Skip to main content

A Rapid and Easy Method for the Detection of Microbial Cellulases on Agar Plates Using Gram’s Iodine


Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram’s iodine instead of the reagents just mentioned. Gram’s iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram’s iodine for the detection of cellulase production by microorganisms using plate assay.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Beguin P, Anbert JP (1993) The biological degradation of cellulose. FEMS Microbiol Rev 13:25–58

    Article  Google Scholar 

  2. Bhat MK (2000) Cellulases and related enzymes in biotechnology. Biotechnol Adv 18:355–383

    PubMed  Article  CAS  Google Scholar 

  3. Camassola M, Dillon AJP (2007) Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation. J Appl Microbiol 103:2196–2204

    PubMed  Article  CAS  Google Scholar 

  4. Coughlan MP (1985) The production of fungal and bacterial cellulases with comment on their production and application. Biotechnol Genet Eng Rev 13:39–109

    Google Scholar 

  5. Gusakov AV, Berlin AG, Popova NN, Okunev ON, Sinitsyn AO, Sinitsyn AP (2000) A comparative study of different cellulase preparations in the enzymatic treatment of cotton fabrics. Appl Biochem Biotechnol 88:119–126

    Article  CAS  Google Scholar 

  6. Hankin L, Anagnostakis S (1977) Solid media containing carboxy methyl cellulose to detect CM cellulase activity of microorganisms. J Gen Microbiol 98:109–115

    PubMed  CAS  Google Scholar 

  7. Hendricks CW, Doyle JD, Hugley B (1995) A new solid medium for enumerating cellulose-utilizing bacteria in soil. Appl Environ Microbiol 61:2016–2019

    PubMed  CAS  Google Scholar 

  8. Ito S (1997) Alkaline cellulases from alkaliphilic Bacillus: Enzymatic properties, genetics and application to detergents. Extremophiles 1:61–66

    PubMed  Article  Google Scholar 

  9. Kasana RC, Sharma UK, Sharma N, Sinha AK (2007) Isolation and identification of a novel strain of Pseudomonas chlororaphis capable of transforming isoeugenol to vanillin. Curr Microbiol 54:457–461

    PubMed  Article  CAS  Google Scholar 

  10. Lamb J, Loy T (2005) Seeing red: The use of Congo Red dye to identify cooked and damaged starch grains in archaeological residues. J Archaeol Sci 32:1433–1440

    Article  Google Scholar 

  11. Mandels M (1985) Applications of cellulases. Biochem Soc Trans 13:414–415

    PubMed  CAS  Google Scholar 

  12. Park J, Park K (2001) Improvement of the physical properties of reprocessed paper by using biological treatment with modified cellulase. Bioresour Technol 79:91–94

    PubMed  Article  CAS  Google Scholar 

  13. Tanaka K, Mii T, Marui S, Matsubara I, Igaki H (1981) Mutagenicity of urinary metabolites of benzidine and benzidine-based azo dyes. Int Arch Occup Environ Health 49:177–185

    Article  CAS  Google Scholar 

  14. Teather RM, Wood PJ (1982) Use of Congo red polysaccharide interactions complex formation between Congo red and polysaccharide in detection and assay of polysaccharide hydrolases. Methods Enzymol 160:59–74

    Google Scholar 

  15. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, New York, NY, pp 315–322

    Google Scholar 

Download references


The authors are grateful to P. S. Ahuja, Director, Institute of Himalayan Bioresource Technology (CSIR), Palampur, India, for providing the necessary laboratory facility. The Council of Scientific and Industrial Research (CSIR) is also acknowledged for providing financial support under the project NWP06. This is IHBT communication No. 0839.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Ramesh Chand Kasana.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kasana, R.C., Salwan, R., Dhar, H. et al. A Rapid and Easy Method for the Detection of Microbial Cellulases on Agar Plates Using Gram’s Iodine. Curr Microbiol 57, 503–507 (2008).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Cellulase
  • Cellulase Production
  • Microbial Type Culture Collection
  • Penicillium Chrysogenum
  • Hexadecyltrimethyl Ammonium Bromide