Nonpathogenic microorganisms are often used as simulants of biological pathogens during the initial phase of detection method development. While these simulants approximate the size, shape, and cellular organization of the microorganism of interest, they do not resemble its surface protein content, a factor particularly important in methods based on immunorecognition. Here, we develop and detect an artificial bacterial spore--B. globigii (BG) Bugbead-a particle mimicking the antigenic surface of BG spores. Two methods of spore protein extraction were compared both quantitatively (by protein concentration assay) and qualitatively (by SDS-PAGE and Western blot): extraction by mechanical disruption and extraction by chemical decoating. The former method was more efficient in producing more protein and a greater number of antigens. BG Bugbeads were made by conjugating the extracted proteins to 0.8-microm carboxyl-coated polystyrene particles via carbodiimide coupling. BG Bugbeads were successfully detected by a bead-based enzyme-labeled immunoassay with fluorescence detection with a detection limit of 6.9 x 10(3) particles/mL. Formation of the Bugbead-capture bead complex was confirmed by ESEM. The concept of a harmless artificial spore can be applied to developing improved simulants for pathogenic spore-forming microorganisms such as B. anthracis, C. botulinum, and B. cereus, which can to be used for method validation, instrument calibration, and troubleshooting.