Inadequacy of Gram's Iodine Assay to Detect Cellulase Degradation during Screening for Lignocellulolytic Bacteria and Fungi
Gram's Iodine is currently used in detection of endoglucanase, a cellulase enzyme which cleaves beta 1-4 glycosidic bonds in CMC (carboxymethylcellulose). However, when a zone of degradation was noticed around non-cellulase containing E. coli ATCC 25922, the Gram's Iodine Assay was further scrutinized. ^ CMC was removed from agar plates, but zones of clearing were still present. This led to testing of bacterial colony, agar and broth media components to determine the objective's target(s) of interest. Agar and broth were deconstructed and (4ml) Gram's Iodine was mixed with each 0.5g CMC, 0.1g NaNO3, 0.1g K2HPO4, 0.1g KCI, 0.05g MgSO4, 0.05g yeast extract, 2.5g LB media, and 0.5g tryptone dissolved in 100ml DD H 2O. Readings were taken with spectrophotometer at 590nm and a change in %T was examined. The growth of bacteria when compared to zone of clearing was also measured when bacterial colony was tested. ^ The major compounds which altered the %T were tryptone and specific amino acids digested by trypsin. These sulfur containing amino acids within LB broth, more specifically L-Methionine, L-Cysteine, and L-Cystine. Thiol groups able to be oxidized will approach a colorless solution when mixed with Gram's Iodine as the cysteine is converted to sulfinic acid. Redox reactions make Gram's Iodine an unsuitable method for plate flooding and cellulase degradation tests. Further endoglucanase testing should be done with other bacterial and fungi staining assays.^
Joshua A OHair,
"Inadequacy of Gram's Iodine Assay to Detect Cellulase Degradation during Screening for Lignocellulolytic Bacteria and Fungi"
ETD Collection for Tennessee State University.