Autoclave spore test: verifying real sterilisation
— Equipo JP Selecta
In steam sterilisation processes, continuous monitoring is essential to guarantee microbiological safety. However, the fact that an autoclave reaches the nominal temperature and pressure values does not absolutely guarantee the elimination of the microbial load.
The autoclave spore test is the reference biological indicator for verifying the real lethal efficacy of a sterilisation cycle, making it the definitive test within quality control protocols.
Unlike physical indicators (which only confirm that the unit ran the temperature ramp) or chemical indicators (which change colour when exposed to certain conditions without certifying the destruction of life), the biological indicator uses living organisms of maximum heat resistance as direct proof of lethality.
What is the spore test and what is it for?
The spore test consists of subjecting a standardised population of bacterial endospores that are highly resistant to moist heat, specifically of Geobacillus stearothermophilus, to the same sterilisation conditions as the real working load.
These spores act as an extreme biological challenge: if the thermal cycle is capable of inactivating them completely, it is concluded that the process has been effective in destroying any other microorganism, virus or vegetative form present in the chamber. If viability or cell multiplication is detected after the subsequent incubation period, the cycle is considered a serious biological failure.
Why is the autoclave spore test the most reliable indicator?
The reliability of the spore test lies in the fact that it directly assesses the final microbiological result and not the physical parameters that condition it. The presence of non-condensable gases, unevacuated air pockets or excess moisture can allow the pressure gauge to show the correct pressure while heat transfer inside the load is insufficient.
For this reason, the autoclave spore test is the reference standard required by Health Authorities and quality inspections, both in the healthcare sector (dental, podiatry, veterinary or surgical clinics) and in the pharmaceutical, biotechnology and food industries.
→ You may be interested in: the Bowie-Dick test and the Helix test, the physico-chemical controls that complement the biological indicator.
How do you carry out the spore test in an autoclave?
To perform the test correctly and obtain reproducible results, the following procedure must be followed:
- Preparation of the biological indicator: place the self-contained vial or the spore-impregnated strip inside a sterilisation pouch or pack identical to the one used with the real working material.
- Placement at the critical point: position the pack with the biological indicator in the thermally least favourable area of the chamber (usually on the lower tray, near the condensate drain or at the geometric centre of a dense load).
- Running the cycle: start a standard sterilisation cycle with the usual temperature, pressure and time parameters (for example, 121 °C or 134 °C).
- Incubation: once the programme has finished and the unit has cooled down, remove the vial carefully. Break the inner ampoule (in self-contained vials) to bring the spores into contact with the culture medium and incubate at 55 – 60 °C.
- Reading the result: inspect the indicator tube after the incubation time recommended by the manufacturer to assess whether there has been bacterial growth.
Interpretation of results
| Type of incubator | Negative result (correct) | Positive result (failure) |
|---|---|---|
| Conventional incubator (24-48 h) | No colour change (keeps the original lilac/purple shade) | Change to yellow/brown (acidification due to the metabolism of live spores) |
| Rapid-readout incubator (1-3 h) | Negative reading / green (absence of enzymatic fluorescence) | Positive reading / red (detection of active enzymatic activity) |
Note: it is essential to include a non-sterilised control vial from the same batch in every incubation process to verify that the spores were viable before the test and that the incubator is working correctly.
What should you do if the spore test fails?
A positive result (presence of bacterial life) immediately invalidates the process. In this situation, the following action protocol must be activated:
- Quarantine the load: withdraw from use and quarantine all material or instruments processed in that cycle or batch. It is considered non-sterile material.
- Rule out procedural errors: analyse whether the chamber was overloaded, whether the vial had expired or whether an incorrect temperature programme was selected.
- Confirmation test: run a verification cycle with the chamber empty, processing only a new biological indicator.
- Technical shutdown: if the second test gives a positive result again, the autoclave must immediately be declared out of service and the intervention of the specialist technical service must be requested to carry out an inspection and recalibration of the sensors.
Testing frequency and applicable regulations
The requirements for biological indicators are regulated internationally by the UNE-EN ISO 11138 standard. In industrial and pharmaceutical environments under GMP guidelines, the biological test is a mandatory part of the periodic qualification protocols for the installation (IQ/OQ/PQ). The usual recommended frequency in clinics and industry is weekly, as well as after any technical repair or prolonged period of inactivity of the unit.
At J.P. Selecta, as manufacturers of scientific equipment since 1947, we understand that the reliability of an autoclave does not end with the precision of its mechanical or electronic components.
Reliability also means in-depth knowledge of the thermal and biological principles that guarantee absolute sterilisation. Integrating these validation requirements into the engineering phase allows us to design safer autoclaves that are reproducible and aligned with the strictest international regulations, driving continuous improvement in laboratory instrumentation.