
<p><strong>Chapter 10: </strong><br><strong>Classification of Microorganisms </strong></p><p><strong>1. The Taxonomic Hierarchy 2. Methods of Identification </strong></p><p><strong>1. The Taxonomic Hierarchy </strong></p><p><strong>Phylogenetic Tree of the 3 Domains </strong></p><p><strong>Taxonomic </strong><br><strong>Hierarchy </strong></p><p>• <strong>8 successive taxa are used to classify each species: </strong></p><p><strong>Domain </strong><br><strong>Kingdom </strong><br><strong>Phylum </strong><br><strong>Class </strong></p><p><strong>Order </strong><br><strong>Family </strong><br><strong>Genus </strong><br><strong>Species </strong><br><strong>**species can also contain different strains** </strong></p><p><strong>Scientific Nomenclature </strong></p><p><strong>To avoid confusion, every type of organism must be referred to in a consistent way. </strong></p><p><strong>The current system of nomenclature (naming) has been in use since the 18</strong><sup style="top: -0.75em;"><strong>th </strong></sup><strong>century: </strong></p><p>• <strong>every type of organism is referred by its genus name followed by its specific epithet (i.e., species name) </strong></p><p><strong>Homo sapiens (H. sapiens) Escherichia coli (E. coli) </strong></p><p>• <strong>name should be in italics and only the genus is capitalized which can also be abbreviated </strong></p><p>• <strong>names are Latin (or “Latinized” Greek) with the genus being a noun and the specific epithet an adjective </strong></p><p><strong>**strain info can be listed after the specific epithet (e.g., E. coli DH5α)** </strong></p><p><strong>2. Methods of Identification </strong><br><strong>Biochemical Testing </strong></p><p><strong>In addition to morphological (i.e., appearance under the microscope) and differential staining characteristics, microorganisms can also be identified by their biochemical “signatures”: </strong></p><p>• <strong>the nutrient requirements and metabolic “by-products” of of a particular microorganism </strong></p><p>• <strong>different growth media can be used to test the physiological characteristics of a microorganism </strong></p><p>• <strong>e.g., medium with lactose only as energy source </strong>• <strong>e.g., medium that reveals H</strong><sub style="top: 0.5001em;"><strong>2</strong></sub><strong>S production </strong></p><p><strong>**appearance on test medium reveals + or – result!** </strong></p><p><strong>Commercial devices for rapid </strong><br><strong>Identification </strong></p><p><strong>Perform multiple tests simultaneously </strong></p><p><strong>Enterotube II </strong></p><p><strong>Such devices involve the simultaneous inoculation of various test media: </strong></p><p>• <strong>~24 hrs later the panel of results reveals ID of organism! </strong></p><p><strong>Use of Dichotomous Keys </strong></p><p><strong>Series of “yes/no” biochemical tests to ID organism. </strong></p><p>• <strong>tests done in a logical order, each test result indicates next test to be done </strong></p><p>• <strong>collective results of multiple tests create a profile allowing ID of microorganism </strong></p><p><strong>Serology (i.e., antibodies) </strong></p><p><strong>Specific antibodies can be used to ID bacteria: </strong></p><p>• <strong>antibodies are produced by animals to anything “foreign” </strong>• <strong>animals (rabbits, goats…) are routinely injected with biological material for which antibodies are needed </strong></p><p>• <strong>antibodies present in the animal serum can then be used in various ID tests: </strong></p><p><strong>e.g., the agglutination test differences in antibody reactivity can reveal different bacterial strains or serovars </strong></p><p><strong>Phage (virus) Typing </strong></p><p><strong>Bacteriophages (viruses that infect bacteria) have very specific hosts and can be use to ID bacteria: </strong></p><p>• <strong>grow a “lawn” of bacteria to be tested on agar plate </strong></p><p>• <strong>“dot” different test phage samples on surface </strong></p><p>• <strong>after ~24 hr, clear zones appear where bacteria have been infected & killed </strong></p><p>• <strong>profile of phage sensitivity can reveal ID of bacteria </strong></p><p><strong>DNA Base Composition </strong></p><p><strong>Members of the same genera or species have nearly identical DNA sequences, and hence the same proportions of G/C base pairs & A/T base pairs: </strong></p><p>• <strong>because they base pair, G = C and A = T </strong>• <strong>G/C + A/T = 100% (e.g., if G/C = 40% then A/T = 60%) </strong></p><p><strong>Determining the G/C content of the DNA from a test organism and comparing to known values is a quick way to eliminate possible identities: </strong></p><p>• <strong>if %G/C is different, cannot be a match! </strong>• <strong>if %G/C is same, might be a match but additional testing is necessary to confirm </strong></p><p><strong>The Use of DNA Hybridization </strong></p><p><strong>With enough heat, DNA strands will separate. Cooling allows complementary strands to base pair. </strong></p><p>• <strong>this technique is used in a variety of ways to see if DNA from two different sources are similar </strong></p><p>• <strong>usually the DNA from one source is immobilized, the other is labeled to allow detection </strong></p><p><strong>“FISH” </strong></p><p><strong>Fluorescent in situ hybridization: </strong></p><p><strong>1) label DNA “probe” (fr. species of interest) w/fluorescent tag 2) chemically treat cells to allow DNA to enter, hybridize 3) wash & view with fluorescence microscopy </strong></p><p><strong>**cells w/DNA complementary to probe will fluoresce!** </strong></p><p><strong>PCR </strong></p><p><strong>Polymerase Chain Reaction </strong></p><p>• <strong>selectively amplifies only desired DNA (if present) </strong></p><p>• <strong>e.g., DNA from suspected pathogen </strong></p><p><strong>PCR is a technique that involves manipulating </strong><br><strong>DNA replication in vitro… </strong></p><p><strong>Overview of PCR Technique </strong></p><p><strong>Every PCR reaction requires the following: </strong></p><p>• <strong>DNA source to be tested (or amplified) </strong>• <strong>artificial primers specific for DNA of interest </strong>• <strong>heat-stable DNA polymerase </strong>• <strong>free nucleotides (dNTP’s) </strong></p><p><strong>Plus an automated thermocycler to facilitate repeated cycles of: </strong></p><p><strong>1) denaturation of DNA (separation of strands) @ ~95</strong><sup style="top: -0.585em;"><strong>o </strong></sup><strong>C 2) hybridization of primers to template @ ~50</strong><sup style="top: -0.585em;"><strong>-</strong></sup><strong>60</strong><sup style="top: -0.585em;"><strong>o </strong></sup><strong>C 3) DNA synthesis @ ~72</strong><sup style="top: -0.585em;"><strong>o </strong></sup><strong>C </strong></p><p><strong>Ribosomal RNA (rRNA) Comparison </strong></p><p><strong>Prokaryotic ribosomes contain 3 different rRNA mol.: </strong></p><p>• <strong>large subunit contains 23S (2900 nt) & 5S (120 nt) rRNA </strong>• <strong>small subunit contains 16S (1500 nt) </strong></p><p><strong>16S rRNA sequence is typically used for ribotyping: </strong></p><p>• <strong>sequence is highly conserved (varies little) </strong>• <strong>degree of difference reflects “evolutionary distance” </strong></p><p><strong>**primary method for classifying prokaryotic species** </strong></p><p><strong>Key Terms for Chapter 10 </strong></p><p>• <strong>dichotomous key </strong>• <strong>serology </strong>• <strong>phage typing </strong>• <strong>hybridization </strong>• <strong>FISH </strong>• <strong>PCR, thermocycler </strong>• <strong>ribotyping </strong></p><p><strong>Relevant Chapter Questions </strong></p><p><strong>rvw: 4-10, 13, 14 MC: 2-8 </strong></p><p><strong>Chapter 3: Microscopy </strong></p><p><strong>1. Types of Microscopy 2. Staining </strong></p><p><strong>1. Types of Microscopya </strong><br><strong>Scale of Magnification </strong></p><p><strong>Light microscopy </strong></p><p>• <strong>limit of resolution* ~0.2 μm </strong>• <strong>sufficient to see most organelles, bacteria </strong></p><p><strong>Electron microscopy (TEM & SEM): </strong></p><p>• <strong>limit of resolution ~2.5-20 nm </strong>• <strong>sufficient to see subcellular detail, large molecular complexes </strong></p><p><strong>*resolution = ability to distinguish objects close to each other </strong></p><p><strong>Light Microscopy </strong></p><p><strong>Most common type is the Compound Light Microscope: </strong></p><p><strong>3</strong></p><p><strong>1) condenser lens focuses light source on sample </strong></p><p><strong>2</strong></p><p><strong>2) objective lens magnifies the image </strong></p><p><strong>1</strong></p><p><strong>3) ocular lens further magnifies image </strong></p><p><strong>Oil Immersion & Light Refraction </strong></p><p><strong>Different media (air, water, glass, oil…) bend light to different degrees. </strong></p><p>• <strong>i.e., have different refractive indexes </strong></p><p>• <strong>the oil immersion lens is too small to capture all light refracted by air </strong></p><p>• <strong>immersion oil has refraction index similar to glass, allows more light to enter the lens </strong></p><p><strong>Bright & Dark Field Microscopy </strong></p><p><strong>Bright Field Microscopy Dark Field Microscopy </strong></p><p></p><ul style="display: flex;"><li style="flex:1">• <strong>standard or “default” </strong></li><li style="flex:1">• <strong>barrier in condenser </strong></li></ul><p></p><ul style="display: flex;"><li style="flex:1"><strong>type of light microscopy </strong></li><li style="flex:1"><strong>eliminates all direct light </strong></li></ul><p>• <strong>only light reflected by specimen enters the objective lens </strong></p><p><strong>Phase Contrast & DIC Microscopy </strong></p><p><strong>Phase-Contrast </strong><br><strong>Microscopy </strong><br><strong>Differential Interference </strong><br><strong>Contrast (DIC) Microscopy </strong></p><p></p><ul style="display: flex;"><li style="flex:1">• <strong>provides internal detail, </strong></li><li style="flex:1">• <strong>variation on phase-contrast </strong></li></ul><p></p><p><strong>contrast, w/o staining </strong></p><p><strong>with a 2</strong><sup style="top: -0.585em;"><strong>nd </strong></sup><strong>light source </strong></p><ul style="display: flex;"><li style="flex:1">• <strong>useful for live specimens </strong></li><li style="flex:1">• <strong>greater detail, contrast </strong></li></ul><p></p><p><strong>Fluorescence Microscopy </strong></p><p><strong>Fluorescent dyes or antibodies with a fluorescent tag stick to specific targets. </strong></p><p><strong>Under UV light, dye fluoresces, only labeled cells or structures are seen. </strong></p><p><strong>standard </strong></p><p><strong>confocal </strong></p><p><strong>Confocal Microscopy </strong></p><p><strong>Only light from a given depth or plane is transmitted, </strong><br><strong>“out of focus” light excluded </strong></p><p><strong>Electron Microscopy </strong></p><p><strong>Electromagnetic lenses focus electron beam onto metal-stained specimen. </strong></p><p>• <strong>electron beams have very short wavelengths </strong></p><p>• <strong>allows far greater resolution than with light microscopy </strong></p><p><strong>Transmission EM (TEM) </strong></p><p>• <strong>thin sections of specimen, highest resolution </strong></p><p><strong>Scanning EM (SEM) </strong></p><p>• <strong>reveals surface features </strong></p><p><strong>2. Staining </strong></p>
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