Monitoring and Verification of Hydrogen Peroxide Disinfection and Sterilization Results in Biosafety Cabinets

After using hydrogen peroxide (especially vaporized hydrogen peroxide VHP) for disinfection and sterilization in biosafety cabinets, rigorous and scientific monitoring and result verification are the only way to confirm the effectiveness of sterilization, as well as the core requirements for quality control and regulatory compliance. This is not simply a 'run the device' process, but a closed-loop process that requires multiple pieces of physical, chemical, and biological evidence.
The following is a systematic analysis of monitoring and validation techniques.
Core principle: Triple verification system
A complete validation should include "process parameter monitoring", "chemical indicators", and "biological indicator challenge testing", all of which complement each other and are indispensable.

This is the first line of defense for verification, which involves real-time recording of key physical and chemical parameters through device sensors, proving that the sterilization cycle operates within the set and effective parameter range.
Monitoring parameters:
Hydrogen peroxide vapor concentration (ppm): the most critical parameter. It must be achieved and maintained at the predetermined target concentration (e.g. hundreds of ppm) for a sufficient period of time. Low concentration can lead to sterilization failure.
Relative humidity (% RH): controlled at a low level (usually<40%) to ensure that VHP is in an efficient "micro condensation" state rather than large droplet condensation. Excessive humidity can dilute disinfectants and may damage equipment.
Temperature: Monitor the temperature of the chamber, which affects the saturated vapor pressure and condensation process.
Pressure/airflow: Ensure the chamber is sealed and the gas circulation is normal.
Exposure time: The duration maintained at the target concentration.
How to execute:
Automatically completed by the sensors and control system built into the VHP generator.
Key requirement: The equipment should be able to generate and store complete cyclic data graphs (concentration time curve, temperature and humidity curve), which should be included as part of the validation report. Any parameter deviation from the preset range should result in an alarm or loop termination.

Used to quickly and intuitively determine whether an item is exposed to a hydrogen peroxide sterilization environment, and can indirectly reflect whether the dosage is sufficient.
Type and principle:
Label type chemical indicator card: Contains dyes sensitive to hydrogen peroxide. When a sufficient amount of hydrogen peroxide dose (concentration x time) is accumulated, irreversible changes from one color to another occur.
Placement location: It should be placed in the most difficult to reach sterilization location, such as:
The deepest corner of the safe cabinet workspace.
Inside the drainage channel.
The joint between the filter and the installation frame (if feasible).
Internal exhaust pipe (if covered by disinfection).
Function and limitations:
Function: Provide immediate results for quick release; Confirm that the steam has been distributed to the designated location; As an identifier to distinguish between "processed" and "unprocessed" items.
Limitations: Can only prove exposure, cannot prove sterility. The color change only indicates that the minimum dose threshold has been reached, but does not guarantee the killing of all microorganisms.

This is the gold standard of verification and decisive evidence. By using standardized microbial spores with known high resistance, it is directly proven that the sterilization process is sufficient to kill the most difficult to kill microorganisms.
Selection of biological indicators:
Standard strain: Spores of Geobacillus stearothermophilus. This is the internationally recognized standard test bacterium with the strongest resistance to hydrogen peroxide.
Carrier form: usually a spore suspension droplet stained paper strip, stainless steel sheet, or installed on a specific carrier.
Known spore count: Each biological indicator contains a known number of spores, typically 10 ^ 5 to 10 ^ 6 CFU (colony forming units).
Placement strategy (the most critical step):
Location: It must be placed at the "coldest point" where sterilization is most challenging or where gas is most difficult to penetrate. For biosafety cabinets, recommended locations include:
Under the workbench, in the corner near the front grille.
The junction between the back wall and the workbench.
On the back or in gaps of internal instruments such as pipette holders.
If the verification scope includes the interior of the exhaust valve or pipeline.
Quantity: Use at least 3 biological indicators and include one untreated positive control.
Testing and interpretation process:
Exposure: Perform a complete VHP sterilization cycle with the BI cabinet.
Recycling and cultivation:
After the cycle is completed, remove the BI under sterile conditions.
Put it into a dedicated recovery tube containing culture medium.
Cultivate in a 56-60 ° C incubator for 24-48 hours (specific time according to the instructions).
Simultaneously cultivate positive control (unsterilized) and negative control (sterile culture medium).
Result interpretation:
Verification successful: All sterilized BI culture media remain clear (no discoloration/turbidity), indicating no spore survival. The positive control must show active growth (yellowing/turbidity of the culture medium), and the negative control must remain clear.
Verification failure: Any processed BI showing growth (yellowing/turbidity) indicates a failure of the sterilization cycle. It is necessary to thoroughly investigate the reasons (such as parameter errors, placement, equipment malfunctions, sealing issues, etc.), adjust the plan, and conduct a complete verification again.

First verification:
After the installation of VHP equipment or the initial sterilization of a new type of safety cabinet, it must be carried out.
This is the most comprehensive verification, which requires determining the 'coldest point' and establishing effective loop parameters.
Regular revalidation:
Usually conducted every six months or annually.
Used to confirm the consistency of equipment, programs, and operators.
Daily monitoring:
Chemical indicators should be used for each sterilization cycle, and process parameter maps should be reviewed.
The frequency of biological indicator testing can be reduced as appropriate (such as monthly or quarterly), but areas with strict regulations may require the use of every batch/cycle.

All monitoring and verification activities must be documented formally, and the report should include at least:
Equipment information: VHP generator model and number.
Target device information: Biosafety cabinet model, number, location.
Loop parameters: set target concentration, time, temperature and humidity, etc.
Process data graph: concentration time curve of actual operation, etc.
Chemical indicators: type, batch number, placement location, color change results (with photos attached).
Biological indicators: strain, batch number, spore quantity, placement location, culture results (attached with photos of the culture tube).
Conclusion: Clearly determine whether the sterilization cycle is effective.
Executor and Date: Signature of operator and reviewer.

summary
Unterilized sterilization is equivalent to no sterilization.
A reliable validation system for hydrogen peroxide disinfection and sterilization of biosafety cabinets is:
Ensure process control through "process parameter mapping", achieve rapid visual release through "chemical indicators", and ultimately obtain microbiological ultimate effectiveness certification through "biological indicator challenge testing".
This triple validation system is the key to elevating hydrogen peroxide disinfection from an "operation" to a traceable, reproducible, and reliable "science", and is also the cornerstone of meeting the requirements of GLP, GMP, and biosafety laboratory accreditation at home and abroad. Laboratory managers must implement this as a mandatory procedure and continuously monitor it.