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6 The MicroscopeBackgroundBiological objects are often so small that they cannot be viewed with the naked eye. We must use microscopes to study them. In this lab we will use the compound light microscope. A light microscope uses light illuminated from below that passes through the object in order to view it. Often, the objects are dyed so you can visualize them better. The compound microscope is “compound” because it uses two magnifiers, or lens. The objective lenses magnify the image first, and then the ocular lens (or eyepiece) magnifies it again. Microscopes do not work like simply turning on a television. You have to know a few things about how they operate to make them work well for you. It is completely possible to look through a microscope and not see the object you are viewing at all. Now that we know the background of the microscope, let’s identify parts and look at some specimen, the object that you are viewing. PurposeIdentify parts of the microscope and their functions.
Use the microscope to view specimen.
Materials Virtual online microscope
Virtual Letter E slide Procedure1. Identify parts of the microscope.Please locate and review the following terms. Label these terms on figure 1 on the next page.Base, Arm: support microscope; use to carryLight source: a bulb that directs a beam of light up through objectLight control: adjusts amount of light emitted from bulbDiaphragm lever: adjusts amount of light passing through for best contrastCoarse adjustment knob: bring specimen into approximate focusFine adjustment knob: bring specimen into final focusStage with clip: holds and secures slideStage adjustment knobs: move slide right/left and towards/awayOcular lens (or eyepiece): magnifies specimen (usually 10x)Nosepiece: rotates objective lensObjective lens: magnifies specimen Scanning Power: magnifies 4X; used to scan the whole slide
Low Power Objective (LPO): magnifies 10X; used to see more detail
High Power Objective (HPO): magnifies 40X or 43X; used to see even greater detail
Label the parts on the figure below that were listed previously. Figure 1. Compound Light Microscope2. Understand terminologyIn order to talk about the microscope in a scientific manner, we need to understand some common terminology.Total Magnification is the final magnification after the image passes through all of the lenses. It is calculated by multiplying the magnification of the objective times the magnification of the eyepiece. Fill in table below:Table 1. Objective Lens and MagnificationObjective LensMagnification of Objective LensMagnification of Ocular LensTotal MagnificationScanning Low High Field of view is what you see when you look into the eyepiece. When drawing what you see in the field of view, be sure to draw the size of the image relative to the field of view and the details of the specimen. As the magnification increases, the area seen in the field of view decreases. Less of the specimen is seen because it is more zoomed in on.Resolution is the shortest distance at which two points can be distinguised. The higher the resolution, the better the discrimination between two points which produces a finer image. As the magnification increases, the resolution also increases.Depth of field is the range of depth that can be in focus on the specimen. When the depth of field is smaller, less depth is in focus on the specimen. As the magnification increases, the specimen is more zoomed in so the depth of field decreases.3. Carrying the microscope Always use both hands, one hand at the arm and the other under the base.
Carry it vertically, close to the body.
Make sure cord is tightly wrapped.
Place the microscope at least 4 inches (about 10 cm) from the edge of the table. Never place it on an uneven surface!
4. Care and storage of microscope Use the microscope assigned to you (usually matching your desk number, if applicable).
Check the microscope for the following conditions at the start and end of use:General cleanliness of the microscope
Scanning power objective clicked in place
Lens & stage at closest point
No slide left on stage
Mechanical stage centered
Cover on the microscope
Cord properly wrapped
Use only lens paper to clean optical parts of the microscope, i.e. lenses.
5. Procedure to view objects These videos will help review parts of the microscope procedure to view specimen:How to use a microscope (7 min 54 sec)
Use and care of microscope (4 min 45 sec)
Microscope for Beginners: Q&A (5 min 18 sec)
Focus specimen on scanning objective (4x):Center specimen: Place slide securely on stage with the clip and center specimen using your naked eye and stage adjustment knobs.
Use coarse adjustment knob: Check to make sure scanning power objective is in place and the lens and stage are at its closest point. Look into eye piece. Slowly turn coarse focus knob. Keep turning the knob in the same direction until image comes into focus.If you turned the knob all the way and missed the image, stop looking into the ocular lens and turn the coarse focus knob all the way back to starting position. Repeat previous step, but this time turn the knob more slowly.
To check if you are focused on the right image, move the slide and you should observe corresponding movement of image.
Use fine adjustment knob to obtain sharp focus of image.
Adjust light: Use your light control and/or diaphragm lever to receive greatest amount of light. As you increase magnification, you receive less light.
Center specimen. Look through the eyepiece and use the arrow pointer in your field of view.
Focus specimen on low power (10x): Switch to Low Power Objective (LPO): Do NOT move any knobs when switching the objectives! Gently rotate the revolving nosepiece and click the LPO in place.
Use coarse adjustment knob, then fine adjustment knob to sharpen focus.
Adjust light and center specimen.
Focus specimen on high power (40x): Switch to High Power Objective (HPO): When on the high power objective, NEVER use the coarse focus knob because the lens can hit the slide.
Then use fine adjustment knob only and adjust light as needed.
Store microscope properly: When you are finished, return microscope to storage conditions (follow points listed above in care for microscope above).
Procedure to use the interactive NC Bionetwork Virtual MicroscopeNow you will practice using a virtual microscope to view the letter “e”.Go to the virtual microscope website.
Start by selecting Guide to read about virtual microscope & overview. Go back to the main menu by clicking “Main”.
Click Learn to find out what each part of the microscope is for. Go back to the main menu by clicking “Main”.
Click Explore to view a variety of slides under different magnifications.
Click the “?” in the microscope slide box.
In the Slide catalogue, select “Sample slides”.
View the slide Inside, select “Letter E”: The virtual microscope loads the Letter E slide at the center of the stage and starts viewing at 4X (scanning power objective lens). You will see a blurry image within the field of view.
Focus the “e.” Use your mouse to adjust the Coarse Focus until the image is in focus. Fine tune it using the Fine Focus. Slowly slide the Light Adjust to the right to obtain optimum brightness. Watch carefullyhow the parts of the microscope move when you are selecting the objective lens & adjusting the Coarse Focus, Fine Focus and Light Adjust controls! Use your mouse to drag on the field of view to center the “e”. Draw the image in the field of view for scanning power.
Click “10X” (low power objective lens). Watch as the nosepiece moves until the low power objective lens clicks in. Adjust the Coarse Focus, Fine Focus & Light Adjust to obtain the clearest image with optimum light. Observe again the microscope parts that move as you adjust the controls. Center the “e.” Draw the image in the field of view for low power.
Click “40X” (high power objective lens). This time, only Adjust the Fine Focus & Light Adjust to obtain the clearest image with optimum light. Moving the Coarse Focus will endanger the lens & slide and make you lose your focus. Center the “e.” Draw the image in the field of view for high power.
Go back to the main menu by clicking “Main”.
Click “Test” to do a short practice quiz.
Discussion/ConclusionYou put the letter “e” on the stage oriented as if you were reading it regularly. You look through the eyepiece.Draw how the “e” appears in your field of view at scanning, low and high power objectives. Be sure to draw the size of the image relative to the field of view and the details of the specimen.
As the magnification increases, what happens to theField of view? Increase or decrease (select one)
Resolution? Increase or decrease (select one)
Amount of light? Increase or decrease (select one)
What happens to the orientation of the “e” in the field of view?
If you move the object to the right on the stage, which way does it move in the field of view? __________
You put the cross fibers on the stage oriented as if you were reading it regularly. You look through the eyepiece.
Observe how the fibers appear in your field of view at scanning, low and high power objectives. Be sure to label the different colors.
How many fibers are in sharp focus simultaneously under theScanning lens? _____
Low power? _____ High power? _____
As the magnification increases, what happens to theDepth of field? Increase or decrease (circle one)
Based on the two exercises above, why should you never start viewing your specimen on the high power objective lens?
7 Eukaryotic Cell Structure BackgroundCells come in all different sizes and shapes. They are classified into two major groups based on structure. Prokaryotic cells are much smaller and do not contain any membrane-bound organelle. Eukaryotic cells are larger and contain numerous membrane-bound organelles. Prokaryotic cells will be examined in the laboratory exercise that will survey the diversity of life on earth. This laboratory exercise will focus on the structure of the two most common types of eukaryotic cells: plant and animal cells.To appreciate the cell in action, watch The Inner Life of the Cellby BioVisions at Harvard University (3 min 12 sec). This video shows movement of organelles and substances inside your cells in real time.PurposePrepare wet mounts of plant and animal cells for microscope observation.
Use the light microscope to compare and contrast the structure of plant vs. animal cells.
Materials (demonstrated in video)Microscope
Microscope slides and cover slips
Water (or 0.9% sodium chloride solution)
Methylene blue (or iodine) stain
Online: Virtual microscope with plant slide
Procedure for plant cellView how to prepare a wet mount of an Elodea leaf(2 min 30 sec) OR
how to prepare a wet mount https://youtu.be/UWbGZMO4o_U(2 min 30 sec) To create a wet mount of an Elodea leaf, this general procedure was followed.Put a drop of water on the center of a slide.
Cut an extremely small piece of an Elodea leaf and place it on the water.
Place a coverslip onto the slide.
Observe the plant cell slide under the virtual microscope by following this procedure.Go to the virtual microscope.
Click Launch and then Explore
Click the “?” in the microscope slide box
In the slide catalogue, select “Plant Slides”.
Inside, select “Plant Cells.” The virtual microscope loads the Plant cell slide at the center of the stage. You will see a blurry green field of view.
Click “10X” (low power objective lens). Watch as the nosepiece moves until the low power objective lens clicks in. Use your mouse to adjust the Coarse Focus until the image is in focus. Fine tune it using the Fine Focus. Slowly slide the Light Adjust to the right to obtain optimum brightness. Click on the field of view to move the plant side. Draw the image of plant cells in the field of view for low power.
Click “40X” (high power objective lens). This time, only adjust the Fine Focus & Light Adjust to obtain the clearest image with optimum light. Moving the Coarse Focus will endanger the lens & slide and make you lose your focus. Draw the image in the field of view for high power.
Drawing of plant cells: label cell and observed organelles. Procedure for animal cell View Cheek Epithelial Cells (3 min 52 sec) to observe how to prepare a wet mount of human cheek cell and view images of the cheek cells under low & high power magnification.
To create a wet mount of cheek cells, this general procedure was followed.Put a drop of methylene blue on a slide. Caution: methylene blue stains clothes and skin.
Gently scrape the inside of your cheek with the flat side of a toothpick.
Stir the end of the toothpick in the stain and then throw the toothpick away.
Place a coverslip onto the slide by lowering it at a 45 degree angle to prevent formation of air bubbles.
Draw the cheek cell images under low & high power magnification as seen in the video.
Discussion/Conclusion Stains may be used but not all organelles are seen, why?
Describe the shape of the plant cell. List the organelles that were visible.
Describe the shape of the human cell. What organelles of the cell were visible?
Describe visible similarities and differences between plant and animal cells.
What observations support the claim that plant and animal cells are eukaryotic?
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