Analysis of cell components

Question 1

Magnification Definiton

Answer 1

How many times bigger the zie of the image is than the actual object. magnification = size of image / over size of real object. Must be both in same units

Question 2

Resolution Defintion

Answer 2

How detailed the image is.
Can the microscope distiniguish between two objects really close together
If the microscope can’t, increasing the magnification will not help

Question 3

Two main types of microscopes

Answer 3

1. Optical Mircoscope

2. Electron Microscope

Question 4

Optical Microscope

Answer 4

1. Uses light to form the image
2. Max resolution 0.2 micrometres. Cannot be used to see objects smaller than 0.2 micrometres, including ribosomes, ER, lysosomes. Can see mitochondria + nucleus
3. Max useful magnification x1500

Question 5

Electron Microscope

Answer 5

1. Uses light to formn image
2. Max resolution 0.0002 micrometres.
3. Higher resolution than light microscope. More detailed image produced
4. Max useful magnification x1500 000

Question 6

Two types of electron microscope

Answer 6

1. Transmission Electron Microscope (TEM)

2. Scanning Electron Microscope (SEM)

Question 7

Transmission Electron Microscope (TEM)

Answer 7

1. Beams of electrons transmitted through specimen
2. Denser parts of specimen abosrb more electrons. These parts are darker on the image
3. Higher resoluion. More detailed image. Can see internal structures of cells very clearly
4. Must be thin specimen
5. Only 2D image

Question 8

Scanning Electron Microscope (SEM)

Answer 8

1. Beams of electrons scan across specimen
2. This produces a 3D image. Can see surface of specimen on the image
3. Lower resolution than TEM. Less detailed image
4. Can be used to view thick specimen

Question 9

Practical: How to view specimen under light microscope

Answer 9

1. Use pipet to add 1 drop of water to slide
2. Use tweezers to add thin section of the specimen to the slide
3. Add a stain. This makes structures inside cells show up. E.g. iodine stains starch, makes starch grains show up
4. Add cover slip. Ensure no air bubbles. This can obstruct view of specimen

Question 10

Cell Fractionation

Answer 10

Seperating organelles from the rest of the cell.
Consists of three stages:
1. Homogenisation (breaking up the cells)
2. Filtration (Filtering the homognate through a gauze, to remove large cell debris and tissue debris)
3. Ultracentrifugation (seperating a particular organelle from the rest)

Question 11

Homogenisation

Answer 11

1. The cells are grinded in a blender
2. This causes plasma membranes to break and therefore, the organelles are released into a solution
3. This solution must be
   - Ice Cold
   - Isotonic
   - Buffered

Question 12

Why should the homogenate solution be Ice Cold?

Answer 12

1. To reduce enzyme activity

2. Which ccan break down organelles

Question 13

Why should the homogenate solution be isotonic?

Answer 13

1. The conc of chemicals in the solution outside the cell should be the same as inside the cell. This is to prevent damage to organelles via osmosis

Question 14

Why should the homogenate solution be buffered?

Answer 14

1. Add buffer solution

2. To maintain stable pH

Question 15

Filtration

Answer 15

The homogenate is filtered through a gauze. This is to remove large cell debris and tissue debris. The organelles are smaller and therefore, pass through the gauze.

Question 16

Ultracentrifugation

Answer 16

1. The filtered homogenate is put in a test tube
2. This test tube is put in a centrifuge (machine which seperates materials by spinning)
3. At first, centrifuge is spun at a slow speed. This forces the heaviest organelles to the bottom of the tube (the nuclei). This forms a thick sedement at the bottom of the tube known as the pellet
4. The rest of the organelles are suspended in the fluid above the pellet, this is known as the supernatant
5. The supernatant is then drained off. It is put in a test tube and put in centrifuge and then spun at a higher speed. This forces heavier organelles to bottom of test tube (ie. mitochondria), forming pellet. The rest of the organelles are suspended in solution above pellet, forming supernatant
6. This supernatant is then drained off, put in test tube,. and spun even faster in centrifuge
7. This process is repeated till all organelles are seperated from the cell

Question 17

Why is the pellet made up of ligher organelles each time the centriguge is spun?

Answer 17

1. Centrifuge is spun at a higher speed each time

2. Organelles are seperated in mass order, from heaviest to lightest