MIMG101 Discussion Week 2 PDF

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MIMG 101 with Professor Peter Bradley and Professor Robert Gunsalus...

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Different Cellular Structures: - Prokaryotic Cell- smaller, lack membrane-bound organelles, lack nucleus (DNA is in a nucleoid), recently discovered: cytoskeleton-like structures, but not like those in eukaryotes - Eukaryotic Cell- larger, ** Know differences between four microscopes Bright Field Microscopy: - What is the max magnification of a traditional light microscope? 1000X What is the max resolution? 0.2 micrometer What is the advantage of using oil immersion? At greater than 100X magnification, the oil aids in gathering the light rays emitted from the specimen at higher angles. Can live cells be stained with cationic dyes like crystal violet and methylene blue? Cells cannot be alive- cells dead so that catonic dyes can penetrate through membranes and wall Objective lens x ocular lens= total magnification - Ocular lens always 10X Gram Stain - Step 1: begin with heat fixed cells - Step 2: flood slide with crystal violet dye for 1 min. - Step 3: add iodine solution for 1 min.- can be decolorized in gram negative cells but not in gram positive cells - Step 4: Wash slide with alcohol for 20 sec - Step 5: Counter stains with safranin - the gram negative cells will hold this other color since they were decolorized Why do Gram positive bacteria stain purple while Gram negative bacteria are decolorized? Because gram positive bacteria have thicker cell walls (peptidoglycan) ; the crystal violet and iodine bind to form complex, harder for them to go through cell wall, when negative, thinner wall so easier for them to go through cell wall Phase Contrast Microscopy - Undefracted light undergoes a -90 degree shift (¼ wavelength) which heightens the contrast and improves the image. This technology enables better visualization of the cell Dark Field Microscopy - In dark field microscopy, it is the refracted rays, not the unrefracted rays, that reach the objective. Hence the background appears dark (where there’s nothing to refract). Like phase contrast, this is a technique that allows heightened image contrast better visualization of live cells Differential Interference Contrast - Polarized light passes through a prism, giving a 3D like appearance - Internal structures- nuclei, vacuoles - Unstained cells Fluorescence microscopy

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Know the light path 1) first barrier filter: lets through only blue light with a wavelength between 450 and 490 nm - 2) beam splitting mirror: reflects light below 510 nm but transmits light above 510 nm - 3) second barrier filter: cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560 nm Under what circumstances would a researcher use GFP fusion proteins instead of a immunofluorescence assay? It will always be dead for immunofluorescence assay and GFP fusion proteins don’t need to be dead- GFP fusion proteins can be tracked in live cells. While it is an added step to transform a cell to express GFP fusion proteins, the researcher can track real time localization of the target of interest in a living cell. Immunofluorescence of structures or proteins inside the cell requires that the cell be killed and fixed so the antibody can enter the cell Why would a researcher choose an indirect immunofluorescence assay over direct? What are some advantages? Low budget, it can be cheaper, as conjugated antibodies against common antigens like the constant region of another antibody can be purchased in bulk, and, as long as the researchers produce their primary antibody in the animal against which the secondary antibody targets, they can just add the secondary antibody without having to conjugate more antibody to the fluorophore. Sensitivity: also the indirect assay can amplify the fluorescence signal, as many secondary antibodies can bind Confocal microscopy - What is the basic principle of confocal microscopy? - capturing multiple 2 dimensional images at different depths in a sample enable the reconstruction of 3D structured within an object Electron Microscopy (EM) - Three types: - Transmission EM- can see internal structures of cells, slices through structures, limited to ‘flat’ sliced image, have to fix/stain sample- dead cells (osmic acid or lead) - Scanning EM- 3d structures, used for revealing external features, specimen treated with heavy metal (gold), external structures of cells, only takes samples’ topography (outsides), have to fix/stain cells (dead) - Cryo electron microscopy- 2D shadows taken from particles, computer creates composite, 3D image, viruses, proteins, images have a lot of noise, must image many targets since shadows are different at different angles, don’t need to stain/fix samples - How does it differ from light micropscopy? - Uses electrons, not light - Greater resolution Cryo ET - In cryo em: - The sample is flash frozen in many configurations - The shadows cast by the electron beam are reconstructed into a 3d image

Microbial DIversity - They must adapt to many different environments RNA hypothesis: - Life started with self replicating RNA - Taken up in lipoprotein vacuoles Methods for classifying microbial diversity - Morph diversity - Metabolic diversity- categorized by their carbon sources, energy sources, electron source - Ecological diversity- temperature, salinity, ph, pressure - Genetic diversity- comparison of genes from different microbes, what features the “molecular clock” gene should have, Universally distributed, functionally homologous, slow to change, ideal size for sequencing/ analyzing- use 16S rRNA - why? All organisms have it, highly conservative, sufficient nucleotides (1500 nt) to see changes throughout evolution compared with 5S RNA (120nt) yet easier for sequencing, association coefficients Ecological diversity - Psychrophiles: extreme cold environments, membranes-, proteins- Halophiles- high saline environment; cells pump high K+ into cytoplasm, many enzymes require high K+, cells actually require salt for cell wall stability, cell wall glycoprotein stabilized by Na+

2 distinct Taxa for prokaryotes - Other support 3 domain of life - RNA polymerase - bacteria : 4 subunits - EUkarya: 10-12 subunits - SDS Page analysis - Inhibitors of translation - sensitivities to inhibitors - Membrane structures (difference in archaea) Phytanyl side chain found only in archaea Ether linkage in archaea and ester linkage in bacteria and eukaryotes Phylogeny Horizontal Gene Transfer - Movement of genetic material between unicellular and/or multicellular organisms other than by the (“vertical”) transmission of DNA from parent to offspring Classification

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Grouping of simmilar organisms Hierarchial subgrouping Linnaeus - domain-kingdom-phylum-class-order-family-Genus-Species Binomial System of taxonomic classification: Genus species...