Studying cells Flashcards
Methods of studying cells
Microscopy
Cell fractionation
What is cell fractionation?
A method of studying cell organelles by separating them from the cell
Step 1 of cell fractionation
Take a sample of cells and HOMOGENISE it in a blender with a buffer solution to break up cells (NOT ORGANELLES) and release organelles that are being studied
Properties of buffer solution
Cold
pH constant
Water potential in solution same as inside the cell (isotonic)
Why does the buffer solution have to be cold?
To keep enzyme activity low so destructive enzymes prevented from engulfing
Why does the buffer solution have to have a constant pH?
Because if pH changed, then the enzymes in organelles would denature and damage organelles
Why does the buffer solution have to have same water potential as inside cell
Prevents osmosis of water into and out of organelles causing them to swell or shrink respectively = damage
What is left after homogenisation?
The homogenate. Contains unbroken cells and different density organelles
What is done with the homogenate?
Put in a centrifuge to spin at relatively high speed
Balance out with same volume of sample in other tube
What is left after spinning at relatively high speed?
The larger organelles which experienced more force sunk to bottom (pellet)
But mid-small organelles remain in solution, not spun fast enough to sink (supernatant)
What is done with the supernatant and pellet?
Pour out supernatant so first tube is left with pellet of separated high density organelle
Why is the supernatant spun at increasingly high speeds?
So smaller density organelles are separated from solution and form a pellet to be separated
What should pellets be kept at?
Ice cold temperature to reduce enzyme activity
Will all organelles be separated perfectly?
No because pellets always contain traces of others
High density organelles
Nucleus
Golgi
Endoplasmic reticulum
Cell surface membranes
High- mid density organelles
Mitochondria
Mid- low density organelles
Lysosomes
Low density organelles
Ribosomes
Light microscopes
Using visible light to pass through specimens to view layers of cells with organelles
Eyepiece lens
View specimen here w eye piece graticule
Coarse focus
Moves stage up and down to focus it