2.7. Detection of Reactive Oxygen Species (ROS)

The ROS that was released by both treated and untreated bacterial cells were detected using an Acridine orange-ethidium bromide (AO/EtBr) staining procedure. A fluorescent microscope was used to measure the antibacterial activity of the GONPs, GO-PEG, and GONPs-PEG-N. sativa on the organisms for the study. To distinguish cell viability after treatments an AO/EtBr staining procedure was done. A total of 50 μL bacterial suspension of both treated and untreated was mixed with 50 μL (prepared from 10 μg/mL AO/EtBr stock solution) and was left for about 2 min. The staining procedure followed, after which a thin film of the mixture was applied on a glass slide and then observed under an immunofluorescent microscope. For the living cells, the Acridine orange-stained fluoresce green while for the dead cells the ethidium bromide-stained fluorescence red [18].