Microbiology Lab Exam Ii

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BLAST

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Basic Local Allignment Search Tool. A bioinformatics tool that allows us to navigate through huge databases and compare an amino acid or nucleotide sequence to the library of published or submitted sequences. Closely related genes give us information about the function of the protein product or the identity of the organism they belong to. Using 16s ribosomal RNA Gene sequence and chromatogram.

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Biochemical Test: Catalase

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Hydrogen peroxide is a strong oxidizing agent that kills susceptible cells and is used by organisms to protect against infectious bacteria. Bacteria have defense mechanisms to counteract H2O2 activity which are facilitated by catalase. Bacteria with catalase make air bubbles when they come in contact with H2O2. To check for catalase H2O2 is added two bacterial smear and observed for bubbles to indicate a reaction.

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Biochemical Test: SIMS

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Sims media which is a semi-solid differential media is inoculated with bacterial sample. If the bacteria is motile and has flagella it will be able to move throughout the media. Indole is produced when tryptophan is metabolized. Indole production can be detected using Kovacs reagent which will turn red. A heavy metal salt which turns black can be added to the media and used to detect hydrogen sulfide produced by certain bacteria.

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Sanger Sequencing

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Chain termination sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. Developed by Frederick Sanger and colleagues in 1977, it was the most widely used sequencing method for approximately 25 years. Resulting DNA fragments are heat denatured and separated by size using gel electrophoresis.

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Phylogenetic Tree

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Cases of relation regarding the evolutionary relationships between taxonomic groups constructing using SSU sequence data or sometimes all available data. Can be constructed based on different criteria. The universal Tree of Life is based on molecular phylogeny where a comparison of molecular features meaning DNA sequence rather than morphological traits. DNA sequences are used more but morphological data is still an important piece used in making predictions about evolutionary relationships. The SSU gene serves as a marker for comparison. This gene is an effectively universal presence and a convenient size of about 1,500 nucleotides. It contains regions of relatively hugh variability interspersed amongst other highly conserved regions. Comparison of the hypervariable region is useful for identification since these regions typically contain nucleotide sequences that are unique to a particular species. When working with bacterial dna we use the 16s rrna Gene for analysis.

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Biochemical Test: Oxidase

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The oxidase test identifies the presence or absence of cytochrome C oxidase. Chromogenic reducing agents are chemicals that develop color as they become oxidized. In an oxidase test the reducing agent is added directly to bacterial growth on solid media and a bacterial colony is transferred to paper saturated with reagent. If the reducing agent is oxidized a color change will occur indicating that cytochrome c oxidase is present.

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Biochemical Test: Starch Hydrolysis

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This test differentiates between bacteria that hydrolyze starch in bacteria that do not. Bacteria are grown on starch agar and when colonies become visible the plate is flooded with iodine which turns blue when it complexes with starch. Without the presence of starch the medium surrounding the colonies will appear red.

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Biochemical Test: Gelatin Hydrolysis

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Some microorganisms secrete an enzyme to hydrolyze gelatin. To detect gelatinases we use nutrient gelatin. The gelatin is inoculated with bacteria. If the bacteria hydrolyzes gelatin, the gel will be liquid below 28 degrees celcius.

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Biochemical Test: Lipid Hydrolysis

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Tributyrin Agar is used for media and the plate is inoculated with bacteria. If the bacteria is lipase positive, clear zones will appear on the growth as evidence of lypolytic activity.

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Antibiotic Resistance

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An antibiotic is a molecule that enters a cell and interacts with a target molecule. Antibiotics that bind to the prokaryotic ribosome have an inhibitory effect on protein synthesis. Any cellular change that prevents the antibiotic from binding or reaching its target confers resistance to the organism. There are two main mechanisms that enable resistance to be acquired.

Spontaneous mutations that arise through error in DNA replication can lead to resistance if the mutation alters the structure of an antibiotic target.

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Transposon Mutagenesis

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Allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Virulence genes in viruses and bacteria can be discovered by disrupting genes and observing for a change in phenotype. This has importance in antibiotic production and disease control. Non-essential genes can be discovered by inducing transposon mutagenesis in an organism. The transformed genes can then be identified by performing PCR on the organism's recovered genome using an ORF-specific primer and a transposon-specific primer. Since transposons can incorporate themselves into non-coding regions of DNA, the ORF-specific primer ensures that the transposon interrupted a gene. Because the organism survived after homologous integration, the interrupted gene was clearly non-essential.

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Replica Plating

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The transfer of cultures from one media to a new media that may be selective or differential.

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Selection vs Screening

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These methods allow us to separate bioactive compounds from other molecules present in a culture which is the first step in determining that compounds physical and chemical properties, mode of action and activity spectrum. Selective media supports the growth of some species but not others. When a screening is done two types of bacteria can grow but can be distinguished phenotypically.

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PBAM1 Vector

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Incomplete

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Efficiency of Conjugation

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Incomplete

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Serial Dilutions

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Used to determine microbial counts for liquid and solid samples. Help microbiologists calculate cfu to give approximate number of viable cells per milliliter or gram of sample. Sample is diluted with water or saline to keep cells in suspension alive. Reduces number of cells per volume to a proportion that is easier to work with and count. Once we have reached the desired dilution for a sample we can add it back to a solid media that supports the growth of bacteria.

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Horizontal Gene Transfer

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This method of acquiring antibiotic resistance involves acquisition of a fragment of DNA harboring genes that confer resistance. Bacteria can acquire DNA from other cells or viruses through HGT. Genes acquired through this method include pumps that expel antibiotics from the cell or enzymes that chemically modify an antibiotic so it no longer functions.