Lecture 6, 9/12 - Microscopy

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Here is the recording for today's lecture: Microlecture912

• Keep in mind a topic to write a paper about. Also he will not be here the 26th and 27th of Sept.

• • Epstein Bar Virus - mononucleosis, burketts lymphoma (found in Africa)
• • Microscopy – different types of microscopes
• Bright field
• • We use it downstairs
• Dark field
• • Cost 3000$ (special condenser that increases price)
• • Detects spirochetes, syphilis, Borrelia burgdorferi
• • Resolving power (RP) ≈ .2um [be able to convert to other units of measurement]
• Ultra violet microscopy
• • Has about half the wave-length of visible light (250nm)
• • RP = 0.1 allows you to see more than BF. As resolving power decreases your ability to resolve increases.
• • Cost $10,000 – lots of special components
• • Fluorescent antibody method
• • Immunofluorescence – used to diagnose. An antigen could be a bacteria or a virus. For instance you can use to determine if something has a rabies virus.
• • NEGRI – pockets of fluorescence indicate presence of antibodies – if it does than the thing has rabies
• Phase contrast microscopy
• • It increases contrast of what you are looking at.
• • Primary use: used to see intracellular structures in eukaryotic cells. You can see things such as mitochondria or yeast
• • Research tool, not diagnostics
• Transmission Electron Microscopy (TEM) – (huge)
• • Electrons have to go through the specimen
• • Hard to use. Operate at high voltage.
• • Cost $100,000 – expensive components and service
• • RP=10ang. You can see molecules at 1ang
• • To stain organisms you need heavy metals (platinum, palladium)
• • You have to have very thin specimens in order for the electrons to go through
• • Has high pressure vacuum pumps
• • Key characteristic: you can see inside of what you are looking at.
• • Electromagnet is used as an illumination source.
• Scanning electron microscopy (SEM)
• • You can get 3 dimensions because it looks at the surface
• • Used for topography
• • Uses electron beam
• • Cost $50,000 to high end of $200,000
• • Stain with gold
• • RP about 100ang
• • Smaller than TEM, could fit on desk
• • Electromagnet is used as an illumination source.
• • REVIEW: Make table that contrasts table between BF, TEM, SEM
• o Cost (see notes above)
• o Resolving power (see notes above)
• o Magnification – BF=1,000x, TEM=50,000x – 1,000,000x (through photography), SEM=similar to TEM but you don’t need as much magnification.
• o Specimen – BF=both living and dead, TEM+SEM=dead
• o Color – BF=yes TEM+SEM=not directly
• o Illumination source – BF=common light TEM+SEM=electron gun
• o Size – BF=small, TEM=huge (need your own room), SEM=medium-small (could fit on a desk)
• o Type of condenser – BF=bright field condenser discovered by Abbe, TEM+SEM=electromagnets
• o Use – BF=general specimens, bacteria TEM(see inside)+SEM(see surface)=viruses
• o Prep time – BF=quick TEM+SEM= take days and weeks, TEM – slice it thin, stain it and then you can look. SEM – you can work with bigger things but still lots of prep time
• o Neisseria gonorrhoeae – the clap, the drip, STD
• Different types of stains and why we do it.
• o to see small transparent structures visible
• o see intracellular organelles like spores
• o We also stain sometimes to see extracellular organelles like flagella
• Simple stain
• • Negative stain
• • Crystal stain
• • Methalyne blue
• Differential complex stains
• Gram stains (see handout “A General Comparison of Gram-Positive and Gram Negative Organisms” from 9/12)
• Gram (+) have primarily one layer- Peptidoglycan
• A lot of lipid – gram (-), ex. E. coli
• A little lipid – gram (+), ex. B. sub
• Crystal violet – primary stain, causes both to become purple. Then comes Mordant which is a complexing agent. Forms a CVI complex. After that you decolor with something like acetone. Come back with safrinin (counter stain) and the gram stains are red and pink.
• It has to do with lipid solubility. Acetone and alcohol are lipid solvents dissolving the CVI in gram (-). The gram (+) there are too few lipids.
• • Acid fast stain
• • Spore stain
• Organisms
  
• • Gram positives
• Require many nutrients to grow
• Cell wall lipid small amounts
• Cell wall peptidoglycan high
• Reacts with penicillin
• Does not react to streptomycin
• If there are stains in the media it will not grow
• • Gram negative
• Grow nearly anywhere
• Cell wall lipids high
• Cell wall peptidoglycan small amount
• Penicillin does not work on it
• Reacts with streptomycin
• If there are stains in the media it will grow