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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.