Methods Of Studying Cells

Question 1

Magnification

Answer 1

How many times bigger the image is compared to the actual object

Size of image/size of real object= magnification

Or Imagine the triangle

Size of image/
Size of real object x magnification

Question 2

Resolution

Answer 2

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

Question 3

What are the 3 stages to Cell fractionation?

Answer 3

1. Homogenation
2. Filtration
3. Ultracentrifugation

Question 4

Cell fractionation… step by step

Answer 4

1. Add 5cm3 cold, isotonic buffer to the sample- to break open cells and release organelles intact.
2. Spin supernatant from first sample at 3500xg for 20mins- supernatant contains lighter organelles for separation on the next spin cycle.
3. Homogenise sample thoroughly- to prevent enzyme action, ph fluctuation (damages proteins) or osmotic damage to organelles
4. Cut tissue into small pieces using a sharp blade- to spin heaviest organelle(nuclei) to the bottom of tube as a pellet.
5. Filter homogenate- to remove cellular debris.
6. Pour equal volumes of homogenate into centrifuge tubes- to make tissue sample easy to homogenise.
7. Decant supernatant into a second centrifuge tube and remove pellet for study- so the tubes are balanced during centrifugation.
8. Spin homogenate for 10mins at 1000xg- next heaviest organelles (chloroplasts) are spun to the bottom of the tube.
9. Repeat previous 2 steps increasing the speed and duration of centrifugation each time- by increasing spin speed and duration, successively lighter fractions of organelles will be isolated.

Question 5

Why is the buffered solution in cell fractionation…..

Answer 5

Cold- to reduce enzyme activity that might break down the organelles.
Same water potential as the tissue- to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.
Buffered- so that the PH does not fluctuate. Any change in pH could alter the structure of organelles or affect the functioning enzymes.

Question 6

The light microscope

Answer 6

Highest magnification=X1500 
Level of resolution= 2um(micrometre)
Waves can pass through or bounce off specimen depending on specimen.
Specimen cannot be damaged by beam.
Image in colour.
Low risk to user.
Specimen can alive or dead.
Very portable.
Mild specimen preparation so low risk of artefacts.
Lenses made from glass.

Question 7

Transmission electron microscope

Answer 7

Highest magnification= x500000
Level of resolution= 1nm (can resolve better than light microscope due to much shorter wavelength)
Waves pass through specimen.
Beam can damage specimen.
2D image.
Black and white( but colour can be airbrushed on)
Heavy metal, toxic stain- hazardous to user.
Specimen will be viewed dead due to preparation.
Not portable.
Dehydration cause distortion of specimen.
Lense made from electromagnetic coils.
Specimens must be kept in a near vacuum to prevent interference of unwanted molecules

Question 8

Scanning electron microscope

Answer 8

Highest magnification= x100000
Level of resolution= 20nm (can resolve better than light microscope due to much shorter wavelength)
Waves bounce off specimen.
Beam can damage specimen.
3D image.
Black and white( colour can be airbrushed on)
Heavy metal, toxic stain- hazardous to user.
Specimen will be dead due to preparation needed.
Not portable.
Lense made from electromagnetic coils.
Specimens must be kept in a near vacuum to prevent interference of unwanted molecules.