CELLS - TOPIC 2 (Cell Structure Chapter 3) Flashcards
METHODS OF STUDYING CELLS
What is cell fractionation
The process where cells are broken up and the different organelles they contain separated out
Homogenisation steps
- the tissue to be studied, cut into small pieces + placed into a cold, buffered, isotonic solution
- cells are broken up in a homogeniser (to release the organelles from the cells)
- the fluid is then called a homogenate
- The homogenate is then filtered ( to remove any complete cells and large pieces of debris)
- A suspension of homogenate is then placed in a test tube and then centrifuged
Why are cells broken up in a homogeniser?
To break open cells
To release the organelles from the cells (breaking the cell membrane)
Why is the homogenate filtered ?
To remove any complete cells and large pieces of debris e.g. cell wall/membrane
Explain the necessary tissue conditions (cold, isotonic, buffered)
Cold - to reduce enzyme activity (to prevent digestion of organelles)
Isotonic - to prevent water being lost or gained, prevents osmosis so no shrinkage of organelles, Same water potential to prevent bursting of the organelle.
Buffered - to maintain a constant pH to prevent protein from denaturing.
What is ultracentrifugation?
The process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge
Ultracentrifugation process
- the tube of filtrate is placed in the centrifuge and spun at a low speed
- The heaviest organelles, nuclei, are forced to the bottom of the tube, where they form a thin sediment
- These larger fragments are then removed and the supernatant (fluid at the top of the test tube) is re-spun at a faster speed than before
- The next heaviest organelles, the mitochondria, are forced to the bottom of the tube
- The process is continued in this way so that, at each increase in speed, the next heaviest organelle is sedimented and separated out
-process continues until there is no sediment left in the tube
THE ELECTRON MICROSCOPE
What is magnification ?
The number of times an image is enlarged compared with the real size of the object
How do you calculate for magnification ?
Magnification = size of image / real size
What is resolution ?
The ability to distinguish between two points that are close together
Why is it not possible to determine the identity of structures labelled X using an optical microscope?
- Low resolution
- Because wavelength of light is too long
(Can with an electron microscope because ….
1. High resolution
2. Wavelength of electrons is shorter)
When to use a light microscope?
- used for specimens above 200nm
- Light microscopes shine light through the specimen
- The specimens can be living (and therefore can be moving) or dead
- Light microscopes are useful for looking at whole cells, small plant and animal organisms, tissues within organs
What are the two types of electron microscopes ?
- TEM ( transmission electron microscope)
- SEM ( scanning electron microscope )
TEM key points:
- The electrons are focussed onto the specimen by electromagnets
- The electrons are passed through (transmitted) the specimen
- specimen must be very thin
- Produces a flat 2D image
( parts of specimen that absorb = appear dark
parts of specimen that transmit = appear bright ) - higher resolution than SEM
Why is the resolving power of the TEM not always being achieved in practice?
- difficulties in preparing the specimen limit the resolution that can be achieved
- a higher energy electron beam is required and this may destroy the specimen
What are the main limitations of TEM?
- the whole system must be in a vacuum and therefore living specimens cannot be observed
- a ‘complex’ staining process is required and even then the image is not in colour
- the specimen must be extremely thin
- The image may contain artefacts (look like real structure, but are the results of preserving and staining )
SEM key points :
- a beam of electrons bounce off specimen, so not as thin as TEM
- in colour
- 3D
What are the limitations of SEM ?
- same limitations as TEM ( except specimens do not need to be as thin, electrons bounce off rather than penetrate the specimen, means we can get a 3D image)
- lower resolving power than TEM
When using a light microscope, how can we measure the size of objects?
Using an eyepiece graticule ( a scale is etched on the glass disc, the scale is visible when looking down the eyepiece of the microscope
What is the calculation of 1 graticule division ?
1 graticule division = no. of micrometers/ no. of graticule division
How to find the measurement of the length of the object ?
graticule divisions X magnification factor = measurement (um - micrometre)
(The specimen slide would be used to replace the stage micrometer and the eyepiece graticule at the same magnification would be used to measure the length of the object)
EUKARYOTIC CELL STRUCTURE