MD105 Molecular and Cellular Biology Lab Exercise 1 PDF

MD105 Molecular and Cellular Biology Lab Exercise 1: Membrane permeability of onion plant cells and human red blood cells Fall Semester 2024 TC.Kyriakou & D. Markantoni COVID-19 Safety Protocol for labs ▪ Be exactly on time to avoid throngs of s...

MD105 Molecular and Cellular Biology Lab Exercise 1: Membrane permeability of onion plant cells and human red blood cells Fall Semester 2024 TC.Kyriakou & D. Markantoni COVID-19 Safety Protocol for labs ▪ Be exactly on time to avoid throngs of students at the lab entrance. ▪ Always curry with you the protective gear (lab coat with cuffed sleeves, mask, goggles). ▪ Students without any of the gear will not be allowed in the lab. ▪ Hand hygiene is essential. Wash your hands with soap, then put on some gloves. ▪ Disinfect working surfaces before and after experimentation. ▪ In case a student has symptoms such as fever, couch or breathing difficulties please report immediately.  Absences ▪ Mandatory participation ▪ Physical presence or synchronous participation (only after permission from the department is granted) ▪ Only two justified absences are excused (severe health condition or another emergency) ▪ In every case justified absences must be announced before the beginning of the class. ▪ Lab experiments are not to be repeated!  Be prepared in advance ▪ Study the uploaded material. Print out the protocol or experimental methodology. ▪ Mark materials and equipment needed on the protocol. ▪ Be mentally prepared for technically demanding applications. Closed-toe shoes! Wear lab coat, goggles and gloves! Have your hair tight (pony-tail)! No food and drinks!  Keep a Laboratory notebook Take a note on everything while being explained to you. Keep a record of protocol applications marked with dates. Make calculations of reagent concentrations: e.g.C1.V1=C2.V2 Evaluate results and spot what is needed to be changed. How to move and communicate in the lab Move as if in a bubble Know your surroundings Work as a team Raise your hand to ask No fast movements Work in silence  Contact information Email: [email protected] (N41) [email protected] (N42)...for any question, clarification or any other issue! Office: N30 (2nd floor – New Building) EUC account policy In case that a student sends email to a faculty member through his/her personal email account please notify them that in order to read the email it must be send through their EUC email account.  Objectives ▪ Observe the effect of different solutes on diffusion of plant and human cells ▪ Explain hypertonic, hypotonic and isotonic in terms of cellular environment ▪ To compare and assess cell transport activity in human and plant cells  Safety Precautions 1. Do not eat or drink in the laboratory. 2. Wear safety glasses, lab coat and gloves when performing the experiment. 3. Scalpels are extremely sharp; caution is advised when using. 4. Hold the scalpel as you would a pencil. 5. Cut with a downward motion, but never push down very hard to make a cut. 6. Watch the placement of your specimen-holding hand. Do not cut toward your holding hand. 7. Do not use for anything other than the intended task. 8. Use caution with glass slides and cover slips.  Diffusion For living organisms to survive, nutrients, water, and waste must be able to move into and out of the cell. This process maintains homeostasis. The movement of materials into and out of the cell is regulated by the membranes that surround both the organelles within the cell and the outer plasma membrane. These membranes are selectively permeable. The cells live in an aqueous or high in water content environment. Dissolved within the water are solutes (salts and organic molecules) needed by the cell to function. The simplest way molecules move is through diffusion, which is when substances move from an area of high concentration to an area of low concentration. Diffusion does not require the expenditure of energy.  Osmosis What is the main difference between osmosis and diffusion?  Onion Plant Cells Osmosis in onion cells If a plant cell is surrounded by a solution that contains a: 1. Higher concentration of water molecules than the solution inside the cell, water will enter the cell by osmosis and the plant cell will become turgid (firm). 2. Lower concentration of water molecules than the solution inside the plant cell, water will leave the cell by osmosis and the plant cell will become flaccid (soft).  Experimental Procedure PART A: Onion plant cell Collect all your materials and take them to your work area. 1. Peel your onion. 2. Place the onion tissue layer on a dry microscope slide with the shiny side facing up. 3. Do not forget to label the slide (group and solution). 4. Add a small drop of solution A directly to the tissue. 5. Carefully cover it with a coverslip and avoid any air bubble from entering the coverslip. 6. Repeat steps 1-3.  Experimental Procedure PART A: Onion plant cell 8. Add a small drop of solution B, and C directly to the tissue. 9. Carefully cover it with a coverslip and avoid any air bubble from entering the coverslip. You must have three slides ! 10. Observe the prepared mount of the peel under the low and high magnification of a microscope. 11. Label the parts you see (cell wall, cytoplasm, nucleus) and take pictures (one for each slide). 12. Compare the three slides and decide as a group which slide has solution A, B, and C. PART B: Red Blood Cells  Red Blood Cells Red blood cells – erythrocytes ▪ Biconcave shape ▪ Produced in bone marrow, released in bloodstream (immature-mature) ▪ Carriage of Oxygen from the lungs to peripheral tissues ▪ Hemoglobin ▪ Anucleate ▪ Cannot divide or replicate (mature) ▪ 4-6 million per microliter in our blood (40%)  Red Blood Cells Osmosis in red blood cells Effects of hypertonic, isotonic and hypotonic solutions on red blood cells If the solution is hypertonic, water will move out of the RBC and cause it to crenate (shrivel or shrink). If the solution is hypotonic, the RBC will swell and lyse (rupture or explode) as water moves into the RBC.  Experimental Procedure PART B: Red blood cells 1. Add a small drop of blood onto a clean slide. 2. Do not forget to label the slide (group and solution). 3. Add a small drop of solution A directly to the drop. 4. Carefully cover it with a coverslip and avoid any air bubble from entering the coverslip. 5. Repeat steps 1-3. 6. Add a small drop of solution B directly to the drop. 7. Carefully cover it with a coverslip and avoid any air bubble from entering the coverslip. 8. Repeat steps 1-3.  Experimental Procedure PART B: Red blood cells 9. Add a small drop of solution C directly to the drop. 10. Carefully cover it with a coverslip and avoid any air bubble from entering the coverslip. 11. You must have three slides ! 12. Observe the blood drops under the low and high magnification of a microscope. 13. Label the parts you see (cytoplasm and membrane) and take pictures (one for each slide). 14. Compare the three slides and decide as a group which slide has solution A, B, and C. QUESTIONS This is what the onion cell looked like under the microscope at about 40x Which of the following is correct? A. Hypertonic, Isotonic, Hypotonic B. Hypotonic, Hypertonic, Isotonic C. Hypertonic, Hypotonic, Isotonic  Which of the following is False? A. When onion cells are placed in a hypotonic solution, water moves out of the cell, resulting in crenation. B. When onion cells are placed in a balanced salt solution, there will be no net movement of water molecules. C. As in all plant cells, the cell of an onion peel consists of a cell wall, cell membrane, cytoplasm, nucleus and a large vacuole. D. When onion cells are placed in a hypotonic solution, water enters the cell, and cell starts to swell. Which of the following is correct? A. Hypertonic, Isotonic, Hypotonic B. Hypotonic, Hypertonic, Isotonic C. Hypertonic, Hypotonic, Isotonic  Which of the following is True? A. When red blood cells are placed in a hypertonic solution, they shrink away from the rigid wall. B. When red blood cells are placed in a hypertonic solution, they shrink, resulting in plasmolysis. C. When red blood cells are placed in a hypertonic solution, water within the cells moves out via osmosis into the surrounding solution, causing the red blood cells to shrink and shrivel. D. When red blood cells are placed in a hypotonic solution, they swell, resulting in plasmolysis.