Methods of studying cells

Question 1

Benefits of optical microscope

Answer 1

Use light to form images

Cheaper

Easy to move

Doesn’t take up a lot of space

Question 2

Limitations of optical microscope

Answer 2

Maximum resolution of 0.2um so cannot see organelles smaller than that

Maximum magnification of x1500

Question 3

Benefits of transmission electron microscopes

Answer 3

Give high resolution images so you can see internal structures of organelles

Question 4

Limitations of transmission electron microscopes

Answer 4

Have to view specimen in a vacuum

Have to be dead organisms

The specimen has to be thin

Question 5

Benefits of scanning electron microscopes

Answer 5

Show the 3D outer surface of the organelles

Can be used on thick specimens

Question 6

Limitations of scanning electron microscopes

Answer 6

Give lower resolution images than TEMS

Can only be used on living specimens

Question 7

Measuring the size of an object viewed with an optical microscope

Answer 7

Requires a microscope slide with a micrometer scale

You will put the micrometer slide on the microscope stage, focus on it with each objective starting from low to high power and measure the field of view (diameter)

Question 8

Magnification

Answer 8

How many times an image has been enlarged by

Question 9

Resolution

Answer 9

The minimum distance by which two objects can be distinguished from one another

Question 10

What is the formula to work out the MAGNIFICATION of an image?

Answer 10

Magnification = image size ÷ object size (actual size)

Question 11

Cell fractionation

Answer 11

This process involves breaking up a suitable sample of tissue and then centrifuging the mixture at different speeds

Cell fractionation can be split into three stages:
Homogenisation
Filtration
Ultracentrifugation

Question 12

Homogenisation

Answer 12

Homogenisation is the biological term used to describe the breaking up of cells

The sample of tissue (containing the cells to be broken up) must first be placed in a cold, isotonic buffer solution

The solution must be:
Ice-cold to reduce the activity of enzymes that break down organelles
Isotonic (it must have the same water potential as the cells being broken up) to prevent water from moving into the organelles via osmosis, which would cause them to expand and eventually damage them
Buffered (have a buffer solution added) to prevent organelle proteins, including enzymes, from becoming denatured

The tissue-containing solution is then homogenised using a homogeniser

This is a blender-like machine that grinds the cells up (the cells can also be vibrated until they break up)

This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate

Question 13

Filtration

Answer 13

The homogenate (containing the homogenised cells) is then filtered through a gauze

This is to separate out any large cell debris or tissue debris that were not broken up

The organelles are all much smaller than the debris and are not filtered out (they pass through the gauze)

This leaves a solution (known as the filtrate) that contains a mixture of organelles

Question 14

Ultracentrifugation

Answer 14

The filtrate is placed into a tube and the tube is placed in a centrifuge

A centrifuge is a machine that separates materials by spinning

The filtrate is first spun at a low speed

This causes the largest, heaviest organelles (such as the nuclei) to settle at the bottom of the tube, where they form a thick sediment known as a pellet

The rest of the organelles stay suspended in the solution above the pellet

This solution is known as the supernatant

The supernatant is drained off and placed into another tube, which is spun at a higher speed

Once again, this causes the heavier organelles (such as the mitochondria) to settle at the bottom of the tube, forming a new pellet and leaving a new supernatant

The new supernatant is drained off and placed into another tube, which is spun at an even higher speed

This process is repeated at increasing speeds until all the different types of organelle present are separated out (or just until the desired organelle is separated out)

Each new pellet formed contains a lighter organelle than the previous pellet

The order of mass of these organelles (from heaviest to lightest) is usually:
Nuclei
Chloroplasts (if carrying out cell fractionation of plant tissue)
Mitochondria
Lysosomes
Endoplasmic reticulum
Ribosomes