2.3 Methods of Studying Cells

• The 2 stages of cell fractionation are:

  1. Homogenisation
  2. Ultracentrifugation
• Cell Fractionation Part 1: -

  1. Tissue is cut into small pieces and placed into a cold, isotonic, and buffered solution.
  2. It's ground into smaller pieces using a homogenizer, which releases the organelles from the cell.
  3. Homogenate is filtered to remove complete cells and large debris.
  4. A suspension of homogenate is then centrifuged. Faster = greater force.
• Cell Fractionation Part 2: -

  1. Slower speeds collect larger organelles at the bottom of the test tube, the smaller organelles remain near the top in the supernatent liquid.
  2. These large organelles (e.g. nuclei) called sediment pellets are removed. The supernatent remaining is re-spun at a faster speed.
  3. By continuing this way, the smaller and smaller organelles will be recovered.
• When the homogenate is being created, why is it important to keep the solution cold?

  To reduce enzyme activity that might damage organelles.
• When the homogenate is being created, why is it important to keep the solution isotonic to the tissue?

  To prevent organelles bursting (lysis) or crenating (shrinking) due to osmosis.
• When the homogenate is being created, why is it important to add a buffer to the solution?

  To maintain a constant pH to prevent enzymes in the organelle from denaturing.
• Why is the homogenate filtered prior to ultracentrifugation?


  To remove any other complete cells and large organelles that we don't want (e.g. cell wall/membrane).
• What order would you expect organelles to be separated out in?

  Nucleus -> Mitochondria -> Lysosomes -> Ribosomes.

  Larger, denser organelles would get separated out first.
• Image - the picture you see.
• Object - The material you're putting under the microscope.
• Resolution - the minimum distance apart that 2 objects can be in order for them to appear as separate items.
• Magnification - how many times bigger the image is when compared to the object.
• Light microscopes: -

  • small and portable
  • view thin sections of plants and animals
  • maximum magnification of x1500
  • maximum resolving power 0.2 micrometres
• Electron microscopes: -

  • can't observe living material
  • difficult preparation process
  • metal salts used as a stain to scatter electrons
  • maximum resolving power 0.05 micrometres
  • high resolution and magnification
• • overall magnification = eyepiece lens magnification x objective lens magnification
• The two types of electron microscopes are: Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM).
• TEMs work by firing a beam of electrons at a very thin specimen. Dense parts of the specimen absorb electrons and these appear dark. Less dense parts allow the electrons to pass through. Electrons that pass through bleach the fluorescent screen.
• SEMs work by producing a beam of electrons that is focused on the specimen by a condenser and electromagnets. The beam is passed back and forth across a portion of the specimen. The e- are scattered by the specimen and the scattered electrons are detected and a 3D image is produced.
• You would expect a mitochondria to be oblong, but why do they sometimes appear circular on TEM photographs?

  They have been cut across the transverse plane.
• Magnification = image size/actual size
• Actual size = image size/magnification
• Image size = actual size x magnification
• Convert measurements into micrometres when carrying out microscopy calculations.
• m to mm --> x1000
• mm to nm --> x1000000
• mm to m --> /1000
• An eyepiece graticule is a measure of how many graticule intervals an object under a microscope is.
• A stage micrometre is a slide with a ruler used to calibrate the graticule to work out the length of different graticule intervals at different magnifications.
• Give one advantage of viewing a biological specimen using a TEM compared with using an SEM.


  You can see the organelles and how the inside of the cell is structured rather than just the outside, which is all you could see using an SEM.