The experiment was repeated using Congo red staining. When Mm plates containing CMC were stained, only five of the fourteen isolates showed carbohydrolytic (cellulase) activity (Fig. 1D). In the absence of substrate, no clearing zones were detected (Fig. 1C).This suggests that hydrolysis of CMC was a prerequisite to clear-ing of Congo red. When Mm agar plates containing xylan were stained with the Congo red dye, no hydrolysis zones were detected(data not shown). This suggests that at variance with Gram’s iodine,Congo red clearing requires that the substrate be present and that sufficient cellulase activity be produced by the microorganisms.Both staining methods were also evaluated using commercial,cell-free enzymes. Following staining with Gram’s iodine, clear zones were produced by enzymes C4 and C5 deposited on both agarose plates as well as Mm plates with and without substrate(CMC or xylan, Fig. 2A). However, when the plates were stained with Congo red dye, clear zones resulting from polymer hydrolysis were detected in the presence of CMC, but not in the absence of CMC (Fig. 2B). These results were confirmed by applying a quantitative method based on reducing sugar detection (with DNS) [11].Using CMC as substrate, cellulase activity recorded for C4 and C5was 24.7 and 15.7 IU/mg, respectively.