Cell fractionation

• Principles of cell fractionation and ultracentrifugation as used to separate cell components
• 1. Homogenise tissue using a blender
• - Disrupts cell membrane / break open cell
• - Release contents / organelles
• 2. Place in a cold, isotonic, buffered solution
• - Cold reduces enzyme activity so organelles aren’t broken down
• - Isotonic so water doesn’t move in/out of organelles by osmosis so they don’t burst / shrivel
• - Buffered keeps pH constant so enzymes don’t denature
• 3. Filter homogenate
• - Remove large, unwanted debris e.g. whole cells, connective tissue
• 4. Ultracentrifugation
• a) Centrifuge homogenate in a tube at a low speed
• b) Remove pellet of heaviest organelle and spin supernatant at a higher speed
• c) Repeated at higher and higher speeds until organelles separated out, each time pellet is made of lighter organelles
• d) Separated in order of mass/density: nuclei → chloroplasts → mitochondria → lysosomes → endoplasmic reticulum → ribosomes
• There was a considerable period of time during which the scientific community distinguished between artefacts and cell organelles

  Repeatedly prepared specimens in different ways

  • If an object could only be seen with one preparation technique, but not another it was more likely to be an artefact than an organelle