Public reporting burden (or this collection of information is estimated AUTHOR(S)Sivananthan Sarasanandarajah, Joseph Kunnil, Burt V. Bronk, Lou Reinisch PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Department of Physics and Astronomy University of Canterbury Private Bag 4800 Christchurch, New Zealand SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES)Air AFRL-HE-WP-TR-2004-0140 SUPPLEMENTARY NOTES 12a. DISTRIBUTION AVAILABILITY STATEMENTApproved for Public Release; distribution is unlimited. 12b DISTRIBUTION CODE ABSTRACT (Max/mum 200 words)Dipicolinic acid (DPA) and the Ca2+ complex of DPA (CaDPA) are major chemical components of bacterial spores. With fluorescence being considered for the detection and identification of spores, it is important to understand the optical properties of the major components of the spores. In this paper we report in some detail on the room temperature fluorescence excitation and emission spectra of DPA and its calcium ion complex and comparison of the excitation-emission spectrum in a dry, wet paste and aqueous form. DPA solutions have very weak, if any, fluorescence and it is only slightly greater in the dry state. After the exposure to a broad source UV light of the DPA, wet or dry, we observe a large increase in fluorescence with a maximum intensity emission peak at around 440 nm for excitation light with wavelength around 360 nm. There is a slight blue shift in the absorption spectra of UV exposed DPA from the unexposed DPA solution. CaDPA in solution and dried show very slight fluorescence and a substantial increase of fluorescence was observed after UV exposure with emission peak around 410 nm for excitation around 305 nm. The detailed excitation-emission spectra presented here are necessary for better interpretation of the fluorescence spectra of bacterial spores where DPA is a major chemical component.

Fluorescence spectroscopy has been used to measure fluorescence quantum efficiency (QE) of dried Bacillus spores (washed and unwashed) fixed to a quartz substrate. Fluorescence spectra and QE of anthracene in ethanol was used as the standard. We measured the absorption and fluorescence signal of the spores as a function of the number of spores. The absorption was measured from 600 nm to 250 nm using the reflectance in an integrating sphere. The fluorescence spectra were measured using excitation wavelengths at 280, 360 and 400 nm at room temperature. The absorption cross sections for the unwashed spores were 1.3 x 10-8, 8 x 10-9, and 5 x 10-9 mm2/spore at 280, 360 and 400 nm, respectively. The fluorescence QE was 0.13 +/- 0.03, 0.33 +/- 0.12 and 0.43 +/- 0.26 at 280, 360, and 400 nm, respectively. The QE decreased by a factor of 2, 4 and 4 at these same wavelengths after washing and redrying the spores.

Fluorescence has been suggested as a method with which to detect and identify bacterial spores. To better understand the nature of the fluorescence signal, we observed the intrinsic steady-state fluorescence and phosphorescence spectra of Bacillus globigii (BG) in both dried and aqueous forms. In vitro, dried, and suspension forms of BG were measured at room temperature in 300-600-nm excitation wavelengths. Also, the phosphorescence of dry BG spores was measured at room temperature at 300-600-nm excitation wavelengths. The wet BG spores exhibited a strong maximum in their fluorescence spectrum, with the peak excitation wavelength near 300 nm and emission wavelength near 400 nm. When the BG was dried, this peak shifted to an approximately 450-nm excitation maximum and an 500-nm emission maximum. The difference between the wet and the dry spore fluorescence spectra cannot be explained by the phosphorescence of the dry spores. Other changes must take place when the spores are wet to account for the large changes observed in the spectrum.

No abstract

The fluorescence spectra of Bacillus spores are measured at excitation wavelengths of 280, 310, 340, 370, and 400 nm. When cluster analysis is used with the principal-component analysis, the Bacillus globigii spores can be distinguished from the other species of Bacillus spores (B. cereus, B. popilliae, and B. thuringiensis). To test how robust the identification process is with the fluorescence spectra, the B. globigii is obtained from three separate preparations in different laboratories. Furthermore the fluorescence is measured before and after washing and redrying the B. globigii spores. Using the cluster analysis of the first two or three principal components of the fluorescence spectra, one is able to distinguish B. globigii spores from the other species, independent of preparing or washing the spores.