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Effect of culture medium on the identification of yeasts of the genus Cryptococcus using mass spectrometry
Jurnečková, Alena ; Vadkertiová, Renata (referee) ; Stratilová, Eva (advisor)
Cryptococccus genus is known for its difficult identification and taxonomical classification in area of clinical microbiology. For this bachelor thesis, total 22 yeast strains of the Cryptococcus genus were chosen. The part of strains was firstly analyzed by D1/D2 domain LSU rRNA gene analysis. Three types of culture medium – YPD, potato dextrose agar and Sabouraud’s medium were selected for cultivation. Samples were prepared according to standardized method of Bruker Daltonik company, Institute of Chemistry of SAS and combination of these two methods. Identification was done by MALDI-TOF mass spectrometry. Obtained spectra were compared using corresponding software and evaluated on the basis of specific algorithm. The most advantageous culture medium for cultivation and biotyping with the largest percentage score was YPD (Yeasts pepton dextrose). On the other hand, the least advantageous culture medium was Sabouraud’s agar, which reached the smallest percentage success due to parameters of Bruker Daltonik algorithm. The most succesfull method of sample preparation and application was the combined method with YPD as a culture medium. The results of complete analysis are dendrograms for each medium showing the genetic similarity of yeast strains. The dendrogram shows categorization of the single strains into appropriate groups. Cr. flavescens (CCY 17-3-28, CCY 17-3-30) and Cr. laurentii (CCY 17-3-2) strains were correctly integrated into phylogenetic group Cr. laurentii I (branch in the dendrogram with designation A). Cr. flavus (CCY 17-3-5) doesn’t belong to this group, although it shows similarity with Cr. flavescens. The strains Cr. carnescens (CCY 17-3-13) and Cr. victoriae (CCY 17-3-26) belong to the phylogenetic group Cr. laurentii II (designated as B). Cr. magnus (CCY 17-4-39, CCY 17-4-40) strains show similarity with these strains, but doesn’t belong to the phylogenetic group II. The strains Cr. gastricus (CCY 17-5-1) and Cr. diffluens (non-attached) form a branch designated as C. Cr. aerius (CCY 17-4-9) strain, which was also put into this group, was proposed to sequence analysis, because its spectrum indicates that it should be rather a Cr. diffluens strain. The group D contains Bulleromyces albus (CCY 17-3-35, CCY 17-3-36) and Cr. saitoi (CCY 17-3-18, CCY 17-4-2) strains. The sequenced Cr. albidus (CCY 17-4-1), non-sequenced Cr. diffluens (CCY 17-4-13) and Cr. terreus (CCY 17-8-1) form the group E. The strain CCY 17-4-13 was proposed to sequence analysis because of occurrence of the Cr. diffluens sequenced strain in the group C. The sequenced Cr. aerius (CCY 17-25-1) is also part of this group, but it represents a separate branch. The last group is named as F and consists of Cr. macerans (CCY 17-19-3) and control strains Cr. neoformans var. neoformans (CCY 17-1-4, CCY 17-1-5).
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Microalgae harvesting
Drozd, Jiří ; Naď, Martin (referee) ; Lošák, Pavel (advisor)
This work describe microalgae and their properties. Within the scope of the work, a research was conducted on the utilized microalgae and the possibilities of cultivation in the Central European region were examined. Furthermore, harvesting and drying methods for microalgae were analyzed, and an experiment was conducted using a promising spray drying method. The research revealed the properties and potential uses of microalgae. Various types of microalgae, their nutritional values, and possibilities of application in a wide range of fields were investigated. The cultivation possibilities of microalgae in the Central European region, including the utilization of wastewater and renewable energy sources, were also examined. Harvesting and drying methods for microalgae were analyzed, considering the advantages and disadvantages of different techniques such as flocculation, centrifugation, and spray drying. An experiment utilizing the spray drying method was conducted, and the results showed that this method is effective and enables the achievement of high quality and purity of dried microalgae. The work also explores the possibilities of further development and utilization of microalgae. It describes the potential use of microalgae as food supplements and as alternatives to traditional protein sources. Additionally, the potential applications of microalgae in the pharmaceutical and cosmetic industries were mentioned. In conclusion, this work provides information on the utilization of microalgae and the methods of cultivation, harvesting, and drying. The results suggest that microalgae have great potential for further development and utilization in various industries.
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Aplikace růstových regulátorů pro multiplikaci Vitis vinifera L. v podmínkách in vitro
Mančíková, Simona
The experiment was done at Faculty of Horticulture in Lednice, at the Mendeleum-Institute of genetics. The plant materil was obtained from the technical isolation in Lednice. Four selected grape varieties ('MT' 25/7, 20/52'PM','CR2' 1/48,'K 5BB') as primary culture and multiplied in selected media. All varieties were grown at the same temperature and light conditions. MS medium combined with BA (0.7 mg.l-1), IAA (0.1 mg.l-1) was chosen for the primary culture. The pH was adjusted to 5.8. After the establishment of primary cultures, only the variety 'CR 2' 1/48 survived. In the other varieties ('MT' 07/25, 20/52 and 'PM''K 5BB') infection by fungal and bacterial infections, hyperhydratation, no proliferation of shoots and necrosed individual segments were observed. Variety'CR2' was (after the establishment of primary cultures) multiplied onfive media; DKW combination of BA (0.6 mg.l-1), IBA (0.01 mg.l-1), MS in combination with BA (3.0 mg.l-1), NAA (0, 2 mg.l-1), C2D combination of BA (1.5 mg.l-1), MS enriched by BA (1.5 mg.l-1), IBA (1 0 mg.l-1), and LQ combination of BA (0.4 mg.l-1) and NAA (0.01 mg.l-1). Individual media were evaluated separately with the calculation multiplier of 4 passage. C0 medium was evaluated as the most suitable medium for in vitro grapevine multiplication . The lowest number of new formed shoots and also the worst results were observed on C1 medium. The results of the thesis evaluaete the health condition of plants and the multiplication factor on different media. New formed shoots during the ever single passaging was evaluated as well.
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Meristem cultures in garlic (Allium sativum) breeding
ŠVEHLOVÁ, Eva
The diploma thesis deals with the use of meristem cultures in garlic (Allium sativum) breeding. The source material was used variety Tantalum of garlic. The using material, before the isolation of meristem, was tested for the occurrence of viral diseases by immunological tests ELISA (Enzyme-Linked Immuno-Sorbent Assay), also known as EIA (Enzyme Immunoassay). The method used to detect antibodies and antigens. The material was tested for viruses onion yellow dwarf (OYDV - Onion Yellow Dwarf Virus) and virus genus Carlavirus (GCLV - Common Garlic Latent Virus). Then the sound material was sterilized, cultured and then it was obtained meristem. Cultivation of preparation meristem was realised according available methodologies. After preparation meristem put on MS medium with vitamins and growth regulator IAA, NAA and BAP.
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Optimization of Culture Medium for HEK293 Cell Line
Čuperková, Romana ; Vaněk, Ondřej (advisor) ; Šácha, Pavel (referee)
HEK293 is a human cell line derived from embryonic kidney cells and is a frequently used system for the production of recombinant proteins. This work dealt with optimization of the composition of serum-free medium for HEK293S and HEK293T cell lines as a compensation for expensive commercial media. The growth of culture and expression of reporter proteins SEAP and GFP was monitored as the markers. I managed to create a new medium which contained, among other compounds, insulin (1 mg/l), transferrin (5 mg/l) and a mixture of trace elements. During the cultivation in a mixture of commercial medium EX CELL 293 with my new medium 293S cells grew faster than during the cultivation in commercial media (doubling time 20,47 ± 2,68 hours (srel = 13,1 %)). It seems that the new medium is suitable for transfection of HEK293 cell lines with a relatively high expression of recombinant proteins. Transfection ratio of DNA:PEI (w/w) for this medium is 1:2 to 1:3.
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Effect of culture medium on the identification of yeasts of the genus Cryptococcus using mass spectrometry
Jurnečková, Alena ; Vadkertiová, Renata (referee) ; Stratilová, Eva (advisor)
Cryptococccus genus is known for its difficult identification and taxonomical classification in area of clinical microbiology. For this bachelor thesis, total 22 yeast strains of the Cryptococcus genus were chosen. The part of strains was firstly analyzed by D1/D2 domain LSU rRNA gene analysis. Three types of culture medium – YPD, potato dextrose agar and Sabouraud’s medium were selected for cultivation. Samples were prepared according to standardized method of Bruker Daltonik company, Institute of Chemistry of SAS and combination of these two methods. Identification was done by MALDI-TOF mass spectrometry. Obtained spectra were compared using corresponding software and evaluated on the basis of specific algorithm. The most advantageous culture medium for cultivation and biotyping with the largest percentage score was YPD (Yeasts pepton dextrose). On the other hand, the least advantageous culture medium was Sabouraud’s agar, which reached the smallest percentage success due to parameters of Bruker Daltonik algorithm. The most succesfull method of sample preparation and application was the combined method with YPD as a culture medium. The results of complete analysis are dendrograms for each medium showing the genetic similarity of yeast strains. The dendrogram shows categorization of the single strains into appropriate groups. Cr. flavescens (CCY 17-3-28, CCY 17-3-30) and Cr. laurentii (CCY 17-3-2) strains were correctly integrated into phylogenetic group Cr. laurentii I (branch in the dendrogram with designation A). Cr. flavus (CCY 17-3-5) doesn’t belong to this group, although it shows similarity with Cr. flavescens. The strains Cr. carnescens (CCY 17-3-13) and Cr. victoriae (CCY 17-3-26) belong to the phylogenetic group Cr. laurentii II (designated as B). Cr. magnus (CCY 17-4-39, CCY 17-4-40) strains show similarity with these strains, but doesn’t belong to the phylogenetic group II. The strains Cr. gastricus (CCY 17-5-1) and Cr. diffluens (non-attached) form a branch designated as C. Cr. aerius (CCY 17-4-9) strain, which was also put into this group, was proposed to sequence analysis, because its spectrum indicates that it should be rather a Cr. diffluens strain. The group D contains Bulleromyces albus (CCY 17-3-35, CCY 17-3-36) and Cr. saitoi (CCY 17-3-18, CCY 17-4-2) strains. The sequenced Cr. albidus (CCY 17-4-1), non-sequenced Cr. diffluens (CCY 17-4-13) and Cr. terreus (CCY 17-8-1) form the group E. The strain CCY 17-4-13 was proposed to sequence analysis because of occurrence of the Cr. diffluens sequenced strain in the group C. The sequenced Cr. aerius (CCY 17-25-1) is also part of this group, but it represents a separate branch. The last group is named as F and consists of Cr. macerans (CCY 17-19-3) and control strains Cr. neoformans var. neoformans (CCY 17-1-4, CCY 17-1-5).
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