2. Cells - Methods Of Studying Cells And Microscopy Flashcards
What is the equation for magnification?
Magnification = size of image/size of real object
What is resolution?
It is the minimum distance apart that two objects can be in order for them to appear as separate items.
Briefly explain what the process of cell fractionation is:
It is the process where cells are broken up and the different organelles they contain are separated out.
What happens before cell fractionation can begin?
The tissue is placed in a cold, buffered solution of the same water potential as the tissue.
For cell fractionation, why is this solution cold?
To reduce enzyme activity that might break down the organelles.
For cell fractionation, why is this solution buffered?
So that the pH does not fluctuate. Any change in pH could alter the structure of the organelles of affect the functioning of enzymes.
For cell fractionation, why is this solution of the same water potential as the tissue?
To prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.
What are the two stages to cell fractionation?
-Homogenisation
-Ultracentrifugation
What is the process of homogenisation?
Cells are broken up by a homogeniser (blender). This releases the organelles from the cell. The resultant fluid, known as the homogenate, is then filtered.
Why is the homogenate filtered during homogenisation?
To remove any complete cells and large pieces of debris.
What is the process of ultracentrifugation?
The fragments in the filtered homogenate are separated in a machine called a centrifuge. This spins the tubes of homogenate at very high speed in order to create a centrifugal force.
What is the first stage of ultracentrifugation?
The tube of filtrate is placed in the centrifuge and spun at a low speed.
What is the heaviest organelles are therefore is forced to the bottom of the tube first?
The nuclei
What do the organelles form once forced to the bottom of the tube?
A thin sediment or pellet.
What is the fluid at the top of the tube called after the sediment is removed?
The supernatant.
What is changed with each spin on the centrifuge?
Speed - increases every time.
What is the next heaviest organelles after the nuclei that are forced to the bottom of the tube once spin at higher speed?
The mitochondria.
Compare how an optical microscope and transmission electron microscope work:
- Optical microscope uses light waves, a TEM uses a beam of electrons
- TEM focuses using magnets, optical uses glass lenses
Contrast the limitations of the use of an optical microscope and a TEM:
- TEM has a greater resolution than an optical microscope
- So TEM can observe smaller organelles/in greater detail
- TEM can only view dead specimens but optical can view live specimens
- TEM does not show colour, optical does
- TEM requires very thin specimens
- Therefore TEM requires a more complex/time consuming preparation
Briefly explain how to calibrate the eyepiece graticule:
- A special microscope slide, a stage micrometer, is required
- Line up the eye-piece graticule scale and the stage micrometer
- Calculate the length of divisions on the eye-piece graticule
