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Detection viability of bacteria population using flow cytometry
TŮMOVÁ, Lucie
This work deals with the detection of viability of Xanthomonas euvesicatoria using a flow cytometer. The center of this work was tp optimize the method and the treatment of bacterial cultures with high temperature at given time intervals. The heat-treated cultures were dyed with an intercalating dye called propidium iodide which marks the permeabilized membranes of the cells and thus provides information on the number of dead (apoptotic) cells. The evaluation of the results confirmed the assumption that the number of surviving cells would decrease with the increasing treatment time. A temperature of 90 ° C was used, which according to other works, seems to be the most evident for the Xanthomonas genus. Flow cytometry has proven to be a simple, fast and efficient method for the viability detection of Xanthomonas euvesicatoria.
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Methods for viability evaluation of Xanthomonas vesicatoria after low temperature plasma treatment
ZEMANOVÁ, Marta
The thesis deals with methods for viability evaluation of the phytopathogenic bacterium Xanthomonas euvesicatoria after low-temperature plasma treatment. Low-temperature plasma produced by Gliding Arc experimental device was used for treatment of X. euvesicatoria. The viability of the bacterial cells was assessed using a scanning electron microscope (SEM) and by measuring of the fluorescence in the Smart-DART device using PrestoBlue chemical reagent. Methodology has been optimised for the sample preparation for the treatment by low temperature plasma and used for evaluation of applied methods. Lethal effect of gliding arc plasma to this gram-negative bacteria was verified by SEM which showed. There is significant structural changes on the cell surface. Viability assessment of X. euvesicatoria using Smart-DART device is a fast, time-saving and inexpensive evaluation of cell viability. The great advantage of this device is its ability to measure the fluorescence in real time. The disadvantage of this method is lower reliability in current stage of research.
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Design and testing of multiplex-PCR primers for detection of bacterial spot of tomato
STEHLÍKOVÁ, Dagmar
The subject of this work is to develop multiplex-PCR assay for specific detection of plant pathogenic bacteria of Xanthomonas genus causing bacterial spot of tomato. PCR primers for detection of groups A (X. euvesicatoria), B (X. vesicatoria), C (X. perforans) and D (X. gardneri) were developed based on the DNA sequences obtained by sequencing and from the GenBank database (NCBI). Four primer pairs - Xe_shotgun_104, Xe_shotgun_1819, Xv_atpD_403, Xp_efP_202 were designed and subsequently thoroughly tested and optimized for parallel detection of these bacteria. Specificity of the primers was tested on a large complex of bacterial strains pathogenic to tomato and related crops. Following the protocol described above X. vesicatoria, X. euvesicatoria, X. perforans and X. gardneri can be quickly and reliably identified in a single multiplex-PCR assay.
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