Detecting Bacillus Spores by Raman and Surface-Enhanced Raman (SERS) Spectroscopy

Jun 02, 2005

Immediately following the September 11, 2001 terrorist attacks, four letters containing anthrax causing spores were mailed through the U.S. postal system infecting 22 individuals, five fatally. The anxiety caused by this bioterrorist attack was exacerbated by the extensive time required for positive identification of the Bacillus anthracis spores and the unknown extent of their distribution along the east coast. The delay in identification was due to the fact that spores had to be germinated and grown in culture media to sufficient cell numbers so that the 16S rRNA gene unique to B. anthracis could be measured. Consequently, the Center for Disease Control and Prevention (CDC) employed a combination of biological analyses of culture grown colonies and polymerase chain reactions to differentiate bacilli from other bacteria and from each other (1,2).

From this bioterrorist attack, it became clear that considerably faster methods of analysis were required. This would expedite assessment of the scale of an attack as well as the extent of facility contamination. This information, in turn, could be used to minimize fatalities, because it was learned that if exposure is detected within the first few days, the majority of victims can be treated successfully using ciprofloxacin, doxycycline, and/or penicillin G procaine (3). However, the challenges of developing such an analyzer are formidable considering that the CDC estimates that inhalation of 10,000 B. anthracis endospores or 100 nanograms will be lethal to 50% of an exposed population (4). An additional challenge has emerged since the 2001 attacks, in that a secondary type of postal-based terrorism has proliferated in the form of hoax letters (5–7). Literally tens of thousands of letters containing harmless powders have been mailed to create additional fear (8). Consequently, an analyzer must not only be able to differentiate B. anthracis spores from other biological materials, but also must be able to identify these harmless powders to eliminate fear and potentially costly shutdowns (9).

In the case of postal-based terrorism, we have been investigating the utility of Raman and surface-enhanced Raman spectroscopy (SERS) to meet the analytical challenges of speed, sensitivity, and selectivity by identifying visible and invisible particles on surfaces, respectively (10–13).


Figure 1. Raman spectra of (a) Bacillus cereus spores and (b) calcium dipicolinate. Conditions: 500 mW of 1064 nm at the sample, 5-min acquisition time.
Raman Spectroscopy – Bacilli Spores and Hoax Materials Raman spectroscopy is attractive because very small samples can be measured without preparation. The sample need only be placed at the focal spot of the excitation laser and measured. Moreover, the rich molecular information provided by Raman spectroscopy usually allows unequivocal identification of chemicals and biochemicals. As early as 1974, the Raman spectrum of Bacillus megaterium was measured and shown to be dominated by calcium dipicolinate (CaDPA, 14). This chemical can be used as a signature because only spore forming bacteria contain CaDPA, at ~10% by weight (15–17), and the most common spores, such as pollen and mold spores, do not. The ability of Raman spectroscopy to measure and identify spores is exemplified in Figure 1. Here an ~1-mm3 spec (~100 µg) of Bacillus cereus spores, a nontoxic surrogate for B. anthracis spores, was placed on a glass surface, positioned at the focal point of a 1064-nm excitation laser, and measured in 5 min using an FT-Raman spectrometer. Comparison of this spectrum to that for CaDPA shows that the characteristic peaks of the latter at 821, 1014, 1391, 1446, 1573, and 3080 cm-1 are observed readily in the former. These peaks can be assigned to a CH out-of-plane bend, the symmetric pyridine ring stretch, an OCO symmetric stretch, a symmetric ring CH bend, an asymmetric OCO stretch, and the aromatic CH asymmetric stretch, respectively.


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