Micro bio session 3

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nhi
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Micro bio session 3
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2011-02-06 20:45:25
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micro bio session 3, Lisa Rabbe
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  1. Explain the principles underlying brightfield microscope. Give ex. of us of each.
    • brightfield: need some sort of staining
    • resolving power: ability to see 2 pts as separate instead of one, measure of how precise the microscope is.
    • good one: 0.2 microns
    • oil drop: refracts light, decreases scatter
    • ex. use of normal classroom
  2. Explain the principles underlying darkfield microscope. Give ex. of us of each.
    • uses condenser that scatter light
    • organism is illuminated against a dark background
    • use for thin organisms like treponema: spirochete (for syphilis)
    • increases resolving power: 0.02 microns
    • disadvantage: can't see internal structure. light goes around it instead of through it.
    • useful for observation of thinner structures such as flagella or microbes not visible in bright-field
  3. Explain the principles underlying phase contrast microscope. Give ex. of us of each.
    • use special objective. light to bounce out of phase. THings that are denser, can see internal structures
    • use on live specimens
    • can see cilia and flagella better
    • no need staining but it looks colored
    • improves contrast between cells andmedium
    • useful for visualizing cells non-fluorescent cells w/o killing them
  4. Explain the principles underlying fluorescent microscope. Give ex. of us of each.
    • emission of light of characteristic wavelength
    • expresses fluorescent protein (GFP)
  5. Explain the principles underlying electron microscopy microscope. Give ex. of us of each.
    • not a form of light microscopy
    • necessary for visualization of viruses, detailed cellular structures
    • only a thin section of a non-living cell can be viewed
    • TEM: ectron beam aim specimen through it. distinguish dense or non-dense area
    • SEM: 3 D image.
  6. Give examples of direct microscopic examinations.
    • simplest
    • sample suspended in water or saline (wet mount)
    • dyes non specifically stain the cellular material, increasing the contrast with background, and permit examination of deatiled structures
    • India ink method, KOH method, iodine
  7. Give examples of differential stains.
    • gram stain: + dark blue, - pink
    • acid-fast: for TB stain pink, counterstain purple if not
    • Fluorescent stains: make Ab specific to some bug and conjugate a fluorescent stain to it
  8. Define the four types of culture media: enriched (nonselective), selective, differential and specialized.
    • enriched: support most non-fastidious (fussy feeders) bugs
    • selective: some grow and some don't
    • differential: 1. most things grow but they look different
    • 2. ID by looks: if it grows, it might turn different color
    • 3. ex. hemolysis of blood agar. a. Alpha hemolytic "green" - partially break down red blood cell. b. beta hemolytic - complete clear zone because blood has been completely lysed c. gamma hemolysis - non, it cannot break down red bloo cells
    • specialized: bugs that you are growing have special growing characteristics. bugs that require specialized media are: rickettsia, chlamydia, unique because they are obligate intracellular parasite: has to live in cells
  9. What factors influence success of a culture?
    • a. depends on biology of organism. Some extremophiles. metabolic reference.
    • b. site of infection. i. hard to get pure culture from mouth. how likely is it to get infection of culture?
    • c. immune response made by patient. infection of heart valves. Bacteria maybe have made a biofilm.
    • d. use the right media. growth conditions
  10. Describe how researchers identify and differentiate between different strains of bacteria (PCR, RFLP, DNA hybridization and in situ hybridization).
    • a. PCR(polymerase chain reaction): copy machine for DNA
    • i. Looking at copies DNA
    • ii. Use bacteria to do the copying (thermal philes)
    • b. RFLP (restriction fragment length polymorphism)
    • i. Cut DNA and run in gel and get “fingerprint”
    • ii. Multidrug resistant bacteria that looks different in RFLP that is spreading around
    • c. DNA hybridization:
    • i. Look for DNA (bacterial/viral) in a sample
    • ii. Common place done is to screen big sample for presence of certain pathogen
    • d. In situ hybridization
    • i. Using DNA probes to look for DNA in tissue sample
    • Ex. Screen for virus in tumor
  11. What is the theory behind serological diagnosis? HINT: what are you detecting?
    • a. Serology: looking for antibody
    • i. Ex. HIV testing
    • ii. Done with monoclonal antibodies
    • 1. Inject Ag into animal and then they will make Ab
    • 2. Collect Ab in serum. Collect B-cells but B cells die in culture.
    • 3. Vs. this way:
    • a. Inject mouse with Ag
    • b. Collect B cells
    • c. Fuse them with tumor cells (put soap on them)
    • d. Screen fused B cells to look for Ab production and immortality
    • e. When found, make clone of them and sale
  12. Review precipitation and agglutination tests.
    • Precipitation: specific ag-ab complexes and cross-reactivity can be distinguished by immunoprecipitation techniques immune complex falls out of solution when antigen is a molecule
    • Agglutination tests: immune complex falls out of solution when Ag is a cell/cell-sized particle.
  13. Be familiar with manufacture and use of monoclonal antibodies. Give examples of antibody use including FLOW cytometry, ELISA, Western blot, RIA.
    • a. FLOW cytometry: cell "sorting"
    • i. Base them on the colors that they have
    • b. ELISA
    • i. Assy where you can use Ab to look for Ag that could be virus
    • ii. Put viral protein in well, add patient serum
    • 1. If pt has Ab, it will bind
    • 2. Wash it away: if theyb ound, it statys there if not, it gets wash awaya
    • 3. Put anti human Ig Ab (fluorescently labeled) that binds to the pt serum
    • 4. If +, you do another test call Western blot
    • iv. Problem: cross reactivity
    • c. Western blot
    • i. Looking for protein
    • ii. Variant in HIV test
    • 1. Run on a gel several proteins from HIV virus
    • 2. Lab takes this paper with the protein on them, add pt Abs to it. If 3 bands binds, you have HIV because those 3 proteins are very specific to HIV
    • d. RIA
    • i. Involves radiation and Ab
    • ii. Blood test for allergy: RAST. Stop using it because radiation (dangerous)
    • iii. More sensitive than ELISA
  14. Differentiate between a microbiological and immunological/ molecular microbiolocial approach to pathogen identification. Describe the role of pathognomonic symptoms in diagnosis.
    • Micro (5 I): Innoculate, incubate, isolate, inspect, identify
    • Immunological/ molecular micro: i. Ab titer or just look at DNA or just look at DNA or just the AB of it. ii. Use ELISA
    • pathignomonic synptoms: indicates a given disease
    • i. Ex. Koplic spots: measles
    • ii. Tetanus: risus sardonicus (pull on face muscles)
    • ii. Diptheria: pseudomembrane on tonsils
    • 1. Block the throat, highly vascular so don't cut it
  15. Review the major steps for microbiology: collecting sample, types of media, blood culturing, aerobic/anaerobic culture conditions.
    • samples obtained from blood, urine, feces, sputum, pus or a specific area of body such as oral cavity through swab and streak onto a surface of agar plate. (do not want contamination.)
    • Different types of media used to grow clinical samples
    • i. general purpose media - support growth of most aerobic and facultatively anaerobic organisms. (blood agar)
    • ii. enriched media - select for particular organisms that will have a growth advantage on that particular medium
    • iii. selective media - ehances for the growth of certain organisms while retarding the growth of others ( antibiotic)
    • iv. differential media - base on visible appearnace or biochemical property
    • blood samples: aseptically removing blood with 1 bottle incubated aerobically and other incubated anaerobically at 35 C
    • Aerobic/anaerobic culture: needed since agents of disease could be an obligate anaerobes
  16. Define idiopathic and iatrogenic diseases.
    • idiopathic: don't know the cause
    • iatrogenic: disease that came from health care procedure or setting
  17. Define bacteremia, fungemia, and septicemia.
    • Bacteremia: bacteria in the blood
    • fungemia: fungi in blood
    • septicemia: fungi or bacteria in blood. blood infection.
  18. Define meningitis and meninges as well as the organisms that cause disease.
    • meningitis: swelling of meninges. can cause either by bacteria or virus
    • meninges: 3 membranes - dura, mater, arachnid
  19. Describe MIC.
    • MIC - minimal inhibitory concentration : test for antibiotic susceptibility
    • i. Purchase tubes that has different dilutions of antibiotic.
    • ii. Inoculate with bacteria in tubes and look for growth.
    • 1. look for growth thru precipitation, turbidity, or any change
    • iii. Smallest amount of antibiotic dilution that can inhibit the bacteria
    • b. anitmicrobial susceptibility testing: use for selecting chemotherapeutic agents active against the infecting organism

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