Methods of Studying Cells

• Magnification formula =


  Magnification = size of image / size of real object
• What is resolution =


  the minimum distance apart that two objects can be distinguished as separate objects in an image - the greater the resolution the more clear the image will be
• What are the 2 types of microscope ?

  1. Light microscope
  2. Electron Microscope
• What are the advantages/disadvantages of the Light and /Electron Microscope...

  1. Light microscopes are relatively cheap and easy to use - Electron microscopes are very expensive and hard to use
  2. Light microscopes rely on light - electron microscopes use electrons instead of light
  3. Light microscope resolution is limited to 0.2um - electron microscopes max resolution is 0.1nm (2000x better than light microscope (can study subcellular structures))
  4. The interior of an electron microscope is a vacuum so you cant view living specimens - also requires staining
• Why might biologists want to use cell fractionation?

  cell fractionation = the process of separating cell organelles from each other

  useful for when biologists want to study one specific organelle to either conduct research on perhaps its function or look at its appearance under an electron microscope

  This process involves breaking up a suitable sample of tissue and the centrifuging the mixture at different speeds
• Cell fractionation can be split into 3 stages :

  • Homogenisation
  • filtration
  • Ultracentrifugation
• What is homogenisaton?

  • the biological term used to describe the breaking up of cells
  • the sample of tissue must first be placed in a cold, isotonic buffer solution
  • The tissue- containing solution is then homogenised using a homogeniser - a blender like machine that grinds the cells up ( the cells can also be vibrated until they break up)
  • This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate
• Why must the sample tissue be placed in a solution which is ice-cold before homogenisation?

  reduce the activity of enzymes that break down organelles
• Why must the sample tissue be placed in a solution which is isotonic before homogenisation?

  • Isotonic (it must have the same water potential as the cells being broken up) to prevent water from moving into the organelles via osmosis, which would cause them to expand and eventually damage them
• Why must the sample tissue be placed in a solution which is buffered before homogenisation?


  to prevent organelle proteins, including enzymes, from becoming denatured - maintains the pH
• What is filtration?

  • The homogenate (containing homogenised cells) is then filtered through a gauze
  • This is to separate out any large cell debris or tissue debris that were not broken up
  • The organelles are all much smaller than the debris and are not filtered out (pass through the gauze)
  • This leaves a solution (known as the filtrate) that contains a mixture of organelles
• what is ultracentrifugation - first steps?

  • the filtrate is placed into a tube and the tube is placed in a centrifuge - a centrifuge is a machine that separates materials by spinning
  • The filtrate is first spun at a low speed - this causes the largest heaviest organelles (such as nuclei) to settle at the bottom of the tube - where they form a thick sediment known as a pellet
  • The rest of the organelles stay suspended in the solution above the pellet
  • This solution is know as the supernatant
• next steps of ultracentrifuging - once it contains supernatant...

  • The supernatant is drained off and placed in another tube, which is spun at a higher speed.
  • once again this causes the heavier organelles (such as the mitochondria) to settle at the bottom of the tube, forming a new pellet and leaving a new supernatant
  • The supernatant is drained off and placed into another tube - which is spun at an even higher speed
  • This process is repeated at increasing speeds until all the different types of organelle present are separated
  • Each new pellet contains a lighter organelle than previous
• Order of the mass of organelles (heaviest to lightest):

  1. Nuclei
  2. Chloroplasts
  3. Mitochondria
  4. Lysosomes
  5. Endoplasmic reticulum
  6. Ribosomes
• Diagram of cell fractionation:

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• 2 main types of electron microscope:

  1. Transmission electron microscopes (TEM's) = use electromagnets to focus a beam of electrons - transmitted through the specimen - Denser parts absorb more electrons, making them look darker on the image. Give high resolution images (good) can see the internal structure of organelles. But can only be used on thin specimens
  2. Scanning electron microscopes (SEMs) = scan a beam of electrons across the specimen - image shows the surface of the specimen - can be 3D. can be good as they can be used on thick specimens - yet give lower resolution images than TEMs
• how to prepare a temporary mount of a specimen on a slide :

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