Cell Structure and Function Flashcards
Principles of Light Microscopy
compound light microscope uses visible light to illuminate cells
Types of Light Microscopy
- Bright-field
- Phase-contrast
- Dark-field
- Fluorescence
Bright-field scope?
- Specimens are visualized because of differences in contrast between specimen
and surroundings - Two sets of lenses form the image
- Objective lens (usually 10x -100x mag.) & ocular lens (usually 10x – 20x mag.)
- Total magnification = objective magnification ✕ ocular magnification
- Maximum magnification is ~2,000✕
Resolution
The ability to distinguish two adjacent objects as separate and distinct
- Limit of resolution for light microscope is about 0.2 µm
Magnification
The ability to make an object larger
How to calculate Magnification?
- Magnification = ocular x objective
- ex. Ocular = 10x, Objective = 40x
- Magnification = 10 x 40 = 400x
Resolution
- The ability of a lens to distinguish small objects that are close together
- Ex) resolving power of 0.2µm - Two points can be distinguished if they are at least 0.2 µm apart
- Light must pass between two points for them to be viewed as separate objects
- As wavelength decreases resolution improves
Throw ink-covered objects at target (“E”)
- Basketballs: Cannot fit between arms, poor resolution.
- Tennis Balls: Fit between arms, resolution improves.
- Jelly Beans & Beads: As diameter of objects thrown decreases, greater numbers pass between the arms & the resolution increases.
How does improving Contrast in light Microscopy help?
Improving contrast results in a better final image
How to improve Contrast?
- Staining improves contrast
- Dyes are organic compounds that bind to specific cellular materials.
- Examples of common stains are methylene blue, safranin, and crystal violet
What are the types of staining
- Simple Staining
- Differential Stains
Simple Staining
- One dye used to color specimen.
- Chromophore - colored portion of a dye
- Two types:
- Basic Dye
- Acidic Dye
What are the two types of simple staining?
Basic Dye: positively charged chromophore.
- Binds to negatively charged molecules on cell surface.
Acidic Dye: negatively charged chromophore
- Repelled by cell surface
- Used to stain background
- Negative stain
Preparing Samples for Staining
- Preparing a smear: spread culture in thin film over slide -> Dry in air.
- Heat fixing and staining: Pass slide through flame to heat fix -> Flood slide with stain; rinse and dry.
- Microscopy: -> place drop of oil on slide; examine with 100x objective lens.
Differential Stains
The Gram Stain
- Separates bacteria into 2 groups based on cell wall structure .
- Gram Positive
- Gram Negative
Gram Positive
Cells that retain a primary stain
- Purple
Gram Negative
Cells that lose the primary stain
- Take color of counterstain
- Red or Pink
What are the two differential stains?
- Acid Fast Stain
- Endospore Stain
Acid Fast Stain
- Detects mycolic acid in the cell wall of
the genus Mycobacterium
- Mycobacterium – retains primary stain
- Fuchsia (pink)
- Anything else on slide – color of counterstain
- Blue
Endospore Stain
- Endospores retain primary
- Green - Cells counterstained
- Pink - Ex. Bacillus anthracis spores.
Phase-Contrast Microscopy
- Phase ring amplifies differences in the refractive index of cell and
surroundings. - Improves the contrast of a sample without the use of a stain.
- Allows for the visualization of live samples.
- Resulting image is dark cells on a light background.
Dark Field Microscopy
- Specimen is illuminated with a hollow cone of
light - Only refracted light enters the objective
- Specimen appears as a bright object on a dark
background - Used to observe bacteria that don’t stain well
- Ex) Treponema pallidum: the causative agent of syphilis
Fluorescence Microscopy
- Used to visualize specimens that fluoresce
- Emit light of one color when illuminated with
another color of light
Cells in Fluorescence Microscopy?
- Cells may fluoresce naturally
- Ex. Photosynthetic Cyanobacteria have
chlorophyll
- Absorbs light at 430 nm (blue-violet)
- Emits at 670 nm (red) - After staining with Fluorescent dye
- Ex) DAPI specifically binds to DNA
Differential interference contrast (DIC) microscopy
- Uses a polarizer to create two distinct beams of polarized light
- Gives structures such as endospores, vacuoles, and granules a three-
dimensional appearance - Structures not visible by bright-field microscopy are sometimes visible by
DIC
Confocal scanning laser microscopy (CSLM)
- Uses a computerized microscope coupled
with a laser source to generate a three-
dimensional image - Computer can focus the laser on single
layers of the specimen - Different layers can then be compiled for a
three-dimensional image - Resolution is 0.1 μm for CSLM
Electron Microscopy
- Electron microscopes use electrons
instead of photons to image cells
and structures - Wavelength of electrons is much
shorter than light → higher
resolution
What are the two types of electron Microscopes?
- Transmission electron microscopes (TEM)
- Scanning electron microscopes (SEM)
Transmission Electron Microscopes
- High magnification and resolution (0.2 nm)
- Specimen must be very thin (20 – 60 nm)
- Unstained cells do a poor job of scattering electrons
- Must be stained with metals → lead or uranium
- Bind to cell structures to make them more electron dense
- Enables visualization of structures at molecular level
Scanning Electron Microscopy (SEM)
- Specimen is coated with a thin film of heavy metal (e.g., gold)
- An electron beam scans the object
- Scattered electrons are collected by a detector, and an image is
produced - Allows an accurate 3D image of specimen’s surface.
Prokaryotes (Before Nucleus)
- No membrane bound nucleus or organelles
- Generally smaller than eukaryotes
- Simple internal structure
- Divide by binary fission
- Most are unicellular
Bacteria (Eubacteria)
- Diverse metabolism
- Live in a broad range of
ecosystems - Pathogens and non-pathogens
Archaea (Archaebacteria)
- Diverse metabolism
- Live in extreme environments
- Non-pathogens
What are the Cell Morphology?
- Coccus (pl. cocci)
- Roughly spherical
- EX. Streptococcus pyogenes
- Bacillus (pl. bacilli)
- Rod shaped
- EX. E. coli
- Spirillum (pl. spirilla)
- Spiral shaped
- EX. Spirillum volutans
Different Cells
- Cells with unusual shapes -> Spirochete -> Treponema pallidum
- Budding & appendaged bacteria -> Caulobacter crescentus
- Filamentous bacteria -> Streptomyces griseus
Prokaryote Size
- Average:
- E. coli ~ 1.0 x 3.0 µm
- Staphylococcus aureus ~ 1.0 µm diameter - Very small:
- Mycoplasma genitalium ~ 0.3 µm - Very large:
- Epulopiscium fishelsonii ~ 80 x 600 µm.
Membrane Structure (Cytoplasmic Membrane)
- Cell or plasma membrane
- Thin structure that surrounds the cell
- Vital barrier that separates cytoplasm from environment
- Highly selective permeable barrier; enables concentration of specific
metabolites and excretion of waste products
Membrane Structure (Composition of membranes)
- General structure is phospholipid bilayer
- Contain both hydrophobic (fatty acid) and hydrophilic (glycerol-phosphate)
components - Can exist in many different chemical forms as a result of variation in the
groups attached to the glycerol backbone - Fatty acids point inward to form hydrophobic environment; hydrophilic
portions remain exposed to external environment or the cytoplasm
Phospholipid Structure (Ester Phospholipids)
- Consist of: glycerol
- 2 Fatty acids
- Phosphate
- Side chain (optional)
Phospholipid Structure (Amphipathic)
Has both polar and non-polar characteristics
Phospholipid Structure (Polar)
- Molecule carries full or
partial charge- Hydrophillic
Phospholipid Structure (Non-Polar)
- Molecule is uncharged
- Hydrophobic
Membrane Structure (Cytoplasmic Membrane)
- 8–10 nm wide
- Embedded proteins
- Stabilized by hydrogen bonds and hydrophobic interactions.
- Mg2+ and Ca2+ help stabilize membrane by forming ionic bonds with
negative charges on the phospholipids - Somewhat fluid
Membrane Structure (Membrane Proteins)
- Integral membrane proteins
- Firmly embedded in the membrane
- Peripheral membrane proteins
- One portion anchored in the membrane
Membrane Structure (Archaeal Membranes)
- Ether linkages in phospholipids of Archaea
- Bacteria and Eukarya that have ester linkages in phospholipids
Membrane Structure (Archaeal Membranes) Part 2
- Archaeal lipids lack fatty acids; have isoprenes instead
- Major lipids are glycerol diethers and tetraethers
- Can exist as lipid monolayers, bilayers, or mixture
Membrane Function (Permeability Barrier)
- Polar and charged molecules must be transported
- Transport proteins accumulate solutes against the concentration gradient
- Prevents leakage and functions as a gateway for transport of nutrients into, and wastes out of, the cell
Membrane Function (Protein Anchor)
- Holds transport proteins in place
- Site of many proteins that participate in transport, bioenergetics, and chemotaxis
Membrane Function (Energy Conservation)
- Generation of proton motive force
- Site of generation and dissipation of the proton motive force
