A - 3 - Analysis of cell components Flashcards
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 types of microscope?
Optical (light)
Electron
How does an optical (light) microscope work?
They use light to form an image
What is the maximum resolution of an optical microscope?
0.2 Micrometres
Which organelles can you not see using a light microscope?
- Ribosomes
- Endoplasmic reticulum
- Lysosomes
What is the maximum useful magnification of an optical microscope?
X1 500
How do electron microscopes work?
The use electrons to from an image
What is the maximum resolution of an electron microscope?
0.0002 micrometres
What is the maximum useful magnification of an electron microscope?
X1 500 000
What are the two types of electron microscope?
Transmission electron microscope
Scanning electron microscope
How do TEM’s Work?
They use electromagnets to focus a beam of electrons which is then transmitted through the specimen. Denser parts of the image absorb more electrons so they will look darker on the image.
What are the advantages and disadvantages of TEMs?
+ Give high resolution images so shows small objects
- Can only be used on thin specimens
- Can only be used on non living specimens
How do SEMs work?
They scan a beam of electrons across the specimen. This knocks off electrons from the specimen which are gathered in a cathode ray tube to form an image
What are the advantages and disadvantages of SEMs?
+ Can be used on thick specimens
+ Can be 3D
- Give lower resolution images than TEMs
- Can only be used on non-living specimens
Describe how to prepare a microscope slide
1 - Pipette a small drop of water onto the centre of the slide
2 - Use tweezers to place a thin section of your specimen on top of the water drop (needs to be able to let light through it)
3 - Add a drop of stain (used to highlight objects in the cell)
4 - Add a cover slip. To do this, stand the slide upright on the slide next to the water droplet and carefully tilt and lower it so it covers the specimen, while trying not to get any air bubbles in it
What is an artefact?
Something you can see down a microscope that isn’t part of the cell
What are the three steps to cell fractionation?
- Homogenisation
- Filtration
- Ultracentrifugation
What is homogenisation?
It means breaking up the cell.
How and Why is it done during cell fractionation?
It can be done by either vibrating the cells, or grinding them up in a blender.
This is done to break up the plasma membrane and release the organelles into the solution
How must the homogenised solution be kept?
Ice-cold
Isotonic
Constant PH maintained
Why must the solution be kept ice cold?
To reduce the activity of enzymes that break down organelles
The solution must be kept isotonic? What does this mean? And why is this done?
The solution should have the same concentration of chemicals as the cells being broken down to prevent damage to the organelles via osmosis.
How is the Solution kept at a constant PH?
A buffer solution is added
Describe the filtration stage of cell fractionation
The homogenised cell solution is filtered through a gauze to separate any large cell or tissue debris from the organelle
What is the supernatant?
The fluid above the sediment
What Is the pellet?
A thick sediment formed at the bottom of the of the solution
How does ultracentrifugation take place?
- The cell fragments are poured into a tube
- The tube is put into a centrifuge and is spun at a low speed, forming a pellet and a supernatant
- The heaviest organelles are flung to the bottom first
- The supernatant is then drained off and poured into another tube and spun at a higher speed
- This is then repeated until all organelles have been separated
What is the order the organelles are usually separated in?
heaviest first - Nucleus - Mitochondria - Lysosomes - Endoplasmic reticulum - Ribosomes Lightest last
