Bacillus atrophaeus vs Geobacillus stearothermophilus: Which Spore Test for Which Cycle

Walk into a reprocessing room and you will usually find one box of spore tests and one incubator. That works right up until the day the facility adds an ethylene oxide sterilizer, a hydrogen peroxide system or a dry-heat oven, and someone orders the same indicators that worked for steam.

The organism inside a biological indicator is chosen to be harder to kill than the bioburden you actually care about, using the sterilant in question. Get the pairing wrong and the test still runs, still produces a colour change, and still gets written in the log - it just does not mean anything.

Which organism belongs with which sterilant?

Two species carry almost the entire workload.

Geobacillus stearothermophilus is the steam organism. It is a thermophile: it grows happily at temperatures that stop most bacteria, which is what makes it a fair challenge for a moist-heat cycle. It is also the organism used for most hydrogen peroxide vapour monitoring, because the resistance profile holds up in that chemistry.

Bacillus atrophaeus is the low-temperature organism. It is used for ethylene oxide and for dry heat, and it incubates in the same range as ordinary laboratory organisms - roughly 30 to 37 degrees Celsius.

Why does incubation temperature break so many tests?

Because it is invisible in the result. An indicator read at the wrong temperature does not announce itself - it either fails to grow when it should, or grows when it should not, and either way the log entry looks completely normal.

This is why dual-chamber incubators exist: the two temperature ranges cannot share a single block. It is also why the incubator should be checked against a reference thermometer rather than trusted on its dial. A dry-block incubator with a calibrated block removes most of that uncertainty, and a four-well fluorescence reader narrows it further where readout time matters.

Where does the choice of organism show up in practice?

Three places, and all three are worth a line in your procedure.

Purchasing. Indicator packaging names the organism, the sterilant and the incubation temperature. If a catalogue entry does not state all three, ask before you order.

Storage and rotation. Spore strips, self-contained vials and ampoules all carry expiry dates, and heat exposure shortens useful life. Rotate stock and read the lot before use.

The record. When a reviewer reads your spore-test log, the combination of sterilizer type, indicator organism and incubation temperature is the fastest way to see that the programme is coherent. A log showing a steam sterilizer, a low-temperature incubation and a passing result is not a passing programme - it is an untested one.

A practical way to keep the pairing straight: label the incubator with the organism and the temperature range, not with the room name. Then the indicator, the incubator and the log all agree, and a new staff member can follow the chain without being told. A five-pack trial at CA $12.99 with shipping included is enough to rehearse the full run - process, incubate, read, record - before you rely on it in a live cycle, and a 50-pack of three-hour fluorescence indicators is the usual next step once the programme is running daily.

References

1. Public Health Ontario - reprocessing of medical devices, infection prevention and control (checked 16 September 2026)
2. CSA Z314:23, Canadian medical device reprocessing standard (CSA Group) (checked 16 September 2026)
3. National Center for Biotechnology Information - Bookshelf, sterilization and disinfection reference (checked 16 September 2026)

Related Reading

• Sterilization compliance hub: monitoring, logs and provincial expectations
• Dry-block incubators for spore testing: SPSmedical, Crosstex, Propper and open options compared
• Chemical indicator classes in practice: a brand-by-brand comparison of Class 4, 5 and 6 products
• Sterilization monitoring supplies and consumables for Canadian clinics
• free Ontario sterilization compliance log
• printable autoclave sterilization log sheet (free)

Frequently Asked Questions

Why does the name keep changing between Bacillus and Geobacillus?

The steam indicator organism was described for decades as Bacillus stearothermophilus. Reclassification moved it into the genus Geobacillus, so both names still appear in catalogues, certificates of analysis and older standard editions. Bacillus atrophaeus went through a similar change - it was long sold as Bacillus subtilis var. niger. The organism on the label is the same; only the genus has moved.

Can one biological indicator cover every sterilizer in the building?

No, and this is the most common mix-up. Spore choice follows the sterilant, not the department. A steam indicator will not give a meaningful result in an ethylene oxide cycle, and an ethylene oxide indicator is not a valid challenge for a steam cycle. If a facility runs both, it stocks both.

Does the incubation temperature matter if the indicator still changes colour?

Yes. Each organism has a growth range, and reading at the wrong temperature can produce a false pass or a false fail. Steam indicators are normally incubated at 55 to 60 degrees Celsius; organism used for ethylene oxide and dry heat grows nearer 30 to 37 degrees. A dual-chamber incubator exists precisely because those ranges do not overlap.

How do biological indicators relate to the ISO 11138 series?

ISO 11138-1 sets the general requirements for all biological indicators. The later parts add requirements by sterilant - ISO 11138-2 for ethylene oxide, ISO 11138-3 for moist heat and ISO 11138-4 for dry heat. When a certificate of analysis cites a part number, it is telling you which sterilant the product is qualified for.

Last updated: September 2026. CliniEco Medical holds MDEL #35334 issued by Health Canada, and supplies clinics, long-term care homes and home-care programs across Canada.