Chapter 10: Classification of Microorganisms

Chapter 10: Classification of Microorganisms

<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 &amp; 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 &amp; 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 &amp; 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) &amp; 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&nbsp;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 &amp; 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 &amp; 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 &amp; Dark Field Microscopy </strong></p><p><strong>Bright Field Microscopy&nbsp;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 &amp; 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>

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    36 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us