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A biological indicator (BI), often called a spore test, challenges a steam-sterilization cycle with highly resistant spores and then checks whether they survived. For steam, the relevant organism is Geobacillus stearothermophilus. To use one correctly, select an FDA-cleared commercial BI made for your sterilizer and cycle parameters, use the matching control required by its instructions, place it as directed for that BI system, run the intended cycle, incubate and read it exactly as labeled, and interpret the result together with mechanical and chemical monitoring.
CDC recommends biological monitoring at least weekly, for every load containing implantable items, and whenever possible quarantining those implantable items until the BI is negative. A negative BI is evidence that the test challenge was inactivated; it is not proof that every item in the load was sterile or exposed to adequate conditions.
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What a biological indicator actually verifies
A BI contains resistant bacterial spores in a defined carrier or self-contained device. The spores are exposed to the sterilizer cycle, then assessed for growth or another product-defined signal. Because the spores are deliberately difficult to kill, the test provides a direct challenge to microbial destruction rather than merely showing that the chamber reached a displayed temperature.
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Outbyte PC Repair FREEClear out junk files and repair common Windows errorsFree Scan →Outbyte Driver Updater FREEScan for outdated or missing drivers - takes under a minuteDriver Scan →For steam sterilization, CDC identifies Geobacillus stearothermophilus spores. The BI must be intended for the exact sterilizer technology and cycle parameters you are testing. A spore product made for another modality or for different cycle conditions is not an interchangeable substitute. CDC’s recommendation is to use an FDA-cleared commercial preparation specifically intended for the sterilizer type and cycle parameters; see the CDC summary recommendations.
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| Monitoring layer | What it tells you | What it cannot establish by itself |
|---|---|---|
| Mechanical monitoring | Whether the recorded cycle variables, such as time and temperature, reached the programmed or required values. | That every location in the load received those conditions or that microorganisms were killed. |
| Chemical indicator | Whether the indicator was exposed to specified process conditions, according to its class and instructions. | That the load is sterile or that the required microbial lethality occurred. |
| Biological indicator | Whether the resistant spore challenge in the BI was inactivated under the test conditions. | That every item in the load was sterile or exposed to adequate conditions. |
Use all three layers as part of one monitoring program. CDC states that a negative BI does not prove all items in the load are sterile or that all items were exposed to adequate sterilization conditions; the wording appears in the CDC glossary.
What to obtain before running the test
Choose a BI matched to the process
Read the current BI labeling before purchase or use. Confirm the intended sterilizer technology, the exact cycle parameters, regulatory clearance and intended use, any required test pack or challenge device, storage conditions, incubation or reader requirements, and the manufacturer’s placement and interpretation instructions. Marketplace descriptions are not a substitute for the product’s controlled instructions.
For a steam autoclave, the label should identify steam use and the cycle conditions for which the device is validated. Do not assume that a generic strip, a BI for another sterilization technology, or a device validated for a different cycle can be used simply because it is described as a “spore test.”
Plan for a same-lot control
CDC dental monitoring guidance says a spore test should be used at least weekly and should include a matching control: the BI and control should come from the same lot number. Follow the product directions for whether the control is incubated or read in a particular way and whether it is processed with the load or kept unprocessed. The control demonstrates that the spores, media, incubation conditions, and reader can produce the expected positive response.
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Have the records and monitoring materials ready
Prepare the load identification, cycle type, sterilizer identifier, operator information, and the mechanical and chemical monitoring records before starting. CDC recommends recording the sterilizer and cycle type, load identification number, load contents, exposure parameters such as time and temperature, operator name or initials, and mechanical, chemical, and biological results. Use your facility’s approved record system and retention rules.
How to run a steam BI: the operational sequence
- Confirm applicability. Verify that the BI is intended for your steam sterilizer and the exact cycle parameters. Read both the BI instructions and the sterilizer manufacturer’s instructions. If either set of instructions conflicts with your planned setup, stop and resolve the discrepancy before processing a test.
- Inspect and identify the test. Check the lot, expiration date, packaging integrity, storage conditions, and any required test pack or holder. Label the BI or its record so it can be tied unambiguously to the load and cycle. Do not use a damaged, expired, wet, or improperly stored device unless the manufacturer’s instructions explicitly permit it.
- Prepare the matching control. Select the control from the same lot when required by the product and facility procedure. Handle it exactly as its instructions specify. A control is not an optional replacement for processing the test BI; it is a separate check on the test system.
- Place the BI as directed. Follow the BI manufacturer’s placement directions and any instructions for a validated challenge pack. The correct location depends on the BI system, sterilizer configuration, load arrangement, and cycle. There is no universal “best spot” established by the CDC pages. Do not invent a location or place the device where it cannot encounter the intended challenge conditions.
- Load the sterilizer correctly. Arrange the load according to the sterilizer and instrument-processing instructions. Keep the BI in its specified challenge configuration and avoid changing the pack, wrapping, container, or load density in a way that invalidates the manufacturer’s directions.
- Run the intended cycle. Start the cycle specified for the items and the BI. Record the cycle identifier and the exposure parameters shown by the sterilizer. Do not shorten, extend, or substitute a cycle and then interpret the result as evidence for the original process.
- Review mechanical and chemical results. At the end of the cycle, check the printout or electronic record and the chemical indicators required for the load. CDC recommends that an unacceptable mechanical or chemical result be treated as a reason not to use the processed items, regardless of the BI result. Resolve the monitoring failure under facility policy.
- Process, incubate, or read the BI exactly as labeled. Some products require activation, crushing, transfer, incubation, or use of a dedicated reader; others use a different readout method. Incubation temperature, reading time, and the meaning of a color, fluorescence, or other signal vary by product. The CDC material does not establish one universal incubation protocol, so use the current BI instructions rather than a generic time or temperature.
- Read the control and test together as required. Confirm that the control produced the expected result and that the test result is valid under the product’s interpretation rules. If the control is invalid, do not treat the test as a valid negative; contact the responsible supervisor or follow the manufacturer’s invalid-control procedure.
- Document the outcome. Record the BI lot, test or control identification, incubation or reader result, operator, date, cycle and load, and any corrective action. Attach or reference the mechanical and chemical records so the three monitoring layers can be reviewed as one event.
Where should the BI go?
Placement is one of the most common sources of incorrect testing. CDC’s dental monitoring FAQ directs users to follow the BI manufacturer’s placement instructions. A challenge location may be inside a specified test pack, container, or portion of the load selected by the product’s validation. It may change with the sterilizer type, chamber configuration, cycle, and packaging system.
Do not copy a placement rule from another BI, another sterilizer model, or an online discussion. If your facility uses a commercial test pack, use the pack exactly as labeled; do not open it, rearrange its contents, or substitute a different wrapping unless the manufacturer has validated that configuration. If the instructions are unclear, obtain clarification from the BI manufacturer, sterilizer manufacturer, infection-prevention lead, or other responsible technical authority before running the test.
How often to run biological monitoring
Routine schedule
CDC recommends BI monitoring at least weekly with an FDA-cleared commercial preparation intended for the relevant sterilizer and cycle. A facility may choose daily monitoring when a sterilizer is used frequently because daily tests can reveal a problem sooner; that is an operational risk decision, not a replacement for the minimum weekly recommendation.
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Loads containing implantable items
Monitor every load containing implantable items. Whenever possible, quarantine those implantable items until the BI result is negative. The quarantine decision should be built into the facility’s release procedure so that an implant is not issued merely because the mechanical display looked normal or a chemical indicator changed as expected.
Other situations requiring attention
Follow facility policy for testing after installation, relocation, major repair, sterilizer malfunction, process changes, or other events that could affect performance. The exact additional testing schedule is determined by applicable regulations, accreditation requirements, the sterilizer manufacturer, and the BI instructions.
How to interpret a negative result
A valid negative BI means the spores in that test challenge did not produce the growth or signal that the product defines as a positive result, under the stated incubation or reader conditions. It is meaningful evidence about that challenge and cycle, especially when the control behaved as expected and the mechanical and chemical records were acceptable.
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It is not a certificate that every item in the load is sterile. Items can be shielded by air pockets, overloading, incorrect packaging, poor cleaning, blocked lumens, wet packs, or other process and handling problems even when one BI is negative. CDC’s exact caution is that a negative biologic indicator does not prove that all items in the load are sterile or that they were all exposed to adequate sterilization conditions.
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Release decisions therefore require the complete record: correct cycle selection, acceptable mechanical parameters, chemical-indicator results, valid BI and control results, proper loading, and compliance with the item and sterilizer instructions. If any required element is unacceptable, hold the affected items and use the facility’s investigation process.
What to do when the BI is positive
A positive steam BI is a process signal that requires prompt escalation. CDC’s suggested protocol for management of a positive BI recommends the following sequence; see the CDC positive-indicator protocol, updated December 20, 2023.
- Take the sterilizer out of service. Stop routine processing in that sterilizer until the initial investigation is complete.
- Notify responsible personnel. Inform the area supervisor and infection-control or infection-prevention department according to local policy.
- Check the test itself. Verify that the BI was the correct product, within date, stored correctly, placed as directed, incubated or read at the specified conditions, and interpreted correctly. Confirm that the matching control was valid.
- Check the cycle and sterilizer. Review the time and temperature settings, cycle selection, load arrangement, packaging, and mechanical record. Look for operator error, an incorrect cycle, overloading, air-removal problems, inadequate cleaning, or a sterilizer malfunction.
- Repeat BI testing in three consecutive cycles. Use correctly prepared tests and controls under the facility’s validated procedure. Document each cycle and result.
- Escalate persistent positives. If repeat tests remain positive, treat the implicated items as nonsterile, recall and reprocess relevant loads as required, and involve maintenance and the sterilizer manufacturer. Apply local reporting, regulatory, and risk-management requirements.
- Return the sterilizer to service only after resolution. CDC’s suggested protocol allows return when the problem is resolved and all three repeat BIs are negative, subject to facility policy and any required technical clearance.
For a single positive BI in a steam sterilizer, CDC notes that nonimplantable items do not need to be recalled solely because of that one result unless a sterilizer or process defect is identified. That steam-specific advice should not be generalized to other sterilization modalities. Implantable items and any load affected by a confirmed process defect require the more conservative quarantine, recall, and reprocessing decisions specified by your policy and applicable rules.
Do not confuse BI testing with a Bowie-Dick test
A Bowie-Dick test is a separate air-removal test for dynamic-air-removal sterilizers. A failed Bowie-Dick result can indicate an air-removal or steam-penetration problem, but the test is not a biological indicator and does not replace biological monitoring. Follow the applicable CDC steam-sterilization guidance and the sterilizer manufacturer’s instructions for daily air-removal testing and failure response.
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Choosing among BI systems
There is no universal fastest readout or preferred brand established by the cited CDC guidance. Compare products on the characteristics that affect your actual process:
| Decision point | Questions to answer from current labeling |
|---|---|
| Process compatibility | Is the BI cleared and intended for steam, your sterilizer type, and the exact cycle parameters? |
| Challenge configuration | Does it require a specific test pack, holder, container, or placement location? |
| Control | Is a same-lot control required, and how must it be handled and interpreted? |
| Readout | What incubation equipment or reader is required, and what is the validated reading time? |
| Interpretation | What signal is positive, negative, or invalid, and what happens if the control fails? |
| Storage and handling | What temperature, humidity, light, expiration, and transport limits apply? |
| Records | What lot, cycle, operator, and reader information must be retained? |
Choose the system that your staff can use consistently and document correctly, not merely the one advertising the shortest nominal readout. Verify every claim against current manufacturer instructions and your jurisdiction’s requirements.
Common mistakes and corrective checks
- Using the wrong BI: Confirm steam compatibility and cycle parameters before processing. A BI for another modality is not suitable.
- Skipping the control: Use the same-lot control when required and verify that it is valid before accepting the test result.
- Guessing the placement: Follow the product’s validated placement or test-pack instructions rather than a generic chamber-location rule.
- Using a generic incubation time: Read the current label; incubation temperature and readout timing differ among systems.
- Releasing implants before the BI is negative: CDC recommends monitoring every implant load and quarantining implants whenever possible until a negative result is available.
- Treating a negative BI as proof of load sterility: Review mechanical readings, chemical indicators, load configuration, packaging, and handling as well.
- Ignoring an unacceptable mechanical or chemical result: Hold the affected items and investigate even if the BI is negative.
- Continuing to run a sterilizer after a positive BI: Take it out of service, notify responsible personnel, investigate, and perform the CDC-suggested three-cycle repeat sequence.
- Failing to connect records: Link the BI lot and result to the specific sterilizer, cycle, load contents, operator, and mechanical and chemical records.
How much confidence does a BI provide?
A BI is stronger evidence than a display reading or chemical color change because it directly challenges the process with resistant spores. Its evidence still applies to the test device and conditions under which it was used. It cannot compensate for poor cleaning, incorrect packaging, blocked lumens, overloading, air-removal problems, wrong cycle selection, or mishandling after sterilization.
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CDC’s sterilizing-practices page cites an older estimate of about 1% steam-sterilization failure. That figure comes from an older underlying reference and is not a current universal failure benchmark; it should not be used to predict the performance of a particular sterilizer or facility. The practical implication is to maintain routine monitoring and a documented response system rather than relying on an assumed failure rate.
Minimum documentation checklist
- Sterilizer identifier and cycle type
- Load identification number and date
- Load contents, including whether implants are present
- Exposure parameters, such as recorded time and temperature
- Operator name or initials
- Mechanical monitoring result
- Chemical-indicator result
- BI product, lot, control, placement or test-pack configuration, and result
- Incubation or reader information and any invalid or delayed result
- Disposition of the load and corrective actions for any failure
Keep these records under the facility’s approved retention schedule and make them accessible for release decisions, investigations, maintenance review, and required reporting.
The practical decision rule
Use a steam-specific, FDA-cleared BI containing the appropriate Geobacillus stearothermophilus spore challenge; pair it with the required same-lot control; place and process it exactly as the BI and sterilizer instructions specify; run it at least weekly and for every implant load; and read it with the prescribed equipment and timing. Release items only when the BI, control, mechanical record, chemical indicators, load configuration, and facility procedure all support release. A positive result means stop, notify, investigate, repeat across three consecutive cycles, and escalate persistent failures before the sterilizer returns to service.
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