Characterization of the Surface Morphology of Bacillus Spores by Atomic Force Microscopy

The surface morphology of Bacillus spores was resolved by atomic force microscopy in order to determine if characteristic surface features could be used to distinguish between closely related species. Four strains were studied: Bacillus anthracis Sterne strain, Bacillus thuringiensis var. kurstaki, Bacillus cereus strain 569, and Bacillus globigii var. niger. The spores were separated from a nutrient agar culture by filtering and centrifugation, suspended in deionized water, and immobilized on a graphite substrate by spin-coating. Atomic force microscopy imaging was done using intermittent contact mode, in air, under reduced humidity. Seven to ten spores of each species were fully characterized at three scan sizes: whole spore, 750nm2, and 500nm2. Images were captured with both height and phase information. Height images showed an irregular topography of subtle grooves, bumps, and steps across the curved upper surface of the spores. Phase images showed a superficial grain structure with different levels of phase contrast. Although some similarities were observed among spores of the same species, there was also significant variability within each species. The four species were not distinguished by observed surface morphology. Surface texture analysis (roughness, power spectral density, and bearing) did not establish quantitative differences between species based on surface topography. Overall, atomic force microscopy revealed a spore surface morphology rich with information. Additionally, it appears that further analysis and a larger sample size could provide sufficient information to allow identification and differentiation between B. anthracis and its close relatives based on spore surface morphology