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 1

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

Answer 1

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

Question 2

What is the supernatant?

Answer 2

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 3

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

Answer 3

• Higher resolution
• Can see internal structures
• Greater magnification

Question 4

Why are EMs used instead of light microscopes?

Answer 4

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

Question 5

What are 3 limitations of using an EM?

Answer 5

• 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 6

How do SEMs differ from TEMs?

Answer 6

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

Question 7

What is meant by the term ‘resolution’?

Answer 7

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

Question 8

In microscopy, what are artefacts?

Answer 8

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

Question 9

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

Answer 9

0.2 micrometres

Question 10

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

Answer 10

0.1nm

Question 11

What is the equation used to work out magnification?

Answer 11

Magnification = size of image/size of real object

Question 12

Briefly outline how a TEM works

Answer 12

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 13

Briefly outline how a SEM works

Answer 13

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 14

Which organelle sinks to the bottom first in unltracentrifugation?

Answer 14

nuclei

Question 15

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 15

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 16

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

Answer 16

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

Question 17

What is the main limitation of a light microscope?

Answer 17

low resolution due to longer wavelength of light