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Analysis of bacrerial cells employing flow cytometry and flurescence microscopy
Müllerová, Lucie ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on fluorescent analysis of viability and PHA content in bacterial cultures, the main methods of investigation were flow cytometry and fluorescent microscopy. In order to determine viability of C. necator H16, several viability probes were tested, nevertheless, only BacLightTM kit and propidium iodide can be used to estimate portion of viable and live bacterial cell in samples. Further, Acridine orange was used to monitor physiological state of bacterial culture and two hydrophobic probes, Nile Red and BODIPY 493/503, were used to investigate PHA content in bacterial cells. Application of BODIPY 493/503 seems to be promising since this probe does not require permeabilization of bacteria cells and it can be used along with propidium iodide. Furthermore, several fluorophores were tested in the microscopic part. In was found that concentrations used in cytometric analyses were too high for microscopic use. Emission from the SYTO9 fluorophore is seen mainly in the green channel but because of the high concentration some emission was visible in the red channel. Cells stained with BODIPY 493/503 had very high fluorescence intensities when the stain concentration was 10 . At the same time, negative amplitudes of fluorescence were measured in both strains of C. necator, but in case of C. necator H16 that amplitude was much more pronounced. In this strain surprising high concentration of BODIPY stain was observed on the surface of PHB granules. Anisotropy of the fluorophore was nearing 0 which is very surprising.
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Study on PHa biosynthesis by cyanobacteria
Drinka, Jakub ; Kouřilová, Xenie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis concerns with the effect of selected environmental stresses towards the viability of cyanobacterial species Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Both strains were studied via flow cytometry using the appropriate fluorochromes while also observing the applicability of these stains and precision of this method in the analysis of cyanobacterial cultures. In spite of the optimization being carried out, fluorescein diacetate was not suitable for the analysis of these cells because of their autofluorescence interfere with the fluorochrome emissions. Therefore, SYTOXTM Blue Dead Cell stain was used for the further experiments. A cryoprotective effect of polyhydroxybutyrate was observed during the freezing-thawing cycles. On the other hand, the culture rich in PHB seemed to be less resistant towards the oxidative stress induced by the hydrogen peroxide, probably justifiable by the state of chlorosis. Furthermore, this thesis deals with the increase/decrease in biomass concentration and intracellular PHB pool as a response to the external carbon source addition, analysed by the gas chromatography paired with FID detection. The most intesive cell growth in Synechocystis sp. PCC 6803 was detected in the presence of sodium acetate combined with gamma-butyrolactone while the highest PHB content in the medium supplied with acetate and 1,4-butanediol. Nevertheless, Synechocystis salina CCALA 192 showed markedly higher polyhydroxybutyrate accumulation under both photoautotrophic and mixotrophic conditions, the only non-suitable substrate was gamma-butyrolactone.
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Connection between stress resistance of bacteria to temperature-mediated stress and PHA accumulating ability
Nováčková, Ivana ; Benešová, Pavla (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the involvement of polyhydroxyalkanoates (PHA) into the stress response of bacteria, specifically when they are exposed to temperature-mediated stress. The theoretical part focuses on the stress response of bacteria in general, the mechanism of response to temperature-mediated stress and PHA involvement into the stress response. The aim of experimental work was to study the correlation between the amount of PHA in the biomass of the bacteria and their viability after exposure to temperature-mediated stress. PHA producing bacterial strains Cupriavidus necator H16, Burkholderia cepacia and Burkholderia sacchari and non-producing mutant strain Cupriavidus necator PHB4 were used for experimental work. Bacteria accumulated poly(3-hydroxybutyrate) (PHB), the amount of PHB in the biomass was determined by gas chromatography with FID. Viability of live bacterial cells and cells after exposure to temperature-mediated stress was determined by using flow cytometry. It turned out that the content of PHB in biomass assists bacteria to resist the freezing shock. By comparing the viability of bacteria C. necator H16 and C. necator PHB4 after exposure to multiple stresses it also has been observed that PHB increased the resistance of cells against temperature-mediated stress and stress caused by low pH.
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Use of microscopy and flow cytometry to molecular characterization of carotenogenic yeasts.
Vacková, Hana ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The aim of presented bachelor thesis is to optimize the methods for study of yeasts using flow cytometry and fluorescence microscopy. The theoretical part deals with the structure of yeast cells, principles and application of flow cytometry and fluorescent microscopy. Use of microscopy and flow cytometry in study of pigmented microorganismswas reviewed too. In the experimental part methods optimization was performed on yeasts Cystofilibasidium capitatum, Rhodotorula glutinis, Sporobolomyce sroseus, Sporobolomyces shibatanus. Further, the growth curve and the viability of yeasts under the influence of stress factors (freezing and the heat shock 60 C) was measured. Cystofilobasidium capitatum has shown to be more resistant to stress than Rhodotorula glutinis. It was found that there is the possibility of using methylene blue instead of propidium iodide in yeast cell analysis by flow cytometry.
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Viability of probiotic bacteria in some foods
Pokorná, Martina ; Bokrová, Jitka (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on viability od probiotic bacteria in some foods and looking for optimal food and beverages, that exhibited a minimal negative effect on the growth and viability of probiotic bacteria. Lactobacillus acidophilus, Bifidobacterium breve and commercially complex of probiotics were tested. In the practical part, probiotics were incubated in the environment of selected types of model and real foods. Further, incubation in model gastrointestinal juices to simulate human digestion was performed. Model foods with a moderate value of concentrations of sugar, protein and alcohol were shown the best alternative for growth of bacteria and their lowest mortality. For salt, the smallest concentration was shown as the most suitable. The chicken broth, chicken and beef meat were evaluated as the best environment for the capsule with complex probiotics. The vegetables and vegetable broth were stated like the best for Lactobacillus acidophilus and Bifidobacterium breve. According to the preliminary results we can conclude that the probiotics should be preferably consumed during meals and with the sufficient amount of liquids for the activation of all cells.
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Comparison of antimicrobial effect of emulsions for cleaning and dissinfection
Chrápavá, Edita ; Dzurická, Lucia (referee) ; Márová, Ivana (advisor)
Currently, hand disinfectants and other disinfectants are in high demand. Due to the pandemic and the spread of Covid-19, they are used by society daily. The reason for their use is not only to prevent the spread of bacteria and other microorganisms from the hands to another part of the body or objects, but also to prevent the spread of the disease. The subject of the bachelor thesis is the control of antimicrobial effects of several commonly available disinfectants using broth and agar dilution methods. From the measured data, the absorbance was processed and subsequently viability of the culture.
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Electrostimulation method for cardiomyocyte life extension
Čermáková, Adéla ; Ronzhina, Marina (referee) ; Čmiel, Vratislav (advisor)
This work deals with electrostimulation cardiomyocytes and design stimulation device for their cultivation and stimulation. First part of work deals with electrophysiological myocytes and the next problems with electrostimulation and preserved their original morphology and physiology structure. The following section is designed and constructed stimulation device to improve cell viability. Stimulation took place under different conditions and the success rate depends on the number of cells that survived.
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Studium změn antimikrobiální aktivity houby Ganoderma adspersum pomocí kultivační strategie OSMAC
Komárková, Lucie
Ganoderma adspersum is a white rot fungus that degrades lignocellulose in nature. However, it has also been proven to have many biological activities, including antioxidant, antitumor and anti-inflammatory. This study is focused on monitoring the antimicrobial activity of G. adspersum against the bacteria Escherichia coli, Staphylococcus aureus, MRSA and Klebsiella pneumoniae. First, the fungus was cultivated in three media with different additives. Using the minimum inhibitory concentration test, the effect of individual cultivation on viability was monitored. The lowest values were measured in YMB medium with tryptophan, which reduced the viability of E. coli up to 23 %. Based on this result, E. coli was chosen for growth curve, where media with additives were compared with pure media. It was found that pure media showed greater inhibitory activity than media with additives. The highest activity was observed in pure PDB medium, in which nearly 40% inhibition occurred at a concentration of 0,25 mg/ml. The results showed that Ganoderma adspersum could be a potential antimicrobial agent.
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The effect of amyloid beta on viability of selected neuropathogens: challenging the infectious hypothesis of Alzheimer disease
Vosála, Ondřej ; Macháček, Tomáš (advisor) ; Černý, Jan (referee)
Alzheimer's disease is the leading cause of dementia worldwide, affecting millions of individuals. Researchers have identified amyloid beta (A ) as the primary agent responsible for neuropathogenicity, but the factors triggering its overproduction remain uncertain. An relatively new theory, known as the infectious hypothesis, sheds light on this phenomenon by associating the excess production of A with its potential role as an antimicrobial peptide-an element of the innate immune response against neuroinfections. This hypothesis posits a transition from acute physiological overproduction during neuroinfection to a chronically dysregulated and pathological process targeting both pathogens and neural tissue. So far, experimental evidence supporting this theory has been primarily obtained through studies involving viruses or bacteria. Aim of this study was to investigate the role of multicellular pathogens in stimulating A overproduction and, on the other hand, A role in elimination of such infections. Toxocara canis was chosen as a main model of neuropathogenic parasite. Although successful infections were achieved in BALB/c mice, there was no observable increase in A production even after 16 weeks of infection. To further challenge the survivability of Toxocara larvae in increased A concentration in...
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