Biological Indicators: Spore Testing Protocols for Clinics

Every autoclave cycle ends the same way: a printout, a colour change on a pouch, and a rack of instruments that looks clean. What most of those indicators cannot tell you is whether anything actually survived. Biological indicators answer that question, and for Canadian clinics that reprocess instruments, weekly spore testing is the step that separates routine habit from real assurance.

What Are Biological Indicators?

Biological indicators (BIs) are sealed vials containing a defined population of Geobacillus stearothermophilus spores, the heat-resistant organism used to challenge steam sterilizers. Because these spores are far harder to destroy than typical microorganisms, they act as the strictest available test of a sterilization cycle. Each vial holds a growth medium and a colour indicator. If the cycle fails, surviving spores germinate during incubation, the medium changes colour, and the vial signals failure. If the cycle worked, the medium stays unchanged.

Why is spore testing the only direct sterility check?

Chemical indicators — the stripes on autoclave tape and Class 4 pouches — confirm that a package was exposed to heat. They do not confirm that anything inside it was actually killed. Temperature, pressure, and time charts show that the chamber performed on paper. A biological indicator shows what the chamber performed on living organisms, which is a different question entirely. CSA Z314.8, the Canadian standard for effective sterilization in health care settings, and AAMI ST79 both treat biological monitoring as the definitive verification of sterilizer function. Physical and chemical monitoring are necessary, but they are not sufficient on their own. Spore testing is the only check that proves lethality.

How to Run a Biological Indicator Test

Running a BI test is straightforward, but the details matter. Place one test vial in the centre of a fully loaded chamber — the area that is hardest to sterilize — and run a normal cycle. Keep a second vial, the control, unprocessed and away from the sterilizer. After the cycle, incubate both vials together at the temperature and for the time specified by the manufacturer, typically 55–60 °C for 24 hours with a self-contained vial such as the CliniEco Medical Biological Indicator — 24-hour Self-Contained, 25-Pack. A colour change in the control confirms that the medium supports growth; if the control does not change, the test is invalid and must be repeated. A colour change in the processed vial means spores survived and the cycle failed.

CSA Z314.8 and the Weekly Testing Requirement

CSA Z314.8 requires biological monitoring of every steam sterilizer at least once per week, with more frequent testing recommended for high-volume practices and after any repair. This is not a suggestion. Health Canada expects facilities that reprocess medical devices to maintain written records of sterilization monitoring, and the weekly BI result belongs in that record. Clinics running multiple loads a day often test daily, following the more conservative guidance in AAMI ST79. A vial is inexpensive; an unrecognized sterilizer failure is not.

Responding to a Positive Result

A positive BI means the load was not sterile, and neither were the loads processed since the last negative test. Act immediately: recall every load processed since the last negative result, quarantine the instruments, and reprocess them once the sterilizer is verified. Notify anyone who may have used items from those loads. Remove the sterilizer from service and have it inspected and repaired by a qualified technician. Retrain staff on loading, packaging, and cycle selection, since operator error is a common cause. Then run three consecutive BI cycles with controls; the sterilizer returns to service only after three negative results. Document every step.

Spore Testing Protocol Checklist

Post this checklist at your reprocessing station and initial each line:

• Run a control vial alongside every test vial.
• Test each sterilizer at least weekly, per CSA Z314.8.
• Place the test vial in the centre of a loaded chamber.
• Incubate at the manufacturer's specified temperature for the full 24 hours.
• Read and record the result: unchanged medium means no growth.
• Log the date, cycle number, operator, and lot number.
• Recall, quarantine, and reprocess on any positive result.
• Service the sterilizer and retrain staff before the next use.

Biological monitoring is not paperwork. It is the difference between assuming a load is sterile and knowing it is. Paired with validated packaging such as the CliniEco Medical Sterilization Pouch — Class 4 Dual Indicator, Assorted Sizes (200-Pack) and CliniEco Medical Autoclave Tape, a weekly spore test gives Canadian clinics a monitoring system that stands up to inspection. Established systems such as 3M Attest and Crosstex have set the benchmark in this category, and self-contained 24-hour vials make that same standard practical for a practice of any size.

Related reading: explore our sterilization compliance hub for every industry that sterilizes. Also see Sterikon vs CliniEco: sourcing options for Canadian clinics.

start with our pillar guide on sterility assurance level (SAL) and what 10⁻⁶ really means.

Frequently Asked Questions

How often should a clinic run a spore test in Ontario?

Every day the sterilizer is used, and for each type of cycle used that day. Weekly spore testing is the CDC and AAMI ST79 baseline followed in much of the United States; on its own it does not meet the Ontario expectation.

How many biological indicators are needed per test?

One BI per sterilizer per cycle type per day. Place it inside a process challenge device within a load that represents your normal instrument mix, and incubate it alongside an unexposed control from the same lot.

How long do spore test results take?

A 24-hour self-contained indicator gives a result the next day. Conventional spore strips incubated at 55-60 °C are read at 48 hours. A load should not be released for patient use while its test is still incubating.

What happens after a positive spore test?

Quarantine the load and keep the instruments out of circulation. Document the result, review the cycle and the way the load was packed, then repeat the test with a fresh in-date indicator. If the repeat is also positive, take the sterilizer out of service until it is repaired and passes monitoring.

Related reading

• Rapid Readout Biological Indicator Readers: Every Major Brand Compared (2026 Canada Price Guide)
• BI vs Chemical Indicators: What's the Difference in Sterilization Monitoring?
• The Real Annual Cost of Biological Monitoring: Locked Ecosystems vs Open-System BIs in Canadian Dental Clinics
• free Ontario sterilization compliance log
• printable autoclave sterilization log sheet (free)