topic 2 - analysis of cell components

Question 1

what is magnification

Answer 1

how much bigger the image is than the specimen

Question 2

how do you calculate magnification

Answer 2

size of image ÷ size of real object

Question 3

what is resolution

Answer 3

how detailed the image is - how well a microscope distinguishes between two points that are close together

Question 4

what are the two main types of microscope

Answer 4

optical (light) microscope and electron microscope

Question 5

what do optical microscopes use to form an image

Answer 5

light

Question 6

what is the maximum resolution of an optical microscope, what does that include

Answer 6

about 0.2 micrometers - meaning you can’t use an optical microscope to view organelles smaller than 0.2 μm - including ribosomes, endoplasmic reticulum and lysosomes

Question 7

what’s the maximum useful magnification of an optical microscope

Answer 7

about x1500

Question 8

what do electron microscopes use to form an image

Answer 8

electrons

Question 9

what microscope has a higher resolution

Answer 9

electron microscopes give a more detailed image of

Question 10

what is the maximum resolution of an electron microscope

Answer 10

about 0.0002 micrometers (about 1000 times higher than an optical microscope)

Question 11

what’s the maximum useful magnification of an electron microscope

Answer 11

about x 1 500 000

Question 12

what are the two types of electron microscopes

Answer 12

(1) scanning
(2) transmission

Question 13

what do TEMs use electromagnets for

Answer 13

to focus a beam of electrons, which is then transmitted through the specimen

Question 14

why do some parts of images look darker in TEMs

Answer 14

denser parts of the specimen absorb more electrons which makes them look darker

Question 15

what can TEMs only be used on

Answer 15

thin specimens

Question 16

what is a pro to TEMs

Answer 16

they give high resolution images - so you see the internal structure of organelles like chloroplasts

Question 17

how do SEMs form an image

Answer 17

SEMs scan a beam of electrons across the specimen - this knocks iff electrons from the specimen, which gathered in a cathode ray tube to form an image

Question 18

what do the images you end up with from SEMs show

Answer 18

the surface area of the specimen and they can be 3D

Question 19

what’s a pro to SEMs

Answer 19

they can be used on thick specimens

Question 20

what’s a con to SEMs

Answer 20

they give lower resolution images than TEMs

Question 21

what’s the first step to preparing a ‘temporary mount’ of a specimen on a slide

Answer 21

starr by pipetting a small drop of water onto the slide (a trip of clear glass or plastic) - then use tweezers to place a thin section of your specimen on top of the water drop

Question 22

what’s the second step to preparing a ‘temporary mount’ of a specimen on a slide

Answer 22

add a drop of a stain - stains are used to highlight objects in a cell (e.g eosin is used to make the cytoplasm show up - iodine in potassium iodine solution is used to stain startch grains in plant cells)

Question 23

what’s the third step to preparing a ‘temporary mount’ of a specimen on a slide

Answer 23

add the cover slip (a square of clear plastic that protects the specimen) - to do so, stand the slip upright on the slide, next to the water droplet - then carefully tilt and lower it so ur covers the specimen - try not to get any air bubbles under there - they’ll obstruct your view of the specimen

Question 24

what are the three steps to cell fractionation

Answer 24

(1) homogenisation - breaking up the cells
(2) filtration - getting rid of the big bits
(3) ultracentrifugation - separating the organelles

Question 25

how can homogenisation be done

Answer 25

vibrating the cells or by grinding the cells up in a blender - this breaks up the plasma membrane and releases the organelles into solution - the solution must be kept ice-cold to reduce the activity of enzymes that break down organelles - the solution should be isotonic - meaning it should have the same concentration of chemicals as the cells being broken down, to prevent damage to the organelles through osmosis - a buffer solution should be added to maintain pH

Question 26

how is filtration done

Answer 26

homogenised cell solution is filters through a gauze to separate any large cell debris or tissue debris (like connective tissue from the organelles) - the organelles are much smaller than the debris so they pass through the gauze

Question 27

what is ultracentrifugation used for

Answer 27

after filtration you’re left with a solution containing a mixture of organelles - to separate a particular organelle from all the others you use ultracentrifugation

Question 28

what’s the first step to ultracentrifugation

Answer 28

the cell fragments are poured into a tube - the tube is put into a centrifuge (a machine that separates material by spinning) and is spun at a low speed - the heaviest organelles get flung to the bottom of the tube by the centrifuge - they form a thick sediment at the bottom (the pellet) - the rest of the organelles stay suspended in the fluid above the sediment (the supernatant)

Question 29

what’s the second step to ultracentrifugation

Answer 29

the supernatant is drained off, poured into another tube, and spun in the centrifuge at a higher speed - the heaviest organelles (this time the mitochondria) form a pellet at the bottom of the tube - the supernatant containing the rest of the organelles is drained off and spun in the centrifuge at an even higher speed

Question 30

what’s the third step to ultracentrifugation

Answer 30

this process is repeated at higher and higher speeds, until all the organelles are separated out - each time the pellet at the bottom of the tube is made up of lighter and lighter organelles