OBJECTIVES: To determine the fragility of the erythrocyte membrane against the hemolytic effect of NaCl solutions of varying concentrations

Authors Avatar by alwahyu (student)

PRACTICAL 4: OSMOTIC FRAGILITY

OBJECTIVES:

To determine the fragility of the erythrocyte membrane against the hemolytic effect of NaCl solutions of varying concentrations.

INTRODUCTION:

Osmosis is the diffusion of a solvent through a semi-permeable from a region of low solute concentration to a region of high solute concentration. The semi-permeable membrane is permeable to the solvent, but not to the solute, resulting in a chemical potential difference across the membrane which drives the diffusion. That is the solvent flows from the side of the membrane where the solution is weakest to the side it is strongest, until the solution on both sides of the membrane is the same strength (that is, until the chemical potential is equal on both sides).

Red blood cells are bound by a membrane, which allows water to pass through while generally restricting the solutes. This process called osmosis, causes cells to shrink due to loss of water when in a hypotonic medium. This results in swelling and ultimately haemolysis as the cell bursts. The osmotic fragility test uses this fact to determine the concentration of solute inside the cell by subjecting it to the hemolytic effects of solutions of different concentrations.

MATERIALS:

  1. Ten test tube of NaCl (0.85%, 0.75%, 0.65%, 0.55%, 0.50%, 0.40%, 0.35%, 0.20%, 0.10% and 0%)
  2. Distilled water
  3. Blood
  4. Pipette
  5. Spectrophotometer
  6. Cuvettes
  7. Centrifuge

PROCEDURE:

  1. Ten test tubes is prepared and labeled as follows: 0.85%, 0.75%, 0.65%, 0.55%, 0.50%, 0.40%, 0.35%, 0.20%, 0.10% and 0% NaCl.
  2. 5 ml of Nacl is pipette in each test tube according t the label and 5 ml distilled water is pipette in test tube labeled 0% NaCl.
  3. 10-µl blood (with added heparin or sitrat, which act as anticoagulant) is pipette at each test tube. The content of test tube mixed carefully and the test tube sentenced to resting period of 20 minutes at room temperature.
  4. Test tube is shaking carefully and centrifuged for 9 minutes at the velocity of 2000 rpm.
  5. Supernatant is transferred to labeled cuvettes.
  6. Using supernatant from the test tube, which is labeled 0.85% NaCl as reference (0% haemolysis) optical density (OD) of each supernatant calculated using spectrophotometer with the wavelength of 540 nm.
  7. Percentage of haemolysis is calculated using the following formula:
Join now!

                            OD sample

                 OD of sample with 100% lysis      X 100%

  1. OD is recorded and percentage of NaCl is converted to mole/L and Osmols/L.
  2. A graph percentage of haemolysis vs. concentration of NaCl is plotted using graph paper. (this graph is known as osmotic fragility curve)

RESULT:

*RMM of NaCl is 58.5g/mole

Since percent weight per volume is grams chemical per 100 mililiter solution, this calculation gives the grams of chemical in ...

This is a preview of the whole essay