Easy Method for the Detection of Cellulases from Halophilic Bacteria on Agar Plates Using Gram’s Iodine
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Mastoore Asadbeygi,1 Ali Mohammadi,2,* Mohammad Reza Soudi,3
1. Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
2. Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
3. Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
- Introduction: The term ‘‘cellulases’’ refers to a group of enzymes that catalyze the hydrolysis of cellulose into sugars. Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulolytic enzymes from microorganisms also have many potential biotechnological and industrial applications. Cellulases are required in large quantities because of their application in many industries, such as textiles, detergent, food, animal feed, bio-fuel, paper and pulp, pharmaceutical, and waste management. The first step in the development of the industrial process for the production of an enzyme is to isolate the potential strains. Isolation and screening of microbes for cellulases are of immense importance keeping in view of the demand for new enzymes and the improvement of their biotechnological applications. An easy, fast, and environmentally friendly qualitative method is described for screening microorganisms producing extracellular cellulase on the agar plate.
- Methods: Soil samples were collected from Howz-E Soltan Salt Lake of Iran, for the isolation of bacteria-producing cellulases. Samples were transported to the laboratory and stored at 4C until used. Five-fold serial dilutions of each soil sample were prepared in sterilized distilled water, and a 0.1 ml diluted sample was spread on the surface of nutrient agar medium with NaCl 20% (pH 7.4). Plates were incubated at 30C for 48 hours for bacteria. Morphologically different colonies appearing on the plates were purified on the respective medium for bacteria. The purified isolates were preserved at 4C and used during the course of the study.
- Results: As a result, The two bacterial isolates T1 and H1-1 producing cellulase were identified as Metabacillus halosaccharovorans sp. based on partial 16S rRNA gene sequencing. The observation on the development of cellulose clearance zone showed Gram’s iodine to be more effective compared with Congo red plus 1 M NaCl for screening and qualitative estimation of cellulase production by microorganisms on CMC agar plates. There was difficulty in differentiating the enzyme-lysed zone from the CMC-containing area in the plates flooded with these reagents. Moreover, there was a waiting period of at least 5 to 10 minutes for the development of clearance zones. Interestingly, the plates flooded with Gram’s iodine showed distinct, clear, and prominent zones of clearance around the colonies showing cellulase production with bluish-black coloration in the nonhydrolyzed part of the medium. It took only 5 minutes for the development of clearance zones after flooding. There is a possibility that the added Congo red could have affected growth and cellulase production by the isolates because of its harmful effects. However, a sharp increase in the color contrast of the hydrolyzed zone and nonhydrolyzed portion of the medium is obtained on flooding with Gram’s iodine because it further enhanced the appearance of the already sharply discernible clearance zone by producing a bluish-black complex with cellulose (polysaccharide) and not with the glucose (monosaccharide). This new method for assaying has the double advantage of being quick as well as based on nontoxic chemicals.
- Conclusion: The present studies showed that the use of Gram’s iodine remarkably enhances the sharpness of the clearance zone,
making the process for screening cellulase-producing microorganisms easy, efficient, and rapid. Although the
new method is a qualitative assay, the greatest advantage lies in its simplicity, quickness of performance, and
effectiveness for screening a large number of microorganisms. Moreover, it avoids the use of toxic chemicals.
- Keywords: Halophiles, Screening, Isolation, Cellulase, Staining
