Assessment of the activity of filamentous fungi using Mag fura

A new staining method for the examination of active regions of hyphae is presented. Using a simple protocol, application of the fluorescent stain Mag fura (tetra-potassium salt) in low pH buffer to two filamentous fungi resulted in bright responses to active hyphal regions. Not only were active apices delineated, but more distal compartments were also found to respond to the stain. Some apices did not stain, presumably because they were inactive. It is believed that all stained regions retained cell membrane integrity and are thought to have had high membrane ATPase activity. All showed high nuclear and mitochondrial complements. The stain was apparently held within the cell wall structure, close to the cell membrane. It is hypothesized that it was responding to a localized flux of divalent cations from the cell membrane. With non-active regions, only a low level of response to the stain remained, at the exterior of the cell wall. This low emission is thought to be a response to contaminating ions in the buffer. It could be clearly distinguished from the bright response associated with the active regions. Counter-staining with other commonly used fluorescent probes showed these regions of low response (unless they were completely degenerated) still contained nuclear material and mitochondria. The fluorescent signal from active regions of fungal hyphae was sufficiently bright and persistent for visualization by a conventional CCD camera. This will allow the development of image analysis protocols to measure the effects of environmental conditions on fungal physiology, using commonly available equipment.