AQA Biology: 3.2.1.3 Methods of studying cells

Define "magnification"

How many times bigger the size of the image is than the size of the object

Define "resolution"

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

Define "cell fractionation"

The process where cells are broken up and the different organelles they contain are seperated out

Why is cell fractionation necessary?

To obtain large numbers of isolated organelles in order to study cell structure and function

Why is the tissue placed in a cold, isotonic, buffered solution before cell fractionation?

Cold - to reduce enzyme activity that might digest/break down the organelles
Isotonic - to prevent organelles bursting/shrinking as a result of osmotic gain/loss of water
Buffered - to maintain a constant pH to prevent any change in the structure of organelles or function of enzymes

What are the 2 stages in cell fractionation?

Homogenation + ultracentrifugation

Define "homogenation"

Blending the cells in a homogeniser to break the cell membranes and release the organelles from the cells. The resultant fluid (homogenate) is then filtered.

Why is the homogenate filtered before ultracentrifigation?

To remove any unbroken cells or large pieces of debris

Define "ultracentrifugation"

The process by which the fragments in the filtered homogenate are seperated in a machine called a centrifuge

What does the centrifuge do?

Spins tubes of homogenate at a very high speed in order to create a centrifugal force

Process of ultracentrifugation (for animal cells)

1. Tube of homogenate placed in centrifuge and spun at slow speed
2. Heaviest organelles (nuclei) forced to bottom of tube, where they form a pellet
3. The fluid at the top of the tube (supernatant) removed, leaving just sediment of nuclei
4. Supernatant trasnferred to another tube and spun in the centrifuge at faster speed than before
5. Next heaviest organelles (mitochondria) forced to bottom of tube
6. Process continues, with lighter organelles being sedimented and spread out at higher speeds

What property of cell organelles allows them to be seperated by ultracentrifugation?

Different densities

How does a light microscope produce an image?

Beam of light shone through the specimen

How does a TEM produce an image?

- Condenser electromagnet focuses beam of electrons from electron gun onto specimen
- Beam passes through thin section of specimen
- Parts of specimen that let electrons pass through appear light, parts that absorb electrons appear dark
- Image produced on screen
- Image can be photographed to produce a microphotograph

How does a SEM produce an image?

- Beam of electrons shone onto surface of metal-salt-stained specimen
- Electrons scattered in pattern depending on contours of surface of specimen
- Electrons reflected back onto detector
- Computer analysis of pattern of scattered electrons + secondary electrons produced
- 3D image produced

Advantages of light microscope

- Inexpensive to purchase + operate
- Can be used with living specimens
- Original colour of specimen can be viewed
- Easy to operate
- Relatively small

Advantages of TEM

- 2000x higher resolution than light microscope
- Detailed 2D images

Advantages of SEM microscope

- 2000x higher resolution than light microscope
- Detailed 3D images

Disadvantages of light microscope

Poor magnification (400-1000x) and resolution

Disadvantages of TEM microscope

- Needs vacuum to work; living samples cannot be used
- B&W image
- Complex staining process
- Image may contain artefacts
- Extremely expensive
- Needs extremely thin specimen
- Only produces 2D image

Disadvantages of SEM microscope

- Needs vacuum to work; living samples cannot be used
- B&W image
- Complex staining process
- Image may contain artefacts
- Extremely expensive
- Lower resolving power than TEM
- Cannot view inside of samples