Comparison of Dyes for Easy Detection of Extracellular Cellulases in Fungi

Abstract

To evaluate which dye is effective in a plate assay for detecting extracellular cellulase activity produced by fungi, four chromogenic dyes including remazol brilliant blue, phenol red, congo red, and tryphan blue, were compared using chromagenic media. For the comparison, 19 fungal species belonging to three phyla, ascomycota, basidiomycota, and zygomycota were inoculated onto yeast nitrogen-based media containing different carbon substrates such as cellulose (carboxylmethyl and avicel types) and cellobiose labeled with each of the four dyes. Overall, the formation of clear zone on agar media resulting from the degradation of the substrates by the enzymes secreted from the test fungi was most apparent with media containing congo red. The detection frequency of cellulase activity was also most high on congo red-supplemented media. The results of this study showed that congo red is better dye than other three dyes in a plate assay for fungal enzyme detection.

Characterization of a strong CCA-treated wood degrader, unknown Crustoderma species

“…The ability of the isolate to produce extracellular enzymes was evaluated by culturing the organism on chromogenic media supplemented with different polymeric carbon substrates (Yoon et al 2007). Briefly, the fungal isolate was precultured on 2% MEA (Difco, USA) at 25°C for 5 days.…”

Production of thermostable xylanase by thermophilic fungal strains isolated from maize silage

“…Xylanase producer strains were selected using the platescreening method, which consists in the detection of clear zones formed as a consequence of the enzymatic activity that hydrolyses the Congo red dye substrate (Yoon et al, 2007). Among the 21 isolated strains, seven were able to growth on xylanCongo red plates; however, two of them were unable to promote discoloration of the agar medium, probably because they did not secrete the enzyme out of the cell.…”

“…The inoculated media were incubated at 55°C for 5 days. Then, the xylanase enzyme activity was determined by analyzing the clear zone formed around the fungal colony as a result of the reaction between the enzymes secreted by the fungi and the chromogenic substances present in the solid medium (Yoon et al, 2007).…”

Screening and detection of extracellular cellulases (endo- and exo-glucanases) secreted by filamentous fungi isolated from soils using rapid tests with chromogenic dyes

“…The crops were washed with 5 mL NaCl (0.5 M) solution for 10 min (Teather and Wood, 1982;Kim et al, 2000). The second and third sets of plates were flooded with Phenol Red and Trypan Blue respectively (Yoon et al, 2007) and it proceeded in the same way as with Congo red solution. The last set of plates was flooded with 10 mL Gram's Iodine (2.0 g KI and 1.0 g iodine in 300 mL distilled water) for 3 to 5 min (Kasana, et al, 2008;Johnsen and Krause, 2014).…”

“…The screening method for the detection of extracellular enzymes using dyes developers or chromogenic such as Congo red, Phenol red, Trypan blue, Gram's iodine, Remazol brilliant blue, it has been commonly used for in vitro selection of polysaccharides degraders microorganisms and characterized as a simple, fast and costefficient technique (Yoon, et al, 2007;Kasana et al, 2008;Jo et al, 2011). Where, the cellulolytic activity is reflected by the appearance of clear halos that surround the colony and not degraded areas arise without exposure or color variation (Johnsen and Krause, 2014).…”