Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis

Mycelial morphology is a critically important process property in industrial fermentations of filamentous micro-organisms, as particular phenotypes are associated with maximum productivity. However, the accurate quantification of complex morphologies still represents a significant challenge in elucidating this relationship. A system has been developed for high-resolution characterisation of filamentous fungal growth on a solid substrate, using membrane immobilization and fully-automatic plug-ins developed for the public domain, Java-based, image-processing software, ImageJ. The system has been used to quantify the microscopic development of Aspergillus oryzae on malt agar, by measuring spore projected area and circularity, the total length of a hyphal element, the number of tips per element, and the hyphal growth unit. Two different stages of growth are described, from the swelling of a population of conidiospores up to fully developed, branched hyphae 24 h after inoculation. Spore swelling expressed as an increase in mean equivalent spore diameter was found to be approximately linear with time. Widespread germination of spores was observed by 8 h after inoculation. From approximately 12 h, the number of tips was found to increase exponentially. The specific growth rate of a population of hyphae was calculated as approximately 0.24-0.27 h(-1). A wide variation in growth kinetics was found within the population. The robustness of the image-analysis system was verified by testing the effect of small variations in the input data.