centrifuga

• organelle is a membrane bound subcellular specialised structure
• debris is the organic waste. scattered pieces of remains/loose natural matter
• pellet is a small compressed mass of substance
• cell fractionation

  1. homogenise (blend) to break down the cell wall/membrane and release organelles POSSIBLE DAMAGE
  2. Filter through a sieve (not filter paper) to remove whole cells or debris
  3. use centrifuge to perform ultracentrifugation
• how to ultracentrifuge

  1. spin the homogenate (solution+organelles) at a relatively slow speed so the most dense organelle settles at the bottom
  2. remove supernatant and spin at a higher speed to cause the next most dense organelle to settle at the bottom
• 1. Tissue is cut into pieces in cold conditions in a buffering isotonic solution.
  2. The tissue is homogenised (in a blender) so that the organelles are released from the cell.
  3. A fluid called homogenate is also formed. The homogenised suspension is filtered.
  4. The filtrate is centrifuged at a low speed.
  5. The supernatant is decanted and re-centrifuged at higher speeds until the desired organelle is separated out.
  6. The contents of the supernatant can then be analysed.
• first step in cell fractionation is placing the tissue in cold, isotonic buffer to reduce enzyme activity that breaks the down organelles.

  isotonic so no shrinkage/bursting due to water potential and osmotic movement.

  buffer keeps the ph constant so no denaturation
• the organelles from most to least dense

  1. nuclei
  2. mitochondira
  3. lysosomes
  4. endoplasmic reticulum
  5. ribosomes