Unit 2 - Microscopes & Ultracentrifugation

Question 1

Define Magnification and resolution.

Answer 1

Magnification - How many times bigger an image gets under a microscope
Resolution - Detail of image, how well a microscope distinguishes between 2 points which are close together

Question 2

Describe how an optical light microscope works.

Answer 2

• Light beam is focused
• using a convex glass lens
• through air & passes beam through a sample
• max magnification of x1500

Question 3

Describe 2 advantages and disadvantages of optical light microscopes.

Answer 3

Advantages
- Coloured images - living organisms seen
- Cheap and easy to use

Disadvantages
- Poor resolution
- 2D image

Question 4

Describe how a transmission electron microscope works.

Answer 4

• A beam of electrons passes through a thin section of a specimen.
• Denser areas absorb more electrons so appear darker on the electron micrograph

Question 5

Describe 2 advantages and disadvantages of a transmission electron microscope.

Answer 5

Advantages
- Higher magnification and higher resolution than OLM & SEM
- Smaller objects can be seen

Disadvantages
-The whole system must be in a vacuum so no living organisms can be observed
- Black and white images, Artefacts
-Specimen has to be very thin, 2D image

Question 6

Describe how a scanning electron microscope works.

Answer 6

• A beam of electrons passes across the surface and scatter
• Electrons bounce of surface of specimen & pattern of scattering builds up a 3D image

Question 7

Describe 2 advantages and disadvantages of a scanning electron microscope.

Answer 7

Advantages
-Can magnify significantly more than the light microscope
- 3D image

Disadvantages
-The whole system must be in a vacuum so no living organisms can be observed
- Black and white images, Artefacts

Question 8

What does cell fractionation involve?

Answer 8

Cell fractionation is the process in which different parts and organelles of a cell are separated to be studied in detail.

Question 9

What is the first stage of cell fractionation?

Answer 9

• Break down tissue into cells - Cutting
  Homogenisation
• Cells blended in homogenizer releasing cell organelles
  (Cold solution and controlled pH)
• Filter Homogenate to remove any large debris and tissue debris

Question 10

What is the second stage of cell fractionation?

Answer 10

Ultracentrifugation
- Centrifuge homogenate at increasing sewuence speeds
- At faster speeds(more force), smaller fragments collected in a liquid called the SUPERNATENT LIQUID, supernantent liquid at top cell free
- At slower speeds, the larger fragments(more dense) collected at bottom of the tube
INCLUDE PELLET INFO (E.G chloroplast 2nd pellet)

Question 11

What are the solutions required for cell fractionation?

Answer 11

Buffered – so that the pH does not fluctuate.
constant pH/ prevent from denaturing

Cold – to reduce enzyme activity that might
break down the organelle

Isotonic (same water potential as the tissue) -
to prevent organelles from bursting under osmotic pressure

Question 12

Why is differential centrifugation needed?
- Order of size of the organelle

Answer 12

• Since it involves centrifuging at different speeds
  1. Nuclei
  2. Mitochondria/Chloroplast
  3. Lysosome
  4. Endoplasmic reticulum/Golgi
  5. Ribosome

Question 13

Outline how you would prepare a temporary mount of tissue for an optical light microscope.

Answer 13

• Obtain a thin section of tissue
• Place plant tissue in a drop of water
• Stain tissue on the slide with IODINE to make structures visible
• Add coverslip using mounted needle at 45degrees to avoid air bubbles
  IODINE IN POTASSIUM IODIDE SOLUTION TO VIEW STARCH GRAINS IN PLANT CELLS

Question 14

Magnification equation

Answer 14

size of an image/size of a real object