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59 Cards in this Set
- Front
- Back
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Taxonomy |
the science of the classification of organisms |
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Taxa |
taxonomic categories = subdivisions used to classify organisms - usually based on similarities |
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Phylogeny |
The study of the evolutionary history of organisms |
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Early Taxonomy 2 kingdoms |
Plants and Animals |
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Early Taxonomy 3 kingdoms |
Plants, Animals, Protists (bacteria, protozoa, fungi, and algae) |
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Early Taxonmy 5 kingdoms |
Plants, Animals, Prokaryotes, Fungi, Protozoa |
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Domains |
3 diiferent cell types based on ribosomal characterisitcs Proposed that these 3 cell types can be placed on a level above kingdom |
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3 Domains |
Eukarya, Bacteria, Archeae |
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Domain Eukarya |
Includes kingdoms: Animals, Plants, Fungi, Protozoa |
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Domain BActeria |
Includes prokartoytes such as pathogens, non pathogens, and miscellaneous other bacteria |
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Domain Archeae |
Includes prokaryotes that do not have peptidoglycan in their cell walls Often live in extreme environments and carry out unsually metbolic processes Halophiles, Thermophiles, Methanogens |
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Binomial Nomenclature |
Every Organism is assigned 2 names Genus / specific epithet (species) |
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Taxnomic Hierarchy |
Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species |
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What taxonomic hierarchy is the least specific? |
Domain |
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Classification of Prokaryotes |
Bergey's Manual of Systematic Bacteriology Bergey''s Manual of Determinative Bacteriology
2 domains: bacteria and archeae |
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Classification of Eukaryotes |
Kingdom Protista Kingdom Fungi Kingdom Plantae Kingdom Animal |
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Kingdom Protista |
Simple Eukaryotic orgnaims, mostly unicellular, approx 200,000 species |
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Kingdom Fungi |
More complex eukaryotes, including molds, yeasts and mushrooms |
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Kingdom Plantae |
includes some algae, mosses, ferns, conifers, flowering plants |
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Kingdom Animalia |
includes sponges, worms, insects, vertebrates |
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Practical reason for identifying organisms |
what is organism we are dealing with so we can treat it |
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Research reason for identifying organisms |
classfication |
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Morphology characteristics |
Shape Presences of flagella Presence of a capsule or slime layer Arrangement of the organisms |
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Differential Staining |
One primary characteristic in identifying microorganisms Classification fro cell wall type Combination of Gram stain reaction and morphology and source of sample may give presumptive identification |
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Bichemical tests |
Metabolic activity of organisms may provide cues. |
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Examples of biochemical tests |
Fermentation of different sugars Production of various enzymes - oxidase, catalase, coagulase, API strips |
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Dichotomous key |
Ferment Lactose? Citric Acid as carbon source? |
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Cannot Ferment Lactose cannot use citric Acid |
Shigella |
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CAnnot Ferment lactose Can use Citric Acid |
Salmonella |
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Can Ferment Lactose Cannot Use Citric Acid |
Escherichia |
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Can Ferment Lactose Can use Citirc Acid Cannot produce acetoin |
Citrobacter |
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Can Ferment Lactose Can use Citric Acid Produces Acetoin |
Enterobacter |
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Serology |
Antigen-antibody complexes Differentiates strains by serotype or biovars If you have a known antibody you can test it on an unknown organism, if a complex forms then they can match. Often use materials such as latex, charcoal, ELISA or red blood cells to visualize the complex through agglutination Slide Agglutination test |
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Other methods for identification and classfication |
Phage typing Fatty acid profies Flow cytometry |
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Phage typing |
Highly specific viruses that infect and lyse specific bacteria |
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Molecular Diagnostics and subtyping |
Monitor disease more accurately Allows earlier detection and treatment Reduces unnecessary treatment Allows discrimination of organims below species level Recognizes outbreaks and monitors reoccurence |
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Molecular Subtyping Capabilities |
PCR Real Time PCR Microspher Array PFGE Gene Senquencing Fragment Analysis - VNTR |
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Polymerase Chain Reaction |
Extraction, denaturing, annealing, and extension of DNA Uses primers to amplify area of interest Exponential growth of desired are occurs the extension stage Faster and more senstiive than culture 18 hr to 21 day culture vs 8 hour PCR Amplicon detected using gel matrix Basis for most molecular techniques
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Current Assays |
Basis for Microsphere assay Basis for sequencing |
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PCR Step One |
Step One: Extract DNA form Sample Sample type is critical Multiple Extraction types |
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Master Mix |
cocktail for reagents required to perform a PCR reaction Does not include DNA Master Mix is made for multiple samples then aliquoted into tubes 1 tube per sample DNA from each sample is addes to corresponding tube |
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Components of Master Mix |
Water Forward Primer Reverse Primer MgCl2 Taq Polymerase dNTPs (A,T,G,C) |
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Basic PCR Step One |
Denature The double stranded DNA is heated to approx 94C so that the strands will separate |
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Basic PCR Step Two |
Annealing: The temperature is cooled so that the specific primers can anneal to the complementary DNA sequence |
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Basic PCR Step Three |
Extension: Taq Polymerase adds the bases needed to complete the complementary strand |
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Basic PCR repeat |
Repeats approx 35 times Exponential amplification 2^36 - 68 billion copies |
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Performing PCR on viruses |
Some viruses have RNA instead of DNA RNA must converted into DNA
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Reverse Transcriptase |
RNA uses this to make a complementary strand of DNA which can then be copied in a PCR reaction |
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End Result of PCR |
Run a gel to visualize PCR product Use product in another assay Use Real-Time PCR to determine results |
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Real Time PCR |
Flourochromes Flouresence probes are used to detect amplicon (FAM, TAMRA, SYBR Green) PCR product detected by lasers during annealing or extension stage Results within 30 mintues to 2 hours after extraction Hnad hel or transportable models available Faster and more sensitive than gel based PCR |
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Real Time PCR Applications |
Multiplexing-Up to 4 assays can occur simulatenously in well or tube Viral screening and subtyping Potential bioterrorism agents identified from environmental and clinical samples US Postal Service BIological detection system |
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Biological Detection System |
Northrop Grumman/Cepheid Automated air sampling real-time PCR Monitors B Antrancis Y Pestis, F, tularensis, & Brucella in develpment Began in 2002 National network notification LRN lab confirms postivie samples |
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Microsphere Assay |
Flow Cytometry PCR or ELISA based Antigen coated beads Red and green laser dectection of 100 targets/well Next generation will allow detection up to 1000 targets/well |
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Bioterrosism Screening |
Microsphere Assay BioWatch LRN Screens B antrhacis, Y pesitis, and F tularensis, SEB and Ricin toxin |
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RVP |
17 respiratory viruses Results <8h |
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Salmonella Serology |
Detects approxiamtely 95% of common serotypes |
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Pulsed-Fie Gel Electrophoresis and PulseNet |
PFGE 1984 CDC established Pulse Net after EColi outbreak in 1996 |
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PUlse Net |
All 50 states, Canada, FDA, USDA members Organisms are embeeded in gell matrix, lysed and restriction enzyme applied to generate a banding pattern Electrical pushes DNA through gel in a zig zag patter Run 17-24 hours |
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Sequencing Applications |
Pathogen identification Strain typing Viral genotyping Antimicrobial susceptibility Partial or whole genome sequencing. |
