Quantifying Metabolic Activity of Filamentous Fungi Using a Colorimetric XTT Assay

Abstract

The filamentous nature and robust cell walls of many fungi render traditional measurements of active biomass (e.g., turbidity, dry cell weight) ineffective for most fungal bioprocesses. To overcome this challenge, an assay for quantification of overall metabolic activity is developed using 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), which in the presence of active mitochondria is converted to a water-soluble formazan derivative that absorbs light in the visible spectrum (430-490 nm). Tests on the model fungus Aspergillus nidulans show that in actively growing cultures XTT absorbance is linearly related to dry cell weight below 0.2 g/kg broth. Validation through growth rate testing shows the developed XTT assay is able to accurately quantify reductions in culture metabolism during damaging physical treatment (heat, high shear, microwaving). Experiments in batch culture demonstrate that the developed XTT assay is capable of reporting on metabolic activity where dry cell weight is not. The developed assay is inexpensive, relatively rapid, and easy to conduct, making it ideally suited for assessment of fungal processes in the biotechnology industry.

Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm

“…The tetrazolium salt XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt) (Sigma, St. Louis, MO) was used to measure the metabolic activity of A. fumigatus. Whereas XTT is a direct measure of the metabolic activity of cells, previous studies of A. fumigatus have indicated that XTT results are linear with mass and equated the XTT result with dry weight (19)(20)(21). Our confocal microscopy studies also address this point.…”

Linkage of Oxidative Stress and Mitochondrial Dysfunctions to Spontaneous Culture Degeneration in Aspergillus nidulans

“…Cellular Metabolic Activity Assay-Colorimetric tetrazolium assay using the dye XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} was conducted to compare the WT and Sec cell metabolic activities as previously described (39). In brief, the WT spores and Sec mycelia were inoculated into SDB for 12 h and then transferred into 1 L flasks containing 300 ml SDB for 48 h at 28°C.…”

Stimulation with lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae maximizes cross-reactivity of anti-fungal T cells

“…Anti-fungal activity was assessed with the use of a calorimetric assay with 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxyanilide sodium salt (XTT; Sigma) and 2-methyl-1,4-napthoquinone (menadione; Sigma) as previously described [24]. Before each experiment, fresh stock solutions of XTT (5.75 mg/mL in PBS) and menadione (0.52 mg/mL in acetone) were prepared and stored at 4°C until required.Working XTT-menadione solution was prepared by combining XTT and menadione stock solutions at a ratio of 4:1.…”

“…The anti-hyphal activity of the expanded panfungal T cells was assessed through the use of a colorimetric XTT reduction assay [24]. The percentages of hyphal damage toward A flavus, C albicans, C krusei, R oryzae and S prolificans were 81.7 ± 0.6 (n = 2), 25.1 ± 9.9 (n = 5), 9.8 ± 5.7 (n = 5), 8.7 ± 6.3 (n = 2), Table IV.…”