Tetrazolium/Formazan Test as an Efficient Method to Determine Fungal Chitosan Antimicrobial Activity

Fungal chitosan was extracted from Aspergillus niger mycelia. The produced chitosan was characterized with deacetylation degree of 89.2%, a molecular weight of 2.4 × 104 Da, and 96.0% solubility in 1% acetic acid solution. The antibacterial activity of fungal chitosan was evaluated against two foodborne pathogens, that is, Salmonella typhimurium and Staphylococcus aureus, using the established antibacterial assays, for example, zone of growth inhibition and agar plat count tests, and using 2,3,5,-triphenyltetrazolium chloride (TTC) as chromogenic marker for qualitative and quantitative determining of antibacterial potentiality. The TTC (0.5% w/v) was added, at concentration of 10%, to cultured broth, containing chitosan with different concentrations then the formed formazan was separated. The formation of red formazan could be considered as a qualitative indication for antibacterial activity, whereas the measurement of color intensity for the resuspended red formazan, using spectrophotometer at 480 nm, provided a quantitative evidence for the strength of the used antibacterial agent. Regarding the rapidity, technical simplicity, and cost-effectiveness, TTC assay could be recommended as an efficient alternative method for qualitative and quantitative determination of chitosan antibacterial activity and could be suggested for general evaluation of antibacterial agents