Protecting Biological Indicators Before They Enter the Sterilizer

Biological indicators (BIs) are an important part of sterilization monitoring because they use highly resistant microorganisms to provide a direct challenge to the microbial-killing capability of a process. However, a BI result is only useful when the indicator has been stored, transported, handled, processed, incubated, and interpreted according to its validated instructions for use (IFU).

That makes BI control more than an incubation task. It is a chain of custody that begins when the product is received and ends when the result is linked to a specific sterilizer cycle and disposition decision.

Why pre-use control matters

CDC guidance identifies improper storage, processing, product contamination, material failure, and variation in spore resistance as possible contributors to false-positive biological indicator results.[1] This does not mean that a positive BI should be dismissed as a product problem. It means that the investigation must examine the entire process, including the condition and handling of the BI before it entered the sterilizer.

WHO guidance for sterile pharmaceutical production similarly states that biological indicators should be stored and used according to the manufacturer’s instructions.[2] The practical implication is straightforward: the BI manufacturer’s IFU is a controlled document, not optional background reading.

A practical control framework

1. Receive and identify the product

At receipt, verify the product name, sterilization method, lot or batch number, quantity, expiration or use-by date, and the condition of the shipping package. The receiving record should make it possible to identify which BI lot was available for a particular period of use.

Do not place damaged, wet, opened, contaminated, or incorrectly labeled BI materials into routine stock without a documented assessment. If the package condition raises doubt about performance, segregate the product and contact the supplier or manufacturer for disposition guidance.

2. Store according to the IFU

Storage requirements differ among BI formats and sterilization technologies. A facility should therefore avoid creating one generic storage rule for steam, ethylene oxide, hydrogen peroxide, or other process-specific indicators. Instead, maintain a current IFU or controlled product specification at the point of use and translate its requirements into a documented storage procedure.

Controls may include temperature, humidity, protection from light, separation from sterilants or chemicals, and protection from crushing or puncture. The storage area should be clean, organized, and arranged so that older stock is used before newer stock when appropriate. Environmental excursions should be documented and assessed rather than silently ignored.

3. Control stock rotation and expiry

Expiration dating is not merely an inventory concern. A BI that is beyond its labeled shelf life may not provide the performance characteristics expected by the manufacturer. Use a stock-control method that identifies approaching expiry dates and prevents expired indicators from being selected accidentally.

For critical monitoring supplies, consider maintaining a simple inventory record containing:

• product and process type;
• manufacturer and catalog or reference number;
• lot or batch number;
• quantity received and quantity issued;
• expiration or use-by date;
• storage location and relevant environmental records;
• disposition of damaged, expired, or quarantined stock.

4. Prevent contamination during handling

Handling practices should prevent contamination of the BI, its culture medium, the test area, and associated documentation. Staff should follow the manufacturer’s instructions for opening, placing, retrieving, activating, incubating, and reading the indicator. Avoid unnecessary manipulation, contact with non-clean surfaces, or mixing components from different products unless the IFU specifically permits it.

When a self-contained BI or other system is used, the operator should confirm that the correct activation and incubation steps have been completed. A result that is difficult to interpret, physically damaged, or processed outside the stated conditions should be treated as an exception requiring documented review.

Linking the BI to the sterilization cycle

A BI result has limited value if it cannot be linked to the load in which the indicator was processed. The monitoring record should connect the BI to the sterilizer identification, cycle or load number, date and time, cycle type, operator, test location or challenge device, and final result.

CDC recommendations call for recording the sterilizer and cycle, load identification, load contents, exposure parameters, operator, and mechanical, chemical, and biological monitoring results.[3] This information supports investigation if the BI is positive, the cycle record is incomplete, or a related load must be placed on hold.

What to do when storage or handling is uncertain

If a storage excursion, damaged package, incorrect product, or handling error is discovered, do not automatically treat all affected BIs as valid or invalid. First, place the questionable stock on hold. Review the manufacturer’s instructions, the duration and magnitude of the excursion, affected lot numbers, and any BIs already used during the period.

If a positive BI occurs, the facility should investigate the sterilizer, load configuration, cycle record, packaging, cleaning, operator technique, incubation system, and BI controls. CDC guidance emphasizes that a BI should not be considered false-positive until the sterilization process has been thoroughly analyzed.[1] Where repeated positive results occur, the affected sterilizer and processed items may require escalation, service review, and recall or quarantine actions according to the facility’s procedures and applicable requirements.[4]

Common failure patterns

• Uncontrolled storage: BIs are kept in a general supply area without documented environmental control or excursion review.
• Lot blindness: the facility records a result but not the BI lot, making retrospective assessment difficult.
• Expired stock in active use: expiry dates are checked only during receiving, not during daily selection.
• Incomplete traceability: the BI result is filed separately from the sterilizer cycle record.
• Premature dismissal: a positive result is attributed to “operator error” or “bad BIs” before the full process is assessed.

Limitations

Storage and handling controls reduce avoidable uncertainty; they do not replace mechanical, chemical, and biological monitoring as a coordinated program. A negative BI also does not by itself establish that every item in a load was sterile, particularly when cleaning, packaging, loading, air removal, sterilant access, or post-process handling is inadequate. Chemical indicators and physical cycle records provide complementary information and should be interpreted within the validated process.

Practical takeaway

For each BI used, a CSSD should be able to answer five questions: Was it the correct indicator for the process? Was it within expiry and stored according to its IFU? Was it protected from contamination and handling error? Can it be linked to a specific load? Can the result support a documented release or investigation decision?

Educational disclaimer: This article is for professional education and does not replace applicable regulations, standards, sterilizer instructions, BI manufacturer IFUs, validation requirements, or facility procedures.

References

1. Sterilizing Practices. Centers for Disease Control and Prevention. 2024.
2. WHO good manufacturing practices for sterile pharmaceutical products: Annex 2. World Health Organization. 2024.
3. Recommendations for Disinfection and Sterilization in Healthcare Facilities. Centers for Disease Control and Prevention. 2024.
4. Table 12: Suggested protocol for management of positive biological indicator in a steam sterilizer. Centers for Disease Control and Prevention. 2024.

Educational overview. Follow the applicable product instructions, validated procedures, local regulations and your facility’s approved policies.