Monitoring of filamentous fungal growth by in situ microspectrophotometry, fragmented mycelium absorbance density, and 14C incorporation: alternatives to mycelial dry weight

Abstract

Monitoring of filamentous fungal growth by spectrophotometry is generally considered not feasible. This report describes the monitoring of growth of the filamentous fungi Trichophyton mentagrophytes, Rhizopus oryzae, and Sporothrix schenckii in broth by two new spectrophotometric methods and by 14C incorporation from [U-14C]glucose. Microcultures (200 microliter) were prepared in 96-well, flat-bottom microtiter trays, and macrocultures (4 ml) were prepared in glass vials proportionally scaled up from microcultures. Mycelium accumulation in microcultures was measured without terminating the cultures by in situ microspectrophotometry. Accumulation in macrocultures was monitored by uniformly fragmenting the mycelium with a Broeck tissue grinder and by measuring absorbance density in plastic cuvettes with a dual-beam spectrophotometer. Absorbance measurements were found to increase linearly with mycelial weight. In situ absorbance correlated with absorbance density of fragmented mycelium, indicating that both methods monitored growth equivalently. Both defined lag-, exponential-, and stationary-growth phases. Increases in 14C incorporation, absorbance, and mycelial dry weight were kinetically identical for macrocultures and microcultures of T. mentagrophytes. For R. oryzae and S. schenckii, with the exception of R. oryzae growing in microcultures, incorporation of 14C also defined lag, exponential, and stationary growth after selection of the appropriate isotope-specific activity. This incorporation correlated directly with absorbance. We conclude that in situ microspectrophotometry, fragmented mycelium absorbance density, and, to a lesser extent, 14C incorporation can be used to effectively monitor filamentous fungal growth.