Bacillus endospores are in standard use as biological indicators for evaluating sterilization processes. We describe a rapid method for monitoring endospore inactivation by enumerating germinable, rather than culturable, endospores. We define endospore viability as the percentage of germinable endospores in a total endospore population. Endospores, when immobilized on agarose doped with terbium (Tb3+), release dipicolinic acid (DPA) in the presence of germinants; this DPA release is imaged using time-gated Tb3+-DPA luminescence microscopy. Germinable endospores are enumerated as green luminescent spots that follow intensity time courses characteristic of stage one germination (approximately 10 minutes). Time-gating greatly improves image contrast enabling imaging of single germinating endospores from environmental samples (e.g., surfaces, soils, ice cores) that contain particulates and a high background of autofluorescence. This method was validated against phase contrast microscopy, where germinating endospores transition from phase-bright to phase-dark, and was correlated to culture-based methods. Finally, we applied this method to monitor  the inactivation of germinable endospore suspensions as a function of thermal and UV dosage in comparison to culture-based methods.

publications

Fast sterility assessment by germinable-endospore biodosimetry.  Yung, P.T.; Ponce, A.  Applied and Environmental Microbiology, 2008, 74(24), 7669-7674. [PDF]

A rapid single spore enumeration assay.  Yung, P.T.; Kempf, M.J.; Ponce, A.  IEEE Aerospace Conference, 4-11 March 2006. [PDF]