3.2.1.3 Methods of studying cells

• Cell fractionation is used to separate cell organelles.
• Magnification makes an object look bigger.
• Resolution is the ability to distinguish between two separate points, how clear the image is.
• Magnification is the size of the image compared to the actual size of the object.
• The resolving power of a light microscope is limited as the wavelength of light is too long.
• The purpose of adding a stain is to add contrast and to distinguish between organelles.
• Units of measurement include Metre (m), Milli metre (mm), Micro metre (µm), Nano metre (nm), Pico metre (pm).
• Organelles are separated according to density using ultra centrifugation in cell fractionation.
• An electron microscope has a higher resolution than an optical microscope, which allows the smaller organelles like ribosomes to be seen.
• There are two types of electron microscopes: scanning and transmission electron microscopes.
• Sample preparation for electron microscopy is more complex than light microscopy.
• How do you prepare a temporary mount for light (optical) microscopes?

  • Start by pipetting a small drop of water onto the slide
  • Then use tweezers to place a thin section of your specimen on top of the water drop.
  • Add a drop of a stain.
  • Finally, add the cover slip.
• To go from picometeres (pm) to nanometers (nm) you divide by 1000
• To go from nanometres (nm) to micrometers (µm) divide by 1000
• Three steps of cell fractionation?

  1. homogenisation
  2. filtration
  3. ultracentrifugation
• Ultracentrifugation uses high speeds to separate different components based on their density and size. The heavier components will sink faster while lighter components remain suspended in the solution.
• What happens in homogenisation?

  • The cells are placed in an ice-cold, isotonic, buffered solution
  • Next, the cells are blended in a homogeniser (blender) to break up the plasma membrane and releases the organelles into solution.
• What happens in ultracentrifugation?

  • filtrate solution is centrifuged at a low speed, causing heavier organelles (e.g. nuclei) to form a pellet at the bottom
  • supernatant is drained off, poured into another tube and re-centrifuged until the desired organelles are separated out.
• The supernatant is rest of the organelles that stay suspended in the fluid above the sediment.
• An artefact is a visible structure caused by the preparation of a sample.