Methods of studying cells

Question 1

What do we do in preparation of cell fractionation in the homoginiser and why?

Answer 1

The tissue is firstly placed in a cold, buffered solution of the same water potential as the tissue
Cold- to reduce enzyme activity that may break down organelles
Is of the same water potential as the tissue- to prevent cells shrinking or enlarging due to osmotic movement of water in or out of the cell
Buffered- to control PH so it does not fluctuate.

Question 2

Describe the homoginisation stage of the cell fractionation

Answer 2

We use a homoginiser to do this which is a glass tube with a plunger, and we place out tissue sample into this glass tube and we cover this with a buffer solution. Now we push the plunger up and down to distrupt the tissue and break open the cells, producing a cell homogenate, and this is then filtered to remove complete cells and pieces of debris

Question 3

What happens during the ultracentrifugation stage of cell fractionation?

Answer 3

Tubes containing the cell homogenate go into the sample holder of the centrifuge, and this now spins the sample. The heaviest organelles are now forced to the bottom of the tube where they force a thin sediment or a pellet, and the fluid at the top of the tube, the supernatant is removed leaving just the sedimnet of the nuclei. The supernatant is transferred to another tube and is span in the centrifuge at a faster speed than before, then the next heaviest organelles are foced to the bottom so that at each increase of speed, the next heaviest organelle is sedimented and separated out

Question 4

Define resolution

Answer 4

The minimum distance apart that two objects can be in order for them to appear as two separate items

Question 5

How many micrometres is in a millimetre?

Answer 5

1000

Question 6

How many nanometres is in a micrometre?

Answer 6

1000

Question 7

Give limitations of using a light microscope

Answer 7

Has a low resolution

Question 8

Describe the principles and limitations of using electron microscopes

Answer 8

Electrons have a very short wavelength so the resolution is 2000 times better than light microscopes, however the interior of an electron microscope is a vacuum so we cannot view living specimens, and electron microscopy has to be very carefully stained and the specimen has to be thin, and there may also be artefacts

Question 9

Describe how electron microscopes work?

Answer 9

An electron gun produces a beam of electrons which pass down the microscope. The inside of the electron microscope contains a vacuum so the electrons can pass through without bouncing off air molecules. The electrons are negatively charged so we can use an electromagnet to focus the electron beam

Question 10

Compare the TEM with the SEM

Answer 10

The TEM produces 2D images however SEM is able to produce 3D images. The specimen for the TEM needs to be extremely thin as electrons do not penetrate whereas in SEM it does not need to be as thin. The SEM also has a lower resolving power than the TEM
In SEM the electrons dont pass through the specimen and instead they are scattered from the surface of the specimen

Question 11

Why cant the resolving power of the TEM always be achieved?

Answer 11

There may be difficulties preparing the specimen and a higher electron beam is required which may destroy the specimen