Microscopy Flashcards
Resolution
ability of a lense to distinguish small objects that are close together
factors affecting resolution
wavelength: shorter= higher res.
numerical aperture
refractive index: greater index=higher rex.
working distance: smaller distance, higher res
Why do we stain
increase visibility of specimen
accentuates specific morphological features
preserves specimen
heat fixation
used with bacteria and archaea
could degrade proteins on subcellular surface
chemical fixation
used with larger, more delicate organisms
Two common features of dyes
chromophore groups
ability to bind cells
chromophore groups
conjugate double bonds that give color
ability to bind cells
must bind via ionic interaction
basic dyes
positive charges
interact with negative things
acid dyes
negative charge
interact with positive things
used for negative staining
What can simple staining determine
size
shape
arrangement of bacteria
Differential staining
divides microorganism into groups based on their staining properties
Gram Stain
Divides bacteria (not archaea) into two groups based on amount of peptidogylcan in cell
gram positive will appear purple
gram-negative will appear pink
Acid fast staining
used for organisms with high mycolic acid (repels crystal violet)
high lipid in cell wall will appear pink
low lipid will appear blue
endospore staining
exceptionally resistant to staining
capsule staining
used to visualize capsules surrounding bacteria
india ink or nigrosin
Flagella staining
very thin and can only see with an electron microscope
will not see if not stained
Disadvantages of TEM
electrons can only penetrate thin specimen
only gives 2D image
specimen must be viewed under high vacuum
the specimen is dead- artifacts
Negative stain
specimen spread out on a thin film of heavy metals
heavy metals dont penetrate specimen but render a dark background
used for viruses, bacterial gas, vacuoles
shadowing
coating specimen with thin film of heavy metals but only on one side
used for viral morphology, flagella, DNA
Freeze-etching
freeze specimen and
fracture along greatest weakness
shows intracellular structures
reduces artifacts
usually done along with shadowing
Electron crytomography
rapidly freeze specimen in liquid nitrogen to form vitreous ice
image along multiple axises to form a tilt series
provides extremely high resolution of ultrastructure
