topic 2 - analysis of cell components Flashcards
cgp (topic 2A) 32 - 33
what is magnification
how much bigger the image is than the specimen
how do you calculate magnification
size of image ÷ size of real object
what is resolution
how detailed the image is - how well a microscope distinguishes between two points that are close together
what are the two main types of microscope
optical (light) microscope and electron microscope
what do optical microscopes use to form an image
light
what is the maximum resolution of an optical microscope, what does that include
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
what’s the maximum useful magnification of an optical microscope
about x1500
what do electron microscopes use to form an image
electrons
what microscope has a higher resolution
electron microscopes give a more detailed image of
what is the maximum resolution of an electron microscope
about 0.0002 micrometers (about 1000 times higher than an optical microscope)
what’s the maximum useful magnification of an electron microscope
about x 1 500 000
what are the two types of electron microscopes
(1) scanning
(2) transmission
what do TEMs use electromagnets for
to focus a beam of electrons, which is then transmitted through the specimen
why do some parts of images look darker in TEMs
denser parts of the specimen absorb more electrons which makes them look darker
what can TEMs only be used on
thin specimens
what is a pro to TEMs
they give high resolution images - so you see the internal structure of organelles like chloroplasts
how do SEMs form an image
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
what do the images you end up with from SEMs show
the surface area of the specimen and they can be 3D
what’s a pro to SEMs
they can be used on thick specimens
what’s a con to SEMs
they give lower resolution images than TEMs
what’s the first step to preparing a ‘temporary mount’ of a specimen on a slide
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
what’s the second step to preparing a ‘temporary mount’ of a specimen on a slide
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)
what’s the third step to preparing a ‘temporary mount’ of a specimen on a slide
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
what are the three steps to cell fractionation
(1) homogenisation - breaking up the cells
(2) filtration - getting rid of the big bits
(3) ultracentrifugation - separating the organelles
how can homogenisation be done
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
how is filtration done
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
what is ultracentrifugation used for
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
what’s the first step to ultracentrifugation
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)
what’s the second step to ultracentrifugation
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
what’s the third step to ultracentrifugation
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
