Cell fractionation/Microscopes/ Mitosis Flashcards
What is the equation of magnification?
magnification = size of image/ size of real object
What is cell fractionation?
the process where cells are broken up and the different organelles they contain are separated out
Describe briefly the process of Cell fractionation
the tissue is placed in a cold buffered solution of the same water potential as the tissue. It then goes through homogenisation and ultracentrifugation
Why is the tissue in Cell fractionation put in a cold, buffered solution (with the same water potential?
cold = reduce enzyme activity to prevent self-digestion of organelles
same water potential = prevent organelles from bursting or shrinking as a result of osmotic gain or loss of water
buffered= so that the pH does not fluctuate. Any change to the pH could alter the structure of the organelles or affect the functioning of enzymes
What happens during homogenisation?
- Cells are broken up by a homogenizer (blender), which releases the organelles from the cell
- this results in homogenate, which is then filtered to remove any complete cells and large pieces of debris
What is ultracentrifugation?
the process by which the fragments in the filtered homogenate are separated in a centrifuge.
What happens during ultracentrifugation?
- the tube of filtrate is placed in the centrifuge and spun at a slow peed
- the heaviest organelles, the nuclei, are forced to the bottom of the tube, where they form a thin pellet
- the fluid at the top of the tube (supernatant) is removed, leaving just the sediment of the nuclei
- the supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before
- the next heaviest organelles, the mitochondria, are forced to the bottom of the tube
- at each increase in speed, the next heaviest organelle is sedimented and separated out.
Tell me about light microscopes
- use light to form an image
- The relatively long wavelength of light rays means that light microscopes can only distinguish between two objects if they are 0.2 micrometers or further apart. = poor resolution
- smaller magnification than electron microscopes
- image in colour
Tell me about electron microscopes
- Use beam of electrons instead of light rays
- higher resolution due to the electron beam having a short wavelength
- Electrons are negatively charged, so the beam can be focused using electromagnets
- Electrons are absorbed or deflected by molecules in the air = must be in a vacuum
How do Transmission electron microscopes (TEMs) work?
it consists of an electron gun that produces a beam of electrons that is focused onto the specimen by a condenser electromagnet.
The beam passes through a thin section of the specimen, which absorbs some electrons and lets some through, producing a black and white image
( the specimen is penetrated from below)
Pros and cons of TEM
- has a magnification up to 1 million and a resolution of 0.1nm
- can examine really small organelles, as well as virus and bacteria
- resolving power sometimes not achieved due to difficulties preparing the specimen and a higher energy electron beam being needed, which could destroy the specimen
- the whole system in a vacuum = needs to be dead
- a complex staining process is required and the image isn’t in colour
- the specimen needs to be extremely thin = 2D image only
- may contain artefacts (things that are a result from the way the specimen was prepared)
How do scanning electrons microscope work?
Directs a beam of electrons on the surface of the specimen from above, the beam is then passed back and forth across a portion of the specimen in a regular pattern.
The electrons are scattered by the specimen and the pattern of the scattering depends on the contours of the specimen surface.
A 3D image is produced by a computer analysis of the pattern of scattered electrons and secondary electrons produced.
Pros and cons of SEM
Pros :
- 3D image is produced
- can be used on thick specimens
Cons:
- do not produce a colored image
- has a lower resolving power (20nm)
- can’t see the internal structure of the cell, because it scans the surface of the object.
What are the stages of the cell cycle?
Interphase, mitosis, cytokinesis
What happens during the interphase? (describe all the phases within this phase
the cell increases in mass and size and carries out its normal cellular functions
G1 phase = Cells make the RNA, enzymes, and other proteins required for growth (high amount of protein synthesis). Make organelles and grow bigger
S phase = DNA replicated (chromosomes will now consist if two sister chromosomes are held together by chromatids**)**
G2 phase = the cell keeps growing until all of the organelles have duplicated. Proteins needed for cell division are needed
The mitochondria produce more ATP which will provide the energy for cell division