Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus. After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen. The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera. Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 microg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells. Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA-stained area) by image analysis. At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA-stained area with its growth rate, a functional indicator of viability. The linear correlation (r = 0.979) was: growth rate (g/L x h) = 2.25 x 10(-3) (% of FDA-stained area). This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10-L bioreactor. Estimated viable biomass during fermentation was strongly influenced by the process conditions. The use of FDA, with computer-aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T. harzianum.