Lab 5.pdf

LABORATORY EXERCISE 5 Introduction to Microscopy and Cells Learning Objectives • Understand proper use and handling of a compound microscope • Measure the size of objects with an ocular ruler • Compare a variety of cell types across living organisms During the mid-1660’s, Robert Hooke (1635b–...

LABORATORY EXERCISE 5 Introduction to Microscopy and Cells Learning Objectives • Understand proper use and handling of a compound microscope • Measure the size of objects with an ocular ruler • Compare a variety of cell types across living organisms During the mid-1660’s, Robert Hooke (1635b–1703d) positioned a piece of very thinly sliced cork on his compound microscope to observe its structure. The microscope was handmade by Hooke and consisted of a leather tube that held two convex glass lenses. Although the microscope Hooke built would be considered rudimentary by today’s standards, in the mid-17th century it was an instrument of the highest quality. Hooke’s detailed observation of cork tissue is noteworthy, as it was from this event that Hooke coined the term cell to describe the repetitive units he observed in cork. Apparently, the structures Hooke observed reminded him of a monk’s cell or room at a monastery. Anton van Leeuwenhoek, a Dutch contemporary of Hooke, had a different design for his microscope. In comparison to the compound microscope used by Hooke, Leeuwenhoek’s microscope consisted of a single, high quality glass lens held in place by a brass plate. The superior quality of Leeuwenhoek’s glass lenses enabled him to observe objects in greater detail than Hooke. Flash forward nearly 170 years later into the 1830’s. Improvements in making glass lenses for the microscope allowed scientists to view small objects with even greater clarity and magnification than ever before. Observations made by the scientists Theodore Schwann and Matthias Schleiden have been commonly attributed to the development of modern cell theory. The cell theory states that: 1) the smallest living unit is a cell 2) all living things are made of one or more cells 3) all living cells arise from pre-existing cells (i.e., no spontaneous generation) The microscope is a commonly used tool by multiple branches of science, not only biology. Geologists use microscopes to view crystals and rock formations. Textile manufacturers view fabrics. Electronic engineers view circuits. Construction engineers view materials, such as cement and steel. And there are many other applications. As such, using a microscope is an important skill for all scientists. A primary objective in today’s lab exercise is for you to learn the basics of using a compound microscope, and how it can be used to measure the size of objects on a slide. Parts of the Microscope Ocular Lenses (Eyepieces) with Micrometer Binocular Observation Tube Clamping Screw for Observation Tube Revolving Nosepiece Objective Lens Stage Condenser Slide Positioning X-Y Control Fine Focusing Illumination Coarse Focusing Light Intensity Dial Power Switch Figure 5.1 Leica TM DM750 compound microscope Routine Care and Handling Storage and handling Always use both hands to carry the microscope. There is a handle on the back of the microscope and an undercut in the front for this purpose. When positioning the microscope on the table surface you will need to gently lift it. DO NOT slide the microscope. The scope is equipped with anti-slide feet, which will prevent sliding. Keep all optical components clean. Cleanliness is important for maintaining good optical performance. Excess oil should be removed with lens paper. Then optical surfaces can be cleaned using lens paper moistened with glass cleaner. Do not use Kimwipes™ for this purpose. The cord wrap allows you to wrap the cord in such a way that only the length you need is extended. The microscope should always be covered with the plastic dust cover (provided with the instrument) when it is not in use. And returned to the correct numbered location in the cabinet. Numbers are on the back of each microscope. Setting the Illumination Intensity Set the illumination to the lowest setting to start with, using the controller on the bottom left of the stand. The illumination control knob allows you to adjust the intensity of light. Plug in and Turn on the Microscope 1. Plug the power cable of the microscope into a corresponding grounded socket. 2. Switch on the microscope using the switch at the bottom right of the microscope stand. Using the Condenser The Leica™ DM750 is equipped for Köhler illumination. To open and close this diaphragm, simply turn the knurled ring on the condenser to the right or left so that the white line on the rotating ring is aligned with the objective magnification in use. Above the light is a slide knob that increase or decrease the amount of light. Two long metal screws (condenser positionscrews) are located under the stage and used toreposition the condenser light cone (Figure 5 .2). IMPORTANT. Please do not move these screws. If the microscope light source does not appear to be centered, ask your instructor to reposition the condenser. Figure 5.2 Condenser cone and stage of the Leica™ DM750 Using the Microscope 1) Gently lower the stage to its lowest position (as far as possible from theobjectives) by gently turning the coarse focus knob. Turn the objective nose piece to position the 4x objective in place above the stage (the microscope should be stored with this objective in place). Position the condenser with the condenser knob (Fig.6.2) so that it is below the level of the stage. Lowering the condenser can provide additional contrast. 2) Place your slide between the stage clips. Use the slide positioning X-Y control to position the center of the slide above the light. 3) Look through the oculars with both eyes open and adjust the distance between the oculars to fit your interpupillary distance, so you see a single bright field.Gently raise the stage using the coarse focus knob until you can bringthe specimen on the slide into view. Close your left eye and sharpen the focus with the fine focus knob. 4) The left ocular can be independently focused. To focus the left ocular, closeyour right eye, pinch the small knob on the left ocular between the thumb andforefinger of your right hand, and gently turn the left ocular with your left hand until it is sharply focused. You may need to repeat these steps to obtain the best focus for both eyes. Once both oculars are in focus for your eyes, you just need to adjust the focus by only using the coarse and fine focus knobs. 5) Once you are focused on the specimen, you will not need to lower the stage or use the course adjust knob when moving to higher magnification. Just gentlyrotate the objective nose piece clockwise to move the next objective into position and use the fine focus knob to bring the sample back into sharp focus. 6) As you increase the magnification. The field of view (how much of the specimen you can see) will get narrower. You will want to position the part of thespecimen you want to focus on in center of the field of view each time and may need to adjust the light intensity as you move to higher magnification. Toincrease the light, use the knurled ring below the condenser to align the “white” mark with the proper magnification value. 7) Follow the same procedure to increase magnification from 40x to 100x. If youneed to add oil, place a drop of oil directly on the slide in between moving the40x objective out of position and moving the 100x objective into position. Oilshould be cleaned off the slide with glass cleaner and a Kimwipe™. Oil must be cleaned off the 100x objective with lens paper and glass cleaner. Observing Samples Under the Microscope How to Use the Compound Microscope Micrometer The left ocular objective of our Leica™ DM750 microscopes have been fitted with an ocular ruler. The ocular ruler is numbered from 0 to 10 so it has 10 units. Each unit has 10 divisions, so the whole micrometer has a total of 100 divisions. Ocular ruler has 10 units and 100 divisions. The figure below shows how you would use the micrometer with a specimen to record the size. The specimen (blue oval) measures 5 divisions from 2.0 to 2.5. You would want to record the size as 5 divisions. To determine the actual size of the specimen in micrometers (µm ) , y o u n e e d to make a note of two other bits of information. First, you need to know which objective is being used for your observation and second, you need to know what the actual size of each division is at that total magnification. Magnification We can calculate the total magnification by multiplying the magnification of the objective lenses (4x, 10x, 40x, 100x) by the magnification of the ocular lens (10x). Magnification is the value of how much larger the object appears through the microscope compared to the size of the object as view by the unaided eye. The total magnification is ocular magnification (10X) multiplied by the objective magnification (10x X ?x). This means when using the 10x objective, the total magnification is 100x, and the object appears 100 times larger to when viewed by the unaided eye. Always make a note of the magnification being used for any drawings or observations. The size of the small divisions at each magnification is shown below in Table 6.1. You will need to refer to these numbers when measuring the size of your specimen under the microscope. Table 5.1 Size of the ocular division at each magnification Objective lens magnification: 4X 10 X 40 X 100 X Total Magnification for that lens: 40 X 100 X 400 X 1000 X Actual size of one ocular division (one small interval space): 25 µm 10 µm 2.5 µm 1 µm When viewing an object with the 100x objective, the total magnification is 1000x(10x ocular X 100x objective), and the actual distance between each ocular division is 1 µm. If an object being viewed with the 100x objective spans a distance of 30 ocular divisions, it would measure 30 divisions by 1.0 µm/ division = 30 µm in size. If the same object was viewed at a lower magnification, the actual size of the object has not changed, but it will look smaller when looking through the microscope. This effect is similar to viewing a large mountain very close up, or from miles away. The mountain does not change its true size. However, your proximity or distance from the mountain changes how large it appears. At 400x total magnification the same object mentioned in the paragraph above would only span 12 ocular divisions. The actual size would be determined by multiplying 12 divisions by 2.5 µm/ division and would still equal 30 µm (same size). You should note that the actual size/distance between ocular divisions is calculated by dividing 100/objective power. Example: 100/4 = 25; hence, the distance between each ocular division when viewing an object with the 4x objective is 25 µm. Experimental Observations Width of a printed letter H Today’s laboratory exercise begins with familiarizing yourself with how to properly use the ocular micrometer. Obtain a strip of paper from your instructor that shows the uppercase letter H printed three times. The font sizes for the five uppercase H letters are 6, 10, and 14. Font size 6 is the smallest, font size 14 the largest. Trim away excess paper and mount the strip of letters on a blank slide with a very small amount of water. Use only enough water to make the strip adhere to the slide and not float. A coverslip is not required. Use the 4X objective and measure the maximum width of each letter and enter your values in the space provided below. Note that you will need to perform a calculation to convert the ocular units that you can see in the microscope to the actual size in µm. Check your values with others at your table or the instructor to make sure you are interpreting the measurements correctly. Size of the letter H at various font sizes. For each of the fonts observe them at 40 X magnification and record the number of divisions for the width of the letter “H”. Calculate the size in µm and mm. Font size Number of ocular divisions Size of division at 40X magnification 6 25 µm 10 25 µm 14 25 µm Size of letter (µm) Size of letter (mm) Remeasure any two of the font sizes with the 10x objective. Using the 10x objective will increase the total magnification to 100X. You should note that the number of ocular divisions has increased, yet when ocular division x division size is calculated, the letter size has not changed. Font size Number of ocular divisions Size of division at 100X magnification 6 10 µm 10 10 µm 14 10 µm Size of letter (µm) Size of letter (mm) Differentiation of Cloth Fibers Obtain a swatch of cloth from the plastic bag at your table. From each swatch (nylon, cotton, wool), separate a single thread, and then a single fiber, if possible. If separating a single fiber is not possible, “roll’ the thread between your thumb and fore finger to fray the thread. Place the cloth fiber on a slide. Add a small drop of water. Place a coverslip on the fiber. Observe and draw the texture of each fiber. Is the fiber smooth? Rough? Flat or round? Does it have scales? Does it have projections or branch? Does the fiber twist? Your drawings (or descriptions) should have enough detail to allow a person to correctly identify the type of thread material. Cotton Nylon Wool Unknown Fiber Arrangement Obtain a prepared slide that contains three cloth fibers (nylon, cotton, wool) and examine the slide to determine the 3-D arrangement of the fibers. I.E. which fiber is located at the bottom, which is located in the middle, and which fiber is located in the upper most position? Use your drawings from above as a guide to recognize each fiber’s unique characteristics. Record your slide number. ______________ Where is the location of each fiber? (bottom/middle/top): Wool fiber is: Nylon fiber is: Cotton fiber is: Whole Mount of Elodea Leaf Take a fresh, young leaf from the tip of an Elodea sprig and make a wet mount slide. Place the slide on the microscope and examine the structure of the leaf cells. Can you see cell walls, nuclei, chloroplasts, and/ or cytoplasm? Note that because the leaf is unstained, some of these structures may be transparent and not as evident as in the stained slides. The chloroplasts (small, round green structures) may actually be moving around the perimeter of the cell. Movement of the chloroplasts and other organelles is caused by cytoplasmic streaming – movement of the cytoplasm within the cell. Make a sketch of a portion of the field of including some cells in the space provided below or describe what you see. Label as many parts as possible and note the approximate size of each. What is the function of each organelle you were able to observe? Magnification: Organelle/Structure Size Function Human cheek cells (prepared slide) Next examine a prepared slide of human cheek cells. Compare the cell structure with what you observed with the Elodea leaf. There will be some obvious differences because this slide is fixed (not a wet mount) and is stained, so some structures will be more visible. Use your knowledge about cell structure to inform your observations. Make a sketch of a portion of the field of including some cells in the space provided below or describe what you see. Label as many parts as possible and note the approximate size of each. What is the function of each organelle you were able to observe? Magnification: Organelle/Structure Size Function Comparison of the Plant and Human cell Beginning by examining the perimeter of the cell. Are these two cell types the same shape? Are the cells similar in overall size? Is the cell perimeter thicker in one of the cell types? If yes, which type is thicker and why? Compare the visible internal structures you can see. Notice approximately how much of the inner cell area is occupied by these structures? In what way are these cells typical of animal cells compared to plant cells. What other differences do you notice? It is possible that the human cheek slide you are viewing may contain cellular material other than the cheek cells. Carefully examine your slide for the presence of ‘spots’ located on the surface of the cheek cells, or in other areas of the slide. If your slide contains this material, what do you think it might be? What is the approximate size of a single spot? Privet leaf cross-section (prepared slide) The privet leaf is a tissue that has different layers of cells. Note that you are observing a very thin slice of a three-dimensional object. The upper surface of the leaf is covered by a waxy cuticle that functions as a barrier to help prevent moisture loss from the plant during hot temperatures. The cuticle is stained pink on this slide. Describe or make a sketch of the privet leaf tissue in the space provided below. Can you see three different layers of cells? Draw the general shape, size and appearance of these cells, and the cell structures you observe in them. Label the different cells and use your drawing to indicate what you measured. Some of the measurements may be taken at different magnifications Use the magnification that allows you to see the most detail in what you are measuring. What is the small round violet structure seen in some of the cells? Why do you not observe this structure in all of the cells? Magnification: Description Record the magnification, number of ocular units and multiplier used to compare the size of some of the different cells you see (label them in the drawing). Determine the size for the length and width of the different cells. Magnification: ___________ multiplier at this magnification _______ µm/ division 1. Description cells: Length: ________ µm Width: ________ µm 2. Description cells: Length: ________ µm Width: ________ µm 3. Description cells: Length: ________ µm Width: ________ µm