay.
Step 3: Add 20 ml to the vial holding 10 ml of medium, seal it, and incubate.
Step 4: Read at 24 hours and at 48 hours. Black means bacteria present. Clear or yellow means none seen. Read each batch’s controls first.
Step 5: Log the result, the place, the time, and the sample volume.
Worked Example: How Often Does A 20 ml Sample Miss?
If organisms are spread at random in the water and the average is λ per 100 ml, the chance that a sample of volume v ml contains none at all is e^(-λ × v ÷ 100). This assumes one organism is enough to start growth, which is generous to the test.
- 1 per 100 ml, 20 ml sample: e^(-0.2) = 0.82. The sample misses 82% of the time and detects 18%.
- 2 per 100 ml: e^(-0.4) = 0.67, so it misses 67% and detects 33%.
- 5 per 100 ml: e^(-1) = 0.37, so it misses 37% and detects 63%.
- 10 per 100 ml: e^(-2) = 0.135, so it misses 13.5% and detects 86.5%.
The drinking-water guideline I am recalling expects no E. coli in any 100 ml. A 20 ml sample is five times smaller than that. To test 100 ml you would use five 20 ml vials from the same place, or scale up the vial. At 1 per 100 ml, 100 ml has e^(-1) = 0.37, so the miss rate falls from 82% to 37%. That is still not safety.
This is why the carrier’s rule, lifting an advisory only after two consecutive negative tests 24 hours apart, is a minimum, not a guarantee, and why the warning “a negative test does not guarantee water is safe” matters.
What A Result Does And Does Not License
A black vial licenses action now: advise boiling, increase treatment, look for the cause (Responding to Failures). A clear vial licenses only this: no sign of faecal bacteria in this sample, in this vial, at this moment. It does not cover viruses, parasites or chemicals, it can miss low-level contamination, and a good sample collected badly is worth nothing. Contamination comes and goes: a clear result the day before an outbreak is normal.
Common Mistakes
- Treating a clear vial as a clearance. Check the miss rate above.
- Dirty collection. A bad sample gives a false positive or a meaningless negative.
- Skipping controls. You cannot tell a working medium from a dead one.
- Waiting a day to put the sample into the vial. Bacteria die or multiply in the bottle.
- Reading after 48 hours. Late darkening is unreliable.
Safety
A positive vial is a culture of faecal bacteria
Never open, shake or smell a black vial. Keep incubating vials away from food, kitchens and children. Dispose of positives by killing the contents with a strong chlorine solution or boiling before they are poured away, and wash hands after handling. Do not mix chlorine with acids, vinegar or ammonia products when doing so.
What’s Next
- Learn next: H2S Test Kit — making the medium, the controls and the vials
- Combine with: Routine Testing — how often and where to sample