Cell Fractionation + microscopy

Question 1

define cell fractionation

Answer 1

a technique which allows scientists to study the functions of organelles

Separating out all of the different organelles/components

Question 2

define homogenisation

Answer 2

Homogenise means to break up the tissue and break open the cells

Question 3

what is ultracentrifugation

Answer 3

ultracentrifugation is the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge

Question 4

what does the centrifuge do

Answer 4

spins tubes of homogenate at very high speeds to produce a centrifugal force

this force causes homogenate to separate

Question 5

describe homogenisation

Answer 5

In the first stage, take a sample of tissue containing the cells that we are interested in e.g. heart muscle tissue

Next, homogenise the tissue

Homogenise means to break up the tissue and break open the cells

note: the ER is a very large organelle - but this tends to get broken up during homogenisation

This can be done in a blender

Or we can use a homogeniser ( a glass tube containing a plunger)

Place the tissue sample into the glass tube

Cover with a buffer solution
Buffers keep the pH constant
This is important because if the pH changes, enzymes in the cells organelles could denature ||(COULD ALTER STRUCTURE OF ORGANELLES OR AFFECT THE FUNCTIONING OF ENZYMES)

The water potential of the buffer is the same as inside the cell
This prevents water from moving into the organelles by osmosis and causing them to burst

PREVENT ORGANELLES BURSTING OR SHRINKING AS A RESULT OF OSMOTIC GAIN OR LOSS OF WATER

The homogeniser is then placed on ice
Cooling the sample means that enzymes work more slowly, preventing any destructive enzymes from damaging the organelles REDUCE ENZYME ACTIVITY THAT MIGHT BREAK DOWN THE ORGANELLES

Now push the plunger up and down, to disrupt the tissue and break open the cells

This produces a cell homogenate
The cell homogenate contains all the organelles that we find in the cell

Question 6

what does the cell homogenate contain

Answer 6

The cell homogenate contains all the organelles that we find in the cell

contains all of the organelles which we would find in a cell

Question 7

what needs to be done to find out what the organelles in the cell homogenate do

Answer 7

In order to find out what these organelles do, we need to separate them

Question 8

describe cell fractionation

Answer 8

This produces a cell homogenate
The cell homogenate contains all the organelles that we find in the cell

In order to find out what these organelles do, we need to separate them (in order to understand this, we need to look at relative sizes of different organelles - nucleus, mitochondria, lysosomes, ribosomes)

Separating out all of the different organelles is called fractionation

ultracentrifugation

This is carried out using a machine called a centrifuge
1) We place the tubes containing the filtered cell homogenate into the sample holder of the centrifuge
2)The centrifuge now spins the sample
The organelles are flung , forced towards the bottom of the tube by the forces generated

Larger organelles…

3)First we start with a relatively low speed spin.

the tube looks like this (at the end of the spin)

https://cdn.savemyexams.com/cdn-cgi/image/w=1920,f=auto/uploads/2021/02/Cell-fractionation-stages-3.png - first picture

4) As the centrifuge spins, the larger organelles/heaviest such as the nuclei are flung, forced to the bottom of the tube by the forces generated forming a pellet (pellet containing nuclei)

5) Larger organelles (with a greater mass) such as the nucleus experience a greater force and move towards the bottom of the tube faster than the smaller organelles

6) The remaining organelles stay suspended in the liquid. This liquid is called the supernatant.

Now transfer the supernatant into a new tube, and centrifuge this at a higher speed

After the higher speed spin, the heaviest organelles (which are now mitochondria) now are forced to the bottom of the test tube and form pellet now contains mitochondria

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

Once again, transfer the supernatant to a new tube and centrifuge again at a higher speed. After this spin, the pellet now contains lysosomes

Take the supernatant one more time and transfer this to another tube for a final very high speed spin.

After this spin, the pellet now contains ribosomes

At this point, all of the organelles have been separated by size

Now we can test each fraction to determine how the organelles work

note: in plant cells the chloroplasts come after the nuclei but before the mitochondria

the order in which organelles are separated out - they are separated out in order of mass -from heaviest to lightest: nuclei, mitochondria, lysosomes, ER, ribosomes

Question 9

Additional points about cell fractionation

Answer 9

Additional - we need to keep the pellets on ice until we use them. this is to slow down enzymes which might damage the organelles

it is extremely difficult to separate the organelles fully e.g. the mitochondrial fraction might contain a very small number of nuclei and lysosomes

Also there are other organelles such as the endoplasmic reticulum and Golgi apparatus which might be present in your fractions

Question 10

How are the organelles separated in ultracentrifugation

Answer 10

In ultracentrifugation, the organelles are separated in order of mass - from lightest to heaviest

Question 11

describe what was happening after each spin

Answer 11

Each time, the pellet at the bottom is made from lighter and lighter organelles

Question 12

what is a micrometre

Answer 12

1 x 10-6 metre

Question 13

1 mm is how many micrometres

Answer 13

1 mm = 1000 micrometre

Question 14

nanometre size

Answer 14

1 x 10-9 metre

Question 15

1 micrometre = how many nanometres

Answer 15

1 micrometre = 1000 nanometres

Question 16

Advantage of using light microscopes compared to other microscopes

Answer 16

Light microscopes can be used on living cells - we can look at living cells
This means that we can explore processes such as cell division or movement of cells / chromosomes moving during mitosis

Question 17

Disadvantage of using light microscopes compared to other microscopes

Answer 17

Sometimes a stain needs to be used (before viewing the cell - to help see the cell) - and these can kill cells

poor Resolution - this is due to the nature of light
the wavelength of visible light is around 400nm to 700 nm
if two objects are closer than 200nm (limit of resolution for a standard light microscope) then we cannot see them

resolution of light microscope 200nm—

because… wavelength of visible light is simply too large to allow us to resolve objects closer than that

In conventional light microscopy, light passes through the sample
when trying to view certain parts of the cell which are further away, the light that we see will already have passed through the parts of the cell before reaching what you’re actually trying to view - makes the image blurred and resolution even worse than it could be

Question 18

define resolution

Answer 18

The minimum distance between two objects where they can still be seen as two separate objects

the ability to distinguish between two separate objects

Question 19

describe how laser scanning confocal microscopy works

Answer 19

In laser scanning confocal microscopy, a laser is used to scan the object that we are interested in

This allows us to view a very precise layer of the object

This means that the image produced by laser scanning confocal microscopy has a higher resolution than conventional light microscopy

using laser scanning confocal microscopy we can produce

Question 20

describe the advantages of laser scanning confocal microscopy over conventional light microscopy

Answer 20

In laser scanning confocal microscopy, a laser is used to scan the object that we are interested in

This allows us to view a very precise layer of the object
________________________
This means that the image produced by laser scanning confocal microscopy has a higher resolution than conventional light microscopy

using laser scanning confocal microscopy we can produce three dimensional images of cells

we can visualise specific proteins and structures within the cell and watch them move.

_________________

this technique also allows us to tag specific proteins and structures within cells by using special dyes and antibodies

with laser scanning confocal microscopy we can watch proteins moving around living cells

This helps us to work out the functions of those proteins

Question 21

formula for magnification

Answer 21

magnification = size of image / size of real object

Question 22

scale bar = 10mm which represents 1 micrometre

nucleus = 63 mm

work out actual size of nucleus

Answer 22

scale bar = 10mm
nucleus = 63mm

63/10 = 6.3

means that the nucleus is 6.3 times the length of the scale bar

1 micrometre x 6.3 = 6.3 micrometres

always do organelle / scale bar

Question 23

scale bar = 37mm
mitochondrion = 21 mm

scale bar represents 2 micrometres

work out actual size of mitochondria

Answer 23

scale bar = 37 mm
mitochondrion = 21mm

21/37 = 0.57
means that the mitochondrion is 0.57 times the length of the scale bar

0.57 x 2 micrometres = 1.14

Question 24

object size of mitochondrion = 0.8 micrometres

length = 30mm

work out magnification

Answer 24

magnification = size of image / size of real object

= 30,000 / 0.8 = 37,500 x

Question 25

describe how electron microscopy works

Answer 25

This microscope uses electrons instead of light

Electrons have a very short wavelength (so the resolution is 2000x better than a light microscope)

An electron gun produces a beam of electrons

These electrons pass down the microscope

The inside of an electron microscope contains a vacuum, so the electrons can pass through without bouncing off the molecules in air

Electrons are negatively charged. This means that we can focus the electron beam using electromagnets called electromagnetic lenses

The specimen is placed in the path of the electron beam
Electrons can pass through some parts of the specimen more easily than other parts

The final image is produced on a fluorescent screen

how can particle have a wavelength
electrons have properties of both particles and waves - so they can have a wavelength

Question 26

draw a diagram of electron microscopy

Answer 26

https://www.researchgate.net/profile/Lakmal-Jayarathna/publication/299750087/figure/fig8/AS:614003847987255@1523401333840/Image-formation-method-on-in-a-one-lens-transmission-electron-microscope.png

electron gun
electron beam
electromagnetic lens
specimen
image on fluorescent screen

Question 27

describe the advantages of electron microscopy over a light microscope

Answer 27

the resolution of an electron microscope is around 2000x better than a light microscope -

resolve up to 0.1nm under good conditions - means we can achieve a far greater level of magnification before the image becomes blurred

because of this higher resolution, the electron microscope has been used to make some major discoveries in biology e.g. ribosomes and structure of cell membrane

Question 28

describe disadvantages of electron microscopy

Answer 28

The interior of an electron microscope is a vacuum. This means that we cannot view living specimens using an electron microscope

Electron microscopy requires very careful staining of the specimen and the specimen often has to be very thin

Electron microscopy can lead to artefacts (false images)

(with electron microscopy we can get artefacts)

Artefacts are false images created by the staining process or the conditions inside the electron microscope

biologists have to be careful to check that what they are seeing with an electron microscope is actually real and not created by the conditions in the microscope or by the stain

Question 29

describe how transmission electron microscopy works

Answer 29

This microscope uses electrons instead of light

Electrons have a very short wavelength (so the resolution is 2000x better than a light microscope)

An electron gun produces a beam of electrons

These electrons pass down the microscope

The inside of an electron microscope contains a vacuum, so the electrons can pass through without bouncing off/ colliding the molecules in air

Electrons are negatively charged. This means that we can focus the electron beam using electromagnets called electromagnetic lenses

The specimen is placed/positioned in the path of the electron beam
Electrons can pass through some parts of the specimen more easily than other parts

The final image is produced on a fluorescent screen

how can particle have a wavelength
electrons have properties of both particles and waves - so they can have a wavelength

Question 30

what are the two types of electron microscopes

Answer 30

transmission electron microscopes
scanning electron microscopes

Question 31

describe how a scanning electron microscope works

Answer 31

The electron beam does not pass through the specimen.

Instead electrons are scattered from the surface of the specimen and detected

Question 32

draw a scanning electron microscope

Answer 32

electron gum
electron beam
electromagnetic lenses
electron detector - one either side
specimen

Question 33

compare transmission electron microscopy with scanning electron microscopy

Answer 33

In a transmission electron microscope, the electron beam passes through the specimen

The transmission electron microscope produces flat 2-dimensional images

The transmission electron microscope only works if the specimen is very thinly sliced

Transmission electron microscope has a very high resolution

VS

The scanning electron microscope produces 3-dimensional images

The scanning electron microscope does not require the specimen to be thinly sliced

The scanning electron microscope has a lower resolution than the transmission electron microscope

The transmission electron microscope also requires that the specimen is coated with metal such as gold

This can lead to artefacts

Question 34

describe the difference of the two types of electron microscopes terms of function

Answer 34

In a transmission electron microscope, the electron beam passes through the specimen

In a scanning electron microscope,
The electron beam does not pass through the specimen.

Instead electrons are scattered from the surface of the specimen and detected

Question 35

1 order of magnitude meaning

Answer 35

10 x bigger

apple 10cm
tree 100cm

tree is 1 order of magnitude bigger than the apple