An image analysis program and protocol for the identification and enumeration of live versus dead cells of the yeast-like fungus Aureobasidium pullulans was developed for both populations on microscope slides and leaf surfaces. Live cells took up CellTracker Blue, while nonviable cells stained with DEAD Red. Image analysis macro programs running under Optimas software were used to acquire images and to differentiate and enumerate viable from nonviable cells. The software was capable of discriminating green as a third parameter for identification and quantification of green fluorescent protein-expressing cells in a wild-type population.