(Division of Chemical & Biomolecular Engineering)

SCHOOL OF CHEMICAL AND BIOMEDICAL ENGINEERING

Experiment : Red Cell Osmosis

Name Matric No. Group Date of Expt : : : :

GRADE:

Osmosis
Purpose: 1. Teach the students importance of isotonic solutions. 2. Teach the students, molarity, osmolarity and tonicity. Safety: Please wear laboratory coats at all times. No Eating or Drinking in the laboratory. Wear Gloves when handling blood or blood products. Clean any blood spills, immediately. Osmosis Cells are very sensitive to the buffer condition thus it is very important that we maintain the cells in isotonic buffers to prevent lysis due to osmosis. Osmosis is the movement of water, through a semi-permeable membrane. Even though water will move through a membrane in both directions, there will be a net movement in one direction if there was a concentration gradient. For example if a cell is placed in distilled water, the concentration of water on the outside the cell will be greater than the concentration of water inside the cell. Thus, there will be a net movement of water molecules into the cell. Similarly if a cell is place in solution with high concentration of solutes there will be a net movement of water from inside the cell to the outside of the cell. Tonicity represents the ability of a solution to change the shape of cells that is immersed in it. The solution changes the shape of cells by altering their internal water volume. The red blood cells are commonly used to study the permeability of living cell membranes. The red blood cells will maintain their size and shape in an isotonic solution. However the red blood cells will burst when placed in a hypotonic solution such as water due to net inflow of water and shrink (crenate) when placed in a hypertonic solution due to net outflow of water. Mammalian blood plasma has an osmotic concentration of about 0.3 Osm. A suspension of red blood cells is opaque and lysis causes the suspension to become transparent. This observation can be used to determine the tonicity of various solutions to red blood cells.

Osmosis 1) You are provided with a 3% (3g/100ml) solution of a salt. Using this solution set up 5 tubes of solution (1 ml) with the following concentration (Water, 0.6%, 0.9%, 1.2% and 1.5%). 2) Place the 5 tubes with 1 ml of solution (Water, 0.6% 0.9%, 1.2% and 1.5%) in a test tube rack. 3) Add 10 ul of blood into each test tube. 4) Cover the test tubes and mix by inverting the tube. 5) Observe the tubes at frequent intervals. 6) Record your observation after 30 min.

Permeability of membranes 1. Transfer1 ml of isosmotic solution of NaCl (0.15M) and Urea (0.3 M) to two separate tubes (label the tubes). 2. Place the tubes on a rack. 3. Add 10 ul of blood into each tube, mix by inversion and observe the tubes at frequent interval. 4. Record your observation.

Effect of toxin on RBC 1. Transfer 1 ml of PBS and 1 ml PBS/PEG. 2. Add 10 ul of 1 M CaCl2 to both tubes. 3. Add 10 ul of RBC to the two tubes and mix by inversion. 4. Add 8 ul of toxin to both the tubes and mix. 4. Incubate the tubes at 37oC for 30 min. 5. Spin down the cells (1 min, 3000 rpm) and record your observation.

Introduction to cells Answer sheet Name: Tutorial group: Student ID: Date: Marks:

Water 3% Stock Lysis

0 1 ml 0 ml +

0.6%

0.9%

1.2%

1.5%

NaCl Lysis

Urea

Toxin Lysis

PBS

PBS/PEG

1. Calculate the molarity and osmolarity of 1.2% NaCl (MW 58.8) solution? 2. Based on your results what is the isosmotic concentration of this salt? 3. Why does the RBC lyse in water? 4. Is there any lysis in the tube with 1.5% salt? Explain. 5. Why does the RBC lyse in an isosmotic solution of urea? 6. Why is the isosmotic concentration of NaCl half that of Urea? 7. What is the effect of toxin on RBC and does the PEG enhance or reduce the effect of the toxin? Explain.