Methods of Studying Cells

Question 1

What conditions are required when homogenising a sample?

Answer 1

• ice cold; to prevent enzymes breaking down organelles
• pH buffer; to maintain a constant pH (any change could damage organelles/ impact protein structure)
• isotonic solution; to prevent osmosis from causing damage to organelles

Question 2

What is done after homogenisation, before a sample undergoes ultracentrifugation?

Answer 2

the sample is filtered through a gauze, separating larger debris from smaller organelles

Question 3

What is the supernatant?

Answer 3

the liquid that is removed from the tube after being spun in a centrifuge.It contains the lighter organelles which did not sink at the previous speed.

Question 4

Why would TEM be favourable to SEM to image an organelle and identify it’s structure?

Answer 4

• Higher resolution
• Can see internal structures
• Greater magnification

Question 5

Why are EMs used instead of light microscopes?

Answer 5

As they have higher magnifications and higher resolutions, therefore the smaller internal structures can be viewed in more detail

Question 6

What are 3 limitations of using an EM?

Answer 6

• thin specimens are needed (TEM)
• needs to be in a vacuum, specimens are not living
• complex staining and preparation, artefacts could potentially be introduced
• (black and white images)

Question 7

How do SEMs differ from TEMs?

Answer 7

-SEMs prodcuce 3D images, TEMs produce 2D images
- SEMs need thinner samples
- SEMs involve electrons ‘bouncing’ off of the sample

Question 8

What is meant by the term ‘resolution’?

Answer 8

the ability to distinguish two very small structures as separate (in a magnified image)

Question 9

In microscopy, what are artefacts?

Answer 9

Things you can see down the microscope that are not part of the specimen (e.g bubbles, dust, fingerprints etc.)

Question 10

What is the max. resolving power of a light microscope?

Answer 10

0.2 micrometres

Question 11

What is the max. resolving power of an electron microscope?

Answer 11

0.1nm

Question 12

What is the equation used to work out magnification?

Answer 12

Magnification = size of image/size of real object

Question 13

Briefly outline how a TEM works

Answer 13

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 14

Briefly outline how a SEM works

Answer 14

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 15

Which organelle sinks to the bottom first in unltracentrifugation?

Answer 15

nuclei

Question 16

Contrast how an optical microscope and a transmission electron microscope work and contrast the limitations of their use when studying cells (give 6 out of 8)

Answer 16

1. TEM use electrons and optical use light;
2. TEM allows a greater resolution;
3. with TEM smaller organelles/greater detail in organelles
4. TEM view only dead specimens and optical can view live specimens;
5. TEM does not show colour and optical can;
6. TEM requires thinner specimens;
7. TEM requires a more complex preparation;
8. TEM focuses using magnets and optical uses lenses

Question 17

When studying a micrograph, why might a cell membrane appear as 2 dark lines?

Answer 17

• phospholipid bilayer
• stains phosphates
• phosphate heads are on the inside and outside

Question 18

What is the main limitation of a light microscope?

Answer 18

low resolution due to longer wavelength of light