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DNA isolation and identification of nonpathogenic species of clostridia isolated from cheeses
Sedláček, Zbyněk ; Eva, Kvasničková (referee) ; Rittich, Bohuslav (advisor)
In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).
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Hydrogen production by bacteria of genus Clostridium
Sedláček, Zbyněk ; Turková, Kristýna (referee) ; Rittich, Bohuslav (advisor)
The bachelor thesis deals hydrogen production by bacteria of the genus Clostridium. Thesis gives an overview about hydrogen production mentioned microoganism, characterized further biotechnological methods and show examples of industrial production of hydrogen. Also there are describes the quantitative characteristic and metabolism of bacteria genus Clostridium and examples of usable substrates. Polymerase chain reaction (PCR) is used to the detection and identification by species and specific primers. DNA isolated by fenol extraction from bacterial culture Clostridium tyrobutyricum DSM 2637 was amplified using genus-specific PCR to form PCR products size 619 bp.
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SStudy of crystalline structure of polyhydroxybutyrate and nucleating activity of selected additives
Sedláček, Zbyněk ; Tocháček, Jiří (referee) ; Bálková, Radka (advisor)
This diploma thesis deals with study of crystalline structure of polyhydroxybutyrate (PHB), which contains different types of additives for studying of their nucleation activity and which were prepared by mixing. It is about boronitrid (BN), sacharin, hydroxapatit, plasticizer Tegmer a tree types of talc. Crystal structure was analysed by differential scanning calorimetry and x-ray diffraction, supramolecular structure was observed by optical microscopy (polarized and confocal laser scanning). Nucleating activity was evaluated by isothermal and non-isothermal crystallization made on calorimeter and heated table of optical microscope. There is not influence of additives on crystallographic structure, but additives affects number and size of spherulites including crystal domains defects, which can have impact on final mechanical properties. BN and talcs react as nucleating agents, other additives during low and high cooling speeds (vc) inhibit nucleation and in middle cooling speeds are without effect. Nucleating activity is not evaluated by numerically, because decrease of crystallization temperature together with vc is not linear. Results of direct methods are based on picture analysis, which is great benefit for understanding of crystal behaviour of PHB.
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SStudy of crystalline structure of polyhydroxybutyrate and nucleating activity of selected additives
Sedláček, Zbyněk ; Tocháček, Jiří (referee) ; Bálková, Radka (advisor)
This diploma thesis deals with study of crystalline structure of polyhydroxybutyrate (PHB), which contains different types of additives for studying of their nucleation activity and which were prepared by mixing. It is about boronitrid (BN), sacharin, hydroxapatit, plasticizer Tegmer a tree types of talc. Crystal structure was analysed by differential scanning calorimetry and x-ray diffraction, supramolecular structure was observed by optical microscopy (polarized and confocal laser scanning). Nucleating activity was evaluated by isothermal and non-isothermal crystallization made on calorimeter and heated table of optical microscope. There is not influence of additives on crystallographic structure, but additives affects number and size of spherulites including crystal domains defects, which can have impact on final mechanical properties. BN and talcs react as nucleating agents, other additives during low and high cooling speeds (vc) inhibit nucleation and in middle cooling speeds are without effect. Nucleating activity is not evaluated by numerically, because decrease of crystallization temperature together with vc is not linear. Results of direct methods are based on picture analysis, which is great benefit for understanding of crystal behaviour of PHB.
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DNA isolation and identification of nonpathogenic species of clostridia isolated from cheeses
Sedláček, Zbyněk ; Eva, Kvasničková (referee) ; Rittich, Bohuslav (advisor)
In the food industry are requested speedy and accurate methods for identification of bacteria in microbiological testing of products. Molecular diagnostic methods are based on isolation of DNA from bacterial cells which is amplified in polymerase chain reaction (PCR). The result is fragment DNA about specific size, characteristic for genus or species of bacteria. The aim of the work was isolation of PCR-ready DNA. DNA has been isolated from 8 strains of genus Clostridium. Procedure of cell lysis was optimized in order to find the optimal concentration of EDTA and proteinase K in lysing buffer. DNA was isolated by phenol extraction and using magnetic microspheres. Concentrations 10 mM of EDTA and 10 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by phenol extraction. Concentrations 10 mM of EDTA and 15 l of proteinase K (100 g/ml) were the best for cell lysis for isolation of DNA by magnetic microspheres. Isolated DNA was checked by gel electrophoresis, quantificated by spectrophotometry and tested in PCR. Individuals species were distinguished in denaturing gradient gel electrophoresis (DGGE).
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Hydrogen production by bacteria of genus Clostridium
Sedláček, Zbyněk ; Turková, Kristýna (referee) ; Rittich, Bohuslav (advisor)
The bachelor thesis deals hydrogen production by bacteria of the genus Clostridium. Thesis gives an overview about hydrogen production mentioned microoganism, characterized further biotechnological methods and show examples of industrial production of hydrogen. Also there are describes the quantitative characteristic and metabolism of bacteria genus Clostridium and examples of usable substrates. Polymerase chain reaction (PCR) is used to the detection and identification by species and specific primers. DNA isolated by fenol extraction from bacterial culture Clostridium tyrobutyricum DSM 2637 was amplified using genus-specific PCR to form PCR products size 619 bp.
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