טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFarber Yair
SubjectDetection of Bacteria and Viruses onto Sol-Gel
Matrices by Synchronous Fluorescence
DepartmentDepartment of Civil and Environmental Engineering
Supervisor Professor Robert Armon
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The present study has been divided into two major aspects. The first one, included water microbiology area, in which the development of an analytical method for detection of viruses in water has been performed. The second one, included soil microbiology area, in which the development of a method for the detection of siderophores secretion by bacteria in limited iron environment has been pursued. The basis for these two aspects are similar: the combination of sol-gel technology and substrate fluorescence in order to obtain visible detection of microorganisms or their activity. The sol-gel process provides a ceramic matrix to be doped with thermosensitive organic/bioorganic constituents at room temperature without thermal or chemical alterations consequently retaining their physical and chemical properties. Appling sol-gel technology, a variety of matrices were synthesized: thin films under acidic conditions and particles under basic conditions (Stöber method). Via synthesis of sol-gel particles viruses could be easily adsorbed with high efficacy on the surface of these particles that were doped with PLL in their matrix. PLL (Poly-L-lysine) is a long cationic chain that promotes adherence of cells and proteins to surfaces via electrostatic interactions. Integration of PLL into sol-gel matrix increased φx-174 bacteriophage adsorption 3-fold compared to sole sol-gel control. Following virus adsorption, they were further detected by synchronous fluorescence spectroscopy (SFS) (Δλ = 60 nm). In synchronous fluorescence spectroscopy method, tryptophan (an aromatic amino acid) was the key factor in φx-174 phage detection, though some other fluorescent amino acids like tyrosine were present in this bacteriophage coat proteins. Presumably, this result can be explained based on the fact that tyrosine and tryptophan are located close to each other, hence the irradiated energy of tyrosine could pass to tryptophan, while only tryptophan appears to be detected when φx-174 was subjected to SFS. As a result of sol-gel thin films synthesis at room temperature a unique matrix for detection of bacterial siderophores secretion was tailored. Free iron is inaccessible in soil, in spite of being an essential element. To resolve this contradiction, soil bacteria secrete siderophores as iron high affinity molecules to bind iron and introduce it into cells. In this study, iron had been entrapped in sol-gel matrices and followed for iron release with confocal laser scanning microscopy (CLSM) using Calcein Blue as an indicator. Use of the above method for bacterial iron uptake by bacteria resulted in fluorescence emission. The two present methods developed in this study, detection of viruses in water and detection of bacterial siderophores secretion, clearly demonstrate the high capacity of sol-gel technology and fluorescence combination. This combination can serve as a powerful instrument in microorganisms and their activity detection so much required these days as real time procedures.