Biological and Chemical Indicators in Sterilization: The Complete Guide

The information contained in this article is provided for informational and educational purposes only. For any questions specific to your practice, we invite you to consult your professional order or professional association.

An autoclave displays temperature, pressure, and cycle time. This data is important as it confirms that the device has functioned mechanically. However, it does not indicate whether microorganisms on the instruments have actually been destroyed. In podiatric care, instruments such as curettes, forceps, burs, and scissors come into direct contact with tissues, making them "critical" medical devices according to the CERDM-INSPQ classification: they require complete sterilization, capable of eliminating all forms of microbial life, including bacterial spores—the most resistant organisms that exist. To confirm that this level of sterilization has been achieved, two types of complementary tools exist: chemical indicators and biological indicators.

In Quebec, the framework for reprocessing medical devices is based on several main sources. The Canadian national standard CSA Z314-2023 defines good reprocessing practices in all healthcare settings, including private clinics. The CERDM-INSPQ, mandated and funded by the MSSS, published in October 2024 a practical guide specifically for out-of-hospital clinics—including podiatry clinics—that brings together all current scientific, normative, and regulatory requirements. The CAN/CSA-ISO standards adopted by the Standards Council of Canada (SCC) govern the technical characteristics of biological and chemical indicators. This article is based on these sources.

The CAN/CSA-ISO 11140-1 standard defines six classes of chemical indicators, each answering a different question about the cycle's progress. Class 1, the tape or marking on the pouch, answers a single question: has this package passed through the autoclave? Classes 3 and 4 verify that certain physical parameters—temperature, time—have been met. Class 5, the integrator, simultaneously monitors all critical cycle parameters (temperature, duration, and steam quality) and is the most commonly used in clinics: it is placed inside each pouch with the instruments. Class 6, or the emulator, is calibrated for a specific cycle, for example, 134°C for 3.5 minutes—if the parameters differ even slightly, the result is invalid. Finally, Class 2 groups specific tests such as the Bowie-Dick test, performed at the beginning of the day on pre-vacuum autoclaves to verify that air is properly evacuated and that steam penetrates the load uniformly, according to ISO 11140-4.

Regardless of its class, a chemical indicator reacts to physical and chemical conditions—temperature, humidity, pressure. It does not directly measure the destruction of microorganisms. A Class 5 or 6 indicator that shows a compliant result indicates that the required conditions have likely been met, but does not prove that bacterial spores have been destroyed. This is an important technical distinction, recognized by all standardization bodies. To obtain this direct microbiological confirmation, a biological indicator is required.

A biological indicator (BI)—often called a spore test—is a small device containing a known population of highly resistant bacterial spores, usually between 10⁵ and 10⁶ spores, deposited on a carrier and accompanied by an integrated culture medium. According to standard CAN/CSA-ISO 11138-1 (2017), it is the reference method for validating the efficacy of a sterilization process. The principle is simple: if the cycle destroys these spores—among the most resistant known—then all other less resistant microorganisms have also been eliminated. After the cycle, the BI is activated to bring the spores into contact with the culture medium, and then incubated: a negative result (no growth) confirms that the sterilization worked properly. A positive result indicates a problem in the cycle.

Each sterilization process has its own reference microorganisms, chosen because their spores are particularly resistant to that process. For steam sterilization (autoclave) — the process used in the vast majority of podiatric clinics — the reference species is *Geobacillus stearothermophilus*. Its spores are exceptionally resistant to moist heat: their D-value at 121 °C is at least 1.5 minutes, making it the most demanding test for validating a steam cycle, in accordance with standard CAN/CSA-ISO 11138-3. *Bacillus atrophaeus* is used for ethylene oxide and dry heat sterilization (ISO 11138-2 and ISO 11138-4), processes less common in podiatric clinics but present in other healthcare settings.

BIs are also distinguished by their read time, which has evolved considerably with technology. The traditional method waits for surviving spores to multiply and change the color or turbidity of the culture medium — which takes 24 to 48 hours according to ISO 11138-1. Modern rapid readout systems no longer detect bacterial growth, but rather specific enzymatic activity released by surviving spores, well before their multiplication: this is the case for the 3M/Solventum Attest™ system, which provides a fluorescent result in 1 hour. Ultra-rapid readout systems, such as STERRAD VELOCITY™ (ASP/J&J) or BIOTRACE™ (ASP), use advanced fluorescence or bioluminescence to produce a result in 15 to 20 minutes. In a low-volume podiatry clinic, a rapid readout BI makes it possible to validate a cycle and release instruments on the same day, without waiting until the next day.

The CSA Z314-2023 standard recommends a biological test each day of autoclave use for out-of-facility clinics. A test is also recommended after any repair or maintenance of the device, as well as before commissioning a new autoclave—the ODQ/ODHQ (2024) guidelines specify that three consecutive full-load cycles must yield negative results before use. The results of all these tests are recorded in a sterilization log. For podiatrists who are members of the Ordre des podiatres du Québec, this log is one of the elements checked during professional clinic inspections.

For a load of podiatric instruments in a steam autoclave, a well-documented cycle generally includes: a Bowie-Dick test at the beginning of the day for pre-vacuum autoclaves, verification and archiving of the cycle diagram printed by the device, a Class 1 chemical indicator visible on the outside of each pouch, a Class 5 integrator inside the packages, and a biological indicator according to the recommended frequency. When all these controls are compliant and the BI is negative, the cycle is considered valid.

A few documents and standards are essential references for understanding and applying good sterilization practices in Quebec. CSA Z314-2023 covers the reprocessing of medical devices in all Canadian healthcare settings. CAN/CSA-ISO 11138 standards (parts 1 to 4 of 2017, part 8 of 2021) define the technical requirements for each type of biological indicator. The CAN/CSA-ISO 11140-1 (2014) standard governs the six classes of chemical indicators. The CERDM-INSPQ Guide on the Reprocessing of Medical Devices in Out-of-Facility Clinics (October 2024) is the practical Quebec reference for podiatrists, nurses, and all private clinics. Finally, the disinfectant and sterilizing solutions used in clinics must hold Health Canada Class II approval and be listed in the MDALL database.

Chemical and biological indicators are two complementary tools, each answering a different question. Chemical indicators — from Class 1 tape to Class 6 emulators — confirm that the physical conditions of the cycle have been met. Biological indicators, or spore tests, go further: they directly prove that the most resistant microorganisms have been destroyed. Together, combined with the physical data from the autoclave, they form a complete monitoring system. Understanding the role of each means understanding why sterilization in podiatric care is a process that goes far beyond simply pressing the autoclave's start button.