Lab 1 Membrane Physiology_Parsa.S - Tagged.pdf

HMF 1 (PCB 3703L) Lab 1: Membrane Physiology Parsa sartipi  Cell organelles and Biological Membrane Cell...

HMF 1 (PCB 3703L) Lab 1: Membrane Physiology Parsa sartipi  Cell organelles and Biological Membrane Cell Membrane Semi permeable - Establishes internal constituency of cell - Maintenance of pH, osmolality and nutrient Made up of: Phospholipid bilayer (Liposome), proteins, glycolipids, and cholesterol that will vary from cell to cell depending upon the function of that cell. Human anatomy, 7th Edition, Ch. 2 Martini Transport through Cell membrane Passive process: Pressure/gradient difference. Diffusing down concentration gradient Active process: ATP (cell energy) required * Created on PhET  Brownian Movement/Motion ❑ Discovered by Robert Brown in 1827 ❑ Brownian movement = Random motion of small particles suspended in solution caused by collision ❑ Kinetic energy – energy of motion, all objects posses it (KE = 1/2 mv 2) ❑ Degree of movement: Gas > Liquid > Solid ❑ Depends on the Temperature, mass, and the environment of the molecules as stated by Fick’s Law ❑ Fick’s law: velocity = temp/ √mass ✔ Procedure: Add a drop of Indian ink and oil immersion unto a glass-slide and view under the microscope at (x40) http://physics.bu.edu/~duffy/HTML5/brownian_motion.html https://www.youtube.com/watch?v=V7VtOa8pHno https://www.youtube.com/watch?v=znGTevIPKHE Solute + Solvent = Solution Solute: The substance that is being dissolved Solvent: liquid capable of dissolving or dispersing one or more other substances Solution: Resulting mixture of solute and solvent Eg. Salt + Water = Salt water or Sugar + Coffee = sweetened coffee Diffusion Net (passive) Movement of molecules (solute) down a concentration gradient (high to low concentration) Driving force is kinetic energy, no ATP is required. So how do we increase diffusion/kinetic energy? Ans: increase the temp. and decrease mass. What other factors determine the rate of Diffusion? Extent of the concentration gradient (The greater the difference in concentration, the more rapid the diffusion), Mass of the molecules diffusing, Temp., Solvent density, Solubility, Surface area and thickness of the plasma membrane, Distance travelled Plasma membrane selectively restricts diffusion. Charged and large particles are restricted (while small and non-polar molecules can diffuse through (CO2, N2, O2 ) https://phet.colorado.edu/sims/html/diffusion/latest/diffusion_en.html Does diffusion take place at equilibrium? NO! Diffusion Cont. Agar gel: is 98.5% water (i.e. 1.5% agar), so little resistance to the movement of the dye molecules Dye molecules: Add a drop to each section of agar gel. – Methylene blue – MW 320g – Potassium permanganate – MW 158g – Congo red – MW 697g – Malachite green – MW 365g Record the diameter (mm) every 15 mins with ruler for 60 mins  DATA REPORT SHEET (EXP II) Input the diffusion diameter (cm) of the dye at 0, 15, 30, 45, and 60 mins time-interval Osmosis Osmosis: diffusion of water across a semi- permeable membrane down a solute concentration. Criteria for Osmosis: An osmotic gradient and a semi-permeable membrane i.e. from high concentration of water/solvent to low concentration of water/solvent across a semi-permeable membrane. Or from low concentration of solute to high concentration of solute via a semi- permeable membrane. Activity Info: – Cellulose (dialysis) tube will act as a selectively permeable membrane Always keep wet and tie off open ends – Weigh (g) each sample and place in water and weigh every 15 min for 60 min  DATA REPORT SHEET (EXP III) Input the weight of the bag (in grams) at 0, 15, 30, 45, and 60 mins time-interval Tonicity Living cells are mini-osmometers – Isotonic: Concentration of solutes is equal (=300 mOsm), water goes in both directions, no shape change. – Hypertonic: Cells immersed in a solution with a higher solute concentration (> 300 mOsm), water will leave the cell, cell shape will shrink. – Hypotonic: Cells immersed in a solution with a lower solute concentration (< 300 mOsm), water will flood through the membrane and inside the cell, cell shape will inflate/swell. Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a semi-permeable membrane. It is also defined as the measure of the tendency of a solution to take in water by osmosis. The pressure that needs to be applied to prevent osmosis taking place. Jacobus van’t Hoff first proposed a "law" relating osmotic pressure to solute concentration. A simple extension of this equation was proposed by Harmon Northrop Morse. ╥ = icsoluteRT ╥ is osmotic pressure, csolutes is the molar conc, equal to the number of moles of solute in the solution divided by the solution volume, the total molar concentration of all solutes that cannot pass through a water-permeable membrane. T is the temperature in kelvins (Celsius + 273); i is dimensionless correction factor; R = 0.08208 L atm/mol If the concentration of solutes on both sides of the membrane is equal, then there is no tendency for water to move across the membrane and no osmotic pressure.  Procedure IV 1. Label ten 1.5 ml test tubes and arrange them sequentially in a rack. 2. Place 0.5 ml of sheep's blood in each tube 3. Centrifuge the blood in a microfuge for 5 mins @ 5000 rpm and remove them carefully 4. Decant the plasma with a Pasteur pipette 5. Add 0.5 ml of each of the following into appropriate tube and then vortex to bring the cells into solution 1. 0.100M NaCl 2. 0.125M NaCl 3. 0.150M NaCl 4. 0.175M NaCl 5. 0.200M NaCl 6. 0.250M NaCl 7. 0.300M NaCl 8. 0.300MGlucose 9. 0.300M Glycerin 10. 0.300M Urea 6. Let tubes stand for 3 minutes and hold the tubes to the light. Compare and record the level of transparency of each solution. If the solution is transparent hemolysis has occurred 7. Fill a hematocrit tube with each sample, plug and centrifuge. Determine and record the hematocrit 8. Prepare a blood smear on glass slide for samples I, III and VII. Observe the slide under oil immersion. Record the results. EXPECTED RESULTS (IV) Hematocrit (HCT) Packed cell volume (PCV): % of blood that is made up of red blood cells. Centrifuging whole blood spins formed elements to bottom, plasma forms top layer – RBC value is generally only reported – But WBC and plasma can also be calculated Normal values – Male 47.0 ± 7 – Female 42.0 ± 5 DATA REPORT SHEET (EXP IV)