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BIOL122L Name: ___________________________ Lab 3: Introduction to Microscopy and Cell Structure Learning Outcomes: · Know the structures and functions of basic organelles of eukaryotic cells. · Students will learn about safe handling and care of the microscope. · Students will use observations to develop an appreciation of the microscopic world. Introduction Cellular Structure Cells are the basic units of life. All living things are composed of cells. Some living organisms are composed of only one cell and some, like you, are composed of trillions of cells. Cells come in two basic forms: Those without defined nuclei are called prokaryotic and those with defined nuclei are called eukaryotic. Human cells are eukaryotic cells, so our focus will be those types of cells. Eukaryotic cells have a complex internal composition filled with defined structures called organelles. The organelles have specific functions within the cell. The following diagram highlights the important parts of a typical eukaryotic cell:(Microfilament) (Intermediate filament) Cytoskeleton (Microtubule) Cytoskeleton Cytoskeleton Microscopy The microscope is of enormous importance to the field of biology and has extended our ability to see beyond the scope of the naked eye. The compound light microscope is a precision instrument – treat it with respect! When carrying it, always use two hands, one on the base and one on the arm. The microscope consists of a stand (the base + the arm), upon which the stage and lenses are mounted. The stage is for holding microscope slides. The lens that you look through is the ocular (paired in binocular scopes); the lens that focuses the specimen is the objective. Your microscope has four objectives of varying magnifications (4x, 10x, 40x, and 60x) mounted on a revolving nosepiece. Positioning the specimen over the light source requires you to turn the mechanical stage controls, which operate the slide bracket on the surface of the stage. One control moves the specimen in the x-direction (horizontally), and the other moves the specimen in the y-direction (vertically). Focusing on the specimen is achieved by using two knobs that move the stage up and down, so that it is closer to or farther from the objective. The outer knob is the coarse focus knob, and it makes large, noticeable changes to the position of the stage. The inner knob is the fine focus knob, and it moves the stage so slightly that you may not be able to see it change position. The substage condenser directs light through the slide into the objective. An iris diaphragm on the substage condenser controls the amount of light reaching the objective, and also affects the contrast of the specimen. Activity 1: Cell Structures and Their Functions The table below lists the names of several important cell structures. In your own words, provide the function of the organelles / cell parts. Chromatin has been done for you as an example. Organelle / Cell structure Found in which type of cell? Job / Function Cell membrane (plasma membrane) All cells Cell wall Prokaryotes and some eukaryotes (fungi & plants) Chromatin All eukaryotes A complex of the cell’s DNA and proteins. Packaging DNA into chromatin helps it to fit within the volume of the nucleus and protects it from breakage. Cytosol All cells Cytoskeleton (Microfilaments and microtubules) All eukaryotes Golgi body (Golgi complex, Golgi apparatus) All eukaryotes Organelle / Cell structure Found in which type of cell? Job / Function Lysosome All eukaryotes Mitochondrion All eukaryotes Nucleus All eukaryotes Nucleolus All eukaryotes Ribosome All cells Rough ER All eukaryotes Smooth ER All eukaryotes Activity 2: Calculating Total Magnification Compound light microscopes contain two lens systems, an objective and an ocular. The total magnification of an image is calculated by multiplying the magnification of the ocular by the magnification of the objective. The microscopes we will use have a 10X ocular lens and up to four different objective lenses, as listed in Table 1 below. Note: Not all scopes will have the “high dry” lens. Complete Table 1. The information for the scanning objective has been done for you as an example. Table 1: Total Magnification by Objective Lenses Objective Lens Name Objective Lens Magnification Ocular Lens Magnification Total Magnification Scanning 4x 10x 40x Low power 10x 10x High power 40x 10x High “dry” power 60x 10x Instructions for Using the Compound Light Microscope (Read only) The compound light microscope should be treated as a very fragile piece of equipment. Failure to follow the procedures below will lead to broken slides and damaged objective lenses. The objectives for your microscopes can cost $200 or more. If you damage any of your objectives, it will not be possible to replace it before the end of the term, meaning that you will be without the benefit of that objective for the duration of the course. 1. Before plugging in the microscope, check to make certain that the on / off switch is in the off (O) position. Also check to make sure that the light intensity knob is at the lowest setting (1). Always plug in and unplug the microscope using the plug, not the power cord. 2. Clean the lenses before you begin viewing specimens. NEVER clean the lenses with anything other than the lens tissue provided! Do not wet the lens tissue. First, wipe (not rub) the ocular lenses, then the objective lenses. Begin with the lowest power objective lens and end with the highest power objective lens. 3. Before placing a slide on the stage, be sure that the scanning lens (4x) is in place over the viewing area. Use the rubber grip on the revolving nosepiece to change objective lenses; never grab the objective lenses directly to change focus. Use the coarse focus knob to place the stage at its lowest position. 4. Obtain one microscope slide containing the item you wish to view. Use the lever to open the side of the slide holder and place the slide flat on the stage. Gently release the lever on the slide holder so that the slide is held in place. 5. Use the x- and y-axis knobs to position the slide such that the portion of the slide you wish to view is under the 4x objective lens and over the opening in the stage. 6. Turn on (I) the on / off switch and rotate the light intensity knob to increase brightness. It is not necessary to turn the light all the way up. Doing so will decrease the lifespan of the bulb and hurt your eyes. 7. Adjust the position of the oculars (the interocular distance) so that a single image can be seen when looking through both oculars at the same time. It is important to keep both eyes open to avoid eyestrain, which can lead to headaches. 8. Vision differs between people and between eyes. To account for this difference, use your right eye to bring the image into sharp focus using the fine focus knob. Once the image is in focus, turn the knurled diopter ring on the left ocular lens to the left or right to achieve matching focus with your left eye. Doing this will help prevent eyestrain. 9. Focus on the specimen using the 4x / scanning objective, first using the coarse focus knob and then the fine focus knob. 10. Most objects will require increased magnification. First, move the specimen to the center of the field of view. Then, use the rubber grip on the revolving nosepiece to move the next highest objective lens into place above the slide. Refocus using the fine focus knob. NEVER use the coarse focus knob when using a magnification other than 4x / scanning objective. As you increase in magnification, the objective lens gets closer to the slide on the stage. Using the coarse focus knob at higher magnifications can lead to objective lens damage. 11. Always increase magnification one objective at a time, re-centering the object and refocusing each time. Adjust the light level (using the iris diaphragm) as necessary. 12. When you finish viewing one particular slide, use the rubber grip on the revolving nosepiece to move the 4x / scanning objective back into place and the coarse focus knob to lower the stage to its lowest position BEFORE removing the slide from the stage. Failure to do so could result in damage to the objective lens. 13. Do not adjust any other items on the microscope without first checking with the instructor. 14. When you are finished using the microscope for the day, follow these procedures: · Clean all lenses using a clean lens tissue. Also clean the stage and any other parts that have become moist or dirty. · Turn the light level to its lowest setting (1) and the on / off switch to off (O) BEFORE you unplug the microscope. · Have the 4x / scanning objective lens in place over the stage. · Center the stage clip on the stage (left to right) and all the way back towards the arm. · Lower the stage · Have a peer check your microscope to ensure all steps have been completed for proper storage. ______________________ (print neatly) · After your peer check is complete, have the instructor check your scope at your desk before you put it away. ___________ (instructor initials) · Gently place the microscope into the cabinet in its correct numbered space with the arm facing out / objectives facing in and the cover on. Activity 3: Plant and Animal Cell Structure Part A: Animal Cells (Human Cheek Cell Wet Mount) Materials Flat-edged toothpickDropper bottle of methylene blue Cover slipDropper bottle of DI water Blank slide Procedure * Care must be taken when doing this part of the lab to handle and dispose of the cells with appropriate concern. 1. Place a drop of methylene blue dye on a clean slide. 2. Gently scrape the inside of your cheek with the blunt end of a clean flat-edged toothpick and stir the material on the toothpick in the drop of dye on the slide. Properly dispose the toothpick. 3. Add a coverslip as directed. 4. Focus and examine the slide using the scanning objective (4x). 5. Next, examine the cells using the low (10x) and high (40x) objective lenses. 6. When finished, place your slide and cover slip in the beaker with the bleach solution provided by your instructor. 1. Record the total magnification of the slide that you are using to answer the questions below. _______________x 2. Describe the shape of the cells. _______________________________________________________ _________________________________________________________________________________ 3. Some of the cells may be folded or wrinkled. What does this indicate to you about the thickness of the cells? ________________________________________________________________________ Part B: Plant Cells (Elodea Leaf Wet Mount) Materials ForcepsBlank slide Cover slipDropper bottle of DI water Elodea Procedure 1. Using forceps (tweezers), carefully remove one leaf from a sprig of Elodea. 2