Laser-excited Raman spectra were examined in lyophilized spores of Bacillus cereus. In a comparison of the spectrum of the dormant spore with that of the germinated spore, we found several Raman bands which occurred in the former but not in the latter. Among these Raman bands, the 1,573, 1,395, 1,017, 822, and 662 cm-1 bands were assigned to the vibrational frequencies of calcium dipicolinate (CaDPA). No Raman bands and peaks due to dipicolinic acid (H2DPA) were observed. This Raman evidence indicates that CaDPA is the predominant DPA species in this spore.We also proposed a tentative assignment for other vibrational frequencies due to several components of the spore.Since laser Raman spectroscopy is a powerful technique for investigating the structure of biological materials in vivo (15, 21, 32), extensive knowledge of the Raman response of biological materials has been accumulated to understand the chemical state at the molecular and macromolecular levels (3, 11, 25, 31, 35). In 1974, Raman spectroscopy was first applied to bacterial spores, but the reported work yielded poorly resolved spectra because of a high fluorescence background (39). Since then, no further investigations have been reported and the precise interpretation of the Raman spectra of bacterial spores remains incomplete.We report here spectra of much higher resolution from lyophilized spores of Bacillus cereus. The results showed quite different patterns from the published spectra. We also propose a tentative assignment for vibrational frequencies due to several components of the B. cereus spore. In particular, identification of vibrations due to dipicolinate (DPA), which is a unique major component of bacterial spores, may be of great use in determining its conformation in the spores. MATERIALS AND METHODSOrganism and culture method. Bacillus cereus T was used. Growth and sporulation were induced in modified G medium (12). Spores were harvested, washed and lyophilized as described previously (34).Preparation of germinated spores. Fifty milligrams of the lyophilized spores were suspended in 10 ml of deionized water and heat-activated at 70 C for 30 min. Then, 307

The laser Raman spectra of lyophilized spores of 5 species (6 strains) of Bacillus were examined. Under the experimental conditions employed, only Bacillus megaterium species showed 3 intense Raman bands at 1515, 1157 and 1007 cm−1. Spores of other species, despite their high content of dipicolinic acid, did not show distinct Raman signals. The strong, scattering bands at 1515 and 1157 cm−1 in the spectra of spores of B. megaterium may be attributable to conjugated double‐bond systems, probably of membrane‐associated carotenoids. Their high intensities are due to resonance enhancement.

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