Cell structure

What does cell fractionation allow scientists to do?

Study the functions of organelles

What do we do during the first stage of cell fractionation?

• We take a sample of tissue containing the cells and homogenise the tissue.

What does ‘homogenising’ a cell mean?

It means to break up the tissue and break open the cells,

What are 2 appliances that can be use to homogenise a cell?

1) Blender

2) Homogeniser

How can we homogenise a cell using a homogeniser?

• Place the tissue sample into the glass tube and cover it with buffer solution.

Why do we need to use buffer solution whilst using a homogeniser?

To keep the pH constant

Why is it important that the pH remains constant whilst homogenising?

Because a shift in pH could lead the enzymes in the cell’s organelles to denature

1) What do we do after homogenising the tissue in cell fractionation?

2) Why do we have to do this?

• Place the homogeniser on ice

• Because cooling the sample means that enzymes work more slowly, preventing any destructive enzymes from damaging the organelles.

1) What do we do after placing the homogeniser on ice?

2) What does this step produce?

1) Push the homogeniser’s plunger up and down to break open cells and disrupt tissue

2) A cell homogenate

What does the cell homogenate (produced from pushing the homogeniser’s plunger up and down) contain?

It contains all the organelles that we find in the cell.

State the largest to smallest of these 4 organelles: Nucleus, Ribosomes, Lysosomes and Mitochondria

1) Nucleus

2) Mitochondria

3) Lysosomes

4) Ribosomes

What is the term used to describe separating the different organelles in a cell?

Fractionation

What machine do we use to carry out fractionation?

Centrifuge

How can we use the centrifuge to carry out cell fractionation?

• Place tube containing cell homogenate into the sample holder.

What happens when the centrifuge spins the sample? (Note the different sizes of the organelles)

• The organelles are flung towards the bottom of the tube.

• Larger organelles (i.e nucleus) experience a greater force and move towards the bottom of the tube faster than smaller organelles.

How does the speed of the centrifuge vary throughout the fractionation process?

• First we start with a low speed spin.

What can we see right after the centrifuge spins at a low speed for the first time with the sample inside?

• The larger organisms such as the nuclei are flung to the bottom of the tube forming a pellet

What is the ‘pellet’ and ‘supernatant’ in the fractionation process?

• Pellet- larger organelles that settled at the bottle of the tube

• Supernatant- remaining organelles suspended in the liquid.

What do we do after the pellet and supernatant have formed in fractionation?

• We transfer the supernatant into a new tube and centrifuge for a second time at a higher speed spin.

• After this spin the pellet would contain mitochondria.

What do we do after the mitochondria has formed in the pellet, made during ultracentrifugation?

• Transfer supernatant to new tube and centrifuge again at a higher speed

• This time the pellet contains lysosomes

What do we do after the lysosomes have formed in the pellet, made during ultracentrifugation?

• Take supernatant for the last time and centrifuge again at the highest speed

• The pellet will contain ribosomes, the smallest organelle

How many millilitres is 1m divided into? (1m=xmm)

1000mm

How many micrometres is 1mm divided into (1mm=x micrometres)

1000 micrometres

How many nanometres is 1 micrometre divided into? (1 micrometre= xnanometres)

1000 nanometres

What 3 features do the early light microscope and modern light microscope have in common?

• Eyepiece lens

• Stage for specimen

• Focusing dial

What is the difference in magnification between the early light microscope and modern light microscope?

• Early light microscope- 300x

• Modern light microscope- 1000x

1) What big advantage do light microscopes have over other types of microscopes?

2) What can this advantage allow us to explore

• Light microscopes can be used on living cells

• Which allows us to explore processes such as (active) cell division and movement of cells

What is the main disadvantage of light microscopes and why does it occur?

• It has a low resolution

• Because visible light has longer wavel

Define the resolution of an object

It is defined as:

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

What is the limit of resolution for a standard light microscope?

200 nm.

What is the range of wavelength of visible light?

• 400nm to 700nm

1) Why can we not see two objects that are closer than 200 nm in electron microscopes?

2) Which microscope would we use instead?

1) Because that is the limit of resolution in standard light microscopes

2) An electron microscope

How can we calculate the diameter of a cell using a scale bar?

• Measure length of scale bar with ruler in millimetres

• Measure length of cell using ruler in millimetres

• Divide length of cell by length of scale bar and multiply by the value on the original scale bar

1) State the formula for magnification

2) Unit used for image and actual size?

Image size/Actual size

• Micrometres- p.s Always keep units the same!!!

What do electron microscopes use instead of light?

Electrons

Why do electron microscopes have a higher resolution than light microscopes?

• Because the beam of electrons have a much shorter wavelength than light

• Enabling them to resolve much finer detail.

How much times better is the resolution of an electron microscope compared to a light one?

2000x better

How do transmission electron microscopes work?

• They start with an electron gun which produces a beam of electrons

• These electrons pass down the microscope.

Why can the electrons in a transmission electron microscope pass through it without bouncing off the molecules in air?

Because the inside of an electron microscope contains a vacuum.

What can we use to focus the electron beams in transmission electron microscopes?

Electromagnetic lenses

How can electromagnetic lenses focus on the specimen in transmission electron microscopes?

• Specimen is placed in path of electron beam

• Electrons pass through the specimens (through some parts more easily than others)

• Final image is displayed on a fluorescent screen

How far can we resolve with an electron microscope?

Up to 0.1nm (under good conditions)

What is one major disadvantage of electron microscopes and why is this the case?

• We cannot view living specimens inside it

• Because the interior of an electron microscope is a vacuum

Besides the fact that we can’t view living specimens, what is another disadvantage of electron microscopes?

• It requires careful staining of the specimen and it has to be thin.

What unwanted result can electron microscopes sometimes display and how do they occur?

• Artefacts (false images)

• due to the staining process

• or conditions inside electron microscope

Name the two different types of electron microscope

• Transmission electron microscope

• Scanning electron microscope

What kind of images does the transmission electron microscope produce?

Flat, black and white 2D shapes

Why do transmission electron microscopes always need thin slices of specimen?

• So that the electron beam can pass completely through the specimen

• And display an image on the fluorescent screen

How does the scanning electron microscope work?

• Electrons are scattered from the surface of the specimen and detected.

What type of images does the scanning electron microscope produce>

3D images which do not need specimen to be thinly sliced

Why does the scanning electron microscope have a lower resolution than the transmission electron microscope?

Because they use lower energy electrons that only scan the surface of specimens instead of passing through them

1) Why do scanning electron microscopes require the specimen to be coated with a metal?

2) What unwanted result can this lead to?

1) So that the specimen can become electrically conductive and thus prevents a build up of charge from the electron beam

2) This can lead to artefacts.

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