Fast Viability Assessment of Clostridium Spores: Survival in Extreme Environments

Abstract

Bacterial endospores are formed in genera such as Bacillus and Clostridium in response to adverse environmental changes. Endospores have remarkable resistance to various extreme conditions and can remain dormant for extended periods of time. Clostridium spores are of particular interest due to their significant importance in several industries, such as food processing, wastewater treatment, pharmaceuticals, and health care. They are also the ideal candidates to study Panspermia and potential extraterrestrial life. However, to date, most endospore research has been conducted on Bacillus, and study of the anaerobic spore former, Clostridium, is not adequate. In this study, we have developed a general protocol to produce and purify Clostridium spores. Spectroscopy and microscopy based Endospore Viability Assay (Spectro EVA and Micro EVA) were developed and validated to assess the viability of Clostridium spores. Germinability was used as an indicator for spore viability. The basic principle of the two EVAs is to measure the release of a unique biomarker, dipicolinic acid (DPA), via germination as a proxy for endospore viability. In particular, a luminescence time-gated microscopy technique (Micro EVA) has been developed to enumerate germinable Clostridium endospores within an hour. Micro EVA is based on energy transfer from DPA to terbium ions doped in a solid matrix upon UV excitation. The distinctive emission and millisecond lifetime enables time-resolved imaging to achieve single endospore sensitivity. Comparing to traditional CFU cultivation, EVA probes the early stage of germination, resulting in a much faster detection rate (within 60 minutes) than CFU measurement (more than 3 days incubation). Micro EVA has also been successfully applied to quantify Clostridium spores in an extreme cold biosphere, Greenland ice core, and a hyper-arid biosphere, Atacama Desert, two Mars analogs on earth. The development of EVA provides a faster way to assess viability of Clostridium spores, which has significant importance in various industries. It also enables the determination of the limit and longevity of life, and provides insight on the search of extinct or extant life on Mars and other celestial bodies.