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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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Properties and production of microbial lipases
Martinková, Patrícia ; Reichstädter, Marek (referee) ; Omelková, Jiřina (advisor)
Bachelor´s thesis is focused on testing the culture media for growing various strains of yeasts, producing lipolytic enzymes and to study the influence of culture media composition on the production of lipolytic enzymes. Theoretical part of this thesis states the characteristics of lipolytic enzymes, their properties, sources, conditions for their production and the possibilities of their application. Together 8 strains of yeasts were used in the experimental part of the thesis, namely 2 strains of Yarrowia lipolytica, 2 strains of Kluyveromyces lactis, Cryptococcus saitoi, Candida intermedia, Candida oleophila and Debaryomyces hansenii, which were cultivated on 4 media with different compounds. During the cultivation the growth of biomass was monitored and growth curves were formed based on these results. Selected strains of yeasts were tested during the process of cultivation for lipolytic activity of two types of enzymes – enzymes bonded to the cellular structure and extracellular enzymes, which were measured by spectrophotometer. Production of lipolytic enzymes varied depending on applied culture medium.
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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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Properties and production of microbial lipases
Martinková, Patrícia ; Reichstädter, Marek (referee) ; Omelková, Jiřina (advisor)
Bachelor´s thesis is focused on testing the culture media for growing various strains of yeasts, producing lipolytic enzymes and to study the influence of culture media composition on the production of lipolytic enzymes. Theoretical part of this thesis states the characteristics of lipolytic enzymes, their properties, sources, conditions for their production and the possibilities of their application. Together 8 strains of yeasts were used in the experimental part of the thesis, namely 2 strains of Yarrowia lipolytica, 2 strains of Kluyveromyces lactis, Cryptococcus saitoi, Candida intermedia, Candida oleophila and Debaryomyces hansenii, which were cultivated on 4 media with different compounds. During the cultivation the growth of biomass was monitored and growth curves were formed based on these results. Selected strains of yeasts were tested during the process of cultivation for lipolytic activity of two types of enzymes – enzymes bonded to the cellular structure and extracellular enzymes, which were measured by spectrophotometer. Production of lipolytic enzymes varied depending on applied culture medium.
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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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Semiquantitative bacteriological examination of urine
VITANOVSKÁ, Alena
Urine is a liquid product of metabolism excreted through the kidneys. It refers to the overall health of the body. Urinary tract infections are the second most common disease in the population caused by various pathogens. E. coli is the most frequent pathogen. The increase in resistance of bacterial pathogens to antibiotics is related to the high incidence of persistent infections and their treatment. The main objectives of this thesis are to introduce the bacteriological examination of urinary tract infections and mastering the art semiquantitative bacteriological examination of urine and other processes leading to the diagnosis of urinary pathogens using in the Medical Microbiology Department of Klatovská nemocnice, a.s.. Then, based on the results taken from LIS OLM to evaluate the obtained results and to compare them with the literature. The first part deals with the definition of basic concepts that are associated with urinary tract infections, and its infections and occurring pathogens. The various phases of laboratory procedure are described. In particular, analytical part of laboratory procedure which deals with theoretical description of methods of laboratory diagnostics. The procedures of identification methods, which are used in the Medical Microbiology Department of Klatovská nemocnice, a.s. are described in the methodology. That means semiquantitative examination, microscopy, various biochemical tests for pathogens identification and process for the determination of sensitivity. The research results are evaluated using simple statistics in tables and graphs. In the year 2014 8623 urine samples were examined. 6267 samples came from hospital patients. The samples from hospital patients were for the statistical evaluation. From these samples 1260 were cases of E. coli, Enterococcus 829 cases and 297 cases of Proteus. For further statistical evaluation is carried out with E. coli which determine sensitivity to nitrofurantoin such were 1030. From that 819 women and 211 men. The highest incidence of this pathogen was among women in the age group over 61 years, it were the 542 patients. For men the capture also the highest over the age of 61 years, with 166 patients. The lowest detection of E. coli in both men and women was the lowest compared to other categories of age 7-17.
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Comparison of bacteriological examination of urine using the cultivation and nephelometric method
LOJÍKOVÁ, Aneta
In population urinary tract infections (UTI) occur as the second most common diseases caused by various pathogens. They manifest themselves by the presence of bacteria in urine called bacteriuria. The objective of the thesis was to compare the bacteriological examination of urine in suspected UTI using cultivation-based methods and nephelometric methods. Two procedures were used for cultivation examination of urine. The first was a gradual dilution technique which is considered to be accurate. The second procedure was cultivation on filter paper which is considered to be less reliable. Two types of culture media - URI Select 4 and COS from the firm BIO-RAD were used.. URI Select 4 is a diagnostic medium designed to cultivate urine samples. Blood agar (COS) is a blood enriched medium on which the majority of medically important bakteria grow. The third measurement was carried out with the automatic system URO-QUICK from ALIFAX S.p.A. Cultivation on filter paper was used only as an extending supplementary method to be compared to the gradual dilution technique and the automatic system URO-QUICK. Total of 50 urine samples were tested.
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