Death by Fluid: DIFFUSION, OSMOSIS and MEMBRANE PERMEABILITY

Lab #2 Fall 2017 Death by fluid:

DIFFUSION, OSMOSIS and MEMBRANE PERMEABILITY

I. DIFFUSION

If you have decided that you are only going to learn one thing this entire semester, learn this: HIGH TO LOW. HIGH. TO. LOW.

Think of a crowded club (or something). If everyone in that room gets shoved into one corner, they are going to immediately begin to spread out to areas with less people. That is, people (like anything else), will tend to move from regions of HIGH concentration to regions of LOW concentration.

Diffusion is the movement of molecules within a fluid (liquid or gas) from areas of HIGHER concentration to areas of LOWER concentration. The energy for the diffusion process comes from the kinetic energy of the moving molecules in the fluid; the molecules in solutions and gases are actively moving and have plenty of kinetic energy, so they bump against each other and eventually spread out evenly to reduce the number of collisions. However, other than the energy that comes from particle movement, NO ENERGY IS REQUIRED to trigger diffusion.

The random nature of molecular movement tends to distribute the molecules evenly over time in the area to which they are confined. Diffusion of substances can also occur across the cell membrane, if the membrane allows that particular molecule to cross.

1 LAB SAFETY AND PROCEDURES You all have had lab before but I will reiterate (and enforce) key points: • No food or drink in the lab. It can’t be out on the table and you can’t be eating/drinking it. • Wear gloves when handling lab crystals and sheep blood.

SPEED OF DIFFUSION

So, diffusion always goes from high to low (and I mean ALWAYS). However, the rate at which diffusion occurs can be changed. You will measure the speed of diffusion of potassium permanganate by dropping a small crystal into a beaker containing a small amount of water. This works better than using a drop of aqueous solution, which spreads over the whole surface of the water when it hits it and makes any sort of measurement difficult.

Procedure:

1. Fill a 250 ml beaker with tap water to a level of ONLY about one centimeter to 1/2 inch.

2. Place the beaker upon a piece of graph paper with small divisions and check to see that the paper is visible and readable through the bottom of the beaker. You may need to measure the divisions if it's not stated on the graph paper.

3. Using the metal forceps, drop JUST ONE crystal of potassium permanganate into the beaker NEAR THE CENTER of the beaker, as shown to the right. Super duper important that you drop it in the center of the beaker. If it’s too close to the edge, it will diffuse to the edge and your measurement will be screwed.

4. Immediately start timing the speed of diffusion by noting the position of the purple 'halo" diffusing away from the crystal every fifteen seconds.

-For example, if the outer edge of the purple ring is ten little squares from the crystal at t=60 seconds, and each square is 1mm across, then the KMnO4 has diffused 10 mm in one minute. After the first minute, you only need to make a measurement every minute. See data table below—record your results in the distance column.

Table 1: Speed of diffusion of potassium permanganate time Distance traveled (mm) speed of diffusion (distance/time)

15 s

30 s

60 s

2 min

3 min

4 min

5 min

5. Calculate the speed of diffusion at each time point. Remember that speed is calculated by dividing the distance traveled by the time it took to travel that distance J #physics

6. After 5 minutes of measurement, you can ignore your crystal and proceed with the other sections of the lab. To throw out your crystal, do so by dumping the solution into the KMnO4 waste receptacle and placing your beaker back into You can check on it later to see if it has diffused throughout the water.

Review questions for the diffusion experiment:

1. a. The largest animal cell is about 1 mm in diameter. Using the fastest speed from column 3 in your table above, how long would it take for KMnO4 to diffuse 1 mm? _____

b. Human cells are much smaller. How long would it take for KMnO4 to diffuse 0.1 mm?

c. Does diffusion seem like a fast or slow process at the cellular level in humans? That is, if you could only rely on diffusion to get something from one side of a cell to another, does diffusion seem like it would suffice? ______

2. If a molecule of oxygen had to get from your lungs to your toes just by diffusion, about how long would it take? (assume the distance travelled is 1 meter and use your fastest speed of diffusion). ______Does this seem fast or slow? ______

3. Do you think diffusion is an efficient way to get molecules throughout the body? So, WHY do multicellular organisms have circulatory systems??

3 II. OSMOSIS AND EFFECTS OF TONICITY ON ANIMAL CELLS

Osmosis is the diffusion of water from regions where it itself is more concentrated, to where it is less concentrated. In other words, it always moves towards regions of higher solute concentration.

Osmosis is a special kind of diffusion. It is the diffusion of water molecules across a membrane that is selectively permeable, that is, permeable to water but less permeable to many other molecules. The cell is surrounded by such a membrane. Remember, diffusion is the process of a molecule moving from where its concentration is really HIGH to where its concentration is really LOW. The basic idea is that water will always move by osmosis towards regions with lower water concentration. See the image above. However, another way to put this is to say that water moves TOWARDS REGIONS OF HIGHER CONCENTRATION OF SOLUTES (where a “solute” is just a dissolved substance in liquid, like sugar dissolved in coffee). It does not matter exactly which substances are dissolved in the solution(s), as far as the water is concerned. A cell or compartment (eg., capillary) containing a higher concentration of dissolved substances contains less water, so water will tend to move into this more concentrated solution.

Who cares??? Am I right? You care. That’s who. Even if you don’t know it, you better care. Water can do some really bad stuff to cells if there is too much or too little of it.

IV. Tonicity

Plasma Plasma Plasma

Tonicity describes if a cell will gain or lose water to the solution. A hypertonic solution draws water from the cell, for example.

Whereas osmolarity specifically describes the concentration of a particular solution (in moles/liter) tonicity describes the effect that this solution would have on osmotic movement of water across the cell membrane. The terms hypotonic, isotonic and hypertonic are used to describe the effect that a solution has on a cell (see above). Note: tonicity is always used to describe the solution outside a cell, not the cell or the solution inside the cell itself. Let’s take an example. A 0.3 Osmolar glucose solution has the same number of particles per ml as the cytoplasm of a red blood cell since the cytoplasm is also 0.3 Osmolar (so it is isosmotic to rbc's). Because the concentration of particles (and of water) is the same inside vs. outside the cell, it will produce no net osmotic movement of water across the rbc membrane, so it is called isotonic ("equal tension"). Talk about this with your group before moving on: a red blood cell has an osmolarity of 0.3 Osm. If it is exposed to a solution that has an osmolarity of 0.1 Osm, what will happen to the cell and why? Make sure all group members have an opinion and can explain that opinion.

5 A. EFFECT OF OSMOSIS ON ANIMAL CELLS

Sheep blood provides excellent cells for use in osmosis experiments because the cells are simple and abundant (about 5 billion per milliliter). Sheep blood cells lack the nucleus and organelles of other cells, so are basically membranes surrounding a bag of hemoglobin.

1. EFFECT OF TONICITY ON SHEEP RED BLOOD CELLS

Procedure:

1. Obtain 3 test tubes, label them 1, 2, and 3, and place them into a test tube rack. There is some white tape on the instructor’s desk which you can apply to the tubes and write on.

2. Fill each with 5 ml of the appropriate NaCl solution as listed in table 2, Solution #1 = add 5 ml of 0.9% NaCl to tube #1 Solution #2 = 5 ml of deionize H2O to tube #2 Solution #3 = 5 ml of 10% NaCl to tube #3

*note: 0.9% NaCl has a concentration of 0.3 osmolar. Thus, it is exactly the same concentration as normal human blood (it is isosmotic).

3. STOP! SUPER IMPORTANT! STOP! FOR THE LOVE OF ALL THAT IS GOOD IN THIS WORLD…STOP! Ok. Before you proceed, or do anything, PREDICT whether a red blood cell placed into solution 1 would swell up (gain water), shrink (lose water), or stay the same (not gain or lose water). Write your predictions below:

Solution 1 will make the cell ______Solution 2 will make the cell ______Solution 3 will make the cell ______

4. Then determine if you think these solutions are hyptonic, isotonic, or hypertonic compared to the cell. Tonicity always compares the solution outside the cell to the cell itself.

5. If a cell gains water, it will actually burst and make the blood solution pretty clear. Based on this, predict if a red blood cell placed in solution 1 would become clear or stay cloudy. What about a red blood cell placed in solution 2? Talk it over with your lab mates and write your prediction below and EXPLAIN.

6 6. Now, proceed to step 6 to see if your predictions were right.

7. Carefully add 3 drops of blood to tube #1.

8. Mix it for 60 seconds in front of a printed piece of paper in good light. If the cells burst (lyse) due to osmotic movement of water into the cell, the solution will become transparent and you will be able to read the paper while looking through the test tube.

9. Record whether or not lysis (bursting) occurred in Table 2.

7. Go on to test tubes #2 and #3 and do the same procedure.

Table 2: Effect of Tonicity on Red Blood Cells

Tube # Contents Tonicity of Lysis (y/n) Explanation (in terms of osmosis) solution 1 0.9% NaCl

2 DI H2O (0% NaCl)

3 10% NaCl

2. EFFECT OF TONICITY ON THE APPEARANCE OF RED BLOOD CELLS

Procedure:

1. Remove a drop of solution from test tube #1 above, using a long skinny pasteur pipette, and place it on a microscope slide. Dont forget the coverslip! Examine the cells under the microscope using the 40X objective.

2. Remove a drop of solution from test tube #2, place it on a slide and examine it under the microscope. Do you see any cells?

3. Remove a drop of solution from test tube #3 and examine it under the microscope. Compare these red blood cells to those seen in tube #1 and record your observations below.

4. Put the slides in the 'dirty slides' container.

Table 3 : Effect of Tonicity on appearance of red blood cells

Tube # Contents Tonicity of Lysis Appearance of red blood cells and solution (y/n) explanation 1 0.9% NaCl

2 DI H2O

3 10% NaCl

Review and exam-style questions:

1.) Under normal circumstances, why should you NEVER ever give a patient an IV of pure water? Explain in terms of osmosis, diffusion, osmolarity, and tonicity of the IV solution.

2.) Let’s say a solution is said to be hypotonic compared to the cell. What does this likely mean about the osmolarity of this solution compared to the osmolarity of the solution inside the cell? (higher? lower? same?) Explain your reasoning. Hint: what does a hypotonic solution mean? Does water go into the cell from the solution, or does it leave the cell?

3.) A patient of yours has come into the clinic and is severely dehydrated. You look at their blood under the microscope and notice that the red blood cells appear a bit shriveled (like they lost water to the surrounding plasma). TO help treat the dehydration, you want to hook them up to an I.V. to help them get fluids directly in their bloodstream. Would you choose an I.V. solution that is hypotonic, hypertonic, or isotonic to this person’s red blood cells? Explain why.

4.) Can you think of a scenario when you might want to give a patient a hypertonic I.V. solution? What might that patient’s blood look like?