3.1 - Methods of studying cells

• Equation for magnification ?


  Size of image / Size of real object
• What’s magnification ?


  How many times bigger the image is compared to the object
• What’s resolution ?


  The minimum distance apart that 2 objects can be in order for them to appear as separate items
• What’s cell fractionation ?


  The process where cells are broken up and the different organelles are seperated
• The 3 conditions needed in the solution for cell fractionation ?


  Cold, isotonic, buffer
• Why does the solution need to be cold ?

  Reduce enzyme activity that might break down organelles
• Why does the solution have to be isotonic(same conc.) ?


  Prevent organelles bursting(lysis) or shrinking due to osmotic gain or loss of water
• Why does the solution need to be buffered ?


  To prevent pH fluctuation which could alter organelle structure and enzyme function
• What are the 2 stages of cell fractionation?


  Homogenisation and ultracentrifugation
• What’s homogenisation ?


  Cells are broken up, releasing organelles creating the homogenate
• What’s ultracentrifugation ?


  Fragments in filtered homogenate are separated in a centrifuge, spun at increasingly high speeds to separate organelles into pellets based of their weight, leaving behind a supernatant
• Steps of cell fractionation before ultra centrifugation ?


  • add cells to cold, isotonic buffer solution and homogenise in a blender to release organelles
  • Filter the homogenate to remove large cell debris
  • Add to a centrifuge
• Steps of cell fractionation after homogenisation ?


  • filtrate is added to centrifuge and spun at low speeds, heavier organelles like nuclei are forced to the bottom and form a pellet
  • Supernatant is removed and spun in the centrifuge at a higher speed (ultracentrifugation) and mitochondria pellet forms
  • This is continued at increasing speeds until the organelles you want collects in a pellet