Lecture 2- Cell Structure and Function Flashcards
how does the compound light microscope work?
uses visible light to illuminate
what are the 4 different types of light microscopy?
bright- field
phase- contrast
dark- field
fluorescence
how do specimens get visualized/ seen using a bright- field microscope?
visualized because of differences in contract between specimen and surroundings
the bright- field microscope uses 2 lenses, what are they?
objective lens
ocular lens
what is the maximum magnification of a bright- field microscope?
~2,000x
how does the bright- field microscope work?
light source passes through the specimen, then through the objective lens, then we see the image through the ocular lens
why might we add oil on top of our specimen?
oil changes the diffraction of light and allows us to see the image clearer
what is magnification?
the ability to make an object larger
how do you calculate total magnification?
objective magnification x ocular magnification = total magnification
what happens in magnification is increased a lot?
we wont necessarily have a more clear image, resolution plays a part aswell
what is resolution?
ability to distinguish two adjacent objects as separate and distinct.
ability of a lens to distinguish small objects that are close together
when you’re walking and you see something in the horizon, it takes you a while to see it, that is resolution
what is the limit of resolution for a light microscope?
0.2µm (micrometer), therefore, two points can be distinguished if they are at least 0.2µm apart
as wavelength decreases, what happens to resolution?
increases
what can we use to improve contrast and result with a better final image?
staining!!!
what are characteristics of dyes that we use?
organic compounds
bind to specific cellular materials
what is simple staining?
one dye used to color specimen
what is chromophore?
colored portion of a dye
what are two types of simple staining?
basic dye
acidic dye
how does basic dye work?
its positively charged chromophore so it binds to negatively charged molecules on cell surface
how does acidic dye work?
its negatively charged chromophore (negative stain) so its repelled by cell surface
what is acidic dye used for?
stain background
with basic due we dye the organisms purple, therefore, the background is white
with acidic dye we dye the background purple, therefore, the light shines through the organisms
how do you prepare samples for staining? (6)
- spread some culture in thin film over slide using a loop
- dry in air
- pass slide over flame gently to melt specimen to slide (without killing them!)
- flood slide with stain
- rinse and dry
- put under microscope
what are 3 types of differential staining?
the gram stain
acid fast stain
endospore stain
what is the goal of the gram stain?
separates bacteria into 2 groups based on cell wall structure
what is the process of the gram stain? slide 12.
flood the heat fixed smear with crystal violet (1min)
add iodine solution (1min)
decolorize with alcohol (20sec)
counterstain with safranin (1min)
what color does gram positive turn?
purple
what color does gram negative turn?
red or pink
what does the acid fast stain do?
detects mycolic acid in the cell wall of the genus mycobacterium
what does the mycobacterium do? in acid fast stain
retains primary stain (pink)
what color does the rest of the slide turn in acid fast stain?
blue (color of counterstain)
what occurs in the endospore stain?
endospores retain primary (green)
cells counterstained (pink)
what does phase- contrast microscopy do?
improves the contrast of a sample without the use of a stain
how does phase-contrast microscopy work?
improves contrast without the use of a stain
visualization of live samples
what do you see when you use phase- contrast microscopy?
live samples
image is dark cells on a light background
what do you see with dark field microscopy?
specimen is illuminated with a hollow cone of light
specimen appears as a bright object on a dark background
how does dark field microscopy work?
only refracted light enters (thats why we see what we do)
what is dark field- microscopy used to observe?
bacteria that dont stain well
what is fluorescence microscopy used to visualize?
specimens that fluoresce
how does differential interference contrast microscopy work?
uses a polarizer to create two distinct beams of polarized light
what does interference contrast microscopy do?
gives structures such as vacuoles a three- dimensional appearance
if structures are not visible by bright- field microscopy what is the next microscopy to try?
differential interference contrast microscopy
how does confocal scanning laser microscopy work?
uses a computerized microscope coupled with a laser source to generate a three- dimensional image
how does the three- dimensional image work with confocal scanning laser microscopy?
the computer can focus the laser on single layers of the specimen, therefore, different layers can be compiled for a three dimensional image
what is the resolution for confocal scanning laser microscopy?
1µm
what is the difference between electron microscopes and light microscopes?
electron microscopes use electrons
light microscopes use photons
why is it better to use electrons instead of photons?
higher resolution
what are the two types of electron microscopes?
transmission electron microscope
scanning electron microscope
how does the TEM work?
electron beam focused on specimen by a condenser
goes through magnets as lenses
electrons pass through two magnetic lenses to see the specimen
electrons strike a fluorescent viewing screen (the energy of the electrons is converted to visible light, forming an image)
what will happen if too many electrons are used?
specimen will get destroyed and diminish (like when you light paper on fire)
what is the high resolution of the TEM?
0.2nm
how thin does the specimen need to be for the TEM?
20- 60nm
why does the sample need to be so thin? tem
so that electrons can pass through it and a clear image is showed
what does the TEM sample need to get stained with?
dense metal such as lead or uranium
why does the TEM sample need to be stained with a dense metal?
metal binds to cell structures to make them more electron dense and enables visualization of structures at the molecular level
what is the big difference between TEM and SEM?
TEM: inside structures are viewed (due to slicing)
SEM: surface areas are viewed in 3D
how does a scanning electron microscope work?
an electron beam scans the object
scattered electrons are collected by a detector and an image is produced
what is the specimen coated with? SEM
specimen is coated with a thin film of heavy metal (ex. gold)
what is cell morphology?
the different shapes we see in cells
what does it mean that morphology does not predict physiology, phylogeny… in prokaryotes?
your shape is just a characteristic of your species. what you look like does not mean who you are. “dont judge a book by its cover”
what are the selective forces involved in setting the morphology? 3
optimization for nutrient uptake
swimming motility
gliding motility
what are the 3 more common cell shapes?
coccus (spherical)
bacillus (rod shaped)
spirillum (spiral shaped)
what are the 3 unusual cell shapes?
spirochete
budding & appendaged bacteria
filamentous bacteria
what is the average diameter of a microbe?
1.0µm
ex. E. coli
what size of a microbe would be considered very small?
0.3µm
what size of a microbe would be considered very large?
80 x 600µm
does the open ocean normally contain small or large cells?
small!
0.2 to 0.4µm
how small can a microbe get? what is an unlikely size?
<0.15µm
what are the advantages to being small?
more SA
support greater nutrient exchange per unit cell volume
tend to grow faster than larger cells
what is the cytoplasmic membrane?
thin structure that surrounds the cell and separates the cytoplasm from its environment
what does the cytoplasmic membrane allow to enter?
its highly selective so it only enables concentration of specific metabolites and excretion of waste products
do we use diffusion across the cytoplasmic membrane?
no, it takes too long so we use channels instead
what makes it so that the membrane structure can exist in many different chemical forms?
variation in the groups attached to the glycerol backbone
what does the cell membrane contain?
phospholipid bilayer
membrane proteins
what does a phospholipid consist of?
2 fatty acids
glycerol
phosphate
ethanolamine (R group)
what does it mean that phospholipids are amphipathic?
hydrophobic and hydrophilic
hydrophobic: polar (tails are inward to form hydrophobic environment)
hydrophilic: non- polar (heads are exposed to external environment or the cytoplasm)
what linkages do phospholipids contain in bacteria and eukarya?
ester linkages
what is a polar molecule? charge
molecule that carries full or partial charge
what is a non- polar molecule? charge
molecule is uncharged
how tall is the cell membrane?
8-10 nm
how is the cytoplasmic membrane stabilized?
hydrogen bonds and hydrophobic interactions
Mg^2+ and Ca^2+ help stabilize membrane by forming ionic bonds with negative charges on the phospholipids
gram- negative bacteria interact with a variety of proteins including periplasmic proteins, what do these proteins do?
bind substrates
process large molecules for transport
what does the inner surface of cytoplasmic membrane interact with?
proteins involved in energy- yielding reactions and other important cellular functions
what types of linkages do archaeal membranes have?
ether linkages in phospholipids of archaea
what do archaeal membranes contain?
no fatty acids, have isoprenes instead
major lipids are glycerol diethers and tetraethers
what form is the phospholipid in, in archaeal membranes?
lipid monolayers
lipid bilayers
mixture of both
what is unique about lipid monolayers in comparison to lipid bilayers?
lipid monolayer membranes are extremely heat resistant grow best at temperatures above 80°C
where are lipid monolayer membranes commonly found?
in hyperthermophilic archaea
(normally in real hot places like hot springs)
what are the 3 functions of the cytoplasmic membrane?
permeability barrier
protein anchor
energy conservation
what is the function of the permeability barrier?
polar and charged molecules (+) must be transported
transport proteins accumulate solutes against the concentration gradient
what is the function of the protein anchor?
holds transport proteins in place
what is the function of energy conservation?
generation of proton motive force (moves protons out so the cell can be negative)
