Survival of bacterial spores in extreme environments: Validation and application of a novel endospore viability assay

PHYS 486

Pun To Yung,, Hannah S. Shafaat,, Stephanie A. Connon,, Wanwan Yang,, Elizabeth D. Lester, and Adrian Ponce, Jet Propulsion Laboratory, 4800 Oak Grove Drive, Mail Stop 300-123, Pasadena, CA 91109
Bacterial spores (i.e., endospores) exhibit remarkable longevity and resistance to environmental extremes. Here we present a novel endospore viability assay (EVA) and its application to enumerating viable endospore populations in samples from two extreme environments: (1) Greenland ice cores (GISP2), and (2) Atacama Desert soils (Chile). Specifically, we are investigating bacterial spore longevity by measuring endospore viability as a function of sample age. EVA detects endospore germination, which serves as an indicator for endospore viability, and is based on imaging Tb3+-DPA luminescence halos that form around spore bodies as DPA is released during germination into a Tb3+-doped agar medium. Tb3+-DPA halos are enumerated and reported as germinating spore units (GSU). The germination process is triggered by L-alanine addition, which is complete in less than 10 minutes. Under pulsed UV excitation, the long-lived (t ~ 1 ms) DPA-triggered Tb luminescence is imaged by a lifetime-gated camera, essentially eliminating fluorescent interferents from the image and enabling GSU determination of environmental extracts. Bacterial spore concentrations were determined to be 280 GSU/ml of GISP2 ice core (~1000 years old) and 70 GSU/g of Atacama soil (>5000 years old). These results are evaluated with respect to those obtained from traditional environmental microbiology assays, including culture based assays, direct enumeration, and DNA analysis. We conclude that while these environments are considered extreme from the human perspective, the combination of spore resilience and lack of microbial activity in these samples serves to preserve bacterial spores on a time scale greater than several thousand years.


Poster Session
7:30 PM-10:00 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster

8:00 PM-10:00 PM, Monday, 11 September 2006 Moscone Center -- Hall D, Sci-Mix

Division of Physical Chemistry

The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006