Microscopy and Cytology Item per class per bench 1. DI Water 6 bottles 1 bottle 2. Methylene Blue 6 bottles 1 bottle 3. Iodine 6 bottles 1 bottle 4. Slide boxes with letter e and thread slides 6 boxes 1 box 5. Slides and cover slips for wet mounts 1box each Varies 6. Toothpicks 6 boxes 1 box 7. Sharpie markers 6 makers 1 marker 8. Model of Cells 2 models Fresh Materials 1. Onion cells 1 onion 1 sample 2. Elodea (aquarium plant) 1 plant 1 leaf 0
Microscopy and Cytology Abstract In today s lab you will be viewing tissue samples from plants and animals under the microscope and identifying the various cellular organelles found in each cell type. Because you may be somewhat unfamiliar with the organelles of the cell, it is highly encouraged that you review your textbook for more information on cellular organelles as well as the cell types that you will be viewing today. 1. Using the Compound (light) Microscope Before you can use the microscope in a laboratory setting, you must first learn how it works as well as the function of each part. In this exercise, you will use your assigned microscope to identify each part as well as write a short description of its function. Results: Stage Power switch Ocular Lens Objective Lenses Base Diaphragm Course Focus Fine Focus Light Source Condenser It is the place where you place your slide Turns on the Microscope The place where you look at your sample You can switch them for greater magnification Base of the Microscope used for support Determines the intensity of light Focus your sample Helps focusing with more detail Where the light comes out Focus light into sample Figure 1. Description and function of the components of a light compound microscope. Be prepared to identify parts of the microscope and be able to describe their function! 1
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2. Orientations of Images Through the Microscope As light passes through the various slides that you will look at today, that light becomes refracted by the pieces of glass placed in the objective and ocular lenses of the microscope. The result is that the orientation of the subject on the slide being viewed is inverted. This means that the particular specimen you are studying will appear to have rotated 180 from the orientation that it is in when viewed with the naked eye. Here you will practice orienting yourself with this concept by studying a slide of the letter e. 1. Open your slide box and find the slide that has the letter e on it. 2. Place the slide on the microscope so that when you look down at it, the e is in the correct orientation as though you were reading it on a page. 3. Based off of the information in the introductory paragraph draw the e in the orientation that you expect it to be in when you view it on the microscope: 4. Turn on the light source, and set the microscope to the objective. 5. Use the ocular lenses and the focus adjustment knob to bring the image into focus. In the space provided, draw the e as it appears when viewed through the microscope: 3. The Objective Lenses & Depth of Focus Differences Between Objectives. Aside from magnification, the resolution (amount of detail), field of view (total visible surface area of the slide), and depth of focus (how much the focus knobs can be moved while still keeping the image in focus) differ between each of the objective lenses. Using the microscope and what you know about each of these characteristics and objective lenses, fill in the table below. Using a numeric scale (1-4 where 1=greatest and 4=least), rank each of the objectives compared to one another in each listed category. Objective Field of View Resolution Depth of Focus 4X 1 4 10X 2 3 40X 3 2 100X 4 1 Figure 2. Differing characteristics between the microscope objective lenses. 3
Using depth of focus, look at a prepared slide with three colored threads (yellow, green, and blue) at 4x and then 10x. 1. Start by viewing the thread using the 4x objective, and bring the slide into focus using the coarse focus adjustment knob. 2. Once the slide is in focus, use the fine focus adjustment knob to focus on the different colored threads. Using this, try to determine which thread is on top, which is in the middle, and which is on the bottom. Color of the thread on top: Color of the thread in the middle: Color of the thread on the bottom: 3. Now change the objective to the 10x objective, and bring the colored thread slide into focus. Again use the fine focus adjustment knob to bring the different colored threads into focus, and attempt to determine which is on top, which is in the middle, and which is on the bottom. Color of the thread on top: Color of the thread in the middle: Color of the thread on the bottom: Questions on depth of focus: 1. When using the fine focus adjustment knob, did each colored thread stay in focus longer when viewed under the 4x objective or under the 10x objective? Depth of focus is defined as how much the focus knob(s) can be moved while still keeping the image in focus. If the depth of focus of an objective lens is high, then the focus knob(s) must be moved more to observe a change in focus of the image. If the depth of focus of an objective lens is lower, the focus knobs do not have to be moved as much to make the image go out of focus. 2. Knowing this and based on your observations of the thread slide, which of the two objective lenses (4x) or (10x) had greater depth of focus, and which had the lesser depth of focus? Greater depth of focus: Lesser depth of focus: 4
3. Based off of your answer to question 2, if you were to have viewed the thread slide under the 10x and 40x objective lenses, which do you hypothesize would have had the greater depth of focus, and which would have had the lesser depth of focus? Greater depth of focus: Lesser depth of focus: 3 Preparing Wet Mounts for Microscopy This exercise is directed towards increasing your understanding about preparing wet mounts on microscopic slides and using the compound light microscope. Because microscopes are utilized in all biological classes that follow Biology 1406, it is crucial that you get hands on time and understand how to correctly and efficiently use the microscope. To ensure that you receive this beneficial training, it is highly recommended that if you and your partner choose to divide up tasks, you each take turns using the microscope, so that you can both practice scanning slides, changing objectives, and bringing the slides into focus. Plant cell Animal cell Elodea leaf: 1. Place a drop of water on a microscope slide. 2. Pluck a leaf from the Elodea and place it in the center of the drop of water with its topside facing upwards. 3. Place a coverslip over the leaf. 4. Add one drop of Iodine to the edge of the coverslip, and allow it to diffuse over the leaf. 5. What objective are you going to initially scan this slide with? 4X Check with your instructor to verify that you have selected the correct objective. 6. After you have verified the initial objective, you may slowly move upward in magnification. However, for the leaf, you should NEVER use the 100X objective or the microscope oil. Results: (Verify the answers to these questions with your partner, then verify them with the other group at your table, and, finally, verify them with your instructor) 5
a) Please draw the cell as you observe it in the area provided, and label any organelles that you can see: b) Observe this cell. Is it a plant or animal cell? What observations did you use to make your conclusion? Plant cells, because it contains a cell wall. c) What organelles can you see at this magnification level? Indicate those you can see with a checkmark in the appropriate box in figure 3. Write out the functions of each organelle listed. Organelle Observed? At What Magnification Function Nucleus 40x Contains all genes needed to control activities Cell Wall 4x Structural support and protection Plasma Membrane 40x Cells outer membrane, not to be confused with cell wall. Chloroplasts 40x Conduct Photosynthesis. Vacuole n/a Contain organic and inorganic molecules such as enzymes. Cytoplasm 40x Fluid that fills cells. Nucleoli Figure 3. Identification and function of cellular organelles in plant cells (leaf). d) Note which organelles you were not able to see. Why do you think that you are unable to see them? Can you think of any steps that you could take that would help you to better visualize them? Vacuole, might need a different staining method to display 6
e) Field of view refers to the diameter of the field, or area of the slide that you are able to observe under a given magnification. When you increase the magnification by changing the objective lens, what do you notice about the breadth of the field or area of the slide that you can see? In other words, do you see more and more of the Elodea leaf as you increase magnification (field of view increasing?) or less and less of the leaf as you increase magnification (field of view decreasing?). You can see less of the Elodea leaf but you are able to see in more detail the sample Take time to identify all of the above mentioned structures in the pictures provided on your bench top. Make sure that you can identify these structures and their functions! Onion cell 1. Remove a fleshy leaf from a red onion and snap the leaf backward to remove a thin layer of epidermis. 2. Place a drop of water on a microscope slide and place the piece of onion in the middle of the drop. 3. Place a coverslip over the slide, add a drop of Iodine at the edge of the cover slip, and allow it to disperse across the sample. 4. Place the slide on the stage of the microscope. 5. What objective are you going to initially scan this slide with? 4x Check with your instructor to verify that you have selected the correct objective. 6. After you have verified the initial objective, you may slowly move upward in magnification. However, for the onion cells, you should NEVER use the 100X objective or the microscope oil. Results: (Verify the answers to these questions with your partner, then verify them with the other group at your table, and, finally, verify them with your instructor.) a. Please draw the cell as you observe it in the area provided, and label any organelles that you can see: 7
b) Observe this cell. Is it a plant or animal cell? What observations did you use to make your conclusion? Plant cell, the cell wall is visible. c) What organelles can you see at this magnification level? Indicate those you can see with a checkmark in the appropriate box in figure 4. Write out the functions of each organelle listed. Observed? At What Organelle Function Magnification Nucleus 40x Contains all genes needed to control activities Cell Wall 10x Structural support and protection Plasma Membrane 40x Cells outer membrane, not to be confused with cell wall. Chloroplasts n/a Conduct Photosynthesis. Vacuole n/a Contain organic and inorganic molecules such as enzymes. Cytoplasm 40x Fluid that fills cells. Nucleoli Figure 4. Identification and function of cellular organelles in plant cells (onion). d) Note which organelles you were not able to see. Why do you think that you are unable to see them? Can you think of any steps that you could take that would help you to better visualize them? Vacuole, might need a different staining method to display Stained Cheek cell 1. Gently scrape the inside of your mouth with a toothpick. 2. Obtain one microscope slide and place a drop of water in the center of it. 3. Stir the end of the toothpick into the drop of water. 4. Place a coverslip over the slide. 5. Place the slide on the stage of the microscope. 6. What objective are you going to initially scan this slide with? 4x Check with your instructor to verify that you have selected the correct objective. 7. After you have verified the initial objective, you may slowly move upward in magnification. However, for the cheek cells, you should NEVER use the 100X objective or the microscope oil. 8. What color are the cheek cells before you perform the staining? Transparent 8
9. Now take the slide and place a drop of methylene blue on the edge of the coverslip; draw the stain across the coverslip by placing a paper towel or tissue on the opposite side of the coverslip. 10. Again place the slide on the stage of the microscope. 11. What objective are you going to initially scan this slide with? 4x Check with your instructor to verify that you have selected the correct objective. 12. After you have verified the initial objective, you may slowly move upward in magnification. However, for the leaf, you should NEVER use the 100X objective or the microscope oil. 13. What color are the cheek cells after the staining? Blue Results: (Verify the answers to these questions with your partner, then verify them with the other group at your table, and, finally, verify them with your instructor) a) Please draw the cell as you observe it in the area provided, and label any organelles that you can see: b) Observe this cell. Is it a plant or animal cell? What observations did you use to make your conclusion? Animal Cell, it lacks cell wall. 9
c) What organelles can you see at this magnification level? Indicate those you can see with a checkmark in the appropriate box in the figure 5. Write out the functions of each organelle listed. Observed? At What Organelle Function Magnification Nucleus 40x Contains all genes needed to control activities Cell Wall n/a Structural support and protection Plasma Membrane 40x Cells outer membrane, not to be confused with cell wall. Chloroplasts n/a Conduct Photosynthesis. Vacuole n/a Contain organic and inorganic molecules such as enzymes. Cytoplasm 40x Fluid that fills cells. Nucleoli Figure 5. Identification and function of cellular organelles in mammalian cells. d) Note which organelles you were not able to see. Why do you think that you are unable to see them? Can you think of any steps that you could take that would help you to better visualize them? In the animal cell, different organelles will not appear due to different type of cell. Take time to identify all of the above mentioned structures in the pictures provided on your bench top. Make sure that you can identify these structures and their functions! 10