Bacterial Endospore Detection

Pestov, D., Wang, X., Ariunbold, G. O., Murawski, R. K., Sautenkov, V. A., Dogariu, A., Sokolov, A. V., and Scully, M. O. 2008. Single-shot detection of bacterial endospores via coherent Raman spectroscopy. Proc. Natl. Acad. Sci. USA 105 422-27. 

Owing to its applications in homeland security, sterilization validation, and astrobiology, bacterial spore detection has become a hot field. However, direct detection of bacterial spores can be challenging for the same reasons that make endospores difficult to irradicate. The tough spore coat is impermeable to staining techniques, so most microscopy and flow cytometry methods are not useful. Endospores are also highly resistant to lysis, meaning that common DNA extraction protocols are difficult to perform. The lack of measurable metabolism renders microcalorimetry and cellular respiration techniques ineffective. 

Fig. 5. The method of bacterial spore detection using terbium. Dipicolinic acid (DPA) is released from endospores via lysis or germination and binds to Tb with high affinity. The resulting [Tb(DPA)]" complex exhibits intense luminescence in the visible region (544 nm) under UV excitation (278 nm) that is much greater than Tb " " alone.

We plan to take advantage of ligand-induced enhancement of dipicolinate binding affinity to improve current bacterial spore detection technologies in two ways. The first involves appending terbium(macrocycle) complexes to solid polymer substrates to improve microscopy-based endospore assays. In a second method, we will bind lanthanide complexes to silica to concentrate dipicolinate from very dilute samples on colmnns. 

A current method for detecting bacterial endospores involves dissolving terbium dipicolinate in either water or a water—glycerol mixture, shining a UV beam on the sample, and observing the luminescence, which, if present, indicates the presence of endospores. ... 

Beside the identification of single bacterial cell or spores by means of Raman spectroscopy, the localization of these cells inside partly complex matrices has to be performed. One approach is the combination of Raman spectroscopy, fluorescence spectroscopy and digital imaging techniques. This method was applied to detect traces of endospores and other biothreat organisms even in the presence of complex environmental matrices like bioaeroso-lic background, nasal mucin [67], or tap water [68], Another fully automated device was built to analyze bioaerosols in clean room environments, where prior to the Raman identification method a particle preselection took place [69]. 

Bacterial spores contain a unique chemical marker— dipicolinic acid, or DPA. DPA is present in nearly all bacterial spores and comprises about 10-15% of a spore s dry weight, or approximately 10 molecules per spore (82-84). Detection of this chemical marker can therefore serve as a positive signal for the presence of bacterial spores, and the ammmt of DPA detected can be used to estimate the approximate endospore concentration (81,85). 

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