Cells: Magnification, Cell Fractionation And Ultracentrifugation Flashcards
Resolution definition
Minimum distance apart 2 objects can be for them to be distinguished as seperate objects. Limited by wavelength of radiation —> electron wavelength shorter than light so higher magnification
Optical light microscope characteristics
A microscope that has:
- A beam of light condensed to create an image
- Poorer resolution due to light having a longer wavelength than electrons
- Lower magnification
- Colour images
- Living samples
Magnification definition
Number of times greater the image is than the size of the real object:
magnification = image size / real size
Electron microscope characteristics
A microscope that has:
- A beam of electrons condensed to create an image
- Electromagnets used to condense the beam
- Higher resolving power as electrons have a short wavelength
- Higher magnification
- Black and white images
- A non-living sample in a vacuum
Homogenisation (Step 1.1)
- Can be done by vibrating the cells or by grinding the cells up in a blender
- This breaks up the plasma membrane and releases the organelles into solution
Ultracentrifugation (Step 3)
- The filtered solution is spun at different speeds in a centrifuge
- Organelles are separated according to their densities
The three steps of the cell fractionation process are…
- Homogenization
- Filtration
- Ultracentrifugation
Cell fractionation (Step 1.2)
- Cells are broken open to release the contents and organelles are then seperated
- The cells must be prepared in a cold, isotonic and buffered solution
- Cold: to reduce enzyme activity. When the cell breaks open enzymes are released which could damage the organelles
- Isotonic: must be the same water potential to prevent osmosis as this could cause the organelles to shrivel or burst
- Buffered: the solution has a pH buffer to prevent damage to organelles
Filtration (Step 2)
- Homogenized solution is filtered through a gauze to separate any large cell debri (connective tissue)
- Organelles are much smaller than the debris, so they pass through the gauze
Order of organelles (size + mass) to form in a centrifuge
- Nucleus (most dense)
- Chloroplasts
- Mitochondria
- Lysosomes
- Endoplasmic Reticulum
- Ribosomes
‘Never Catch Monkeys Living Epically Right’. Or ‘Now Call My Lawyer, Evil Rascal’
Differential centrifugation
- The centrifuge spins and the centrifugal forces cause pellets of the densest organelles to form at the bottom
- A centrifuge is first spun at a a low speed and the process is repeated at increasingly faster speeds
- Each time the supernatant (liquid)is removed, leaving behind a pellet of organelles
- The supernatant is then spun again to remove the next pellet of organelles
