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Multiple processes are available for sterilizing medical devices; however, steam sterilization remains the most widely used in healthcare settings, dental practices, and the pharmaceutical industry.

Steam sterilization cycles rely on defined critical variables, primarily temperature, exposure time, and the presence of saturated steam. These parameters must be specified, validated, and continuously monitored to ensure that the conditions required for an efective sterilization are achieved and maintained.

Chemical indicators: Monitoring of steam sterilization

Routine monitoring of steam sterilization cycles may be performed using biological indicators (BIs) or chemical indicators (CIs). CIs incorporate inks that undergo a defined and observable chemical or physical change when exposed to specific sterilization variables. The nature of this change is specified by the manufacturer and must be interpreted by the user as acceptable or unacceptable (fail or pass).

ISO 11140-1:2014 defines six types of CIs, intended to provide evidence of sterilization failures resulting from improper packaging, incorrect loading, or sterilizer malfunctions. It is important to note that CIs cannot be used alone to assess sterility; rather, they constitute one component of a comprehensive monitoring approach.

According to the standard, Type 1 indicators, also known as process indicators, are used externally, i.e., on the outer surface of packages or containers to be sterilized, and their sole purpose is to distinguish processed from unprocessed items. These external indicators provide evidence that an item has been exposed to the process but do not ensure that specific sterilization parameters are met. Type 2 indicators are designed for specific sterilizer performance tests, such as the Bowie–Dick test, which is intended to evaluate air removal and steam penetration in sterilizers with dynamic air removal. Type 2 indicators shall be used exclusively to monitor the specific tests for which they were designed.

Types 3 through 6 CIs are classified as internal indicators and must be placed inside sterilization packs to verify that the sterilizing agent has penetrated the packaging materials and that appropriate conditions have been met at the point of placement. Type 3 indicators respond to one critical process variable, while Type 4 indicators, also known as multivariable indicators, respond to two or more critical process variables. Type 5 CIs, referred to as integrating indicators, are designed to respond to all critical process variables and are calibrated to correlate with the performance of a BI. Type 6 indicators, known as emulating indicators, are intended to simulate specific steam sterilization cycles with well-defined parameters and are also required to
react to all process variables.

The following table summarizes the different types of CIs as defined in ISO 11140-1:

Type	Description	Inteded use
Type 1 “exposure” or process indicator	Used to distinguish processed from unprocessed items.	External indicator
Type 2 specific test indicators	For use in specific tests, e.g. Bowie and Dick-type test.	Sterilizer tests
Type 3 single variable indicator	Reacts to one critical process variable only	Internal indicator
Type 4 multivariable indicator	Reacts to more than one critical process variable	Internal indicator
Type 5 integrating indicator	Reacts to all critical process variables and is calibrated with a biological indicator (BI)	Internal indicator
Type 6 emulating indicator	Reacts to all critical process variables for specified sterilization processes	Internal indicator

Many international guidelines recommend using both internal and external CIs. For instance, the World Health Organization’s Surgical Safety Checklist requires operating room personnel to verify the sterility of each instrument set before use, which can be fulfilled by inspecting both external and internal CIs.

Terragene manufactures Type 1, 2, 4, 5, and 6 indicators for monitoring several sterilization processes, including steam. These CIs are available in a variety of formats and designs. Below is a brief overview and comparison of the CIs manufactured by Terragene for steam sterilization processes, excluding Type 2
indicators, as their design, validation, and intended use involve different concepts. That is why ISO 11140 addresses Type 2 indicators in dedicated parts. Terragene manufactures the CIs in two main formats, differentiated by the physical and/or chemical mechanism through which the indicator response is expressed.

The first one includes printed-ink CIs, in which the chemical reagents are formulated into an ink and printed onto a solid substrate. Depending on the product design, the substrate may consist of a self-adhesive label, a laminated strip, or an indicator tape. When exposed to the sterilization process, the indicator ink undergoes a chemical reaction that results in a defined color change specified by the manufacturer, corresponding to the
indicator endpoint, a concept described in a later section of this document.

The second format consists of “moving front” CIs, or migrating CIs, which have a more complex construction. These indicators incorporate a paper wick and a steam and temperature sensitive chemical pellet contained in a paper/film/foil laminate. Because the ink pellet is sensitive to steam and temperature, it melts upon
exposure and migrates along the paper strip by capillary action. This migration is visible through a window and allows assessment of whether the ink reaches the ACCEPT or REJECT zones marked on the front layer of the indicator. The extent of migration depends on steam quality, temperature, and exposure time.

Accordingly, Terragene offers Type 1, Type 4, Type 5, and Type 6 chemical indicators for steam sterilization in the following formats:

Type 1 | Process Indicators

Type 1 indicators are available in printed-ink formats. Terragene manufactures these indicators as self-adhesive labels (e.g., CD23) and indicator tapes (e.g., CT22). This variety of formats allows the user to select the most appropriate option based on the nature, material, and geometry of the items to be sterilized.

Type 4 and Type 6 | Multivariable and Emulating Indicators

Type 4 and Type 6 indicators are manufactured as printed-ink strips. Available formats include strips with two reactive ink lines (Type 4; e.g., CD29), double-dot strips (Type 4; e.g., CD20), and single-dot strips (Type 6;e.g., IT27-5YS).

Type 5 | Integrating Indicators

Unlike the above indicators, Terragene’s integrators are available not only as printed-ink strips (e.g., IT26-1YS), but also as moving front format (e.g., IT26-C).

Key concepts for CIs: Endpoint and Stated Values

The performance of internal CIs is based on two fundamental concepts defined in the Terms and Definitions section of ISO 11140: the endpoint and the stated values (SV), which are intrinsically related. The endpoint of a CI refers to “the point of the observed change defined by the manufacturer, occurring after the indicator has been exposed to specified SV”, the SV being defined as “the minimum value or values of a critical process
variable at which the indicator is designed to reach its endpoint as defined by the manufacturer”. What does this mean in practical terms? The endpoint is the manufacturer-defined visual change that corresponds to a “pass” condition or acceptable result. In other words, the endpoint is achieved when the critical variables, or the required sterilization conditions, have been met. This change may involve a color shift or, in the case of moving-front indicators, the migration of a dye beyond an accept/reject line.

Performance requirements for Type 1, Type 4, Type 5, and Type 6 indicators

Unlike the other types of CIs, Type 1 indicators do not have manufacturer-defined Stated Values; instead, their performance is evaluated against exposure conditions specified in ISO 11140-1, which define the criteria for acceptable and unacceptable responses. In accordance with the standard, the indicator must reach its endpoint after exposure to saturated steam at 121 °C (+3/0 °C) for 10.0 minutes ± 5 s, or at 134 °C (+3/0 °C) for 2.0 minutes ± 5 s. No visible color change shall occur under reduced exposure to saturated steam or exposure to dry heat at 140 °C (+2/0 °C) for 30 minutes ± 1 minute. The dry heat test is intended to verify that process indicators for steam respond specifically to saturated steam rather than elevated temperature alone.

Regarding internal indicators, Type 4 CIs should reach their endpoint when exposed to the stated values declared by the manufacturer. Terragene produces Type 4 CIs that react to all critical process variables. For fail conditions, 11140-1 requires exposure to SVs reduced by the combined tolerances: a temperature reduction of 2 °C and a time reduction of 25%.

The requirements for Type 5 CIs in ISO 11140-1 are far more prescriptive, as their performance is linked to the inactivation of a BI compliant with ISO 11138-3. Type 5 CIs must give a “pass” result when exposed to saturated steam at 121°C for not less than 16.5 minutes, 135°C for not less than 1.2 minutes, and a third temperature and time combination specified by the manufacturer, which falls within the range 121 to 135°C. Additionally, the standard requires characterization of Type 5 CIs temperature coecient, which serves to demonstrate that
the behavior of the CI is equivalent to the thermal resistance characteristics of the reference BI. This coecient is derived from the slope of the curve obtained by plotting log SV and/or SV versus temperature. The indicator temperature coecient shall be not less than 10 °C and not more than 27 °C, and the correlation coecient of the curve established by least squares linear regression analysis of the data shall be not less than 0,9. When evaluated at SVs reduced by the combined ISO 11140-1 tolerances, the indicators are required to show a “fail” result, corresponding to a temperature decrease of 1 °C and a time reduction of 15%. The tolerances for the detection of a pass and a fail in a Type 5 CI are therefore stricter than for a Type 4.

As for Type 6 CIs, these indicators shall produce a “fail” result when tested at exposure conditions equal to the stated values minus the combined tolerances, defined as a reduction of 1 °C in temperature and 6% in exposure time. As Type 6 indicators are cycle-specific and calibrated to narrow performance windows, the tolerances for differentiating pass and fail outcomes are significantly tighter than those applicable to Type 4 or Type 5 CIs.

In compliance with ISO 11140-1, both Type 5 and Type 6 indicators for steam exposed to dry heat at 140 ºC ±2 ºC for 30 min ± 1 min shall not reach their end points. Again, the dry heat test is designed to ensure that these indicators require the presence of steam in order to respond.

The following table summarizes and compares the different SVs for steam CIs according to indicator type:

Final considerations

CIs, together with physical monitoring and BIs, constitute the three essential technologies used for the routine monitoring of steam sterilization processes. They support effective processing by enabling the differentiation between processed and unprocessed items, demonstrating penetration of the sterilizing agent into the load, and indicating whether the required exposure conditions have been achieved at the point of placement. CIs are therefore indispensable tools for ensuring the reliability and consistency of sterilization workflows in both healthcare and industrial settings.

The selection of the appropriate type of CI is a key aspect of steam sterilization monitoring. The outside of each pack should have a Type 1 process indicator present to provide immediate evidence that a pack has been exposed. The selection of an internal CI is more complex and should take into account clinical requirements and workflow considerations. Importantly, no single type of CI is inherently superior to another. Each type is designed to fulfill a specific function and offers distinct performance characteristics. Consequently, the appropriate selection of a CI depends on the intended application, the sterilization cycle employed, applicable regulatory requirements, and the level of assurance required.

Sterile processing departments generate enormous amounts of data every single day — cycle times, indicator results, load contents, operator IDs, equipment status. The question most CSSDs still haven’t answered is simple: what happens to that data once it’s recorded?

In many facilities, the answer is: not much. It sits in a logbook, a spreadsheet, or a filing cabinet, disconnected from the people who could actually use it to improve how the department runs.

The Real Cost of Manual Data Capture

Paper-based or fragmented digital records don’t just slow teams down — they create blind spots. When indicator readings are transcribed by hand, small errors compound. When records live in separate systems that don’t talk to each other, nobody has a full picture of a load’s journey from washer to shelf. And when an audit or an infection-control investigation happens, teams spend hours reconstructing a timeline that should have been available in seconds.

This isn’t a technology problem in the abstract — it’s a daily operations problem. Manual data capture limits what a CSSD team can actually do with the information they’re already collecting.

From Recording Data to Acting on It in the CSSD

Digitalization changes the role data plays in a CSSD. Instead of being something you log defensively — “just in case someone asks” — it becomes something the team actively uses:

• Real-time visibility into cycle results means deviations get caught during the process, not after the fact.
• Automatic linking between indicators, loads, instruments, and even patients closes the traceability loop end to end.
• Centralized records mean any authorized person can reconstruct a load’s full history without hunting through multiple logs.
• Less manual entry means fewer transcription errors and more time for the tasks that actually require clinical judgment.

This is the shift Bionova® Q is built around: a cloud-based traceability platform that uses AI to read chemical indicators automatically and connects directly to existing incubators to capture biological indicator results — removing manual entry from the equation entirely, with no new servers or IT infrastructure needed.

What Digital Traceability Looks Like Day to Day

For a CSSD team, the practical difference shows up in small moments that add up:

• A biological indicator result appears automatically, even outside working hours, instead of waiting for someone to check the incubator.
• Multiple technicians can work in parallel on different loads without bottlenecking a shared logbook.
• An audit-ready report generates itself, fully digital, instead of being assembled the night before an inspection.
• Every indicator is already linked to its process, load, and user — so tracing an issue back to its source takes minutes, not a full-day investigation.

None of this requires the department to change what it does. It changes how much friction sits between doing the work and having usable data about that work.

Data as a Clinical Decision, Not Just a Record

The deeper shift is this: sterilization data isn’t just a compliance artifact. Captured consistently and connected properly, it becomes a source of operational intelligence — where deviations cluster, which shifts or instruments need attention, where a process can be tightened before it becomes an incident.

That’s the gap between a department that records sterilization and one that manages it. Digitalization, through platforms like Bionova® Q, is what makes that second version possible — turning everyday CSSD data into decisions that improve infection control outcomes.

Ready to see what digital traceability looks like in your CSSD? Discover how Bionova Q works. 

A biological indicator (BI) is a microbiological test system that carries a defined, known population of highly resistant bacterial spores and is used to confirm that a sterilization cycle killed living microorganisms — not just that it reached certain parameters. Where a chemical indicator only reacts to a certain group of chemical and/or physical conditions of the cycle, a biological indicator measures the one outcome that actually matters: whether life survived the process.

That distinction is why biological indicators for sterilization remain the reference method across hospitals, sterile processing departments, and pharmaceutical manufacturing, This note walks through the science behind biological indicators, how self-contained biological indicators (SCBIs) evolved, and how Terragene’s Bionova® product line applies rapid fluorescence detection to shorten a result that used to take a week into one that can take seconds.

What Is a Biological Indicator?

Bacterial endospores are the foundation of biological indicator technology. Sporulation is a dormancy strategy that bacteria trigger under nutrient stress, and the resulting spore form is extraordinarily resistant to heat, chemical agents, and radiation while remaining stable in storage and able to germinate reliably once conditions turn favorable. Those three properties — resistance, storage stability, and reliable germination — are exactly what make bacterial spores suited to monitoring a sterilization process.

Biological indicators are manufactured using species from the genus Bacillus and Geobacillus. International standards (the ISO 11138 series) and pharmacopeial references (USP) specify which organism should challenge which sterilization process:

Geobacillus stearothermophilus is the predominant organism for steam and hydrogen peroxide monitoring: a thermophile with an optimal growth range of 55–60°C. For steam BIs conforming to ISO 11138-3, the minimum D-value must be at least 1.5 minutes with a Z-value of at least 6°C. Bacillus atrophaeus, a mesophile optimal at 30–39°C, is the reference species for EO and Dry heat, with minimum D-values set by ISO 11138-2 and ISO 11138-4 respectively.

The Science Behind Biological Indicators

D-value, Z-value, and the survivor curve

Two parameters quantify how resistant a biological indicator’s spore population is. The D-value is the exposure time or dose needed to achieve a 90% (1-log) reduction in the viable spore count. The Z-value, which applies to thermal processes, is the temperature change that produces a tenfold change in D-value. Plotted together, they describe the survival curve — a semi-logarithmic plot of viable spore count against exposure time that, under ideal conditions, follows first-order inactivation kinetics.

These aren’t abstract numbers: they’re the basis for calculating sterility assurance and for designing cycles with an adequate safety margin. Regulatory guidance recommends that D-values be established on the final finished product with a resistometer tested across at least three different spore lots from different spore crops.

From Spore Strips to Self-Contained Biological Indicators (SCBIs)

Self-contained biological indicators are a design step beyond the original spore-strip format. In an SCBI, the spore carrier and a growth medium sit sealed inside a single hermetic unit. After exposure, the user activates the unit — typically by crushing an internal ampoule to release the medium — and incubates it at a validated temperature. this sealed unit removes the risk of cross contamination and means every user reads the result under the same controlled conditions the manufacturer validated.

Conventional Bionova® SCBIs use pH-sensitive chromogenic indicators in the growth medium: surviving spores produce acid byproducts that trigger a visible color change. The rapid Bionova® line instead uses enzyme-based fluorescence detection, generating a fluorescent product within minutes — well before the turbidity or pH shift a colorimetric read depends on, without giving up sensitivity or specificity. The fastest model, BT225, works differently again: it targets the structural denaturation of heat-stable spore proteins as a direct proxy for spore inactivation, which is what gets its readout down to 7 seconds.

Rapid Readout Technology: From 7 Days to 7 Seconds

The reference incubation period for a biological indicator is 7 days, per ISO 11138-1 §7.3. Terragene’s Bionova® rapid-readout portfolio spans a much wider range — from 7 seconds for steam up to 4 hours for ethylene oxide — letting a facility pick the product that fits its process type, throughput, and regulatory setting.

Steam sterilization (autoclave)

An unexposed control BI with a full spore population typically shows positive growth within 3–4 hours using a conventional colorimetric method. BT225’s 7-second readout and BT224’s 20-minute readout compress that timeline by orders of magnitude by detecting the relevant marker long before visible turbidity or pH change appears, while BT222’s 1-hour readout offers a lower-cost middle ground.

Hydrogen peroxide sterilization

For heat-sensitive devices like endoscopes, BT98’s 5-minute readout can confirm sterility faster than it takes to move the processed load to the point of use; BT96’s 30-minute readout is the option when that slightly longer window isn’t a constraint.

Ethylene oxide and formaldehyde (LTSF)

BT110 brings EO confirmation down to 4 hours, a sharp cut from the 48-hour or 7-day incubation these loads have traditionally required before release. BT102 reads formaldehyde (LTSF) cycles in 2 hours, supporting facilities running low-temperature steam-and-formaldehyde sterilization.

Reduced Incubation Time (RIT): How It’s Validated

A validated reduced incubation time is what allows an SCBI to report a result well before the 7-day reference period elapses, and it isn’t an informal shortcut — it follows a defined methodology. The FDA CDRH’s 2007 guidance on biological indicator 510(k) submissions requires exposing a minimum of 300 BIs (100 from each of three separate manufacturing lots) to partial sterilization cycles engineered to leave 30–80% spore survival. Every BI is incubated for the full 7-day reference period with daily scoring, and the validated RIT is set as the greatest number of incubation days needed for any single lot to reach 97% or more of that lot’s total 7-day positive count.

Results are never averaged across lots — the most conservative lot sets the minimum incubation time. That conservatism matters because it’s designed to hold even in a worst case: if only a single spore survives a near-lethal exposure, the validated incubation window still gives that lone survivor enough time to germinate, multiply, and produce a detectable positive signal.

Sterility Assurance Level (SAL) of 10⁻⁶, Explained

The Sterility Assurance Level is defined as the probability of a single viable microorganism remaining on a product after sterilization. For terminally sterilized medical devices and pharmaceutical products, the internationally accepted target is an SAL of 10⁻⁶ — a probability of no more than one non-sterile unit per million units processed.

The overkill method

The most widely used validation strategy is the overkill approach. A BI carrying a known population — typically 10⁵–10⁶ CFU of an organism far more resistant than any expected bioburden — is used to show that a half-cycle (50% of the full exposure time) is already enough to fully kill every BI. The full cycle then delivers at least double that lethality, which is the safety margin behind the SAL claim.

Regulatory guidance sets minimum spore populations and D-values for cleared BIs: for steam at 121°C, a minimum of 10⁵ spores with a D-value of at least 1.5 minutes and a survival time of at least 5 minutes; for EO at 600 mg/L, 54°C, 60% RH, a minimum of 10⁶ spores with a D-value of at least 2.5 minutes. Terragene’s rapid Bionova® models (BT225, BT224, BT98, BT110, BT102) are manufactured to meet or exceed these benchmarks, with every lot characterized against ISO 11138 across multiple spore crops before release — a faster readout does not mean a smaller resistance challenge.

Why Biological Indicator Placement Matters

A reliable SAL result depends on where the biological indicator sits inside the sterilizer, not just on the indicator itself. In a published investigation it was shown, in a real production cycle (6,480 units), that the equivalent process time (F-value) varied across chamber positions from 19.5 to 23.5 minutes. With a D-value of 2 minutes, that 4-minute spread already corresponds to a 2-log difference in spore kill between the least and most lethal locations. In large or thermally massive loads, the gap can reach 10–12 equivalent minutes — a difference of several orders of magnitude in SAL within the very same cycle.
The practical takeaway: placing a BI without systematic lethality mapping is, in the author’s own words, a dangerous hypothesis. Only mapping identifies the true worst-case (minimum-lethality) location, and only there does a BI give a trustworthy SAL validation. A precise, standardized BI is what allows biological inactivation to be accurately correlated with that mapped physical lethality — placed incorrectly, it can overestimate the true SAL by two or more log orders.

Practical Applications

Routine monitoring in hospital CSSDs

Day-to-day monitoring places one SCBI per sterilization load alongside an unexposed control unit; both are activated and incubated after the cycle. The control has to show positive growth to confirm the spore population was viable, while the exposed BI should read negative at the end of the validated incubation window. With BT225, a steam load can be biologically confirmed before the instruments even reach the point of use; for H₂O₂ sterilization of heat-sensitive devices, BT98’s 5-minute readout can beat the time it takes to move the load to the operating room.

Sterilization validation with the overkill approach

Initial validation typically runs three consecutive half-cycle exposures with BIs placed at the identified worst-case locations; all of them need to show no growth to demonstrate the half-cycle alone is lethal enough. Because the full cycle doubles that exposure, this establishes the safety margin behind the SAL 10⁻⁶ claim. Where a traditional protocol required a 7-day hold between validation runs to confirm results, rapid products such as BT224 or BT98 let a facility confirm a run and move on to the next one the same day.

When a biological indicator tests positive

One of the harder situations in sterilization practice is a positive BI in a cycle where every physical parameter looks normal. That discordance usually points to something the instruments alone can’t see — trapped air pockets in a porous steam load, or insufficient humidification inside an EO load, for example. The right response is never to dismiss the biological result: a BI integrates every lethal and sub-lethal condition it actually experienced at its location, which makes it the most complete single indicator of how the process really performed. A rapid readout just means that investigation can start within minutes instead of days.

From spore biology to regulatory compliance, one principle holds: a biological indicator is only as trustworthy as its science, its placement, and its validation. Terragene Bionova® SCBIs bring rigor and speed together — turning sterility assurance from a multi-day wait into a near-real-time certainty.
Read the full article here.

Developing an OEM solution is about much more than finding a manufacturer. The right OEM partner can contribute technology, technical expertise, manufacturing capabilities, and regulatory knowledge throughout the entire development journey — helping transform an idea into a reliable solution ready for the market.

But how do you know which partner is right for your project? Here are eight key factors to consider.

1. End-to-End Development Capabilities

An effective OEM partnership should extend beyond manufacturing. From the initial concept and product development to validation, production, and delivery, having a partner capable of supporting multiple stages of the process can simplify project management and reduce the complexity of coordinating different suppliers.

At Terragene, multidisciplinary teams work across the development process to support OEM projects from concept to production. The goal: one partner, a more connected development journey.

2. Technology-Driven Innovation

Technology should be at the core of an OEM partnership. The right partner should bring more than production capacity — it should have the scientific and technological capabilities to develop solutions that respond to specific market needs.

Terragene combines biotechnology, engineering, and applied technology to develop solutions for infection prevention and healthcare environments. This technological foundation allows OEM partners to explore differentiated solutions rather than relying solely on standard products.

3. Customization and Flexibility

Every OEM project has different objectives, markets, and technical requirements. A strong partner should be able to adapt its capabilities to the project instead of forcing the project into an existing product model.

Customization may involve product characteristics, specifications, packaging, documentation, technology integration, or other elements required by the customer and target market. Your requirements should shape the solution — not the other way around.

4. Integrated Manufacturing Capabilities

Manufacturing capabilities matter, but manufacturing control matters even more. An integrated production model can provide greater control over processes, quality, and scalability while helping ensure consistency throughout the product lifecycle.

Terragene’s vertically integrated capabilities support multiple stages of development and manufacturing, creating a more controlled path from raw materials and technology development to finished solutions. For OEM partners, this can mean greater visibility and a more reliable supply chain.

5. Quality Built Into the Process

Quality should not be something checked only at the end of manufacturing — it should be considered throughout development and production. For OEM projects, this means working with a partner that understands the importance of controlled processes, validation, documentation, and consistency.

Terragene develops and manufactures solutions under international quality and regulatory frameworks, supporting the requirements of healthcare and infection prevention markets. Quality by design helps create confidence long before the product reaches the customer.

6. Regulatory Expertise

Taking an OEM solution to market can involve complex regulatory requirements. Different countries and markets may require specific documentation, registrations, technical information, and compliance processes.

An OEM partner with regulatory expertise can help anticipate these requirements during development rather than addressing them only once the product is ready for launch. This is particularly important for companies looking to scale their solutions across multiple markets. Regulatory strategy should be part of product development — not an afterthought.

7. Multidisciplinary Expertise

Successful OEM projects rarely depend on one area of expertise. They require collaboration between science, engineering, product development, quality, regulatory, manufacturing, and commercial teams.

Working with a partner that brings these capabilities together can accelerate decision-making and create better alignment throughout the project. At Terragene, multidisciplinary teams work together to connect technical knowledge, innovation, and market requirements.

8. Global Experience and Market Understanding

A solution designed for one market may need to adapt to another. Understanding local requirements, customer expectations, and regulatory environments is therefore essential for companies developing products for international markets.

Terragene combines international experience in infection prevention with a global commercial network, supporting OEM projects with a broader understanding of healthcare markets and their requirements. This global perspective can help OEM partners build solutions with scalability in mind from the beginning.

The Right OEM Partner Is More Than a Manufacturer

An OEM project is a long-term collaboration. The right partner should bring the capabilities, technology, and expertise required not only to manufacture a product, but also to contribute to its development and evolution.

At Terragene, we bring together technology, biotechnology, manufacturing, quality, regulatory expertise, and multidisciplinary teams to support OEM projects from concept to market.

Because your brand deserves more than a manufacturer. It deserves a partner who can help bring your innovation to life.

Your brand. Our innovation.

Explore Terragene OEM Solutions

Effective VH₂O₂ Sterilization Monitoring Starts with a Standardized Challenge

Effective sterilization monitoring is a critical component of infection prevention programs in healthcare facilities. In vaporized hydrogen peroxide (VH₂O₂) sterilization processes, biological indicators (BIs) play an essential role in demonstrating process efficacy. However, routine monitoring practices in many Sterile Processing Departments (SPDs) continue to rely on standalone biological indicators placed inside sterilization pouches, an approach that introduces variability and provides a lower level of process challenge than current best practice recommendations.

Bionova® PCD98 was developed to address these limitations. Designed specifically for VH₂O₂ sterilization monitoring, it combines a Self-Contained Biological Indicator (SCBI) with a standardized Process Challenge Device (PCD), creating a defined and reproducible challenge without requiring manual assembly or the use of a sterilization pouch. Results are available in just five minutes using the Bionova® Hyper auto-reader, while digital traceability is achieved through the Bionova® Q platform.

What Is Bionova® PCD98?

Bionova® PCD98 is a single-use device that integrates biological monitoring and process challenge into a single solution for vaporized hydrogen peroxide sterilization.

Each unit consists of:

• A polypropylene tube containing a spore-inoculated carrier with Geobacillus stearothermophilus ATCC® 7953 (ATCC® is a registered trademark of American Type Culture Collection.).
• A glass ampoule containing liquid culture medium.
• A plastic cap with a permeable barrier that allows VH₂O₂ penetration.
• A process indicator printed on the label that changes from violet to green after exposure to hydrogen peroxide.

The device provides fluorescence-based readout in five minutes at 60 ± 2 °C using the Bionova® Hyper auto-reader. Visual confirmation remains available through color change, while every unit includes a DataMatrix code for integration with the Bionova® Q platform, enabling digital registration and traceability. Storage conditions, shelf life, and product specifications are standardized to support routine sterilization monitoring.

Understanding the Difference Between a Process Challenge Device and a Standalone Biological Indicator

According to ANSI/AAMI ST58:2024, a Process Challenge Device is a system containing a biological indicator—or a biological and chemical indicator combination—that evaluates the effective performance of a sterilization process by providing a challenge equal to or greater than the most difficult item routinely processed.

This distinction is fundamental.

A standalone biological indicator placed inside a sterilization pouch cannot provide the same defined challenge because its performance depends on multiple external variables. A ready to usea Process Challenge Device, by contrast, is specifically designed to reproduce a worst-case sterilization scenario by creating controlled barriers to sterilant penetration, air removal, and exposure of the biological indicator. If sterilization is effective under these more demanding conditions, it provides stronger evidence that the processed load has been adequately sterilized.

Conventional Monitoring Versus Bionova® PCD98

One of the main differences between conventional monitoring and Bionova® PCD98 is the level of standardization.

With a biological indicator placed inside a sterilization pouch, the challenge level depends on factors such as pouch permeability, the assembly technique, and operator practice. Manual preparation introduces variability between users and between sterilization cycles.

Bionova® PCD98 eliminates these variables through a fixed architecture designed to provide the same challenge every time.

Unlike conventional monitoring methods, PCD98:

• Requires no manual pouch assembly.
• Is ready to use immediately.
• Provides a fixed and reproducible challenge.
• Delivers fluorescence-based results in five minutes.
• Includes an integrated process indicator on the device label.

This standardized design allows every monitoring cycle to be performed under consistent conditions, reducing variability associated with conventional biological indicator placement.

Why the Challenge Level Matters in VH₂O₂ Sterilization

The principle behind every Process Challenge Device is to simulate conditions that are equal to or more difficult than those presented by the most challenging devices routinely sterilized.

In VH₂O₂ sterilization, sterilization efficacy depends on exposure time, temperature, and hydrogen peroxide concentration. Physical barriers such as lumens, enclosed cavities, cables, cameras, and complex instruments restrict sterilant penetration, making these devices significantly more difficult to sterilize than a biological indicator simply placed inside a pouch.

Because a standalone biological indicator is exposed through a relatively permeable surface, it does not reproduce these demanding conditions.

Bionova® PCD98 was specifically developed to provide a greater and standardized challenge representative of these more complex sterilization scenarios

How Bionova® PCD98 Creates a Standardized Process Challenge

The effectiveness of a Process Challenge Device depends on its ability to reproduce conditions that are equal to or more demanding than those presented by the most difficult medical devices routinely processed during sterilization.

Bionova® PCD98 achieves this objective through its self-contained architecture. The biological indicator containing Geobacillus stearothermophilus ATCC® 7953 is enclosed inside a polypropylene tube sealed with a permeable barrier cap. Before the sterilant reaches the spore carrier, vaporized hydrogen peroxide must diffuse through this controlled barrier.

Unlike conventional monitoring approaches, the geometry and material properties of the device create a defined restriction to sterilant penetration. This restriction is established by design and manufacturing specifications rather than by pouch selection or operator technique, providing the same challenge in every sterilization cycle.

The Role of the Permeable Barrier Cap

The cap design is central to the performance of Bionova® PCD98 as a Process Challenge Device.

Because vaporized hydrogen peroxide has relatively limited diffusion capacity, the pinhole-sized aperture creates a meaningful challenge during sterilization. This restricted opening performs two simultaneous functions throughout the sterilization cycle.

During the vacuum phase, air must evacuate the internal cavity through the same narrow pathway before sterilant can effectively reach the biological indicator. During the exposure phase, vaporized hydrogen peroxide must diffuse inward through that identical opening until it reaches the spore carrier.

By simultaneously challenging air removal and sterilant penetration, the device creates substantially more demanding conditions than an open configuration. This physical design closely reproduces the challenge presented by cannulated or lumen-bearing instruments, where trapped air must first be displaced before sterilant reaches the most distant internal surfaces.

The standardized geometry of PCD98 provides a reproducible format that delivers the same internal cavity and the same controlled restriction in every unit manufactured. A passing result under these conditions provides meaningful evidence that the sterilization process was capable of overcoming the type of geometric challenge presented by the most difficult instruments routinely processed.

Why Challenge Standardization Matters

One of the principal limitations of monitoring with a biological indicator inside a sterilization pouch is that the challenge level is not fixed.

Several variables influence the performance of the monitoring system, including:

• The pouch manufacturer and material composition.
• Lot-to-lot variation of sterilization pouches.
• Manual assembly performed by the operator.
• Device orientation and sealing.
• Position within the sterilizer chamber.

As a result, different operators or different sterilization cycles may not produce identical monitoring conditions.

Bionova® PCD98 eliminates these variables through standardized manufacturing specifications. The polypropylene tube, permeable cap, and self-contained architecture provide a fixed challenge without requiring pouch selection or manual assembly. Every unit is designed to deliver the same reproducible process challenge, regardless of the facility or operator.

Key Advantages of Bionova® PCD98

1. Ready to Use Without a Sterilization Pouch: Bionova® PCD98 is placed directly inside the sterilizer without assembling a pouch. Eliminating this manual preparation step reduces the potential for operator-dependent errors while simplifying routine workflow in the Sterile Processing Department.
2. Greater and Standardized Challenge: The process challenge is defined by the architecture of the device itself rather than by external variables. This allows Bionova® PCD98 to provide a higher and more consistent challenge than a standalone biological indicator placed inside a pouch.
3. Five-Minute Fluorescence Results: Using the Bionova® Hyper auto-reader, fluorescence detection provides results within five minutes at 60 ± 2 °C. Faster result availability supports earlier load release decisions when compared with conventional biological indicator systems.
4. Integrated Process Indicator: Every device incorporates a process indicator directly on the product label. After exposure to vaporized hydrogen peroxide, the indicator changes from violet to green, confirming sterilant contact without requiring a separate chemical indicator.
5. Digital Traceability: Each Bionova® PCD98 includes a DataMatrix code that enables integration with the Bionova® Q platform. Automatic registration reduces manual transcription while creating audit-ready digital records for every sterilization cycle.
6. Reproducible Placement: The device is placed directly in the most challenging location inside the sterilizer chamber, as identified by the sterilizer manufacturer or previous validation, without constructing a test pack or placing the device inside a tray.
7. Reduced Operational Variability: By eliminating manual pouch preparation and reducing operator-dependent variables, Bionova® PCD98 delivers consistent monitoring conditions from cycle to cycle.

Using Bionova® PCD98

Routine use begins by identifying the sterilizer number, cycle number, and processing date on the device. Bionova® PCD98 is then placed in the most challenging location inside the sterilizer chamber, following prior validation or the sterilizer manufacturer’s instructions. The device should not be placed inside a pouch or package.

Following completion of the sterilization cycle, the process indicator is verified to confirm the color change from violet to green. The biological indicator is then activated by crushing the internal glass ampoule, allowing the culture medium to contact the spore carrier.

The processed unit and an unprocessed positive control from the same lot are incubated in the Bionova® Hyper auto-reader for a maximum of five minutes at 60 ± 2 °C. Fluorescence indicates a positive result (sterilization FAIL), while the absence of fluorescence indicates a negative result (sterilization PASS). Results are recorded before disposal according to local waste regulations

Regulatory and Standards Alignment

Bionova® PCD98 has been developed to comply with internationally recognized standards applicable to biological and chemical indicators used in sterilization monitoring.

The device complies with:

• ISO 11138-1:2017 – Sterilization of health care products — Biological Indicators — Part 1: General Requirements.
• ISO 11140-1:2014 – Sterilization of health care products — Chemical Indicators — Part 1: General Requirements.
• ISO 11139:2018 – Sterilization of health care products — Vocabulary of terms used in sterilization and related equipment and process standards.

In addition, Bionova® PCD98 facilitates compliance with standards and guidance documents that recommend the use of biological indicators within Process Challenge Devices for routine VH₂O₂ sterilization monitoring, including:

• ANSI/AAMI ST58:2024 – Chemical sterilization and high-level disinfection in health care facilities.
• ISO 11138-7:2019 – Guidance for the selection, use and interpretation of biological indicator results.
• ISO 14937:2009 – General requirements for characterization of a sterilizing agent and the development, validation and routine control of a sterilization process for medical devices.

Integration with the Bionova® Hyper Ecosystem

Bionova® PCD98 is designed as part of the Bionova® Hyper integrated solution for VH₂O₂ sterilization monitoring. Within this ecosystem, each component performs a specific function:

Component	Role
Bionova® PCD98	Standardized sterilization challenge and biological detection
Bionova® Hyper (BHY)	Incubation at 60 ± 2 °C with five-minute fluorescence readout
Bionova® Q	Automatic data capture, digital traceability and audit-ready records

Improving Consistency in VH₂O₂ Sterilization Monitoring

Routine monitoring methods based on standalone biological indicators placed inside sterilization pouches may introduce variability because the challenge level depends on pouch characteristics, assembly technique, and operator practice.

Bionova® PCD98 replaces these variable conditions with a standardized architecture specifically developed for vaporized hydrogen peroxide sterilization monitoring.

Its self-contained design provides a defined and reproducible process challenge without requiring manual assembly or sterilization pouches. Combined with five-minute fluorescence results, an integrated process indicator, reproducible placement, and digital traceability through the Bionova® Hyper ecosystem, the device supports a consistent approach to routine monitoring in Sterile Processing Departments. 

By providing a greater challenge than a standalone biological indicator in a pouch, Bionova® PCD98 aligns with ANSI/AAMI ST58:2024 recommendations while helping facilities standardize monitoring practices, reduce operational variability, and maintain complete digital traceability for every sterilization cycle.

Standard cleaning protocols leave measurable Residual Protein in most cannulated instruments. Here’s what three leading hospitals discovered — and what reprocessing teams should do about it.

The reprocessing of reusable medical instruments is one of the most consequential — and most undermonitored — activities in any healthcare facility. While visible soil is easy to address, the invisible threat of residual protein deep inside narrow instrument channels is far harder to detect and far easier to overlook. Residual protein shields microorganisms from disinfectants and sterilants, creating a latent pathway for healthcare-associated infections (HAIs) that standard visual inspection or ATP bioluminescence simply cannot catch.

To quantify this risk under real clinical conditions, a multicenter evaluation was conducted across three leading hospitals in Buenos Aires, Argentina: Hospital Italiano de Buenos Aires, Fundación Favaloro (University Hospital), and Hospital El Cruce Dr. Néstor Kirchner. The study assessed 19 distinct cannulated instrument types using the Chemdye® Pro1 Endo quantitative protein detection system — a BCA-based colorimetric assay adapted for internal lumens.

Why Residual Protein in Cannulated Instruments Is a Patient Safety Issue

Cannulated devices — instruments with narrow internal lumens such as ureteroscopes, hysteroscopes, suction tubes, and aspiration cannulas — present unique reprocessing challenges. Their complex geometry limits fluid dynamics, reduces the shear forces needed for soil removal, and creates dead spaces where biofilm and proteinaceous residue can accumulate and persist.

The consequences are well-documented: residual proteins and biofilm impair the efficacy of high-level disinfection (HLD) and steam sterilization. Among cannulated devices, endoscopes represent the greatest cleaning challenge because of their complex geometries, narrow lumens, and diverse material composition. Several outbreaks of multidrug-resistant organisms have been traced directly to inadequate cleaning of these devices, prompting FDA safety communications and accelerated regulatory scrutiny of automated endoscope reprocessors (AERs).

“The persistence of protein residues — even at levels well below visual detection thresholds — constitutes a latent patient safety risk, as these residues can shield microorganisms from disinfectants and sterilants.”

— MULTICENTER STUDY CONCLUSIONS

Guidelines from AAMI ST91 and the CDC support routine, preferably daily, monitoring of manual cleaning processes, and emphasize ongoing staff training and gap analysis. Yet in practice, most reprocessing departments lack the tools to verify cleaning effectiveness at the level of individual instruments and individual lumens.

The Limits of Current Detection Methods

Conventional post-cleaning verication methods each carry signicant limitations when applied to cannulated instruments.

• Visual inspection: cannot detect sub-visible protein Ims or biolm within internal channels
• ATP bioluminescence: not able to detect viruses or prions or even dead cells, which still represent an organic contamination.
• Ninhydrin-based tests and TOC analysis: lack the sensitivity for low-level residues or are impractical for internal lumen access
• Fluorescence imaging: cannot reach or quantify contamination within long narrow, or tortuous lumens.

How the Chemdye® Pro1 Endo System Works

The Chemdye® Pro1 Endo Hygiene Monitoring System is built around the well validated BCA (Bicinchoninic Acid) assay, adapted specically for internal lumen sampling. The system uses Chemdye® SWE high-absorption swabs — available in four diameters (1.7, 2.0, 2.7, and 3.0 mm) and 2.5 meters in length — that traverse the full length of an instrument’s internal channel in a single pass.

DETECTION WORKFLOW

1. A size-matched SWE swab is passed through the instrument’s internal channel in a single direction, collecting any residual protein.
2. The swab is immersed in the Chemdye® Pro1 Endo reactive solution.
3. Proteins reduce Cu²⁺ to Cu⁺, which forms a purple BCA–copper complex proportional to protein concentration.
4. The device is incubated at 60 °C for 4 minutes in the Bionova® MiniPro reader.
5. Absorbance at 562 nm is measured against a bovine serum albumin (BSA) calibration curve, delivering a quantitative result in μg of protein (range: 1–50 μg; LOD: 0.5 μg).

This design addresses a fundamental limitation of prior methods: no commercially available system could previously access and quantify contamination within long, narrow, or complex lumens. The 2.5 m swab length covers flexible endoscopic channels exceeding 120 cm in a single sampling pass.

Key Findings from the Multicenter Study

1. PROTEIN CONTAMINATION IS WIDESPREAD AFTER STANDARD CLEANING

Across all three hospitals and both cleaning modalities, the Pro1 Endo system detected protein residues in a signicant proportion of instruments that had already completed their institutional reprocessing protocol. Results ranged from 0 μg (undetectable) to 6.3 μg of protein per instrument conrming that standard cleaning does not universally eliminate internal organic contamination.

2. NARROW-LUMEN, LONG-CHANNEL INSTRUMENTS POSE THE GREATEST RISK

When protein results are normalized to internal surface area (μg/cm²), narrow instruments display disproportionately high contamination per unit area. The Olsen Cholangiography Forceps (2 mm diameter, 32 cm channel) showed an average surface density of 0.48 μg/cm², compared to 0.028 μg/cm² for the Ureteroscope and 0.010 μg/cm² for the Cystoscope. Reduced fluid shear, limited mechanical access, and laminar-to-turbulent flow transitions inside narrow channels all contribute to this pattern.

3. Ultrasonic Cleaning Significantly Outperforms Manual Cleaning

Instruments processed via automated ultrasonic cleaning showed an average residual protein level of 0.23 μg, compared to 1.31 μg for manually cleaned instruments — a 5.7-fold difference. Directed cavitation energy accesses complex internal surfaces more reproducibly than manual techniques, which are inherently subject to operator variability.

Critically, however, automated cleaning was not infallible. The Laparoscopic Forceps Sheath (ultrasonic) still registered 1.3 μg in one replicate, and the Cannula Pump showed 1 μg. Instruments with multi-section or multi-lumen architectures may include areas of incomplete cavitation access. Post-cleaning verification remains essential regardless of the cleaning modality.

CLINICAL IMPLICATION

Without post-cleaning verication, instruments carrying residual protein loads
could proceed directly to sterilization — possibly reducing the sterilant penetration and efficacy—
without any opportunity for corrective action.

4. PRO1 ENDO ENABLES REAL-TIME CORRECTIVE ACTION

The most operationally signicant finding of the study was the system’s role as an active feedback tool. At Hospital El Cruce, two instruments with initial readings above 5 μg were agged, re-washed, and retested: 

• The Frazier 9 Fr Suction Tube started at >50 μg. Aer one additional manual
  wash, the result dropped to 0.5 μg — a greater than 98% reduction.
• The Frazier 13 Suction Tube started at 6.3 μg. Two additional manual washes
  still le it at 4.9 μg. Only aer switching to ultrasonic cleaning did the result
  reach 0 μg. 

These cases illustrate the system’s unique value: it does not merely conrm a pass or fail — it identfies when a cleaning method is inadequate for a particular instrument and guides escalation to a more eective approach.

Six Evidence-Based Conclusions for Reprocessing Departments

1. Standard cleaning is not universally suficient: Residual protein was found across all three institutions and both cleaning methods, confirming that compliance with FUS alone does not guarantee internal cleanliness in cannulated instruments
2. Automate where possible: Ultrasonic cleaning reduces average residual protein by a factor of 57 compared to manual cleaning. Where instrument compatibility permits, automated cleaning should be the preferred modality.
3. Prioritize narrow-lumen instruments for enhanced protocols: Instruments with internal diameters s2 mm and longer channels particularly cholangiography forceps, ureteroscopes, and neuro aspiration cannulas-require dedicated monitoring programs.
4. Use quantitative, not qualitative, monitoring: Only a quantitative system can guide corrective action and document the degree of improvement aer re-washing.
5. Pass/fall Indicators are insucient for complex instruments: Verify every cleaning episode, not just periodic audits.
   Inter-procedural variability in soil load and cleaning thoroughness means that a single clean instrument does not predict the next
6. Document results for QMS and regulatory compliance: Quantitative records from the Bionoval MiniPro reader create a traceable audit trail supporting compliance with 150 15883 AAMI ST79, and accreditation frameworks that require evidence-based reprocessing quality monitoring.

Conclusion

This multicenter study provides clear, real-world evidence that residual protein contamination in reusable cannulated medical instruments is not a theoretical concern — it is a confirmed, measurable phenomenon occurring under standard institutional conditions. The Chemdye® Pro1 Endo system demonstrated superior sensitivity, broad instrument compatibility across all 19 device types evaluated, and direct clinical utility as a real-time corrective tool that no conventional method can replicate.

Adopting quantitative protein monitoring as a routine component of the reprocessing verication workflow represents one of the most actionable steps a facility can take to reduce the risk of device-associated infections in patients undergoing procedures with cannulated instruments.

“Pro1 Endo functions not merely as a passive quality indicator, but as an active component of the cleaning verication loop — one that prevents contaminated instruments from proceeding to sterilization and subsequent patient use.”

Why is it necessary to sterilize reusable materials?

In healthcare environments, BPH-Photon Incubator plays a critical role in steam sterilization control by enabling rapid and reliable release of sterilized loads. In operating rooms, central sterile services departments, and emergency settings—where every second matters—immediate biological indicator results are essential to protect patient safety and reduce the risk of healthcare-associated infections (HAIs).

Steam sterilization remains one of the most widely used and effective methods for reprocessing medical devices. However, a load can only be considered safe when sterilization has been biologically verified. Unlike chemical indicators, biological indicators directly challenge the process using highly resistant spores such as Geobacillus stearothermophilus, providing the highest level of assurance.

For years, biological monitoring required long incubation times, delaying load release and impacting workflow efficiency. Today, new technologies are changing that paradigm.

How the BPH-Photon® Incubator Enables 7-Second Results

The Photon® system, developed by Terragene, combines speed and safety to support early, confident decision-making. At the center of this system is the BPH-Photon® Incubator, an automatic incubator and reader designed to deliver a fully automated biological indicator readout in just 7 seconds.

When used with the Photon® BT225 self-contained biological indicator, the system provides immediate results by detecting irreversible protein damage in spores caused by an effective steam sterilization cycle. This innovative approach eliminates the need to wait for microbial growth while maintaining high reliability and regulatory compliance.

Validated against international standards, the 7-second readout correlates with the traditional 7-day incubation result and demonstrates a sensitivity of ≥97%, supporting faster and safer release of sterilized loads in demanding healthcare environments.

According to ISO 11138-8 and the FDA biological indicator guidance, a rapid reading is considered reliable when RIT studies yield sensitivities above 97%. Studies conducted with Bionova® Photon® BT225 demonstrate that its 7-second reading is sensitive enough to ensure safe load release.

Photon® BT225 demonstrate that its 7-second reading is sensitive enough to ensure safe load release. The Photon® system combines groundbreaking speed and precision, offering an effcient solution for optimizing sterilization processes without
compromising patient safety. Its high sensitivity and instant results make it a powerful tool for making immediate and reliable decisions in critical medical situations.

Watch Photon® Quick User Guide