Centrifugation & Osmotic Fragility Chapter 5

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

Centrifugation & Osmotic Fragility Chapter 5

Determining Centrifugal Force (RCF or xg) RPM speed of the rotor in revolutions per minute

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)

Centrifugation & Osmotic Fragility Chapter 5

Table provided by centrifuge manufacturer

https://www.thermo.com/eThermo/CMA/PDFs/Various/File_661.pdf

Centrifugation & Osmotic Fragility Chapter 5

Nomograph

http://www.corning.com/lifesciences/us_canada/en/technical_resources/doc_library/nomogram_computing_rcf.aspx

Centrifugation & Osmotic Fragility Chapter 5

The Experiment We are going to be testing the effect of salt concentration on red blood cells. Why? Diseases such as sickle cell anemia and spherocytosis cause red blood cells to have a different shape than a healthy red blood cells. The change in shape can be detected by testing the reaction of a blood sample to salt solutions at different concentrations. Consider what would happen to each of these cells in an isotonic solution:

Which of the cells would be most sensitive to a hypotonic solution? Least sensitive?

Centrifugation & Osmotic Fragility Chapter 5

The Experiment
RBC in isotonic solution. Haemoglobin is inside the cell RBC in hypotonic solution. Haemoglobin is released form the cell

Decreasing NaCl concentrations

When you centrifuge each tube, what will you find in the pellet? What will you find in the supernatant?

Centrifugation & Osmotic Fragility Chapter 5

Preparation of red blood cell solutions at various NaCl concentrations NaCl Tube concentration (%) 1 2 3 0 0.1 0.3 Total Volume (mL) 10 10 10 Volume of 1% NaCl (mL) Volume of dH2O (mL) Volume of blood stock (mL)

4
5 6 blank

0.5
0.7 0.9 0.9

10
10 10 10

Centrifugation & Osmotic Fragility Chapter 5

NaCl Tube concentration (%) 6 0.9 Total Volume (mL) 10 Volume of 1% NaCl (mL) Volume of dH2O (mL) Volume of blood stock (mL)

Calculations Volume of sodium chloride:

C1V1 = C2V2 (1%)(V1) = (0.9%)(10mL) V1 = 9mL

Volume of blood stock:

dilution x concentration = volume 1/50 x 10mL = volume 0.2mL= volume

Volume of distilled water:

Volume of water = total volume - volume of NaCl - volume of blood Volume of water = 10mL - 9mL - 0.2mL Volume of water = 0.8 mL

Centrifugation & Osmotic Fragility Chapter 5

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?

Centrifugation & Osmotic Fragility Chapter 5

Tube 1 6 NaCl concentration (%) 0 0.9 % Transmittance 20% 90% Absorbance 0.70 0.05

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%