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What Are Biological Indicators (Spore Tests)?

Written by: Bill Warren

What Is a Biological Indicator & How Does It Work?

A biological indicator is a test system that’s embedded within a process challenge device (PCD) and used to verify the efficacy of certain sterilization processes — such as those that rely on sterilizing agents like steam and vaporized hydrogen peroxide.

Biological indicators are also commonly referred to as “spore tests.” This is because they contain viable organisms — typically Geobacillus stearothermophilus spores — that are used to measure sterilizer efficacy. This particular strain of bacteria is naturally resistant to both agents, meaning if sterilizing units are able to effectively kill all spores within the biological indicator during a validation cycle, technicians can also assume that they are capable of achieving effective sterilization during routine use.

To verify a validation test, the biological indicators that underwent the test cycle are incubated within growth media. If no spore growth occurs by the time the incubation period ends, the test is deemed successful. However, if spore propagation is observed at the end of the incubation period, that means sterilization has not successfully been achieved, translating to a failed test result.

What Is Autoclave Validation & Why Is It Important?

In most healthcare and pharmaceutical manufacturing environments, the validation of autoclaves is a critical process to ensure that sterilization cycles are both repeatable and effective. This minimizes the risk of accidentally exposing patients to contaminated materials during their practical use.

Overall, the validation process is made up of a broad set of activities that begin with equipment validation steps, including the following:

  • Calibration Ensures that sensor response falls within a specified range in relation to reference standards.
  • Cycle development A process that defines the parameters of different sterilization cycles, which will be tested during the remainder of the validation process.
  • Installation qualification Evidence that shows autoclaves have been built and installed correctly, including the successful connection of all supporting services (e.g. electricity, water, and steam).
  • Operational qualification A process that shows whether autoclaves perform up to a lab or sterile processing department’s standards.
  • Steam quality testing Ensures that the measurements of the physical aspects of the steam used during sterilization cycles (e.g. temperature, dryness, and non-condensable gas content) fall within established ranges.
  • Factory acceptance testing A process that evaluates an autoclave prior to and after assembly and verifies that it operates as intended.

Autoclave Spore Testing & Validation

Once these equipment validation activities are complete, the next step is to validate the sterilization cycle. This process, also commonly referred to as “performance qualification” or “cycle validation”, incorporates the use of biological indicators to test autoclave efficacy. Cycle validation typically includes the following steps:

  1. Calibrate the temperature probe.
  2. Run test cycles with an empty chamber, being sure to strategically place several PCDs with biological indicators in challenging locations within the chamber.
  3. Run test cycles with a full load, placing multiple biological indicator PCDs within the load itself and in locations throughout the chamber — the more challenging, the better.
  4. Once each cycle is complete, incubate the biological indicators for the prescribed period, noting the presence or lack of spore growth.
  5. Record the results.
  6. If the tests fail (determined by the presence of spore growth), confirm that the autoclave is operating properly, and then modify your cycle parameters.
  7. For each chamber/load configuration, all biological indicators must be inactivated, and the cycle must be repeated successfully three times.

Biological Indicators & Regulatory Compliance

In medical and drug production settings, staying compliant with the regulations that govern sterilization standards is especially crucial. Equipment validation, cycle validation, and the routine use of biological indicators are essential for facilities to meet these standards and to ensure that effective sterilization processes are developed and maintained.


Q: How frequently should spore testing be done?

A: Frequency of spore testing depends on how often an autoclave is used. If it runs multiple loads each day, for example, the Centers for Disease Control and Prevention (CDC) recommends daily testing with biological indicators.

Q: What are the next steps after a failed autoclave spore test?

A: After a failed result, it’s crucial to make sure the autoclave is working properly. If there are no impediments to operation — such as a clogged strainer drain or a blocked drain port — adjust sterilization parameters (e.g. time, temperature, etc.) and run the cycle again, adjusting as needed to achieve a passing result.

Q: Do the types of biological indicators used vary between sterilization methods?

A: Biological indicators are typically used in steam, vaporized hydrogen peroxide, and ethylene oxide (EO) validation processes. While tests of steam and vaporized hydrogen peroxide sterilizers use biological indicators that contain heat-resistant Geobacillus stearothermophilus spores, EO sterilizers are validated using biological indicators that include live Bacillus atrophaeus spores instead.

Q: How do biological indicators compare to chemical indicators?

A: While biological indicators contain live bacterial contaminants chosen because of their high resistance to certain sterilizing agents, chemical indicators are used to monitor the presence of a sterilizing agent during test cycles.

Validation & Your Autoclaves

Validation is a crucial part of ensuring your autoclave lineup stays online and your sterilization standards remain compliant with regulatory requirements. Download your copy of our free eBook: “How to Validate an Autoclave” for more about the tests you may need to perform in order to properly and effectively validate your sterilizers.

Or get in touch with our team of autoclave experts to start the conversation about your validation needs.

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