Chapter 3: A tour of the cell Flashcards
(117 cards)
What is the difference between Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)?
- SEM uses the application of a metallic layer onto a sample which deflects the electron beam onto the detector and is used to study the external cell surface.
-TEM passes the electron bean through a thin sample and can be used to study internal cell structure.
What are the most common reagents used for protein and lipid fixing in TEM?
Protein fixing: Glutaraldehyde
Lipid fixing: Osmium tetroxide
Why are heavy metals used for sample staining in TEM?
Heavy metals preferentially bind to specific molecules and increase the contrast of images by preventing electrons passing through due to their large size (causing darker areas)
What are the two most common reagents used for sample staining in TEM and what do these elements bind to?
- Uranium: Nucleic acids and proteins
-Lead: Lipids
What piece of equipment is used to cut ultrathin EM sections?
Ultramicrotome
What is the approximate thickness of a sample suitable for TEM?
How does this compare to the thickness on EM samples?
- approx. 70 nm thick
-Much smaller than the 5-50 µm samples used in EM.
What does the electron beam hit and what occurs to make the visible image in TEM?
The electron beam hits a fluorescent screen that emits visible light on impact.
Why is the interior of an electron microscope a vaccuum?
To prevent scattering of the electron beam on impact with air molecules.
What controls the direction of the electron beam in an electron microscope?
Magnets
What is Immunoelectron microscopy?
The use of immunolabelling to localise specific molecules to particular organelles using an electron microscope.
What is cell fractionation?
A technique using disruption and ultracentrifugation of a cell/s to separate its components
When cells are disrupted (i.e. before centrifugation) what is formed?
A cell homogenate
In centrifugation, what does the rate of settlement depend on?
- Particle size, shape and density
- Liquid density
- Rotational speed of centrifuge
What is the name for the particles that have collected together at the bottom of a tube during centrifugation?
A pellet
Describe the process of differential sedimentation.
Centrifugation taking place at increasingly higher speeds to separate particles according to their size and density. Each pellet is removed from the supernatant (remaining suspension) before the next speed increase.
Describe density gradient centrifugation
A cell lysate is laid upon a pre-prepared density gradient sucrose solution. Movement through the density gradient optimises separation into bands and can be removed as fractions with a pipette or pierced hole.
How does the nucleoid of a prokaryote appear under an electron microscope - light or dark?
Light
What shape does the DNA molecule form in a prokaryote?
Circular
What is the function of glycogen granules within prokaryotic cells?
Energy storage
What does a gram-negative bacteria have that a gram-positive bacteria does not?
Periplasmic space separating the inner and outer membrane
In bacteria, what is the cell wall composed of?
Peptidoglycan (polymer of sugars linked by amino acids)
What is the difference between gram-positive and gram-negative bacteria?
Gram positive
- thick layer of peptidoglycan at surface
Gran-negative
- Thinner layer of peptidoglycan within the periplasmic space (separates inner and outer membrane)
How can Gram stain be used to distinguish and how?
Gram stain can be used to distinguish between gram-positive and gram-negative bacteria.
Gram stain is absorbed and held by gram-positive bacteria’s thick peptidoglycan layer
Gram stain is not absorbed and held by gram-negative bacteria’s thin peptidoglycan layer
After staining the gram stain is dissolved to dissolve cell membranes and decolourise the cell. Gram positive bacteria will present as purple.
What do the capsules that some bacteria possess protect them from?
Desiccation and phagocytosis by other cells