ISO 11138 Series: BI Standards for Steam, EO and VHP Explained

September 5, 2026

Biological indicators (BIs) are the practical proof that a sterilizer cycle actually killed resistant spores, not just reached a temperature. The ISO 11138 series is the international framework that defines how these indicators are built, tested, and labelled — organized by process so you can match a BI to your sterilizer.

Quick facts: ISO 11138 is a multi-part standard for biological indicators. Steam BIs use Geobacillus stearothermophilus; ethylene oxide BIs use Bacillus atrophaeus. ISO 11138-3 covers moist heat and is the part most often referenced for steam sterilizers in Canadian clinics.

What ISO 11138 does

Part 1 sets general requirements for every biological indicator: the test organism, the spore count per unit, resistance characteristics such as the D-value, packaging, labelling, and required documentation. Later parts adapt those requirements to one sterilization process each, which is why a steam BI is defined differently from an EO BI.

A biological indicator is only useful if its performance is known. A stated spore population and resistance let you interpret a negative result with confidence. Buy from manufacturers that declare conformity to the relevant part of ISO 11138; that declaration is what makes the printed certificate meaningful.

Part 3: moist heat and the Geobacillus standard

ISO 11138-3 covers moist heat: steam sterilization. The reference organism is Geobacillus stearothermophilus, a spore-former whose heat resistance sits well above ordinary bacteria. When a steam cycle inactivates these spores, the load reached lethal conditions.

For dental offices running steam autoclaves, this is the monitoring tool Ontario clinics run on each day the sterilizer is used. Ontario clinics run one BI in a routine load on each day the sterilizer is used — the CliniEco Medical Biological Indicator 24h Rapid Readout (25-Pack) gives a clear result in 24 hours without mailing samples to a lab.

CliniEco 24-hour rapid readout biological indicator 25 pack for steam sterilizers

Part 2: ethylene oxide and Bacillus atrophaeus

Ethylene oxide (EO) is a low-temperature gas process for devices that cannot take steam. ISO 11138-2 governs EO biological indicators, and the reference organism is Bacillus atrophaeus. EO cycles run long and the gas is hazardous, so facilities rely on BI results before releasing a load.

VHP and low-temperature cycles

Vaporized hydrogen peroxide (VHP) is widely used for low-temperature reprocessing of electronics and heat-sensitive instruments. Newer parts of the series cover low-temperature processes such as VHP; most VHP biological indicators are built on Geobacillus stearothermophilus spores, though with a different resistance profile.

VHP chambers vary by design, so use the BI the sterilizer manufacturer recommends. A steam BI bought for an autoclave will not validate a VHP cabinet.

Why matching the BI to the cycle matters

A biological indicator is tuned to one process: organism, spore load, and resistance are chosen so the unit fails if the cycle falls short. A steam BI cannot validate an EO cycle. Before you buy, confirm:

the BI matches your sterilizer type — steam, EO, or low-temperature;
organism and spore population are stated on the label;
D-value and expiry date are printed and legible;
conformity to the relevant ISO 11138 part is declared.

Process	Challenge organism	Standard part	Where you usually see it
Steam (moist heat)	Geobacillus stearothermophilus	ISO 11138-3	Steam autoclaves in dental and medical clinics
Ethylene oxide	Bacillus atrophaeus	ISO 11138-2	Hospitals reprocessing heat-sensitive devices
Vaporized hydrogen peroxide	Geobacillus stearothermophilus (common formats)	Newer parts of the series	Low-temperature cabinets for sensitive instruments

Between BI runs, chemical indicators track each load. CliniEco Medical Sterilization Pouches — Class 4 Dual Indicator, Assorted Sizes (200-Pack) pair the pouch with a multi-parameter chemical monitor, a practical fit for clinics running a BI on each day the sterilizer is used.

Incubation matters too. Steam BIs are incubated near 56–60 °C and EO BIs near 35–37 °C, with the exact range on the label. A dry-block incubator holds that temperature steadily — the CliniEco 24-Hour Biological Indicator Incubator (15 Wells) covers a steam BI plus its control, run on each day the sterilizer is used.

CliniEco 24-hour biological indicator dry-block incubator with 15 wells

Reading a BI certificate

A certificate of conformance names the organism and lot number, states the population per unit, gives the D-value with its test conditions, and shows the expiry date. Compare it with the box label; the two should agree.

Missing details are a red flag: no lot number, no stated population, no conformity reference means the BI's performance cannot be verified. Keep certificates with your sterilization logs so each run is traceable.

FAQ

Which biological indicator is used for steam sterilizers?

Geobacillus stearothermophilus. Steam BIs carry spores of this organism under ISO 11138-3 and are incubated after the cycle to confirm inactivation.

Can I use the same BI for EO and steam?

No. EO biological indicators use Bacillus atrophaeus under ISO 11138-2, while steam BIs use Geobacillus stearothermophilus. Each is validated for its own process, so match the BI to the sterilizer.

Do Canadian dental clinics need ISO 11138 BIs?

Provincial dental regulators expect routine spore testing where instruments are reprocessed — in Ontario the RCDSO requires a biological indicator on each day the sterilizer is used, while a weekly cadence is the CDC / AAMI ST79 baseline in most U.S. states. Choosing BIs declared to recognized standards such as ISO 11138 keeps that monitoring defensible.

How do I read a BI certificate?

Check the organism, lot number, population per unit, D-value with its test conditions, and expiry date. Confirm the label matches the certificate, then file it with your sterilization records.