Thesis (Ph.D.) - Cairo University - National Institute of Laser Enhanced Science - Department of Laser Application in Biotechnology

The conventional quantitative and qualitative methods for microbial detection are complex and require a lot of skills and time. There is a widespread need for cultivation-free methods to quantify the viability of microorganisms. Adenosine tri-phosphate (ATP) is the energy currency of all living organisms; therefore it is a rapid indicator of viability. A bio-sensing assay was developed to detect microbial viability using a bioluminescent recombinant E.coli. Luciferase extracted from transformed bacteria was used to develop ATP bioluminescence assay. Results showed that there is a direct correlation between luminescence intensity of the developed sensing assay and the total bacterial counts. Transformed E. coli was irradiated with He-Ne laser at 632 nm for 1, 2, 3, 4 min. The relation between irradiation times measured by minutes and total bacterial count (CFU) shows that the number of cells in irradiated samples exceeds the control level eventually till irradiation for 3 min. The maximum bacterial growth was observed at 3 min whereas the percentage of survival relative to control increased ten folds. By increasing the irradiation time to 4 min, the bacterial growth decreased slightly to five folds. It was concluded that He-Ne laser pretreatment accelerates cell division where is the mechanism by which He-Ne laser pretreatment changes the activities of enzymes, microcalorimetric parameters, and biophoton emission. He-Ne laser irradiation causes an enhancement of enzyme activities and accelerates enzyme-mediated reactions. Food samples were screened for bacterial counts by the developed bioluminescence assay and confirmed by the traditional bacterial count method. Compared with the plate count method, bacteria counts in samples could be detected rapidly by using ATP bioluminescent assay

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