Nitrogen Dioxide sterilization follows log-linear microbial inactivation kinetics using Geobacillus stearothermophilus biological indicators.

Aim: The primary purpose of this study was to determine the inactivation kinetics of Geobacillus stearothermophilus biological indicators (BIs) exposed to Nitrogen Dioxide (NO₂) gas in the presence of humidity.

Methods: BIs inoculated with 6 log₁₀ G. stearothermophilus spores were used as a test substrate. Three BI Lots manufactured from each of three different BI spore crops were evaluated. Test cycles were run at room temperature with 80% relative humidity. Direct Enumeration methods were used to quantify the resistance of spores with surviving populations greater than 2 log₁₀ Fraction Negative methods were used to calculate spore populations in the quantal region. The methods were combined in order to show spore inactivation from 6 log₁₀ to approximately -2 log₁₀ The D-Value and least-squares regression (R²) were calculated.

Results: Over 100 Direct Enumeration and Fraction Negative Cycles were completed at a fixed NO₂ concentration varying only time. Critical process parameters were maintained over all cycles. Empirical data confirmed a log-linear relationship over an 8 log₁₀ population range with R² values greater than 0.8, allowing for extrapolation of the curve to -6 log₁₀ Study outcomes were comparable for all manufactured BI Lots.

Conclusions: NO₂ sterilization follows first-order log-linear microbial inactivation kinetics, which is consistent with a mechanism of action based on a single active species.

Significance and impact of study: This is the first study to report on the microbial inactivation kinetics of NO₂ sterilization. Further, this is one of the few studies to demonstrate inactivation kinetics applying ISO methodology.