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Characterization of surface and mechanical properties of microbial cells
Khýrová, Markéta ; Lukeš,, Jaroslav (referee) ; Plichta, Tomáš (advisor)
The subject of this diploma thesis is the characterization of bacterial strains capable of polyhydroxyalkanoates (PHA) production in terms of morphology and mechanical properties. With this aim, an atomic force microscope (AFM) was used together with a nanoindentation instrument to perform microcompression tests on selected microorganisms. The model bacterial strains, Cupriavidus necator and Rhodospirillum rubrum, were tested in their PHA-producing form and in their mutant non-producing variant, in order to study the effect of PHA amount on bacterial morphology and mechanical properties. In the experimental part of this work, the preparation of native bacterial samples for both techniques were first optimised. Glass substrates were treated with plasma and the cells were subsequently fixed with poly-L-lysine. In addition, sample preparation including fluorescent staining was optimized for the microcompression tests in order to easily detect cells and determine their size. Finally, the impact of different measurement parameters and mathematical models for calculating Young's modulus on the AFM data were evaluated. The results revealed the effect of PHA on bacterial cell morphology and showed that PHA producers exhibit higher Young´s modulus on average compared to their mutant. It was also shown experimentally that microcompression tests provide significantly higher Young's modulus values than those obtained by AFM.
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Study of biological samples using atomic force microscopy
Khýrová, Markéta ; Smilek, Jiří (referee) ; Slaninová, Eva (advisor)
The purpose of this bachelor thesis was to study Gram-negative bacteria with the ability to accumulate polyhydroxyalkanoates using atomic force microscopy. During the experimental part were studied bacterial strains Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, Rhodospirillum rubrum, and Halomonas halophila. The main aim was to optimise this microscopy method to study either bacteria in the air or bacteria in liquid medium under physiological conditions. The optimisation was done considering the procedure's simplicity, time demands, and reproducibility. 10× diluted bacterial suspense was dried on a glass surface for measurements in the air. Scanning was performed using TESPA-V2 tip with lowered Spring constant in QITM mode. Various procedures for immobilising bacteria on a glass substrate using Poly-L-lysine and Poly(ethyleneimine) were tested for measurements in liquid. The method using Poly-L-lysine was chosen as the most sufficient way of immobilisation. The tips MLCT – A or SNL – B with the spring constant around 0,1 N/m were applied for measurements in liquid using QITM mode. Finally, an attempt was made to obtain information about the adhesion and Young's modulus of dried and live bacteria concerning the high difference of polyhydroxybutyrate content between the bacterial strains Cupriavidus necator H16 and Cupriavidus necator PHB-4.
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Study of biological samples using atomic force microscopy
Khýrová, Markéta ; Smilek, Jiří (referee) ; Slaninová, Eva (advisor)
The purpose of this bachelor thesis was to study Gram-negative bacteria with the ability to accumulate polyhydroxyalkanoates using atomic force microscopy. During the experimental part were studied bacterial strains Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, Rhodospirillum rubrum, and Halomonas halophila. The main aim was to optimise this microscopy method to study either bacteria in the air or bacteria in liquid medium under physiological conditions. The optimisation was done considering the procedure's simplicity, time demands, and reproducibility. 10× diluted bacterial suspense was dried on a glass surface for measurements in the air. Scanning was performed using TESPA-V2 tip with lowered Spring constant in QITM mode. Various procedures for immobilising bacteria on a glass substrate using Poly-L-lysine and Poly(ethyleneimine) were tested for measurements in liquid. The method using Poly-L-lysine was chosen as the most sufficient way of immobilisation. The tips MLCT – A or SNL – B with the spring constant around 0,1 N/m were applied for measurements in liquid using QITM mode. Finally, an attempt was made to obtain information about the adhesion and Young's modulus of dried and live bacteria concerning the high difference of polyhydroxybutyrate content between the bacterial strains Cupriavidus necator H16 and Cupriavidus necator PHB-4.
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