Autoclave Spore Tests: Biological Indicators Explained
A spore test is the only routine check that shows a cycle actually killed something very hard to kill. What is inside a biological indicator, how to run, incubate and read one, how often, and exactly what to do when a vial turns positive.
What a biological indicator is
The spores
Steam biological indicators (BIs) carry spores of Geobacillus stearothermophilus, a heat-loving bacterium whose spores are among the most resistant to moist heat. ISO 11138-3 sets a minimum challenge: at least 100,000 spores per indicator and a D-value at 121 °C of at least 1.5 minutes.
D-value
The time at a given temperature that kills 90% of the spores. With a 121 °C D-value of 1.5 minutes, every 1.5 minutes at 121 °C cuts the surviving population tenfold, so a correct cycle leaves a wide margin.
Why spores as well as chemistry
Chemical indicators react to conditions. A BI shows the biological result — whether highly resistant life survived — so it backs up the printout and the colour changes with direct evidence.
Read next Where spore tests fit with Bowie-Dick and chemical indicators
Types of biological indicator
| Type | How it works | Result in |
|---|---|---|
| Self-contained BI (SCBI) | A spore carrier and a sealed ampoule of growth medium in one vial. After the cycle the ampoule is crushed and the vial incubated; a pH dye changes colour if spores grow | Typically 24 hours to 2 days, as the maker states |
| Rapid-readout BI | A self-contained vial read in a dedicated auto-reader that detects a spore enzyme by fluorescence | From under an hour to a few hours, depending on the product |
| Spore strip | A paper strip in an envelope; after the cycle a laboratory transfers it into growth medium under aseptic conditions | Several days, usually through a laboratory or mail-in service |
Running a spore test
- Use a BI made for steam and for the temperature you run, within its expiry date, stored as the label says.
- Put it inside a pouch or test pack and place it where the steriliser or BI maker specifies — normally the part of the load steam reaches last.
- Run it in a normal, full load on the cycle you use most, not in an empty chamber.
- Let it cool for the time the maker states, then activate it — usually by crushing the media ampoule — and check the medium has wetted the spore carrier.
- Activate an unprocessed control BI from the same lot and incubate it alongside.
- Incubate at the temperature the maker specifies — usually somewhere between 55 °C and 60 °C for steam BIs — for the full stated time.
- Record the result of the test and the control, the cycle, the date and who read it.
The control vial and the process challenge device
Why the control matters
The unprocessed control from the same lot must turn positive. That proves the spores were alive, the medium works and the incubator was at temperature. If the control does not grow, the test tells you nothing — repeat it with a new lot.
Process challenge device (PCD)
A PCD holds the BI behind a defined resistance to steam — a test pack, a tray system or a hollow device — so the test is at least as hard as the hardest item in a real load. Use the PCD your steriliser maker or infection-control policy specifies for your loads.
Hollow loads
Clinics sterilising handpieces and lumened instruments may use a hollow-load PCD that holds the indicator at the far end of a long, narrow path, so the test reflects the load that is hardest to sterilise.
Dental handpiece sterilisationHow often to test
| When | Spore test |
|---|---|
| Routine | At least weekly, the CDC benchmark for dental settings; some infection-control policies require more often |
| Every load with an implant | Yes — and implants ideally held until the BI reads negative (CDC) |
| After installation, relocation or major repair | Before the steriliser goes back into patient use, as the maker or your policy specifies |
| After a positive result | Repeat at once; after a repair, three consecutive fully loaded cycles must pass before return to service (CDC) |
| New packaging, load type or cycle | Test before it goes into routine use |
Read next Validation: IQ, OQ and PQ
When a spore test is positive
A single positive is a reason to stop and investigate, not to panic. Check the control first, then follow your written protocol. CDC’s protocol for dental settings, in short:
- Take the steriliser out of service and review how the load was packed, loaded and run.
- Check the cycle printout and chemical indicators — if they all passed, a single positive often points to a test or handling error rather than a faulty steriliser.
- Recall and quarantine any implantable items from the load until they are shown to be sterile.
- Repeat the spore test straight away on the same cycle.
- If the repeat is negative and procedures were correct, the steriliser can return to service.
- If the repeat is positive, keep it out of use until it has been inspected or repaired and has passed spore tests in three consecutive fully loaded cycles. Where possible, recall, rewrap and re-sterilise items from suspect loads back to the last negative test.
Read next Autoclave troubleshootingTracing packs back to a cycle
Records to keep
- Date, steriliser, cycle number and cycle type for every test.
- BI lot number and expiry date, the test and control results, the incubation time and who read them.
- Every action taken after a positive, including recalls, repairs and retests.
- Keep spore test records with the cycle printouts and chemical indicator records, for as long as your policy and the regulations that apply to you require.
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