Micro bio session 3
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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
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
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
Explain the principles underlying fluorescent microscope. Give ex. of us of each.
- emission of light of characteristic wavelength
- expresses fluorescent protein (GFP)
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.
Give examples of direct microscopic examinations.
- 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
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
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
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
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
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
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.
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
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
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
Define idiopathic and iatrogenic diseases.
- idiopathic: don't know the cause
- iatrogenic: disease that came from health care procedure or setting
Define bacteremia, fungemia, and septicemia.
- Bacteremia: bacteria in the blood
- fungemia: fungi in blood
- septicemia: fungi or bacteria in blood. blood infection.
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
- 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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