What to Do When a Spore Test Fails: The CDC Protocol, Step by Step

A biological indicator (BI), or "spore test," contains highly resistant bacterial spores (Geobacillus stearothermophilus for steam autoclaves). It is the most accepted method for confirming sterilization, because it directly tests whether the process kills highly resistant test organisms — ones far harder to kill than the microbes normally found on your instruments. The CDC recommends running a spore test at least weekly, alongside a matching-lot control (a BI from the same lot number) that is not sterilized.

When the processed indicator comes back positive — meaning the spores survived and grew — it tells you the sterilization cycle may not have worked. The unsterilized control from the same lot should come back positive (the spores grow); that is the result that confirms the spores were viable to begin with. If your control did not grow (came back negative) or was invalid, the test itself is unreliable and should be repeated.

A single positive BI is a warning, not a verdict on every patient. But CDC guidance is clear: treat it as a potential sterilization failure and follow the protocol below until you have proof the sterilizer is working again.

Before you assume the machine is broken, confirm the failure is real and look for a procedural cause — CDC specifically directs you to review your sterilization operating procedures to determine potential reasons for the failed test. Most failures trace back to how the load was run, not to a mechanical fault.

Check, in this order:

• The control indicator. The unsterilized matching-lot control should grow (positive). If it didn't, the test is invalid — repeat it.
• Mechanical readouts. Did the cycle reach the correct time, temperature, and pressure? A printout or display that shows a short or cold cycle points to operator error or a malfunction.
• Chemical indicators. Did the internal and external chemical indicators change color as expected?
• Loading and packaging. Overloading the chamber, packs that are too tight or too large, wrapping that blocks steam penetration, or selecting the wrong cycle are the most common culprits.

If you find a clear operator error, you'll still complete the CDC retest and documentation steps — but you now know the likely cause and how to prevent it.

Follow these steps in order. They come directly from CDC dental infection prevention guidance.

1. Remove the sterilizer from service. Stop using the machine immediately and label it clearly so no one runs a load by mistake. Then review your sterilization operating procedures to look for the cause.

2. Recall and quarantine any implantable items. Any implantable devices processed in suspect loads must be set aside and not used until they are shown to be sterile.

3. Repeat the spore test immediately using the same cycle that produced the positive result.

• If the repeat test is negative and your review shows procedures were performed correctly, the sterilizer can be returned to service.
• If the repeat test is positive, the sterilizer must stay out of service until it is inspected or repaired and the reason for the positive test is determined. Per CDC dental guidance, it should then be re-challenged with spore tests in three consecutive fully loaded chamber sterilization cycles that all return negative before you rely on it again. (Follow your BI manufacturer's instructions for incubation and reading results.)

4. Recall and reprocess the affected instruments. When possible, items from suspect loads dating back to the last negative spore test should be recalled, rewrapped, and re-sterilized. This is why keeping accurate, dated logs matters — they define exactly how far back your recall must reach.

5. Document every action in your sterilization monitoring log as you go (covered in detail below).

Call the sterilizer manufacturer or an authorized service technician when the failure isn't explained by a correctable procedural error — specifically:

• The repeat spore test is also positive. CDC guidance says the sterilizer should not be used until it has been inspected or repaired and the exact reason for the positive test has been determined.
• Mechanical or chemical indicators show the machine isn't reaching correct temperature, pressure, or time.
• You've corrected obvious operator issues and the sterilizer still fails.

Have your sterilizer model and serial number, the cycle parameters, and your recent monitoring records ready when you call. Keep the service ticket, diagnosis, and any repair or calibration paperwork — it becomes part of your corrective-action record. Do not return the unit to service on the strength of a repair alone; confirm it with the three consecutive fully loaded, negative biological-indicator cycles first.

Documentation is what turns a scary event into a compliant one. CDC emphasizes that monitoring records establish accountability and help determine whether an instrument recall is necessary. An inspector wants to see that you recognized the failure and followed the protocol.

Record all of the following, with dates and initials:

• The positive result — date, sterilizer ID, BI lot number and control result, and the cycle involved.
• Sterilizer removed from service — when, and how it was labeled/isolated.
• Cause review — what you checked (loading, packaging, cycle selection, mechanical/chemical readouts) and what you found.
• Implantables quarantined — which items, and their disposition.
• Repeat spore test(s) — dates and results, including the three consecutive fully loaded chamber negative cycles if the unit was repaired.
• Instrument recall — the loads reprocessed back to the last negative test, and confirmation they were rewrapped and re-sterilized.
• Manufacturer/service contact — ticket number, diagnosis, repairs.
• Return to service — the date and the negative result(s) that justified it.

Keep these records with your routine monitoring logs, and retain them long enough to comply with state and local regulations. A free, dated tool like the State Log at /tools/sterilization-log makes the last-negative-test date — and therefore your recall boundary — easy to prove.

Most spore-test failures are preventable and procedural. To reduce recurrence:

• Don't overload the chamber, and leave space between packs so steam can circulate.
• Package correctly — right pouch size and orientation, sealed properly, not stacked in ways that block penetration.
• Select the correct cycle for the load and always allow full cycle time; never open early.
• Use mechanical and chemical indicators on every load so you catch problems before the weekly BI does.
• Maintain the sterilizer on the manufacturer's schedule, including gaskets, water quality, and cleaning.
• Train and cross-train staff who load and run the autoclave, and keep the operating procedure posted.
• Run the weekly biological indicator with a matching control and log it every time.

Consistent loading, packaging, and monitoring habits are the difference between an isolated event and a recurring compliance problem. State rules vary on required test frequency and recordkeeping — check your state's page under /requirements and verify specifics with your state dental board.