Studying cells

• TEM

  • Beam of e- that is focused onto specimen by electromagnetic condenser
  • Beam passes through thin layer of specimen
  • Some parts of specimen absorb beams making them darker
  • Image is produced onto screen and then can be photographed to give photomicrograph
• SEM

  • Directs beam of e- onto specimen from above
  • Beam passed back and forth across portion of specimen in regular pattern
  • Electrons scattered by specimen and pattern of scattering depends on contours of specimens surface
  • We can build up 3D image by computer analysis
• Conditions for cell fractionation

  • Cold- reduce enzyme activity
  • Isotonic- prevent bursting/ shrinking of organelles due to osmosis
  • Buffered- prevent pH fluctuation
• Homogenation

  • Tissue cut into small pieces and placed in C,I,B solution
  • Ground into smaller pieces using homogeniser
  • Homogenate- filtered, remove any complete/large debris
• Ultracentrifugation

  • Homogenate is placed in centrifuge & spun at high speed

  For animal cells

  • Tube put in centrifuge and spun at low speed
  • Heaviest organelle (nuclei) forced to bottom
  • Fluid on top (supernatant) removed and spun at higher speed
  • Next heaviest organelle (mitochondria) forced to bottom
  • Process is continued and each time speed increases so next heaviest organelle forced to bottom