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-_4C]glucose. Microcultures (200 ,lh) 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 "4C 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.

Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence‐based digital image analysis

“…Biomass growth performance is one of the known procedures (Granade et al, 1985) to estimate microbial viability. In our experiments, the inocula were quantitatively the same but qualitatively different, i.e., damaged to different extents.…”

“…Currently, there is no quantitative, fast, and reliable method for the determination of fungal viability, especially for those of the filamentous type. The most frequently used methods are those measuring the culturability as the colony forming unit, CFU (Jolicoeur et al, 1999;Muñoz et al, 1995) and the growth performance test, i.e., capacity of reproduction in liquid cultivation (Granade et al, 1985). The use of dilution and incubation in the CFU method increases the probability of error, especially in the case of filamentous organisms where the particle size is not well defined.…”

“…Indeed, the measurement of FDA hydrolysis has been used to estimate biomass activity, also referred to as cell esterase activity. In the case of unicellular microorganisms, this activity has been compared with other cellular activities such as biomass growth (Granade et al, 1985) and substrates (glucose, O 2 ) utilization (Desgranges et al, 1991;Ingham and Klein, 1982) as well as to measure "microbial activity" in soil (Schnürer and Rosswall, 1982) and water samples (Holzapfel-Pschorn et al, 1987;Ziglio et al, 2002). This technique was also used for the differentiation of live and dead/unhealthy cells of microalgae (Gilbert et al, 1992), as well as mammalian (Ross et al, 1989), bacterial (Bunthof et al, 1999), and plant cell cultures (Steward et al, 1999;Thomas and Gina, 1997).…”

Nutrient and Physiological Requirements for Biomass Production of Pestalotiopsis sp. UMAS P14 and Pseudopestalotiopsis sp. UMAS P2005/2592

“…Evaluation of the physiological requirements for mycelial biomass growth of the isolates were done following the method of Granade et al (1985) and Prasher & Chauhan (2015) in which the biomass was assessed based on their dry weight. The basal liquid medium was prepared following the protocol used by Lateef et al (2015).…”

Quantifying Metabolic Activity of Filamentous Fungi Using a Colorimetric XTT Assay

“…However, filamentous fungi have rigid cell walls which can maintain cells (i.e., mycelia) intact long after death. As a result, commonly employed biomass quantification methods, such as dry cell weight (DCW) and turbidity, can provide erroneous information regarding culture activity or viability (1). As filamentous fungi are used in the bioprocess industry to produce billions of dollars of pharmaceuticals and foodstuffs annually, a straightforward and accurate activity assay would greatly benefit bioprocesses employing filamentous fungi (2).…”