Exercise 6 - DIFFUSION AND OSMOSISStudent Learning Outcomes At the completion of this exercise you should: (l) Be able to define the terms diffusion and osmosis. (2) Be able to list and discuss four mechanisms that cells use to move molecules across their plasma membranes. (3) Be able to explain what Brownian motion tells us about atoms and molecules. (4) Be able to explain the relationship between molecular weight and the rate of a molecule’s diffusion.(5) Be able to list the characteristics of molecules that can, and those that cannot, move passively across a cell’s plasma membrane. (6) Be able to describe how the solute concentration, inside of a cell, affects the rate (speed) of osmosis. (7) Be able to define the following terms: concentration gradient, selectively permeable membrane, hypertonic, hypotonic, isotonic, and homeostasis.Introduction Virtually all life forms are composed of cells. The cell is called the fundamental unit of life because within it occur most of the biochemical life processes. One of the phenomena of life is that the chemical composition of a cell remains fairly constant, in spite of the fact that the cell continually uses substances from its external environment and at the same time discharges other substances into its environment: This state of chemical constancy in living systems is called homeostasis. This homeostasis, in addition to the fact that a cell's surroundings are always of relatively different chemical composition from its inside, leads us to hypothesize that there must be some very selective means of chemical exchange across a cell's plasma membrane. Today, we will investigate the processes of movement of some substances into and out of cells. Question 1. Most "cells" do not appear to have an obvious "mouth" or other visible structures in their cell ("plasma") membranes. Suggest one other way in which materials might be able to pass through the cell's membrane:Replace this text with your answer. Question 2. Cell biologists tell us that there are 4 basic mechanisms that cells use to get molecules across their membranes. You need to learn these 4 strategies. Go to your textbook, or other reference source, and define the following 4 mechanisms: 1.)Osmosis2.)Facilitated diffusion3.)Endocytosis and exocytosis4.)Active transport Brownian Motion Robert Brown made an interesting observation in 1827 that led to the principle that "all atoms and molecules" are in constant motion. Dr. Brown was a botanist and army surgeon who was looking at particles inside pollen grains when he noticed the "rapid oscillatory motion of microscopic particles." He later observed the same movement when looking at substances, like India Ink. India Ink is made of water and billions of suspended clumps of carbon atoms. Under high magnification, Brown observed that the clumps of carbon atoms were vibrating wildly in all directions. He hypothesized that moving water molecules, which cannot be seen, must be colliding with the clumps of carbon, forcing them to move. Further study has shown that Dr. Brown was correct and, today, we call this kind of observation "Brownian motion". A physical scientist would tell you that particles (atoms and molecules) are moving because "they have heat energy (Kinetic energy)". If you ask what kinetic energy is, you will be told that it is "random molecular motion" which, of course, is a circular argument. The point here is that we really do not know the ultimate reason why all atoms and molecules on earth are moving, only that they are and that the more heat energy ("kinetic energy") atoms or molecules have, the faster they move. You are about to make this observation under the compound microscope yourself. The proper way to carry the compound microscope will be demonstrated. Always use two hands. Make sure that the cord is not dangling to prevent a tripping hazard. One hand should be holding the base of the microscope, while the other should hold the arm of the microscope. Before using the microscope, check for the condition the microscope was left in from the prior class. Was the microscope placed back in its assigned compartment with the arm facing out toward you?Was the cord wrapped between the stage and objectives with the plug tucked inside the cord?Was the cord relatively untangled?Was the ocular lens clean? (If not, clean the dirty lens with the appropriate lens cleaner and lens paper (not a paper towel).Was the light turned off?Check that the Condenser is at its highest point, directly below the stage. There is a knob connected to the Condenser that allows it to be moved up and down.Was the mechanical stage centered so that the stage clips don’t hang over the edge of the stage?Was the scanning objective lens (4x) (not another objective) placed over the stage? If not, rotate the nose piece until the scanning objective is facing the stage. Was the stage lowered to the lowest setting possible position? If not, use the coarse focus knob to do so, not the fine focus knob.Were there any slides remaining on the stage? If so, remove the slide and notify your instructor. It is important that the slide is placed in the correct box, or it may get lost.If any of these conditions were problematic, please let your instructor know. Procedure: Plug in the cord and turn up the light intensity.to its maximum value and adjust the iris diaphragm to its most closed setting. As you proceed you can increase the light passing through the specimen by gradually opening the iris diaphragm. Adjust the distance between the oculars: Without placing the prepared slide on the stage yet, look through the oculars. You are likely going to see two circles of white light. Do not try to focus your eyes on any one thing, as nothing is in focus yet. Slowly, move the two oculars together and/or further apart until the two circles of white light become one circle of light, the Field of View. Lower the stage using the coarse focus knob, and make sure the (shortest) scanning objective is facing the stage, so that there is no chance of the slide scratching any objective lenses. Prepare your wet mount slide. Wash and dry a glass slide. Place a small drop of India ink on a clean slide. Place a cover slip on the slide. Coverslip: Carefully cover the preparation with a clean plastic coverslip as follows: Place one edge of the coverslip near to the drop. The stain and water with which you mixed the cells will flow along the junction of the edge of the coverslip and the slide. Carefully lower the coverslip over the specimen keeping the edge of the coverslip in contact with the slide. In this way, the water will flow slowly and uniformly about the specimen and force out air bubbles from beneath the coverslip. (A few air bubbles are not a serious problem for your first slide.) Excess fluid: If liquid spills out or may spill out from under the coverslip, gently blot the excess with a towel, so that it will not later drip onto the stage of the microscope. Before looking through the eyepiece (ocular), open the stage clip, and place the slide on the stage of the microscope beneath the objective, with the coverslip visible on the upper side. The stage clip should be holding the slide in place, not pressing the slide under it. Using the left and right/up and down stage knobs), center the object below the objective without looking through the oculars. Nothing is in focus yet. Coarse adjustment with scanning lens: With the scanning lens in place, move the stage up to its highest point without looking through the oculars. Nothing is in focus yet. Looking through the ocular with your right eye only (squint or cover your left eye), bring the specimen into focus by turning the coarse focus adjustment knob slowly until the specimen is generally in focus. Then turning the fine adjustment knob will bring the specimen into sharper focus. Focus your left eye: Viewing the specimen with both eyes through both oculars, turn the left ocular diopter until the specimen is clear in both eyes. Iris diaphragm: The light coming through the microscope may be either too bright or too dim. If the amount of light is not satisfactory, it can be adjusted by carefully regulating the size of the opening of the iris diaphragm by moving the lever beneath the stage. The iris diaphragm is part of the condenser which concentrates the light coming from the light source. Adjusting on low and high power objectives - use fine adjustment knobs only: If you wish to view the specimen using higher magnification, center the specimen in the field, and carefully rotate the revolving nosepiece to bring the next higher power objective into place beneath the body tube. The specimen will no longer be in focus. In order to sharpen the image of the specimen, adjust the focus using only the fine focus adjustment knob. (Again, the light may have to be adjusted with the iris diaphragm.) Each time you move to the next higher power objective, be sure you center the specimen beforehand. Examine the drop first under scanning, then low power, then under high power. Be sure you can see the individual particles of India ink. Focus your attention on one particle (under high power) for several seconds. Look for a slight but vigorous movement of this particle, independent of the other particles. (Note: You may need to wait a few minutes. Then, if you see a mass "flowing" movement of all the particles in one direction like a small river, this is not Brownian motion. Wait for the flowing to subside, then carefully observe one particle.) Question 3. Describe the Brownian motion in your own words: Replace this text with your answer. Question 4. Describe, in your own words, how this observation indicates that all visible and invisible molecules are in motion? Replace this text with your answer. Remove the slide: Once everyone in your group has viewed the Brownian Motion and you need to remove the slide, be sure to rotate the nosepiece to the scanning objective. Then using the coarse adjustment knob, lower the stage to its lowest position. Then open the stage clip and remove the slide. Clean up.Rinse any wet mount slides and place them in the container marked “Used slides.” Wash and dry the coverslips and place them in their original container.Throw away any Kimwipes or other paper.You will need your microscope for other parts of this lab, so leave it available. However, make sure it is not near where there is sugar solution. Keep it safe.II. DIFFUSION Diffusion is the movement of particles from an area of high concentration to an area of low concentration. This results from the continuous random motion that is characteristic of all molecules in liquid or the gas states. A few observations about diffusion will help us to understand how molecules can move from one location to another, perhaps even across cell membranes. A. Diffusion through a Colloid The contents of a cell (the cytoplasm) may be described as a colloid rather than a liquid or solid. Large protein molecules are present in a cell's cytoplasm that allow it to be in a transitional state of matter called a colloid (somewhere between a liquid and a solid). A special kind of colloid, agar gel, is available in the laboratory and will be used to demonstrate how molecules diffuse from one place to another once they are inside a cell. Agar is a carbohydrate extracted from algae in powder form. A gel is prepared by mixing the powder with water, then heating followed by cooling--similar to the preparation of a gelatin (animal protein) dessert. The result is a gelatin-like substance composed of intertwined molecules with water trapped among them. Two compounds (molecules), potassium permanganate and methylene blue, have been selected to illustrate diffusion through a colloid. Unlike most components of living cells, these compounds are brightly colored, allowing us to watch their diffusion. Procedure: 1. Working with your team, obtain a disposable Petri dish containing agar gel. 2. Using a No. 5 cork borer (a "punch"), make two holes in the agar approximately 5 centimeters apart (See diagram below). Remove the plugs of agar with a toothpick and place in the trash. 3. Bring your agar dish, the dropper bottles of potassium permanganate solution and the dropper bottle of methylene blue back to your lab bench. 4. Place two drops of 1% potassium permanganate solution in one of the holes. In the other hole, place two drops of 1% methylene blue solution. Start a timer for 60 minutes. Return the bottles back to the side lab benches for others to use.5. After approximately one hour, measure the diameter (in millimeters) of the circle that the solutions diffused. Potassium permanganate:mmMethylene blue:mm 6. Examine your Petri dish. Shade the agar in the diagram to demonstrate the movement of the solutions in the diagram below. Shade to show where the concentration of the potassium permanganate is higher and where it is lower. Top view of Petri dish: Side view of Petri dish: