Report on ribosomes

Process

1.  The liver tissue is cut and fluid is added. The fluid is ice-cold to minimise enzyme reaction as if the mixture was warmer, enzymes such as proteases could digest and destroy the organelles being investigated, it is a buffer solution to keep the pH of the liver cells contents constanr as any change is pH could denature the enzymes in the mixture, and this would have an effect on our investigation on the mitochondria and ribosomes of the cell. The solution is also isotonic to to avoid the distortion of any organelles.
3.  The cells are then broken down using a blender called a homogeniser and the liquid that is produced is called the homegenate. This liquid should be filtered to remove any debris.
5. This homegenate is then put in a test tube, which is then put in the centrifuge, ready for spinning.
7.  The mixture is spun at a low speed (800-1000g) for 5-10 minute, the denser parts of the nuclei get spun to the bottom of the test tube, where they form a pellet called the sediment. At this speed, the densest organelle is the nuclei and so this will form a sediment at the bottom of the test tube.
9.  The liquid layer on top, called the supernatant, is then poured into a fresh test tube, leaving the nuclei sediment behind. This supernatant is then spun at a faster speed (10000-20000g) for 15-20 minutes, which results in a sediment of mitochondria. The supernatant is poured into a new test tube and the mitochondria sediment is stored for later use.
11. This new supernatant is then spun at 50,000-80,000g for 30-50 minutes to form a rough endoplasmis reticulum sediment. The supernatant is then poured into a new test tube and then spun at an even faster speed (80000 - 100000g) for 60 minutes. to form a sediment of plasma membranes and the smooth endoplasmic reticulum.
13.  This supernatant, which is now contains no nuclei, mitochondria, rough endoplasmic reticulum, plasma membranes and smooth endoplasmic reticulum is spun again at    150000 - 300000g for 3 hours, and then a sediment of ribosomes will form at the bottom of the test tube. These are stored for later use.

Observation:

Both the Ribosome and mitochondria pellet will be observed using the electron microscope.

The electron microscope will be used over a light microscope for various reasons, which will now be explained.

An electron microscope relies on an electron beam instead of light rays. Electron beams have a much smaller wavelength than light (0.005nm compared to 550nm of light), which means that the electron microscope can show two objects that are very close together as separate objects. This therefore means that the electron microscope has much better resolution than a light microscope, which will allow us to see the structures of the mitochondria and ribosomes much clearer than a light microscope as it can resolve objects just 1 nm apart.

The following table compares the use of electron microscopes and light microscopes:

How does it work?

•  The source at the top of the microscope emits electrons (-ve charge)
•  These electrons are attached to the +ve anodeand are then concentrated into a beam by a -ve cathode.
•  The condensor and objective are electromagnets, which straighten the beam
•  The projector focuses the image on the screen.

Conditions required:

•  Vacuum - otherwise electrons would hit air molecules.
•  The sample must be dead - since being placed in a vacuum.

We believe that we will see the following images.

 

Colour has been added to make the images more clearer to see.