The metabolic activity of EBST was assessed using Alamar Blue® reagent (Life Technologies, Carlsbad, CA). Alamar Blue® system incorporates a cell permeable, non-fluorescent, blue redox indicator, resazurin. Metabolically active cells maintain a reduced cell environment that converts resazurin to resorufin (red fluorescent compound). The increase in fluorescence can be quantitatively measured to determine the metabolic activity of cells. After eBeam irradiation, bacterial samples were stored at 4°C and the persistence of metabolic activity in the eBeam–inactivated S. Typhimurium was monitored for 10 days. Live S. Typhimurium and HKST were used as controls. Ninety microliters of EBST, HKST and live ST samples were mixed with 10μl of Alamar blue reagent and incubated at 37°C for 1 h. Fluorescence was measured at 530–560 nm excitation wavelength and 590 nm emission wavelength using Wallac 1420 VICTOR 2™ plate reader (PerkinElmer, Waltham, MA).

Biochemical assays were performed to confirm the presence of metabolic activity in eBeam-inactivated S. Typhimurium. The ability of bacteria to ferment carbohydrates in media was tested by incubating EBST and HKST in Phenol Red Broth supplemented with sucrose at 37°C for 2 days. Overnight cultures of live ST and E. coli were used as controls. The color change in the media, turbidity and gas production as a result of bacterial metabolism were monitored. Presence of catalase enzyme in the inactivated cells was detected using the catalase test. On clean glass slides, EBST and live ST cells were smeared and a drop of hydrogen peroxide was added. Bubble formation from the bacterial preparations was monitored to detect catalase activity.

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