The effects of the following variables on the occurrence of colony masking (the indistinguishable merging or overlap of sufficiently close colonies) were evaluated experimentally using the bacterium Bacillus subtilis: spore density on a collection surface, concentration of nutrients in the culture medium, sample incubation time, and ability of an observation system to distinguish overlapped colonies. Increasing spore surface density and incubation time increased colony masking, whereas lowering nutrient concentration decreased colony diameter and, therefore, masking but also limited spore germination and growth. Overall, full-strength medium was best for accurate counting of early microcolonies examined with the aid of a microscope, whereas half- or quarter-strength medium was better for counting older readily observable macrocolonies. Masking bias was determined for varying spore surface densities and colony diameters and was applied to two widely used slit-to-agar bioaerosol impactors. Appropriate collection times have been determined for these samplers to minimize colony masking for expected bioaerosol concentrations. It was found, for example, that 6-min samples collected from an environment with an air concentration of 10(3) CFU m-3 would result in colony surface densities, for 3-mm colonies, of 1.5 and 3.9 microorganisms cm-2 for the two samplers with respective masking biases of < 10% and < 20%.