Make the hydrogen sulfide test medium by dissolving 20 grams of peptone, 10 grams of ferric ammonium citrate and 5 grams of sodium thiosulfate in 1 liter of water and boiling it for 20 minutes, pour 10 milliliters into each sealable vial, add 20 milliliters of sample, and incubate at 25-35 degrees C for 24-48 hours: black means gut bacteria are present. Boiling does not kill bacterial spores, so run an uninoculated blank from every batch and throw the batch away if it turns black.
Part of Community Water Testing
The H2S test kit is a set of sealed vials of nutrient medium that turns black when a water sample contains hydrogen-sulfide-producing bacteria, used as a field screen for faecal contamination.
The Kit Is Only As Good As Its Controls
The recipe and procedure below are those on the community water testing page. Written down alone they leave out the checks that tell you whether the kit is working, which is where a home-made test goes wrong. Peptone is a digested protein sold for culture media; if you cannot get peptone, ferric ammonium citrate and thiosulfate, you cannot make the kit to this recipe. The ingredients carry three roles: peptone is food for the bacteria and a source of sulfur, thiosulfate is a further sulfur source that the bacteria turn into hydrogen sulfide, and the iron in the ferric ammonium citrate reacts with hydrogen sulfide to form black iron sulfide.
The page also names a simplified version, rusty iron nails dissolved in vinegar for two weeks, then mixed with gelatin and water. I could not source that variant and I do not know whether it works. Treat any result from it as unvalidated until it has been checked against known positives and blanks, as described below.
What You Need
- Peptone, ferric ammonium citrate and sodium thiosulfate, with a scale that reads grams
- Clean sealable vials of glass or sturdy plastic, about 40 ml
- A pan, a measuring jug and a clock, or a pressure cooker if you have one
- A warm place, 25-35 °C, and a thermometer
- Boiled and cooled water for blanks
- A permanent marker, labels and a log
Method: Make It, Prove It, Use It
Step 1: Make the medium. Dissolve 20 g peptone, 10 g ferric ammonium citrate and 5 g sodium thiosulfate in 1 L of clean water. Boil for 20 minutes.
Step 2: Fill the vials. Pour 10 ml into each. Seal at once. Label with the batch and date.
Step 3: Make the blank. Keep several vials unopened from every batch and incubate one with the rest. It must stay yellow or clear. If it blackens, the batch is contaminated: boiling does not kill spores, and spore-forming gut-type bacteria can survive 20 minutes of boiling. Throw the batch away, and, if possible, sterilise in a pressure cooker next time. I am recalling the spore point from general microbiology.
Step 4: Make a positive control. Stir a pinch of garden soil into a cup of boiled, cooled water, let it settle, and add 20 ml of the clear liquid to a vial. It should blacken. If it never does, the medium is not working. This control is my suggestion and has not been validated here; use it only to show that the medium can turn black.
Step 5: Run the sample. Add 20 ml of the sample, seal and incubate at 25-35 °C for 24-48 hours, with a blank and a positive alongside.
Step 6: Read at 24 and 48 hours against white paper. Black is positive. Clear or yellow is negative.
Worked Example: How Much To Make
- Per vial: 10 ml of medium holds 0.2 g peptone (20 g per 1,000 ml × 10 ml), 0.1 g ferric ammonium citrate and 0.05 g thiosulfate.
- One liter makes 1,000 ÷ 10 = 100 vials.
- A monthly program of 5 samples (source, tank outlet and three taps), plus one blank and one positive control, uses 7 vials a month, 84 a year. One liter lasts about 14 months, but make the batch in smaller amounts, since its shelf life is unknown.
- If you test 100 ml at each site as five 20 ml vials (Coliform Detection), a month uses 5 × 5 = 25 vials plus two controls = 27, and a liter lasts about 3.7 months.
A half-liter batch needs 10 g peptone, 5 g ferric ammonium citrate and 2.5 g thiosulfate and makes 50 vials. Date every batch; make a fresh blank each time you start a new one; and throw away any vial that has turned dark or cloudy before use.
Sensitivity Check
Take the positive-control liquid and dilute it in steps with boiled, cooled water, say 1 part in 10, 1 in 100, and so on, running 20 ml of each. The last dilution that still blackens shows how weak a contamination your kit can see. Record it, because it changes with the batch.
Interference And Failure Modes
- Black blank: contaminated ingredients or vials, or spores that survived the boil. Discard the batch.
- All samples black, including clean ones: the water or container carries sulfide or iron, or the medium is contaminated.
- Nothing ever blackens, including the positive: the medium is dead, too old, wrongly weighed or kept too cold or too hot.
- Water with natural sulfide or high iron: may darken the vial without bacteria. Run a blank of the same water, treated by boiling and cooling, in a second vial.
- Chlorinated sample: residual chlorine kills bacteria in the bottle; the excess thiosulfate in the medium neutralises it once mixed, but move the sample into the vial promptly.
Common Mistakes
- Reporting a result without a blank and a positive alongside.
- Using sample volumes other than the recipe’s. The ratio of sample to medium matters.
- Trusting a clear vial. The test misses low contamination (Coliform Detection).
- Letting the vials sit warm in the sun.
Safety
Incubated vials are cultures of gut bacteria
A black vial contains grown faecal bacteria in high numbers. Never open or shake it, never use the vials as drinking containers, keep them away from food and children, and wash hands after handling. Kill the contents before disposal with a strong chlorine solution or by boiling, never mixing chlorine with acids, vinegar or ammonia products. Do not incubate near cooking or sleeping areas.
What’s Next
- Learn next: Test Log — recording each result with its controls
- Combine with: Routine Testing — how many vials the schedule needs