Abstract
Gram’s Iodine Assay is currently used to detect endoglucanase, a cellulase enzyme which cleaves β-1,4-glycosidic bonds in carboxymethylcellulose (CMC). When a zone of degradation was observed around non-cellulolytic Escherichia coli JM109 with the use of Gram’s Iodine, the assay was further examined. Additional cellulolytic bacteria were chosen to display these inconsistencies. Bacillus thuringiensis subsp. Kurstaki (Btk) and Cellulomonas persica, known to possess endoglucanase, were incubated at 30 °C for 48 h on several different media; 1.5 % agar only, Davis minimal media, CMC agar, non-CMC agar, and Phytagel CMC. After the incubation period all plates were flooded with Gram’s Iodine for 3–5 min. A clear ring of degradation was identified around colonies in plates containing no cellulose and plates containing agar only. When agar was removed as the solidifying agent and substituted with Phytagel, zones of clearing around cellulolytic organisms were altered. These findings indicate that agar alone can cause additional artifacts and should be replaced by other solidifying agents as well as supplementation of the Congo Red Assay. Taking these precautions can overcome inconsistency problems for future cellulolytic screenings.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Elberson MA, Malekzadeh F, Yazdi MT, Kameranpour N, Noori-Daloii MR, Matte MH, Sowers KR (2000) Cellulomonas persica sp. nov. and Cellulomonas iranensis sp. nov., mesophilic cellulose-degrading bacteria isolated from forest soils. Int J Syst Evol Microbiol 50(3):993–996. doi:10.1099/00207713-50-3-993
Hankin L, Anagnostakis SL (1977) Solid media containing carboxymethylcellulose to detect CX cellulose activity of micro-organisms. J Gen Microbiol 98(1):109–115. doi:10.1099/00221287-98-1-109
Johnsen HR, Krause K (2014) Cellulase activity screening using pure carboxymethylcellulose: application to soluble cellulolytic samples and to plant tissue prints. Int J Mol Sci 15(1):830–838. doi:10.3390/ijms15010830
Kasana RC, Salwan R, Dhar H, Dutt S, Gulati A (2008) A rapid and easy method for the detection of microbial cellulases on agar plates using Gram’s iodine. Curr Microbiol 57(5):503–507. doi:10.1007/s00284-008-9276-8
Maki ML, Broere M, Leung KT, Qin W (2011) Characterization of some efficient cellulase producing bacteria isolated from paper mill sludges and organic fertilizers. Int J Biochem Mol Biol 2(2):146–154
Saraswathi S (2011) Characterization and optimization of agarase from an estuarine Bacillus subtilis. Afr J Microbiol Res 5(19):2960–2968. doi:10.5897/AJMR11.480
Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43(4):777–780
Zitomer SW, Eveleigh DE (1987) Cellulase screening by iodine staining: an artifact. Enzym Microb Technol 9:214–216
Acknowledgments
The authors would like to acknowledge the Department of Biological Sciences at Tennessee State University for their facilities and funding to accomplish this work. Several doctoral students; Hui Li, Mailene King and Letimicia Fears were also helpful for their expertise advise.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
OHair, J.A., Johnson, T.L., Ejiofor, A.O. et al. Reducing inconsistent cellulolytic screenings during the Gram’s Iodine Assay. Cellulose 23, 3389–3392 (2016). https://doi.org/10.1007/s10570-016-1027-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10570-016-1027-6