3.2.1.3 methods of studying cells

Question 1

describe the method to calculate mean number of organelles using an optical microscope.

Answer 1

1. measure diameter of field of view and calculate area
2. using stage micrometer and eyepiece graticule
3. count the number of capillaries in large number of fields of view and calculate mean
4. select fields of view randomly

Question 2

What is resolution?

Answer 2

+ how much detail the image has; the better the resolution is the microscope is able to distinguish between two points that are close together

Question 3

What is magnification?

Answer 3

+ how much bigger the image is compared to the specimen sample

magnification = image size/actual size

Question 4

What is an optical microscope?

Answer 4

+ uses light to form the image and focuses this light using glass lenses
+ maximum resolution 0.2µm
+ cannot view smaller organisms such as; ribosomes, ER and lysosomes, mitochondria may be visible but not clear
+ maximum magnification is approx x1500

Question 5

How do you prepare a temporary mount?

Answer 5

1. pipette a drop water onto the slide (so the specimen sticks)
2. use tweezers to place a thin section of specimen on the damp
3. add a drop of stain (e.g eosin for cytoplasm or iodine for starch grains)
4. add a cover slip to protect the specimen (making sure not to get any air bubbles in)
5. thin (single/few layers of cells) to allow light to pass through the specimen

Question 6

What are the advantages of optical microscopes?

Answer 6

+ can see living specimens
+ cheaper
+ show colour
+ less time consuming to prepare slides

Question 7

What are the disadvantages of optical microscopes?

Answer 7

+ lower magnification and resolution
+ 2D
+ can’t view smaller organelles in detail

Question 8

What is a TEM?

Answer 8

+ transmission electron microscope
+ specimen must be very thin and placed in a vacuum
+ a beam of electron passes through the specimen and is dispersed by the structures there
+ scattered electrons are then captured on a photographic plate
+ electrons pass through denser part of sample less easily so areas that are darker are more denser
+ 2D
+ magnification up to x250 000
+ resolution 2nm

Question 9

What are the advantages of electron microscopes?

Answer 9

+ can have a x200 better resolution that light microscopes - because electrons have a shorter wavelength
+ produced detailed images
+ SEM produces 3D images

Question 9

What is SEM?

Answer 9

+ electrons ‘bounce’ off metal-salt-stained sample
+ uses an electromagnet to focus the beam of electrons
+ 3D image is built up on a screen
+ magnification x100 000
+ resolution between 3 to 20nm

Question 10

What are the disadvantages of electron microscopes?

Answer 10

+ the interior is a vacuum - cannot view live specimens
+ requires very careful staining and the specimen often has to be very thin however image is only black and white
+ can lead to artefacts
+ very expensive

Question 11

What are some limitations of TEM?

Answer 11

+ specimen must be very thin to allow the electrons to penetrate
+ therefore flat 2D image
+ can overcome by taking series of sections

Question 12

What are some limitations of SEM?

Answer 12

+ lower resolution than TEM but still 10 times better than light microscope

Question 13

What are artefacts?

Answer 13

+ things that you can see down the microscope that aren’t part of the specimen you are looking at
+ e.g. dust, air bubbles, fingerprints
+ usually made during the preparation of the specimen

Question 14

What is ultracentrifugation?

Answer 14

+ process where cells are broken up and the different organelles they contain separated out
+ used to help us study cell structure and function
+ two stage process: homogenation and ultracentrifugation

Question 15

Step one of ultracentrifugation

Answer 15

+ tissue to be studied is cut into smaller pieces (minced) and placed in a cold, isotonic and buffered solution

Question 16

Step two of ultracentrifugation

Answer 16

+ these are then ground into smaller pieces using a homogensier

Question 17

Step three of ultracentrifugation

Answer 17

+ this releases the organelles from within the cell membrane

Question 18

Step four of ultracentrifugation

Answer 18

+ the homogenate is filtered to remove any complete cells and large debris

Question 19

Step five of ultracentrifugation

Answer 19

+ a suspension of homogenate is then placed in a test tube and then centrifuged
+ the faster the speed at which the tube is spun, the greater the force generated

Question 20

Step six of ultracentrifugation

Answer 20

+ at sloer speeds the larger fragments collect at the bottom of the tube and smaller ones remain near the top suspended in a liquid called the SUPERNATENT LIQUID

Question 21

Step seven of ultracentrifugation

Answer 21

+ these larger fragments (sediment pellets) are then removed and the supernatent remaining is respun at a faster speed
+ some of the smaller fragments collect at the bottom forming a new pellet

Question 22

What is the order of organelle collection?

Answer 22

1. whole cells, nuclei, cytoskeletons
2. mitochondria, lysosomes, peroxisomes
3. microsomes, other small vesicles
4. ribosomes, viruses, large macromolecules