Development and Application of Lyophilized Bacillus Atrophaeus Spore Pellets as an Reference Material of Enumeration for Onsite Validation of Medical Sterilization Equipment

Abstract Aims To develop a user-friendly and portable reference material (RM) for onsite validation of medical sterilization equipment, addressing the limitations of conventional biological indicators that require specialized laboratory facilities. Methods and Results A lyophilized microsphere-based RM containing Bacillus atrophaeus spores was developed by optimizing lyoprotectant formulation. The RM demonstrated excellent homogeneity (F statistic < F critical) and satisfactory stability over 14 days at 4°C and 25°C, rigorously according to ISO 33405: 2024 standard method. When applied to validate UV sterilization devices by quantifying viable spore reduction, it further provided precise and reliable log reduction value measurements. The simple reconstitute-and-culture procedure enabled non-specialists to perform onsite checks. For instance, in UV validation, optimal conditions (e.g., ≥3-log reduction within 30 min at a distance of 150 cm) were easily determined using this RM. Preliminary assessments also confirmed its applicability for ozone and ethylene oxide sterilizers. Conclusions This study presents the first preparation of a convenient, lyophilized spore RM that fulfills ISO 33405: 2024 criteria. Its successful application in validating multiple sterilization modalities demonstrates significant potential for routine quality control and compliance testing in healthcare settings, particularly where advanced laboratory facilities are unavailable.