• Biologists and other scientific researchers may sometimes want to study?one specific?organelle
• This may be to:
  • Look at the appearance of the specific organelle under an electron microscope
  • Conduct research on the specific organelle (for example, to learn more about its function)

   

• To do this, a?pure sample?is needed (containing only the specific organelle being studied)
• The process of separating cell organelles from each other is known as?cell fractionation
• This process involves?breaking up?a suitable sample of tissue and then?centrifuging?the mixture at?different speeds
• Cell fractionation can be split into three stages:
  • Homogenisation
  • Filtration
  • Ultracentrifugation

   

Homogenisation

• Homogenisation?is the biological term used to describe the?breaking up of cells
• The sample of tissue (containing the cells to be broken up) must first be placed in a?cold, isotonic buffer solution
• The solution must be:
  • Ice-cold to?reduce the activity of enzymes?that break down organelles
  • 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
  • Buffered (have a buffer solution added) to prevent organelle?proteins?from becoming?denatured

   

• The tissue-containing solution is then homogenised using a?homogeniser
  • This is 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

Filtration

• The homogenate (containing the 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 (they pass through the gauze)
  • This leaves a solution (known as the?filtrate) that contains a?mixture of organelles

   

Ultracentrifugation

• 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 the 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 known as the?supernatant

   

• The supernatant is?drained off?and placed into 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 new 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 out (or just until the desired organelle is separated out)
• Each new pellet formed contains a lighter organelle than the previous pellet
• The order of mass of these organelles (from heaviest to lightest) is usually:
  • Nuclei
  • Chloroplasts (if carrying out cell fractionation of plant tissue)
  • Mitochondria
  • Lysosomes
  • Endoplasmic reticulum
  • Ribosomes

   

The three stages of cell fractionation (homogenisation, filtration and ultracentrifugation) for separating organelles