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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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Characterization of morphological and physico-chemical properties of microorganisms and components of cell by atomic force microscopy
Blaňka, Ondřej ; Smilek, Jiří (referee) ; Slaninová, Eva (advisor)
The aim of this bachelor thesis was to characterize Rhodospirillum rubrum bacterial cells that produce polyhydroxyalkanotate (PHA) and Rhodospirillum rubrum phaC without the ability to produce intracellular PHA granules using atomic force microscopy. Furthermore, isolated polyhydroxyalkanoate granules in their native state were also characterized by this method. Poly-L-lysine was used in the preparation of the samples, specifically for fixation of bacterial cells and isolated PHA granules, and was applied to slides that had been exposed to plasma at a concentration of 0.01 mg/ml. The tip tested was the MLCT - A tip. The first characteristics obtained were topographic images of Rhodospirillum rubrum and Rhodospirillum rubrum phaC cells and isolated PHA granules. The bacterial cells and PHA granules were further characterized using Young's modulus. To obtain it, a method was designed in which whole cells and PHA granules were first imaged, respectively, and then, in the case of cells, measurements were made targeting individual locations on the cell. The measured data were first evaluated in JPK Data Processing software using the Hertzian data approximation model. This evaluation proved to be unreliable, so a second method was devised using the directive lines obtained with the steep parts of the "extend" curve to evaluate. Whole-cell measurement was chosen as a more reliable measurement because it yielded smaller deviations and provided information about the entire surface of the cell or granule. The Rhodospirillum rubrum directive was (4.5±1.5) nN/µm and for Rhodospirillum rubrum phaC was (14.1±4.4) nN/µm. This confirmed the hypothesis that Rhodospirillum rubrum cells containing PHA granules exhibit different stiffness compared to Rhodospirillum rubrum phaC cells without the presence of PHA granules. The directive for isolated PHA granules was determined to be (9.0±1.0) nN/µm. When compared with the directives of bacterial cells, it shows that the PHA granules exhibit a stiffness that lies between that of Rhodospirillum rubrum and Rhodospirillum rubrum phaC.
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Metody dekontaminace rekombinantních proteinů od bakteriálního lipopolysacharidu
CHARVÁTOVÁ, Lucie
In this work, three decontamination methods based on two-phase micellar system and affinity chromatography were used to decontaminate recombinant proteins from bacterial lipopolysaccharide and to determine which method is the most effective. The efficiency of this method was measured using various recombinant proteins at several protein concentrations. Three different assays, two chromogenic and one fluorogenic, were used to measure the concentration of endotoxins in samples. The most accurate method for measuring the concentration of endotoxins was determined.
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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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Modification of boron doped diamond electrode by poly-L-lysine and its influence on electrochemical behaviour of dopamine and serotonin
Pejzlová, Michaela ; Fischer, Jan (advisor) ; Vyskočil, Vlastimil (referee)
This bachelor's thesis deals with the redox behavior of two neurotransmitters, dopamine and serotonin, at bare anodically activated (O-terminated) and poly-L-lysine-coated boron doped diamond electrode in two media, namely phosphate buffered saline and Neurobasal (medium commonly used for neuron cultivation experiments). Oxidation of dopamine in tested environments has a quasi-reversible character, while serotonin oxidizes irreversibly on boron doped diamond electrode, both O-terminated and poly-L-lysine-treated. Clearly, poly-L-lysine modification of boron doped diamond electrode leads to a decrease in peak currents and a positive shift in oxidation potentials compared to the O-terminated boron doped diamond electrode; interestingly, serotonin is much more sensitive to polymer coating than dopamine, particularly in Neurobasal medium. During successive measurements, a considerable decrease in recorded signals of both neurotransmitters can be observed. Hence, possibilities how to ensure stable current values were investigated: for dopamine, stirring the solution between the individual scans is sufficient method for obtaining repeatable signals (sr = 1.4-11.9 %, n = 5). However, in case of serotonin it is necessary to reactivate the electrode surface anodically in H2SO4 (Eact = +2400 mV) for 5...
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