1.2 Studying Cells

Question 1

Why is an isotonic solution used in homogenisation?

Answer 1

To prevent net water movement via osmosis, which could damage organelles by causing them to burst or shrivel.

Question 2

Define differential centrifugation and its purpose.

Answer 2

Differential centrifugation is where a supernatant is spun at increasingly higher speeds. It is used to separate cell organelles of different densities.

Question 3

Describe the purpose of a hypotonic solution and how it is suited to this purpose.

Answer 3

The solution has a higher water potential than the cell organelles. After the outer membrane is broken down, water moves into the the organelles, causing them to swell and lyse.

Question 4

Why is a cold solution used in homogenisation?

Answer 4

To reduce the kinetic energy, rate of reaction and ACTION OF ENZYMES that could damage organelles. It also prevents enzymes from denaturing.

Question 5

Why are electron microscopes better equipped for seeing smaller organelles than light microscopes?

Answer 5

Electron microscopes have a higher resolution than light microscopes. This is because the beam of electrons used had a smaller wavelength than light, allowing them to distinguish between smaller objects.

Question 6

How can you gain a greater understanding of cell structure?

Answer 6

Magnify and study the structures once they have been isolated.

Question 7

How do you prepare a sample for under a light microscope?

Answer 7

• Add a drop of water onto a slide.
• Place the specimen on the water.
• Add stain if necessary to help visualise organelles and cell structure.
• Place the edge of the coverslip do that it touches the edge of the water.
• Slowly lower the coverslip to prevent the formation and entrapment of an air bubble.

Question 8

Define resolution.

Answer 8

Resolution is the ability to distinguish between things that are close together.

Question 9

Define magnification.

Answer 9

Making things appear larger.

Question 10

Light microscope.
Specimens are illuminated with ______ focussed by _________. They can be viewed by ______ or by using ___________.
Specimens can be ______________, but often need to be ________ with ______ to be seen.
Magnification is limited to _______ as any more would compromise the ___________.
Resolution is limited by the _____________ (_____ to ______). The _______ the ________, the better the _____________. Resolution is about _______ (_______ the __________ of ________). Objects ________ cannot be _____________.
Light microscopes can be used to see up to the _________, but nothing ________.

Answer 10

Light microscope.
Specimens are illuminated with LIGHT focussed by GLASS LENSES. They can be viewed by EYE or by using PHOTOGRAPHIC FILM. Specimens can be DEAD OR ALIVE, but often need to be STAINED with DYE to be seen.
Magnification is limited to 1500X as any more would compromise the RESOLUTION.
Resolution is limited by the WAVELENGTH OF LIGHT to (400nm-600nm) The SMALLER the WAVELENGTH, the better the RESOLUTION.
Resolution is about 200nm (the HALF the WAVELENGTH of LIGHT) Objects SMALLER cannot be DISTINGUISHED.
Light microscopes can be used to see up to the NUCLEUS, but nothing SMALLER.

Question 11

Electron microscope.
This uses a ______ of ________.
_________ can behave like _______.
They are easily _______, such as by a _________.
They are focussed using ___________.
They are detected using ____________ or ______________.
A _________ of ___________ has a very ___________, and so can be used to see objects as small as _______ with a ___________ of _____.
It’s ___________ is ___________.

Answer 11

Electron microscope.
This uses a BEAM of ELECTRONS.
ELECTRONS can behave like WAVES.
They are easily PRODUCED such as by a HOT WIRE.
They are focussed using ELECTROMAGNETS.
They are detected using A PHOSPHOR SENSORS or PHOTOGRAPHIC FILM.
A BEAM of ELECTRONS has a very SMALL RESOLUTION, and so can be used to see objects as small as RIBOSOMES (20nm) with a RESOLUTION of 1nm.
It’s MAGNIFICATION is 500 000x.

Question 12

What are the limitations of electron microscopes?

Answer 12

• Specimens must be fixed in resin and viewed in a vacuum, and no must therefore be dead.
• Specimens can be damaged by the electron beam.
• Specimens must be stained with an electron dense chemical such as lead or gold.
• Images are black and white.
• Much more expensive than a light microscope.
• The preparation of the specimen could lead to the production of artefacts.
• Specimens must be very thin.

Question 13

Define artefact.

Answer 13

An observed structure that is due to the preparation of a specimen, and so is not real.

Question 14

What are the two types of electron microscope?

Answer 14

• TEM - Transmission electron microscope.

- SEM - Scanning electron microscope.

Question 15

What is a TEM? How does it work, and what sort of image does it form?

Answer 15

A transmission electron microscope is the most common form of electron microscope. It works much like a microscope, transmitting a beam if electrons through a thin specimen, and focussing the electrons using electromagnets to for an image on a phosphor screen or photographic film. It has the best resolution of all microscopes compared, and produces a 2D image. It is often used to look at the internal structure of a cell.

Question 16

What is a SEM? How does it work, and what sort of image does it form?

Answer 16

A scanning electron microscope is an electron microscope that fires a beam of electrons across a specimen and collects the electrons scattered by its surface. It has a poorer resolution than the TEM, but produces a 3D image if a cell’s surface. The specimen does not have to as thin as with an TEM.

Question 17

Compare a TEM and an SEM.

Answer 17

• TEM transmits a thin beam of electrons, while SEM collects scattered electrons from the surface of a cell.
• TEM produces a 2D image, SEM produces a 3D image.
• TEM has a greater resolution.
• TEM is used to look at cell structure, whilst SEM is used to look at the cell surface and shape.
• Specimen must be thinner for TEM.

Question 18

What is the equation for magnification?

Answer 18

Magnification = Image size / mm

Actual size / mm

Question 19

What is the conversion between mm, um and nm?

Answer 19

mm-um = x10^3
mm-nm = x10^6
Etc.

Question 20

Compare electron and light microscopes.

Answer 20

Illumination:
L - Light.
E - Beam of electrons.
Focussed by:
L - Lens.
E - Electromagnets.
Max magnification:
L - x1500.
E - x500000.
Resolution:
L - 200nm.
E - 1nm.
Specimens:
L - Living and dead.
E - Dead.
Cost of equipment:
L - Low.
E - High.
Image:
L - Colour.
E - Black and white.

Question 21

Whet is cell fractionation, and what are the steps?

Answer 21

The separation of organelles for study.

Homogenisation, filtration and ultracentrifugation (differential centrifugation).

Question 22

What is homogenisation, and his is it carried out?

Answer 22

Homogenisation is the preparation of suspended organelles to undergo differential centrifugation to separate them.
Homogenisation is carried out by breaking open cells and releasing the organelles in an isotonic, ice cold, buffered solution.

Question 23

Why is the solution used in homogenisation buffered?

Answer 23

To prevent pH changes that could denature proteins.

Question 24

What kind of solution is used in homogenisation?

Answer 24

Cold, isotonic and buffered.

Question 25

Why is the solution filtered before differential centrifugation?

Answer 25

To remove cell, debris, complete cells or tissue.

You could also discard the first pellet from ultracentrifugation to do the same.

Question 26

Describe and explain the process of ultracentrifuge/ differential centrifugation.

Answer 26

The solution of suspended organelles is centrifuges at a low speed.
Larger, denser organelles are forced into a pellet which can be removed.
The solution can then be resuspended and centrifuges again at at higher speed to remove another pellet.
Eventually a final supernatant containing cytoplasm can be isolated.
The pellets can be identified using microscopy.

Question 27

Describe the full process of cell fractionation.

Answer 27

The solution of cells in homogenised in a ice-cold, isotonic and buffered solution.
The solution is then filtered. (Or the first pellet in differential centrifugation is removed)
The solution of suspended organelles is centrifuges at a low speed.
Larger, denser organelles are forced into a pellet which can be removed.
The solution can then be resuspended and centrifuges again at at higher speed to remove another pellet.
Eventually a final supernatant containing cytoplasm can be isolated.
The pellets can be identified using microscopy.

Question 28

Order in which organelles are separated in ultracentrifugation.

Answer 28

It goes in order of density, with the most dense organelles coming first.
Nucleus.
Mitochondria.
Chloroplasts. (Similar density to mitochondria, only slightly less dense).
rER, sER and Golgi apparatus.
Ribosomes.