What Are Biological Indicators (Spore Tests)?

Key Takeaways

• A biological indicator measures an autoclave’s ability to kill microbial life.
• Biological indicators are used alongside physical monitoring and chemical indicators to support autoclave validation.
• Autoclaves need to be validated to ensure cycles are repeatable and effective. Validation also includes calibration, cycle development, qualification, and steam quality testing.
• If a spore test fails, check to see whether the cycle was programmed correctly, whether the drain was blocked, or if there are issues with your steam supply. If a spore test fails a second time, take the autoclave out of service for repair.

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), a tool that simulates a highly complicated load to verify the efficacy of sterilization processes that rely on agents such as 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 was not successfully achieved, translating to a failed test result.

Types of Biological Indicators

Biological indicators for autoclaves typically come in one of three formats:

• Spore strips: A spore strip is a small paper strip inoculated with spores and sealed within a glassine envelope. After a sterilization cycle is complete, the technician transfers the strip into growth media under aseptic conditions or ships it to a laboratory for culturing.
• Self-contained biological indicators: This type of indicator includes both a spore carrier and a sealed glass ampule of growth media, which are contained in a single plastic vial. The technician crushes the ampule after the cycle is complete, which releases media onto the spore carrier without exposing it to the air, which eliminates the contamination risk that comes with handling a spore strip. Healthcare environments commonly use self-contained biological indicators for this reason.
• Rapid-readout indicators: Rapid-readout indicators use the same design as self-contained indicators, with an added enzyme detection system. Rather than wait for spores to multiply, an auto-reader detects fluorescence produced when an enzyme tied to spore outgrowth breaks down a substrate in the media. This type of biological indicator can deliver results in as little as 30 minutes.

How Autoclave Spore Tests Compare to Other Sterilization Indicators

Spore testing is one of three ways to monitor a sterilization cycle. The other two are:

• Physical monitoring: Autoclaves log temperature, pressure, and time throughout the cycle, either through a printout or digital record. A technician can refer to this record to determine whether the cycle ran as programmed.
• Chemical indicators: A chemical indicator is an ink or dye, printed on a strip, label, or tape, that changes color when exposed to the conditions inside a sterilizer. Some respond to temperature alone, while others respond to a combination of temperature, time, and the presence of a sterilizing agent. All can be read the moment a load comes out of the autoclave chamber.

Autoclave tape is one of the most common types of chemical indicator. Technicians use this adhesive tape, which has heat-sensitive stripes that darken during a cycle, to seal packages, helping them distinguish between processed and unprocessed packs. Because the color change happens on the outside of the pack, it confirms exposure rather than sterility of the contents.

Facilities typically run all three tests together to tell whether the autoclave ran correctly, that steam fully penetrated the load, and that the autoclave met the necessary conditions to kill off all microbial life.

What Is Autoclave Validation & Why Is It Important?

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

Facilities typically validate an autoclave during installation, and again after a relocation, major repair, or before running a new load type. Production environments and facilities subject to Good Manufacturing Practice (GMP) may also require periodic requalification. Otherwise, cycles are monitored rather than revalidated.

Overall, the validation process consists 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 period prescribed by the manufacturer, alongside an unprocessed vial from the same lot as the control.
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.

What to Do When a Spore Test Fails

A failed spore test means the indicator did not reach the set temperature or hold it for the necessary amount of time to achieve lethality conditions. Should this happen, start by checking the autoclave’s settings to determine whether the cycle was programmed correctly for the load.

If the issue isn’t with the autoclave’s settings, it could point to a mechanical issue, such as a clogged chamber drain strainer or a load positioned over the drain port. If the issue persists, check your steam supply, as deviations in dryness, superheat, or non-condensable gas content can produce biological and chemical indicator failures even if the sterilizer operates correctly.

Once you’ve identified and addressed the potential point of failure, run a second spore test. If the test fails again, take the sterilizer out of service until it can be repaired.

Biological Indicators & Regulatory Compliance

Complying with the regulations that govern sterilization standards is crucial in medical and drug production settings. 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.

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 a free copy of our eBook, How to Validate an Autoclave, for more about the tests you may need to perform 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.

Frequently Asked Questions

How often should you spore test an autoclave?

How often you need to spore test depends on how often you use your autoclave. The Centers for Disease Control and Prevention (CDC) recommends daily testing with biological indicators for facilities that run multiple loads each day.

Why do biological indicators use Geobacillus stearothermophilus?

Autoclave biological indicators use Geobacillus stearothermophilus because its spores resist moist heat better than almost any other organism that might be present on a load. A cycle that inactivates them will also kill everything else less resistant.

Do all sterilizers use the same kind of biological indicator?

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.

How should you store biological indicators?

Store biological indicators according to the manufacturer’s instructions. Ampules and spore suspensions hold spores in contact with growth media, so refrigeration prevents them from germinating on the shelf. Spore strips and self-contained vials keep spores separate from media until activation, so they can generally be stored at room temperature. Keep all autoclave biological indicators clear of sterilizing agents, chemicals, and UV light.

Can you run a spore test without an incubator?

While you can’t run a spore test without the incubation step, you can still run a spore test even if your facility doesn’t have an on-site incubator by using a mail-in monitoring service.

How do you dispose of spore test vials?

Used biological indicators have live cultures and need to be sterilized before disposal. Run them through a sterilization cycle validated for that purpose, following the manufacturer’s instructions, then discard them with your facility’s biological waste. Remove positive vials from the incubator and handle them as soon as you have test results rather than leaving them to incubate further.