Week 6 Lab 6 Cell Structure and Function

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Note: All your answers to questions must be in Red or other color (not including blue) for easier grading. Points will be deducted if you do not distinguish your answers. Lab 6. Cell Structure and Function Objectives:  Distinguish between eukaryotic and prokaryotic cells.  Describe the structure and function of plant and animal cells.  Define the following terms: diffusion, osmosis, equilibrium, tonicity, turgor pressure, plasmolysis.  Describe what drives simple diffusion (why do the molecules move?).  List the factors that may affect the speed of simple diffusion.  List which molecules, in general, can freely diffuse across the plasma membrane of a cell.  Describe what drives osmosis (why do water molecules move?).  Explain why water moves out of a cell when the cell is placed in a hypertonic solution.  Explain why water moves into a cell when the cell is placed in a hypotonic solution.  Describe what physically happens to a cell if water leaves the cell.  Describe what physically happens to a cell if water enters the cell. Vocabulary:  Cell theory  Plasma membrane  Cytosol  Plasma membrane  Chromosomes  Ribosomes  Prokaryotic cells (Prokaryotes)  Eukaryotic cells (Eukaryotes)  Central Vacuole  Cell wall  Lignin  Chloroplast  Chlorophyll  Centrosome  Lysosomes  Diffusion

 Osmosis  Solute  Isotonic  Hypertonic  Hypotonic  Turgor Pressure  Plasmolysis Introduction: Cell theory states that the cell is the fundamental unit of life. However, cells vary significantly in size, shape, structure, and function. At the simplest level of construction, all cells possess a few fundamental components. These include cytosol (a gel-like substance composed of water and dissolved chemicals needed for growth), which is contained within a plasma membrane (also called a cell membrane or cytoplasmic membrane); one or more chromosomes (condensed DNA and proteins), which contain the genetic blueprints of the cell; and ribosomes , organelles used for the synthesis of proteins. Beyond these basic components, cells can vary greatly between organisms, and even within the same multicellular organism. The two ( main type or categories of cells are) largest categories of cells— prokaryotic cells ( ex: bacteria; usually smaller & simpler ) and eukaryotic cells (ex: plant & animal cells; bigger than prokaryotic & more complicated b/c there are more organelles inside of them) —are defined by major differences in several cell structures. In this exercise, you will examine the semipermeable nature of the cell membrane (aka, plasma membrane). The cell membrane controls what enters and exits the cell, and therefore serves a very important cellular function. You will also explore the concept of tonicity, which refers to the solute concentration of a solution, and its inherent ability to influence the rate and direction of osmosis.

Prokaryotic Cells: Prokaryotic cells are cells without a nucleus. The DNA in prokaryotic cells is in the cytoplasm rather than enclosed within a nuclear membrane. Prokaryotic cells are found in single-celled organisms, such as bacteria, like the one shown in Figure 1 . Organisms with prokaryotic cells are called prokaryotes . They were the first type of organisms to evolve and are still the most common organisms today. Bacteria are described in the following video: https://youtu.be/TDoGrbpJJ14 Figure 1: This diagram shows the structure of a typical prokaryotic cell, a bacterium. Like other prokaryotic cells, this bacterial cell lacks a nucleus but has other cell parts, including a plasma membrane, cytoplasm, ribosomes, and DNA. Identify each of these parts in the diagram.

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Eukaryotic Cells In nature, the relationship between form and function is apparent at all levels, including the level of the cell, and this will become clear as we explore eukaryotic cells. The principle “form follows function” is found in many contexts. For example, birds and fish have streamlined bodies that allow them to move quickly through the medium in which they live, be it air or water. It means that, in general, one can deduce the function of a structure by looking at its form, because the two are matched. A eukaryotic cell is a cell that has a membrane-bound nucleus and other membrane-bound compartments or sacs, called organelles, which have specialized functions. The word eukaryotic means “true kernel” or “true nucleus,” alluding to the presence of the membrane-bound nucleus in these cells. The word “organelle” means “little organ,” and, as already mentioned, organelles have specialized cellular functions, just as the organs of your body have specialized functions. Plant Cells are squarish rectangular in size in shape b/c it contains a cell wall to maintain the cell shape. Main difference btwn the animal & plant cell, are 3 structures: plasma membrane, chromosome, & ribosomes. Plant cells resemble other eukaryotic cells in many ways. For example, they are enclosed by a plasma membrane and have a nucleus and other membrane-bound organelles. A typical plant cell is represented by the diagram in Figure 2b .

Figure 2. Plant cells (b) have all the same structures as animal cells (a), plus some additional structures. Structures found in plant cells, but not animal cells include a large central vacuole, cell wall, and chloroplasts.  The large central vacuole is surrounded by its own membrane and contains water and dissolved substances. Its primary role is to maintain pressure against the inside of the cell wall, giving the cell shape and helping to support the plant.  The cell wall is located outside the cell membrane. It consists mainly of cellulose and may also contain lignin , which makes it more rigid. The cell wall shapes, supports, and protects the cell. It prevents the cell from absorbing too much water and bursting. It also keeps large, damaging molecules out of the cell.  Chloroplasts contain the green pigment chlorophyll and carry out photosynthesis. Chromoplasts make and store other pigments. They give flower petals their bright colors. Animal Cells are circular-ish sometimes similar shape but closely related to a circle & organelles you see here are also found on the plant cell. Main difference btwn the animal & plant cell, are 3 structures: plasma membrane, chromosome, & ribosomes. At this point, you know that each eukaryotic cell has a plasma membrane, cytoplasm, a nucleus, ribosomes, mitochondria, peroxisomes, and in some, vacuoles, but there are some striking differences between animal and plant cells. While both animal and plant cells have microtubule organizing centers (MTOCs), animal cells also have centrioles associated with the MTOC: a complex called the centrosome. A typical plant cell is represented by the diagram in Figure 2a . Animal cells each have a centrosome and lysosomes, whereas plant cells do not.  The centrosome (the organelle where all microtubules originate) replicates itself before a cell divides, and the centrioles appear to have some role in pulling the duplicated chromosomes to opposite ends of the dividing cell.  In addition to their role as the digestive component and organelle- recycling facility of animal cells, lysosomes are considered to be parts of the endomembrane system. Lysosomes also use their hydrolytic enzymes to destroy pathogens (disease-causing organisms) that might enter the cell. Diffusion

Diffusion is defined as the net movement of molecules or ions from a region of high concentration to a region of lower concentration. Diffusion continues until a state of equilibrium is reached, which means that the molecules are randomly distributed throughout the system. Diffusion is considered a form of passive transport because no energy is required in the process. Diffusion can occur in a gas, a liquid, or a solid medium. Diffusion also occurs across the selectively permeable membranes of cells. Osmosis Osmosis is a special case of diffusion in which water molecules pass through a selectively permeable membrane, but larger molecules do not. Osmosis proceeds from a region of high water concentration, across a semi- permeable membrane, to a region of lower water concentration until equilibrium is reached. A solute is a solid substance , such as salt or sugar that is dissolved in a solvent. Water is usually the solvent in living systems. A typical animal cell contains a salt concentration of 0.9%. A solution of equal solute concentration is referred to as isotonic . A cell placed in an isotonic environment will experience movement of water inside and outside the cell, but there will be no change in the biology of the cell. A hypertonic solution contains a high solute concentration with respect to cells. For example, a solution containing 10% salt is hypertonic. When a cell is placed in a hypertonic environment, there is a net movement of water to the outside of the cell (from the higher water environment inside the cell). The cell shrinks in response . A solution of low solute concentration is referred to as hypotonic . A solution containing 0.5% salt is hypotonic with respect to the cell. When a cell is placed in a hypotonic environment, there is a net movement of water into the cell. The cell swells in response .

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Part 1. Molecular Weight and Diffusion Rate Molecular weight is an indication of the mass and size of a molecule. The purpose of this experiment is to determine the relationship between molecular weight and the rate of diffusion through a semisolid gel. You will investigate two dyes, methylene blue and potassium permanganate. Table 1: Properties of Methylene Blue and Potassium permanganate. Molecule Molecular Weight Color Methylene blue 300 grams/mole blue Crystal Violet 407.97 grams/mole purple Question: Which molecule will have the fastest diffusion? Hypothesis: How might these factors influence the rate of diffusion? 1. Temperature: Temperature is a critical factor in determining the rate of diffusion because it directly affects the energy and movement of molecules. Higher temperatures promote faster diffusion, while lower temperatures lead to slower diffusion. 2. Concentration gradient: The greater the difference in concentration, the more rapid the diffusion. 3. Medium molecules diffuse in (gas, liquid, solid): In the medium, there will be different rates of diffusion of the different substances. Diffusion is slowest in solids because particles have less mobility and are tightly packed. Then followed by liquids, the particles in liquids have more freedom to move around since they are not tightly bound together; and the fastest is gases, as the particles in gas have a high degree of freedom and move rapidly in random directions. 4. Molecular weight of the molecule: Heavier molecules move more slowly; therefore, they diffuse more slowly. Lighter molecules move faster, so they will diffuse more quickly. 5. Charge: A difference in charge can influence the rate and direction of diffusion such as charged particles like ions. 6. Solubility: Nonpolar or lipid-soluble materials pass through plasma membranes more easily than polar materials, allowing a faster rate of diffusion. Materials:  Petri dish of agar semi-solid gel  Methylene blue solution  Potassium permanganate solution

 Cork borer  Small plastic metric ruler ❗ C AUTION : C RYSTAL V IOLET : Eye contact may result in permanent eye damage. Poison! Flammable liquid and vapor. May be fatal or cause blindness if swallowed. Harmful if inhaled. May be harmful if absorbed through the skin. May cause eye and skin irritation. May cause respiratory tract irritation. May cause central nervous system depression. May cause liver and kidney damage. May cause fetal effects based upon animal studies. Cannot be made non-poisonous. ❗ CAUTION: M ETHYLENE BLUE : Avoid ingestion, inhalation, and contact with skin, eyes, and mucous membranes. If any should spill on your skin, wash the area with mild soap and water. Methylene blue will also stain clothing. Figure 3. Placing a drop of dye into a small well on an agar plate Procedure:  Obtain a Petri dish of agar  Take the cork borer and gently stick down into the agar. Lift up withdrawing a small plastic plug of agar. Repeat.  Place a single drop of each dye into the agar well. ( Figure 3 ).  Leave the dyes to diffuse overnight. (Your instructor will have one prepared to visualize)  The next day, place a small, clear metric ruler underneath the Petri dish to measure the distance (diameter) that the dye has moved. Enter the data in Table 2 . Results: (Watch the Lab 6. Cell Structure and Function video) and fill the table below: Table 2: Methylene blue and Potassium permanganate Diffusion Results

Molecule Molecular weight (grams/mole) Diameter after overnight (millimeters) Methylene blue 300 grams/mole 1.9 cm = 19 mm Crystal Violet 407.97 grams/mole 1.6 cm = 16 mm Conclusion: 7. Describe the relationship between molecular weight and speed of diffusion. The heavier the weight the slower the rate of diffusion and the lighter the weight the faster the rate of diffusion. Part 2: Diffusion across a selectively permeable membrane (means that the cell membrane the outside layer will allow some structures molecules to come in & out of the cell it depends on several characteristics). Cells acquire the molecules and ions they need from their surrounding extracellular fluid. In living cells, the ability of a molecule to cross the cell membrane is influenced by its size, charge, lipid solubility, and other characteristics. Small molecules such as water, oxygen, amino acids, and ions easily cross the membrane by passive transport processes that do not require energy (diffusion and osmosis). Other molecules do not easily fit through the lipid bilayer and the cell must expend energy to bring them across . You will investigate two molecules, starch, and iodine, for their ability to cross a selectively permeable membrane. A colorimetric test is employed to assess the movement of these molecules. Dialysis tubing is a transparent material with microscopic pores that allow only small molecules to pass. It provides a model of the cell membrane and has many uses in industry and medicine. Question: Which molecules will diffuse into or out of the dialysis tubing? Methylene blue since it is less in molecular weight, and it is placed in a hypotonic environment causing it to swell in response. Hypothesis: Larger molecules will not diffuse as easily as smaller molecules, for example starch . Materials:

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 (2) 250 mL beakers  Dialysis tubing  Glucose solution  Starch solution  Water  Iodine (IKI)  String to tie  Serological pipet  Benedict’s solution  Test tube  Boiling water (water bath)  Paper  Wax pencil Procedure:  Obtain a piece of dialysis tubing that has been pre-cut by the instructor. Thoroughly wet the tubing and open the ends. Tie a knot at one end.  Add 4 mL of starch solution and 4 mL of glucose solution to the dialysis bag.  Add a small piece of paper with the letter A inside the dialysis bag.  Tie a knot at the top of the tubing. Rinse the bag briefly with tap water to remove any traces of starch.  Repeat with a second piece of dialysis tubing inserting a piece of paper with the letter B.  Fill both beakers with 125 mL of tap water. Label them A and B.  Add iodine (IKI) to the beaker of water until a deep yellow (tea-like) color is obtained about 4 full droppers squirts to beaker A. Beaker B will not have iodine .  Submerge the dialysis bag in the water and incubate at room temperature until a color change is observed (15 – 30 minutes).  Record your results in Table 3 .  After you finish recording your color, place the dialysis tubing that was on Beaker B back into it.  Wait an additional 30 min before proceeding.  Obtain a test tube and take about 3 mL of water from the beaker (near the dialysis bag) and preform a benedict’s test.  Mix 3 mL of water from the beaker with 3 mL of benedict’s solution and incubate in boiling water for 5 minutes. Record your results in Table 3 .

Results: (Watch the Lab 6. Cell Structure and Function video) Table 3: Diffusion across the selectively permeable membrane results. Contents Initial Color Final Color Benedict’s Test Conclusion Bag A (1) Glucose Starch Non- transparen t, murky Clear Not Applicable Only glucose and water diffused by passive transport process. Bag B (2) Glucose Starch Non- transparen t, murky Dark blue, almost black Not Applicable Only Iodine and glucose diffused across the dialysis tubing membrane . Beaker A w/bag (2) Water Iodine Tea color, yellowish Yellow Not Applicable Beaker solution is unchange d since the molecules in starch are too large to pass. Beaker B w/bag (1) Water See- through, clear See- through, clear Negative The color of the beaker changed to a murky green. Conclusion: 8. Did starch diffuse across the selectively permeable membrane? How do you know? No, starch (stayed trapped inside the bag), it did not diffuse across the selectively permeable membrane. The permeable membrane allows for

some substances to pass, and blocks others based on the molecular size. We know that iodine played a role inside the bag, but it also contains itself outside the bag because the water in the beaker was yellow. Otherwise, if the iodine had diffused across then both solutions would mix, turning both purple. 9. Did glucose diffuse across the selectively permeable membrane? How do you know? Small molecules such as water, oxygen, amino acids, and ions do not require energy (diffusion/osmosis) so they can easily cross the membrane by passive transport processes. Yet, other molecules do not easily fit through the lipid bilayer so the cell must expend energy to bring them across. In the matter of glucose, it did diffuse across the selectively permeable membrane but only with the help of facilitated diffusion. 10. Which is the smaller molecule glucose or starch? Smaller substances will diffuse across in contrast larger substances will not and we know that diffusion occurs based on molecular size. So, in this case, the glucose molecule is small enough to pass through the membrane and starch molecules are too large to fit through the pores of the dialysis tubing. In summary, diffusion established on the molecular size between the two. 11. Is diffusion a passive or an active transport process? The simplest forms of transport across a membrane are passive; therefore, diffusion is a passive transport process since it does not require energy. Part 3: Observation of Osmosis in Aquatic Plant Plants have cell walls that can prevent lysis if too much water flows into the cell. Plant cytoplasm tends to be hypertonic to the outside environment, which results in an inflow of water and a high amount of pressure (turgor pressure ) inside the cell. When a plant is placed in a hypertonic environment, the water will leave the cell. This causes the cell to shrink and detaches the plasma membrane from the cell wall ( plasmolysis ). Turgor pressure can hold plants upright, while plasmolysis can cause plants to wilt.

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Question: How will exposing plant cells to hypertonic affect their chloroplast arrangement? The chloroplast will react by clustering at the center of the cell. Hypothesis: The rate of osmosis will be faster based on the greater difference between the concentration of water inside and outside of the cell. Materials:  Elodea leaves in water  Microscope slide  Coverslip  Tweezers  NaCl solution Procedure:  Observe two Elodea leaves under the microscope.  Prepare a wet mount in another slide is a leaf in NaCl solution: let the leaf soak in the NaCl solution for at least 40 minutes before viewing it under the microscope  Prepare a wet mount with one leaf in isotonic solution: you can use the water the Elodea is in. You can view this slide immediately afterwards.  Draw what you see, make sure to label the various parts. Results: (Watch the Lab 6. Cell Structure and Function video) 12. Draw your Elodea leaf in isotonic solution and write the total magnification on your drawings. Label the cytoplasm, cell wall, and chloroplasts. Total magnification (40x) x (10x) = 400x

Draw your Elodea leaf in NaCl solution and write the total magnification on your drawings. Label the cytoplasm, cell wall, and chloroplasts. Total magnification (40x) x (10x) = 400x Conclusion: 13. What is the difference between a hypertonic solution and a hypotonic solution? Hypertonic solution contains a high solute concentration, and a hypotonic solution contains a low solute concentration. If a cell is placed in a hypertonic solution, water will leave the cell and the cell will shrink in response from the higher water inside the cell because there is a net movement of water to the outside of the cell. When a cell is placed in a hypotonic environment, water will enter the cell, and the cell will swell. 14. What will happen to plant cells that were placed in a hypertonic (NaCl) solution? When a plant is placed in a hypertonic environment, the water will

leave the cell, in other means, the large central vacuole loses water. The plasma membrane pulls away from the cell wall, the cytoplasm shrinks, and the chloroplasts are found in the center of the cell. So, because the cell shrinks, it also detaches the plasma membrane from the cell wall (plasmolysis), that is why plants wilt. 15. What will happen to animal cells placed in hypotonic solution? Why should this be different from plant cells? Animal cells are different from plant cells . When animal cells are in hypotonic solutions, water enters the cell, the cell will swell/expand, and eventually burst (cytolysis). When plant cells are in hypotonic solutions, large central vacuole gains water, the cytoplasm expands, and the plasma membrane pushes against the cell wall, but the swollen cell does not burst. 16. Why are dehydrated patients given saline intravenously instead of water? Vital vitamins and minerals are the key in saline solution which help restore your body’s water-electrolytes balance; therefore, it offers a quick and effective dehydration relief than ordinary water. A saline solution is isotonic, in other words it has a lower concentration of salt, equaling a perfect balance for your body. Do it At Home (Optional): Osmosis in potato cells ? Question: How does putting potatoes in hypertonic solution affect their rigidity? Materials:  Two slices of potato pre-cut  NaCl (table salt)  2 disposable cups  Water  Marker Procedure:  Obtain two pieces of peeled potato, 2 cm X 0.25cm.  Label two cups with a marker with “Cup 1” and “Cup 2” o Cup 1: Add one piece of the potato and water to the cup so that the potato piece is completely submerged. o Cup 2: Add the second piece of potato and about 4 tablespoons of NaCl (table salt) in water , enough to submerge the potato piece. Swirl to dissolve the NaCl.

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 Incubate at room temperature for about 15 minutes.  Pour off the solution and feel each potato piece. Record your results. Observations:  Which potato piece is stiff? Explain why with respect to osmosis. Cup 1, potato, and water. The potato swelled and got stiff because of the greater concentration of water outside its cells. As a result, the water entered the potato’s cells by osmosis. Turgor pressure is when there is an inflow of water and a high amount of pressure inside the cell. Therefore, the potato is stiff. Hypotonic, water went in.  Which potato is soft? Explain why with respect to osmosis. Cup 2, potato, and table salt (NaCl) in water. The potato became soft because both the water and salt played a role in the process. Water left the cell, causing the cells to shrink and detach, so without pressure the potato goes limp/soft, plasmolysis. Hypertonic, water went out. Licenses and Attributions:  " Cell Structure " by Melissa Ha, Maria Morrow, & Kammy Algiers , LibreTexts is licensed under CC BY-NC .  " Comparing Prokaryotic and Eukaryotic Cells " by OpenStax , LibreTexts is licensed under CC BY 4.0 .  " Plant Cells " by Lumen Learning , LibreTexts is licensed under CC BY 4.0 .  " Unique Features of Animal and Plant Cells " by Lumen Learning , LibreTexts is licensed under CC BY 4.0 .  " Diffusion " by Ellen Genovesi, Laura Blinderman, & Patrick Natale , LibreTexts is licensed under CC BY .  " Osmosis " by Ellen Genovesi, Laura Blinderman, & Patrick Natale , LibreTexts is licensed under CC BY .  " Osmosis and Diffusion " by Lumen Learning , LibreTexts is licensed under CC BY 4.0 .

Week 6 Lab 6 Cell Structure and Function

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