A theoretical study and a micro-scale sensing platform for the detection of germination in Bacillus stearothermophilus spores

Electrical sensing techniques offer reliable and inexpensive solutions for detecting various activities of microorganisms. However, this technology has not yet been extensively applied in health-care industry to verify the success of sterilization processes. One of such processes includes high temperatures, often combined with elevated pressures, where Bacillus Stearothermophilus bacteria spore germination is monitored for sterilization verification through optical means. This project aims to investigate the change in medium conductance during the germination of Bacillus Stearothermophilus spores, and to design a micro-scale device for detecting this biological event. Germination is a process of spore transformation from endospore to vegetative cell in favorable environment. During germination, spores release most of its ions content including DPA2-, Ca2+, Mn2+, Mg2+, K+ and Na+ into the medium and absorb water for core hydration and expansion. The released ions cause a change in the medium conductance. Bacillus Stearothermophilus spores contain a considerable amount of DPA2-compared to other ions, hence it is the major electrically conductive element. The DPA2- electrical conductivity was calculated by using a theoretical model based on two equations, the Stokes-Einstein equation for particle diffusion and the molar conductivity equation. For diffusion calculation, single DPA2- molecule was approximated as a spherical particle, whose radius was calculated based on its crystal data. Using the dimensions of the spore, maximum coverage for 1 cm2 area was calculated to be 45.45 × 106, yet, due to expected experimental artifacts, only 10% of the total spore coverage was taken into account. In addition, it is been reported that, due to the lack of suitable conditions for a specific number of spores' activation, only a partial number of them would germinate. Therefore, only 10% of the spore coverage germination was assumed, thus 1% of the calculated total spore coverage was considered for the conductance calculations. The conductance change value at 1% of spore germination was found to be 7.7 mS/m. The same theoretical model was again applied for 5%, 10%, 25%, 50% and 100% of spores DPA2 yield to investigate the sensitivity of the micro-chip design. Three micro-chip designs with 1 cm2 substrate area and different electrode geometries were developed each loaded with 0.05 mL food solution. These designs were simulated in COMSOL Multiphysics® software to observe the conductance change during germination. It has been shown that, 1% spore germination can change the solution resistance from 124.01 kΩ to 66.03 Ω. This work demonstrates that solution resistance can be monitored for the early detection of Bacillus Stearothermophilus spore germination and can be used in industrial sterilization verification systems.