Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var. niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose. These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods. Large green rods were rarely observed. An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature. This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination. In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation. Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time. Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose. The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator. These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs. vegetative cells.