Detection of Aerobic Bacterial Endospores: From Air Sampling, Sterilization Validation to Astrobiology

Bacterial endospores are formed in genera such as Bacillus and Clostridium in times of incipient stresses. Derivative of their remarkable resistance and ubiquity, endospores are delivery vehicles for anthrax attack, biological indicators for checking sterilization efficacy, and candidates for Panspermia and potential extraterrestrial life, thereby underscoring the significance of their rapid detection. In this thesis project, spectroscopy and microscopy methods are studied to measure the release of a unique constituent, dipicolinic acid (DPA), via germination as a proxy for endospore viability. In particular, a luminescence time-gated microscopy technique (called microscopy endospore viability assay, acronym: μEVA) has been developed to enumerate germination-capable aerobic endospores rapidly based on energy transfer from DPA to terbium ions doped on a solid matrix upon UV excitation. The distinctive emission and millisecond lifetime enable time-resolved imaging to achieve a sensitivity of one endospore. Effective air sampling of endospores is crucial in view of the potential catastrophe caused by the dissemination of airborne anthrax endospores. Based on time-gated spectroscopy of terbium-DPA luminescence, the Anthrax Smoke Detector has been built to provide real-time surveillance of air quality for timely mitigation and decontamination. This technology also finds application in the monitoring of airborne endospore bioburden as an indicator of total biomass in a closed spacecraft system in order to safeguard the health of astronauts. Sterilization validation is of prime concern in the medical field and planetary protection to prevent cross-contaminations among patients and planets. μEVA has yielded faster and comparable results compared with the culture-based NASA standard assay in assessing surface endospore bioburden on spacecraft materials and clean rooms surfaces. The current analysis time has been expedited from 3 days to within an hour in compliance with planetary protection requirements imposed on landers and probes designed for life detection missions. From the perspective of astrobiology, endospores are time capsules preserving geological history and may exist as dormant lives in analogous extraterrestrial environments. μEVA has successfully recovered ancient endospores in cold biospheres (Greenland ice core, Antarctic Lake Vida, polar permafrost) and hyper-arid biospheres (Atacama Desert) on Earth as templates for determining life longevity and the search of extinct or extant life on Mars and other icy celestial bodies. Result authenticity has been validated by a comprehensive suite of experiments encompassing culture-based and culture-independent techniques such as epifluorescence microscopy, flow cytometry, fluorometry, bioluminescence and 16s rRNA analysis. In conclusion, μEVA is a sensitive analytical tool that opens a new realm in microbiology to provide insights into air sampling, sterility assessment and exobiology.