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Centrifugation Spinning solutions at high speed to separate out different components How does it work? Centrifugal, frictional and buoyant forces act together to separate objects according to mass and density Frictional Force prevents particles from moving in the medium Buoyant Force prevents particles from moving in the medium
Centrifugal Force Pushes out from the centre of rotation to move particles
*When the particles reach the bottom of the tube or a steady velocity, the three forces add up to zero* Centrifugal force - frictional force - buoyant force = zero
r distance from the centre/axis of rotation to the end of the tube (measured in cm)
t the time of centrifugation RCF = [(rpm x 2)/60]2 x (r/980)
https://www.thermo.com/eThermo/CMA/PDFs/Various/File_661.pdf
http://www.corning.com/lifesciences/us_canada/en/technical_resources/doc_library/nomogram_computing_rcf.aspx
Which of the cells would be most sensitive to a hypotonic solution? Least sensitive?
When you centrifuge each tube, what will you find in the pellet? What will you find in the supernatant?
4
5 6 blank
0.5
0.7 0.9 0.9
10
10 10 10
Experimental considerations 1) Keep in mind that you are testing the effect of NaCl concentration on cell lysis not the effect of mechanical damage. Be gentle. 2) What order should the water, blood and sodium chloride be added? Why? 3) Should the tubes all have the same total volume? Why is this important? 4) If the cells do not lyse, will they be in the supernatant or in the pellet? 5) If the cells do lyse, what colour would the supernatant be? Why?
Calculation of % lysis:
% lysis =
absorbance of tube X absorbance of tube 1 absorbance of tube 6 absorbance of tube 1 0.05 0.70 x 100
x 100
Why is the absorbance of tube 1 used as the denominator? Hint: what is the % lysis of tube 1?
% lysis =
% lysis =
x 100
% lysis = 7%