Screening Cellulase-Producing Microorganisms

The medium used for the second screening step using the Congo red test was similar to that described above (Section 2.2), except that the carbon source was low viscosity carboxymethylcellulose (CMC) (Sigma, USA). Only those strains that showed substantial growth in the initial screening with Avicel were selected for the Congo red test. Inoculation was carried out by using a platinum needle to transfer the spores from the PDA plate to the center of the plates containing the CMC medium [18 ]. The inoculated plates were incubated for 96 h at 30°C and the growth of the microorganism was measured by the diameter of the colony. A 10 mL aliquot of Congo red dye (2.5 g·L⁻¹) was then added to each plate. After 15 min, the solution was discarded and the cultures were washed with 10 mL of 1 mol·L⁻¹ NaCl. Cellulase production was indicated by the appearance of a pale halo with orange edges, indicative of areas of hydrolysis. This halo was measured for subsequent calculation of the enzymatic index (EI) using the expression:
$\begin{array}{c}\text{EI}=\frac{\text{diameter}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{of}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{hydrolysis}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{zone}}{\text{diameter}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{of}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{colony}}.\end{array}$
The strains that showed an EI higher than 1.50 were considered to be potential producers of cellulases. Three independent experiments were performed for this screening step, with two replicates per strain. For each strain the average EI of the three experiments was calculated, together with the standard deviation.