Chapter 2 BIOL 2460

interference

similar to ripple effect

interacts w/ another wave

diffraction

bending/scattering of wave by object opening

refraction

changes lights direction/speed wave

when light enters higher refractive index it slows down and bends toward normal line (away from boundary)

convex lens

used for microscopy

works on curved boundary to meet focal point

concave lens

refract light away from focal point

ex.) flash light

electromagnetic radiation spectrum

know the higher and lower frequency’s of light

higher frequency waves have higher energy (photons move faster)

magnification

ability to enlarge image

contrast

creation of stark difference in coloration

colors

resolution

ability to make picture crystal clear

ability to tell 2 separate points/objects are separate

shorter wave length = higher resolution

longer wave length = lower resolution

galileo galilei

used compound microscope (uses 2 lens)

robert hooke

first to describe cells (small rooms) using compound microscope and observed cork cells

microscope types

light microscopes

electron microscopes

scanning probe microscopes

brightfield microscope

bright background for dark specimens

ocular lens

10x

objective lens

4, 10, 40, 100x lens

total magnification

ocular x objective = total magnification

darkfield microscopy

good for visualizing unstained or living specimens

dark background bright specimen

phase contrast microscopes

no need to stain or killing specimen gives extra layering for image

differentiates parts of cells

uses annular ring

uses refraction & interference for high resolution image

differential interference contrast microscope

light spins one/other direction (polarize/non-polarize)

good for live unstained specimens

fluorescent microscope

2 types of immunofluorescence (direct/indirect)

transmit excitation light

used to identify pathogens or specific species in environment or find location of certain molecules in cell

indirect - better than direct, adds secondary anti-body, more secondary than primary anti-body

confocal microscope

scans successive z planes with laser

creates multiple 2D pictures then layered for depth to 3D pic

for thick specimens (ex. biofilms)

avoids bleaching

2 photon microscope

good for viewing thicker material

ex) organs, brain slices

uses infrared light

minimizes light scattering through tissues

electron microcopy

resolution limited by wavelength

uses electron and short wavelength

used for DNA

transmission electron microscope

uses magnets to refract electrons

specimen cut thin

uses electron gun

stains specimen with heavy metal

scanning electron microscope

electrons bounce off specimen w/ coating

easy to observe surfaces

does not use light/electrons

uses sharp probes

views individual atoms

staining specimens

wet mount - good for live specimens

fixed mount - good for staining

basic stain

positive charged ions

methylene blue

crystal violet

malachite green

basic fuchsin

carbolfuchsin

safranin

acidic stain

negatively charged ions

eosin

acid fuchsin

rose bengal

congo red

positive stain

stain is absorbed to cells (specimen)

negative stain

stain absorbed to background

india ink

simple stain

1 stain

differential stains

2+ stains

gram stain

allows to separate bacteria (gram positive/negative)

differentiate cell wall components

steps:

primary stain (crystal violet)

mordant (iodine)

decolorizer (alcohol)

counter stain (safranin)

differential stain

acid fast stain

detects mycolic acid and mycobacterium (acid fast bacteria)

ziehl-neelson method (w/ heat)

kinyoun method (w/o heat)

steps:

primary stain (carbolfuschin)

decolorizer

counter stain (methylene blue)

capsule stain

detects protective coats

used to detect cells with capsule from those w/o

dyes dont pentrate capsule

simple stain

steps:

no heat smear

primary stain (india ink)

has halos to visualize

endospore stain

endospore formers

detects organisms with endospores and those w/o

schaeffer fulton method

steps:

heat fix smear

primary stain (malachite green)

decolorizer (water)

counter stain (safranin)

flagella stain

detects flagella appendages

used to view flagella in bacteria

ex.) bacteria, archaea, eukaryotes

steps:

no heat smear

primary stain (specialized)

decolorizer (water)

counter stain (carbon fuschin)

prep electron microscopy

transmission electron microscope (TEM)

very thin specimens

embedded in plastic resin & dehydrated

stained with heavy metals

scanning electron microscope

hydrated

sputter coated with metal

reflection

wave bounce off material

absorbance

wave captured

transmission

wave travels through

leeuwenhoek

used 1st simple microscope