Microscopy/Cell fractionation

Question 1

Cell fractination

Answer 1

The process in which different parts and organelles of a cell a separated so that they can be studied in detail.

Question 2

What is the most common cell fractionation

Answer 2

Differential centrifugation.

Question 3

Homogenation steps

Answer 3

1. The cells are first blended in an homogeniser forming the resultant fluid called the homogenate. This tube of homogenate is then placed in a centrifuge and spun at a slow speed.
2. The heaviest organelles, the nuclei, are forced to the bottom of the tube where a thin sediment or pellet forms.
3. The fluid at the top, called the supernatant, is removed which leaves just the sediment of the nuclei. The supernatant is then transferred to another tube and spun at a slightly faster speed. This time the pellet that forms contains the next heaviest organelle, the mitochondria.

4.This process continues so that each time the speed is increased the next heaviest organelle is
sedimented and separated out.

Question 4

What must the conditions be for cell fraction to take place:

Answer 4

Cold - To reduce enzyme activity that might break the organelles

Same water potential as the tissue - to prevent organelles from bursting or shrinking as a result of osmotic gain or loss of water

Buffered - So pH does not fluctuate. any change could alter structure of organelles or functioning of enzymes

Question 5

Light microscope

Answer 5

• Have poor resolution as a result of relatively long wavelength of light
• use a pair of convex glass lenses that can resolve images that are 0.2um
  apart

Question 6

Magnification equation

Answer 6

size of Image /Size of Actual

Question 7

What are the two types of Electron microscope

Answer 7

Transmiison electron microscope

Scanning electron microscope

Question 8

Transmisson Microscope

Answer 8

A beam of electrons passes through a thin section of a specimen. Areas that absorb the electrons appear darker on the electron micrograph that is produced.

Question 9

Scanning electron Microscope

Answer 9

In a scanning electron microscope a beam of electrons passes across the surface and scatter. The pattern of scattering builds up a 3D image depending on
the contours of the specimen.

Question 10

Limitations of Both electron microscopes

Answer 10

• The whole system must be in a vacuum so living specimens cannot be observed.
• A complex staining process is required which may introduce artefacts into the image.
• Specimens have to be very thin, particularly for TEM so that the electrons can pass through.
• SEM has a lower resolving power than TEM, but both have greater resolving power than a light microscope.