A significant difference between two closely related Bacillus spores has been discovered using optical chromatography. This difference can be harnessed for the separation of microscopic particles using opposing laser and fluid flow forces. Particles of different size, composition, and shape experience different optical and fluid forces and come to rest at unique equilibrium positions where the two forces balance. Separations in excess of 600 mum have been observed between Bacillus anthracis Sterne strain and its genetic relative, Bacillus thuringiensis. These findings open new possibilities for detection and characterization of the biological warfare agent, B. anthracis, the causative agent of anthrax, the deadly mammalian disease. The large optical separation between these species is surprising given their close genetic relationship but may be explained by differences in their shape and exosporium morphology, which may result in differences in fluid drag force. The observation of large differences due to less common variables indicates the complex nature of the force balance in optical chromatography, which may in the future be used to separate and characterize microbiological samples. In general, the discovery of such large differences between such closely related biological species suggests new possibilities for the separation and characterization of microorganisms using the full range of emerging techniques that employ radiation pressure (optical filtering, laser tweezers, optical chromatography, etc.).