Cell Fractionation And Microscopy

Question 1

What must be done before cells can undergo cell fractionation?

Answer 1

Cell tissue is cut up and placed in a cold, buffered solution with the same water potential.

Question 2

What happens during homogenisation?

Answer 2

• Cells are blended in a homogeniser, causing organelles to be released from cells
• The homogenate that forms is filtered to remove complete cells and large pieces of debris.

Question 3

Why must the solution that tissue is placed in be cold?

Answer 3

To reduce enzyme activity that may break down organelles

Question 4

Why must the solution that tissue is placed in be pH buffered?

Answer 4

To prevent the structure of organelles, or the function of enzymes, being altered

Question 5

Why must the solution that tissue is placed in have the same water potential?

Answer 5

So the organelles of the cells do not burst or shrink as a result of osmosis

Question 6

What happens during ultracentrifugation?

Answer 6

• A homogenate is placed into a centrifuge and spun at a slow speed. This causes heavy organelles, the nuclei, to be forced to the bottom of the tube and form a pellet.
• The fluid at the top, the supernatant is separated from the pellet and transferred to a new tube. The new tube is spun at a slightly faster speed so that the next heaviest organelles, the mitochondria form a pellet
• The process is repeated to separate the next heaviest organelle with each increase in speed.

Question 7

What is the resolution of each type of microscope?

Answer 7

Light microscope: 0.2µm

Transmission electron microscope: 0.1nm

Scanning electron microscope: 20nm

Question 8

How does a TEM work?

Answer 8

A beam of electrons are passed through a thin section of a sample from below. Areas that absorb electrons will appear darker while areas that allow electrons to pass through will appear lighter.

This produces an image on a screen that can be photographed to give a photomicrograph

Question 9

How does an SEM work?

Answer 9

A beam of electrons passes across the surface of the specimen from above. The electrons scatter, the patterns of which are used to build a 3-D image depending on he contours of the specimen.

Question 10

What are the limitations of a TEM?

Answer 10

• The whole system must be in a vacuum so that air particles do not deflect electrons out of the beam alignment. Means that living specimens cannot be observed.
• Complex staining process is required, which may produce artefacts for the image.
• Specimen must be very thin so that electrons can pass through
• Using high energy electron beams to achieve higher resolution may lead to destruction of the specimen.
• More expensive and time consuming than light microscopy

Question 11

What are the limitations of an SEM?

Answer 11

• Specimen must be in a vacuum to prevent particles deflecting electrons out of beam alignment. Means that living specimens cannot be viewed.
• Lower resolving power than a TEM
• Complex staining process required, which may introduce artefacts to the photomicrograph
• More expensive and time consuming than light microscopy