Biological Indicator Steam Sterilization Steps

This guide explains how to use a biological indicator (BI) to verify that a steam sterilization cycle actually kills microorganisms, not just that the machine reached temperature and pressure. You’ll learn the step-by-step process from placing the BI in a load to incubating and reading the result, plus what to do if the test fails. Before you begin, confirm your autoclave is working normally and that you have the correct type of BI for your sterilizer (typically self-contained vials with Geobacillus stearothermophilus spores for steam).

What You’ll Need

• Biological indicators (self-contained vials or strips with spore disks and growth medium)
• Positive control BI (same lot, not exposed to the cycle)
• Incubator set to the BI manufacturer’s specified temperature (usually 55–60°C for steam)
• Personal protective equipment (heat-resistant gloves, lab coat, safety glasses)
• Sterilization packaging (e.g., pouches or wraps) if you are testing a wrapped load
• Logbook or tracking system to record results
• Timer or clock

Step-by-Step

1. Check the BI lot and expiration date. Use only BIs that are within their shelf life and have a control lot number. Do not use expired or damaged indicators.
2. Place the BI in the most challenging location. Typically, this is the center of the load or the coolest part of the chamber (often the lower front, near the drain). For wrapped items, place the BI inside a package that is representative of the load. For liquid loads, place the BI in a liquid-filled container that mimics the slowest heating point.
3. Run the sterilization cycle. Close the autoclave door and start the cycle you intend to verify (e.g., 121°C for 30 minutes or 134°C for 4 minutes). Let the cycle complete fully, including the drying phase if used.
4. Remove the BI after the cycle. Wait until the chamber pressure is zero and the load has cooled enough to handle safely. Use heat-resistant gloves. Remove the BI from the load and allow it to cool to room temperature for at least 10 minutes before handling further.
5. Activate the BI. Follow the manufacturer’s instructions – for self-contained vials, crush the glass ampoule inside the plastic tube to bring the growth medium into contact with the spore strip. For strip BIs, aseptically transfer the spore disk to a tube of sterile growth medium.
6. Incubate the test BI and a positive control. Place the test BI and the positive control (an unprocessed BI from the same lot) into the incubator at the specified temperature (usually 55–60°C). Incubate for the full recommended time (often 24 or 48 hours). Do not open the vials during incubation.
7. Read the results. After incubation, observe the color change in the growth medium. A positive control should show a color change (e.g., from purple to yellow) and turbidity, confirming that the spores are viable and the medium supports growth. The test BI should show NO color change (remains purple) and no turbidity, indicating that all spores were killed.
8. Record the results. Document the date, cycle number, load contents, BI lot number, incubator temperature, and the result (pass/fail) in your logbook. Keep records per your facility’s policy.
9. Dispose of used BIs. After reading, autoclave the used BIs and positive controls before discarding them as biohazardous waste, as they contain live spores (the positive control) or potentially viable spores if the test failed.

Common Mistakes

• Placing the BI in the wrong location. Putting it near the steam inlet or in an easy-to-reach spot may not represent the hardest-to-sterilize area, giving a false pass.
• Not using a positive control. Without a positive control, you cannot be sure the BI was viable and the medium was working – a false negative could occur.
• Reading the result too early. Some BIs require 48 hours of incubation; reading at 24 hours may miss a slow color change, leading to a false pass.
• Forgetting to cool the BI before crushing. Crushing a hot ampoule can cause the glass to shatter or the medium to boil, ruining the test.
• Using the wrong BI type. BIs for ethylene oxide or dry heat are not suitable for steam; always use G. stearothermophilus for steam cycles.
• Not recording the lot number. If a failure occurs, you cannot trace the batch or recall other loads.

Tips

• Run a BI test at least weekly, or per your facility’s protocol (often daily for implantable devices).
• Use a biological indicator test pack (a standardized challenge) if available, to ensure consistent placement and challenge.
• Keep a spare incubator thermometer to verify the incubator temperature is correct.
• Label the BI with the load number and date before placing it in the sterilizer.
• Store BIs according to the manufacturer’s instructions – usually in a cool, dark place.
• If a cycle fails, quarantine the load and reprocess all items before releasing them.

Troubleshooting

If the test BI shows a color change (positive): The sterilization cycle failed. Immediately quarantine the load – do not use any items. Notify your supervisor or infection control. Check the cycle parameters (temperature, time, pressure) from the printout. Check for common causes: overloading, improper packaging, air leaks, or a malfunctioning steam supply. Re-run the BI test with a new BI after correcting the issue. If the failure persists, the autoclave may need maintenance or calibration.

If the positive control does not change color: The BI lot may be defective, or the incubator temperature is wrong. Do not interpret the test BI result – the test is invalid. Repeat the test with a new BI from a different lot and verify the incubator temperature.

If the medium is cloudy but not discolored: This could indicate contamination. Treat as a failed test and repeat.

If the BI vial leaks or breaks: Discard it and repeat the test. Do not attempt to interpret a damaged BI.

Frequently Asked Questions

Q: How often should I run a biological indicator test?
A: At least weekly, but many guidelines recommend daily testing, especially for implantable devices. Always follow your facility’s policy and regulatory requirements.

Q: Can I use a chemical indicator instead of a biological one?
A: Chemical indicators only show that certain conditions (like temperature) were reached; they do not prove that spores were killed. Biological indicators are the gold standard for sterility assurance.

Q: What is the incubation temperature for steam BIs?
A: Typically 55–60°C (131–140°F) for G. stearothermophilus. Always check the manufacturer’s instructions for your specific product.

Q: How long do I incubate the BI?
A: Usually 24 to 48 hours. Some rapid-readout BIs provide results in a few hours, but they still require full incubation for definitive confirmation. Follow the product’s instructions.

Q: What should I do if the BI test fails?
A: Quarantine the load, do not use any items, and reprocess them. Investigate the cause (cycle parameters, loading, equipment) and repeat the test. If failures continue, contact your sterilizer service provider.

Q: Can I reuse a biological indicator?
A: No. Biological indicators are single-use. Reusing them will give invalid results and risks contamination.

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