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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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