Lab 5 Worksheet - osmosis

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Dec 6, 2023

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Lab 5: Microscopes, Osmosis, and Cell Structure Objectives: At the end of this lab, you should be able to: 1. Demonstrate the proper procedures to use a compound and a stereoscopic microscope. 2. Identify the parts of a compound and a stereoscopic microscope. 3. Identify cell structures. 4. Compare simple diffusion to osmosis. 5. Discuss the characteristics of the plasma membrane and how it influences the types of molecules that can diffuse through it. 6. Differentiate between hypertonic, isotonic and hypotonic solutions and their effect on cell morphology. Introduction The cell is the fundamental unit of life; it is the simplest biological structure able to divide and metabolize. The Cell theory establishes that all cells come from other cells and all organisms are composed of cells. It is necessary to understand the structure and function of cells to be able to understand the organization of living organisms. Prokaryotic (from the Greek Pro= before, Karyon= nucleus) cells are unicellular; they don’t have a nucleus or membrane bound organelles. Prokaryotes however have DNA (contains genes) and ribosomes that read the RNA transcribed from the DNA. Eukaryotes (from the Greek Eu=true, Karyon=nucleus) possess a defined nucleus with a nuclear membrane. Prokaryotes appeared on Earth approximately 3.5 billion years ago, whereas Eukaryotes appeared 1.5 billion years ago. Eukaryotic cells contain organelles with various functions, including nucleus, mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, etc. Some of these structures can only be observed with an electron microscope. The nucleus is one of the biggest organelles; it contains the genetic material and it can be easily observed with a bright field microscope. Generally, the ER, mitochondria, Golgi apparatus and lysosomes are not observed with a bright field microscope. Mitochondria can be observed under oil immersion but without much detail. Figure 1 : Cell structure

In order to study cells, we need microscopes. There are different types of microscopes, with different resolutions which are used for various purposes. These include compound microscopes also called bright field microscopes (figure 3), electron microscopes (to study the ultrastructure of the cell, with a resolution of 0.2 to 0.3 nm, magnification of around 300,000X), fluorescent microscopes (to observe cells labeled with fluorescent dyes) and other more specialized microscopes. Parts of a compound (bright field) microscope (refer to figure 3)  Ocular: Lenses in the eyepiece, usually 10X magnification.  Objectives: Lenses on the revolving nosepiece, usually 4X (scanning lens) and 10X (intermediate lens), 40X and 100X magnification (high power lenses).  Revolving nosepiece: Holds the objectives. This is the part that should be used to move the objective lenses.  Scanning lens: The lowest magnification objective (4X or 10X depending on the microscope).  Condenser lens: Used to focus the light from the lamp.  Condenser diaphragm : Controls the amount of light that passes through the specimen.  Stage: Where the slide with the specimen to be viewed is positioned. There are adjustment knobs to correctly position the slide and clips to secure the slide.  Fine and coarse focus: Adjust the distance between the stage and the object. Diffusion and Osmosis For an organism to function properly, it is important that the organism maintain an internal environment that will meet certain requirements needed to sustain life. Maintaining a constant internal environment is termed homeostasis . The plasma membrane plays a crucial role in homeostasis, by influencing the type of molecules which are able to pass the membrane. This membrane is made predominantly of phospholipids. These molecules possess fatty acid tails that are hydrophobic (water -fearing) and a charged phosphate head group that is hydrophilic (water-loving). Thus, the plasma membrane allows small, hydrophobic molecules to pass through the membrane while large, hydrophilic molecules are prevented from entering. Due to the differential permeability of the plasma membrane, the membrane is said to be selectively permeable . Dissolved ions and molecules are found in the aqueous solutions in the internal and external environments of the cell. Passive transport processes allow for the movement of the molecules and ions through the aqueous solutions or the plasma membrane. Passive transport processes require no input of energy into the system and include diffusion, osmosis and facilitated diffusion. The processes studied in this lab will be osmosis. Diffusion is a physical process in which the kinetic energy of molecules and ions moves them from region of high concentration to low concentration. After perfume is sprayed into the air, it is diffusion that moves the perfume from an area where the perfume can be smelled (high concentration) to an area that the perfume cannot be detected any longer (low concentration). Osmosis is a specific form of diffusion involving the movement of water. Specifically, it is the movement of water across a semi- permeable membrane from a region of low solute concentration to a region of high solute concentration (see figure to the right). If a red blood cell is placed in an isotonic solution (i.e., one that has the same solute concentration as that found in the plasma and the cytoplasm of red blood cells), then the cell will retain its normal shape (figure below). If a red blood cell is placed in a hypotonic solute solution (i.e., one that has a lower solute concentration than the

plasma or cytoplasm), then water will enter the cell more rapidly than it leaves. This will cause the cell to swell (see figure below) and possibly burst or lyse . If a red blood cell is placed in a hypertonic solute solution (i.e., one that has a higher solute concentration than the plasma or cytoplasm), then water will exit the cell faster than it will enter. This causes the cell to shrink and have a bumpy appearance or crenate (see figure below). Part I: Manipulating the compound microscope Exercise 1 1. Turn on your microscope. Using the light adjustment knob, adjust the amount of light to mid-range to avoid damage to your eyes . 2. By moving the revolving nosepiece (the ring where the objectives are located), put the lowest objective (scanning lens) into position. This could be the 4X or 10X, depending on your microscope. NEVER move the objectives by holding the objectives themselves , always use the ring of the revolving nosepiece . If you rotate the objectives by exerting pressure over them, you could damage the alignment of the objectives. ALWAYS use the scanning lens as the first objective for each slide you need to observe. Never start trying to find the specimen using 40X magnification. It will not only be more difficult for you, but you could also scratch the lenses and break the specimen slides. 3. To make a wet mount, place a tip of an elodea tip on a clean glass slide. Add one drop of water, and put a cover slip on top of the leaf. To do this, touch the glass slide with the border of the cover slip and gently lower the other side of the cover slip until the leaf is covered (see figure below; using forceps will make this task easier). Eliminate the excess water by gently touching the border of the cover slip with a Kimwipe. Figure 2 : Illustration of how to prepare a wet mount Cover slip Forcep s Slide

Figure 3: Parts of a compound microscope (also called a brightfield microscope) 4. Position the slide on the stage. Put the lowest objective (4X – scanning lens) into position. Bring the leaf into focus by using the coarse focusing knob. Gently move the oculars until you see only one image of the leaf. This will adjust the oculars to your pupils and now you can see with both of your eyes. Anytime you need to use the microscope, you may need to adjust this distance if somebody else has changed your settings. 5. Close the field diaphragm (if using the CX31 microscope) to block the most amount of light as possible. 6. Change the height of the condenser to a position that allows you to view a polygon with sharp edges as you look through the oculars using the knob the left side. 7. If using the CX31 microscope, you can use the two metal screws on the condenser to center the light in the field of view. 8. Now you can open the field diaphragm until light extends just beyond the field of view. 9. Now focus the leaf by using the fine focus knob. Then, using the revolving nosepiece (NOT THE OBJECTIVES THEMSELVES), go to the next magnification. 10. SLOWLY move the coarse focusing knob until the image is almost in focus. 11. Next adjust the fine focus. *** Never skip magnifications without going through this step in each one of the intermediate magnifications. Never turn the fine adjustment knob more than 2 revolutions. You could damage the lens and break the slide. If you cannot focus, go back to the previous magnification and focus again . ***

Part II: Focusing on a Specimen Exercise 1 Prepare one or two wet mount slides of pond water. In order to do this, take a drop of pond water and put it on a clean slide. Carefully put the cover slip on top. Eliminate the excess of water by carefully touching the edge of the cover slip with a piece of paper towel. Focus the microscope. If the organisms are moving too fast, gently add ProtoSlo to the side of the cover slip . Exercise 2: Effect of solute concentration on the rate of osmosis The rate of diffusion is influenced by many factors including the concentration gradients of solutions. In this exercise, you will investigate the effect of solute concentration on the rate of osmosis. 1. Obtain a piece of dialysis tubing and fold over 3 cm of one end of the tubing, pleat the folded end (similar to an accordion) and close the tube with a clamp. 2. Fill the tubing until it is half full with one of the colored solutions: 3. After filling half full, gently squeeze the bubbles out of the tubing. 4. Press the sides of the bag together so that the air does not reenter. 5. Close the top end of the bag as you did in step 1. 6. Weigh the bag (including the clamps) and record the weight. 7. Place the bags in separate beakers of distilled water. 8. Start your timer 9. Allow them to sit in the beaker for 15-20. 10. After removing the bags and wiping off the excess water, weigh each bag separately. 11. Record the weights in your lab report. Exercise 3: Osmosis – Animal cells Red blood cells (erythrocytes) are cells made of plasma membrane and large quantities of hemoglobin which is required to carry oxygen and carbon dioxide in the blood. Their membranes are permeable to water, oxygen and carbon dioxide, but impermeable to proteins, sodium chloride and glucose. Normally these cells have a biconcave shape which enhances their ability to carry the blood gases. Today, you will expose your own red blood cells to various solutions to determine the effect of osmosis on cell shape. Students should wear gloves to perform this experiment and ANY materials ending up with blood on it should go in the proper waste container provided 1. Obtain three slides and label each one, off to the side, with a grease pencil as: solution A, solution B, and solution C. 2. Using an alcoholic wipe, clean the area of your finger you wish to withdraw blood from. Let the area dry. 3. (Only the person donating the blood should handle their blood. Otherwise, you might catch a life- threatening disease). 4. Using a sterile blood lancet gun, prick your finger and drop a small amount of blood onto the middle of each division of the slide. 5. Work quickly to add a drop of each solution to the correct division while being careful not to touch the slide or the blood already on the slide. 6. Use a toothpick to mix the blood with each solution (using a fresh toothpick for each sample). *** Dispose of the bloody toothpick in the Sharps Container!!! 7. Place cover slips over the blood sample and observe the blood cells under the microscope.

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