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Name: __________________________________ Date: ___________________ Class Period: ____

The Microscope and Cellular Diversity Lab

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OBJECTIVES: 1. Learn the proper care and handling of the compound microscope.

2. Learn how to use the compound microscope.

3. Learn the names of the parts of a microscope and functions of each.

4. Learn how to prepare a wet mount slide for microscopic observation.

5. Learn to estimate the sizes of the field of view and objects observed in the

microscope.

6. Learn to distinguish in the microscope prokaryotic and eukaryotic cells.

THE MICROSCOPE: There are two major types of microscopes based on the kind of energy used by the device. A light microscope uses visible light that is magnified as it passes through glass lenses. The electron microscope uses an electron beam that is passed through magnetic lenses before the magnified image is projected on photographic film or on a fluorescent screen similar to the television. You will be using two different types of light microscopes. The compound microscope is “compound” because there are two lenses that collect and focus the light from the source as it is transmitted through the sample. For light to pass through the specimen, the specimen must be very thin. The image of the specimen seen in the microscope is not only magnified but the resolution is improved. Resolution is defined as the ability to reveal detail or to distinguish two objects that are close to each other as being two rather than one. Our eyes can “resolve” two objects that are at least 0.1 millimeters (mm) apart, whereas a good light microscope has a resolution up to 1,000 times closer or 0.1 micrometers (µm). The ability to distinguish detail also depends on contrast. Contrast is the differential absorption of light by parts of the object being viewed. Contrast in the microscope can be increased by decreasing the light intensity or by staining of the specimen. The proper selection of stains can also increase our ability to identify specific structures within the specimen. MATERIALS NEEDED: Compound Microscope Stereomicroscope Lens Paper

Lens Cleaner Newsprint Transparent Ruler

Microscope Slides Cover Slips Dropper Bottle with Water



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Looking at the Letter “e” Under the Compound Microscope

DIRECTIONS: 1. Clear an area on your desktop for the microscope. Obtain the compound microscope assigned to you

from the cabinet. Your instructor will demonstrate the proper methods for handling and using the microscope.

2. Plug in the microscope and make sure the excess cord is out of the way. 3. If it is not already in place, rotate the nosepiece until the scanning lens (i.e. 4x) is above the opening

in the stage. 4. Locate and then gently rotate the larger coarse adjustment knob on the side of the body. Notice

that the stage moves a relatively large distance when you turn the knob. The coarse adjustment knob should only be used when you are viewing a specimen under low magnification.

5. Obtain a glass slide, cover slip, and a piece of newsprint from the supply table. Cut out a single

letter “e” from the newspaper and place it right side up on your slide. Add a drop of water to the newsprint. Place a glass cover slip over your letter to hold it in place. Place the slide on the horizontal stage of the microscope. Make sure the slide is held securely in place with the thumb clip. Center the letter under the scanning lens with the stage motion knobs.

6. Adjust the sliding eyepieces to match the distance between your eyes. 7. Turn on the light switch and adjust the illumination with the brightness control dial. Scanning

lenses require less illumination than high-power lenses. On the Olympus scopes, lower the condenser lens until a grainy image is obtained. Then go slightly lower.

8. Turn the coarse adjustment knob until the stage is in its uppermost position. 9. While looking through the eyepiece gently turn the coarse adjustment knob to lower the stage.

When your letter becomes visible, use the smaller fine adjustment knob to sharpen the focus. You should now see a single image of your letter in the circular field of view. If not, align your letter in the center of the field of view. To insure that both ocular lenses are in focus, first close your left eye and focus the image in the right eyepiece with the focus knobs. Next, open your left eye and use the focusing ring on the left eyepiece to bring the image into focus.

Adjust the iris diaphragm lever under the stage to obtain the best contrast possible. The distance between the slide and the bottom of the scanning lens is the working distance for that lens.



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Draw your letter in the space below exactly as it appears in the field of view.



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QUESTION 1-1: How does the microscopic image of your letter differ from its appearance on the slide? 10. Without moving the stage, rotate the nosepiece until the 10x (low power) objective lens is above

your letter. If the microscope is parfocal and parcentric, the image will remain in focus and centered when you move from one magnification to another. Otherwise, use the fine focus knob to bring your letter into clear focus and the stage motion knobs to center the letter. Remember to adjust the light intensity and the iris diaphragm to obtain the best image possible.

QUESTION 1-2: How is the working distance for the low power lens different from the working distance on the scanning lens? (Hint: This field of view magnifies 100x and the previous power was 40x. It is 100x/40x = 2.5 times the magnification. So the field of view = 1/2.5 or 0.40x the previous field of view.) Draw your letter as it appears in the field of view. 11. With the image centered and in focus, turn the nosepiece until the 40x (high power) lens is above

your letter. Do not move the stage up or down before changing the objective lenses. The lenses are designed to easily rotate over the slide without hitting it.

12. Center and focus your letter as before. (Note: Never use the coarse adjustment knob for focusing

when the high power lens is in use!) Remember to adjust the light intensity and diaphragm to obtain the best resolution possible.

Note the working distance for the high power lens. This magnification (400x) is 10x the magnification of the first image (40x), so it only capture 1/10th of the original field.



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Draw your letter as it appears in the field of view. Magnification The total magnification for each objective lens on your microscope can be calculated using the following formula:

Total Magnification = Objective Magnification x Ocular Magnification QUESTION 1-3: Record the magnification values in Table 1-A. Table 1-A. Calculation of magnification and field of view

Total Magnification and Field of View Dimensions for the Compound Microscope Objective Power Scanning Low High Objective Magnification Ocular Magnification Total Magnification Field of View Diameter mm mm mmField of View Diameter µm µm µmField of view area (µm2)



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Estimating the Size of an Object as Seen in the Microscope 1. Return to the scanning lens on your microscope. Remove your letter slide and carefully place the

metric side of a transparent ruler on the stage across the field of view of the scanning lens. Slowly focus with the coarse adjustment until the lines on the ruler are clear. Align on of the millimeter lines on the ruler such that it is at one edge of the field of view.

2. Measure the diameter of the field of view in millimeter. _________ mm

What is the diameter in micrometers? _________ µm QUESTION 1-4: Record the diameter in micrometers in Table 1-A, on the assignment sheet, and beside your drawing. 3. The ruler markings are too far apart to be useful in calculating the diameter of the field of view at

higher magnifications. However, the diameter can be estimated using the formula: Diameter of field of view of scanning lens X Magnification

of scanning lens = Diameter of field of view of high power lens X Magnification of high

power lens QUESTION 1-5: Calculate and record the diameters of the field of view for your low power (10x) and high power (40x) lenses in Table 1-A. 4. With your newly calculated diameters, estimate the diameter of your letter “e”. _________ µm 5. Use your field diameters to calculate the area of the field of view at each magnification. QUESTION 1-6: Record the area of each field of view in Table 1-A.



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CELLULAR DIVERSITY Two major types of cells can be described based on their internal organization. Prokaryotic cells have no membrane-bound nucleus. In contrast, eukaryotic cells have a membrane-bound nucleus. These two cell types differ in some other important ways. For example, eukaryotic cells are generally larger and possess a variety of membrane-bound compartments in addition to the nucleus. These compartments, or organelles, specialize in such functions as energy production, synthesis, and internal digestion. Similar activities take place in the prokaryotic cell; however, all of these take place in the cytoplasm. You will observe and learn to recognize representative examples of pro- and eukaryotic cells and observe cellular diversity within each group. PROKARYOTIC CELLS MATERIALS NEEDED: Prepared Slide of Stained Bacteria Microscope Slides and Cover Slips

Culture of Live Cyanobacteria (Anabaena) Culture of Live Cyanobacteria (Oscillatoria)

Bacteria have a variety of cell shapes (e.g.: rods, spirals, spheres), but knowledge of these does not provide insight into bacterial function. Bacteria vary greatly in their metabolic pathways. The classification of bacteria is primarily dependent on the type of chemical reactions carried out in their cytoplasm. You will investigate cyanobacteria, a type of photosynthetic bacteria that dominated the earth from 2.5 billion years ago until about 600 million years ago. Thousands of different species of cyanobacteria live today. Cyanobacteria are common inhabitants of surface waters in ponds, lakes, and streams. The photosynthetic cyanobacteria were originally classified in the plant kingdom and were called the blue-green algae. Like plants, the cyanobacteria perform photosynthesis which produces sugars for energy and oxygen as a by-product. Cyanobacteria play a crucial role in nitrogen fixation, which is taking gaseous nitrogen (N2) from the air and making it available to other organisms in more usable forms such as -NO3

- and NH3+. Cyanobacteria that perform this process have a specialized structure called a

heterocyst which contains the necessary enzymes to carry out the reactions of nitrogen fixation.



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Wet Mount of Cyanobacteria: 1. Obtain a clean slide and cover slip from the supply area. 2. Place a small drop of water containing cyanobacteria in the center of

your slide. 3. Gently touch the edge of the cover slip to the edge of the drop of water

(Figure 1-B). The drop will spread out evenly along the side of the cover slip.

4. Lower the cover slip slowly onto the slide. Avoid air bubbles under the

slide. If there is too much water under the cover slip, place the edge of a piece of paper towel next to the cover slip. Excess water will be drawn into the paper.

5. Place your slide on the microscope stage and center the cover slip

under the scanning lens. 6. Try to locate the cyanobacteria. The cyanobacteria are blue-green in

color. Adjust the iris diaphragm for better contrast. 7. Center the cyanobacteria in your field of view and move to low power.

Focus, center, and adjust the light for the best image. 8. Move to the high power lens. 9. Note the overall shape of the cyanobacteria. Can you see any sheath

around the outside of the cells? Are there any heterocysts present? QUESTION 1-7: Draw and label the cyanobacterium on your wet mount. Be sure to include the specific name of the cyanobacterium. Figure 1-C show the species we have in lab today. Microscopic Observations of Other Bacteria 1. Obtain a prepared slide of bacteria from the supply table. 2. Place the slide on the stage and try to center the area containing the

bacteria directly under your scanning lens. 3. Move to your low power lens (10x objective) and focus and center the

bacteria in the field of view. You still will not be able to see much more detail at this magnification.

4. Now examine the bacteria under high power (40x objective).

Remember, only use the fine focus knob with the high power lens. 5. Bacteria are routinely classified according to their shapes. How many

different shapes can you find on this slide?



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QUESTION 1-8: Draw and label at least 3 different shapes of bacteria observed on your slide. 6. Compare your drawings with the shapes in the diagram below.



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EUKARYOTIC CELLS (PROTISTS, FUNGI, PLANTS, ANIMALS) MATERIALS NEEDED: Microscope Slides and Cover Slips Protoslo (methyl cellulose) Culture of Amoeba (live) Culture of Paramecium (live) Mixed Culture of Protists Elodea Plants

Onions Dropper Bottle of I2KI (Iodine Solution) Toothpicks Dropper Bottle of 0.9% NaCl Dropper Bottle of 0.5% Methylene Blue

Eukaryotic cells contain a membrane-bound nucleus and many different organelles. The cytoplasm forms the internal substance of the cell. In this section, you will observe a variety of prokaryotic cells. Note the difference from bacteria and not the differences and similarities of eukaryotic cells. Wet Mount of Protistans: 1. Obtain a clean slide and cover slip from the supply area. 2. Draw a few drops of culture medium from the bottom of the dish containing the protistan culture. 3. Place one drop of the medium on the slide and then add a drop of methyl cellulose. Methyl cellulose

increases the viscosity of the medium to slow the swimming speed of the protistans. 4. Gently add a cover slip. 5. Look for the protistans under scanning power. If you are having trouble finding any, try looking along the

edges of the cover slip. Observe them under low and high power. QUESTION 1-9: Draw and label at least on protist you observed on the slide. Be sure to include any cellular structure you could identify. Compare your sample with Figure 1-E, which show some common freshwater protists.



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Wet Mount of Plant Cells: Plant cells are easily recognizable in the microscope because of their rigid cell walls. In green plants, numerous chlorophyll-containing chloroplasts and a large vacuole will be obvious. 1. Obtain a clean slide and cover slip from the supply area. 2. Use a pair of forceps to remove a small leaf from the growing tip of a sprig of the common aquatic plant

Elodea. 3. Place the leaf on the slide in a drop of fresh water; then cover with a cover slip. 4. Find the leaf under scanning power. Center in the field of view an area at the edge of the leaf where a spine is

present and the leaf is thinner. 5. Switch to low power and observe the regularly-shaped, rectangular cells. A wall surrounds each cell.

Observe the arrangement of the chloroplasts in the cell. They may be pushed up against the wall by the water-filled central vacuole that occupies about 90% of the cell.

6. Next, examine the cells at high power. Observe the chloroplasts carefully. In a healthy plant they will be

moving. This movement of the cytoplasm in a cell is called cyclosis or cytoplasmic streaming. Add a drop of methylene blue stain to the edge of the cover slip and draw it under by touching a piece of towel to the opposite side. Be careful not to stain your skin or clothes. The nucleus should appear as a faint circle at the edge of the cell.

QUESTION 1-10: Draw a cell for the Elodea leaf under high power. Label the cell wall, chloroplasts, and nucleus on your drawing. Using your knowledge of the diameter of the field of view, estimate and record the size of a typical Elodea cell. Wet Mount of Onion Cells: 1. Obtain a clean slide and cover slip and a small piece of onion from the supply area. 2. Break and peel off a piece of the clear, inner epidermis (skin) of the fleshy scale. Your instructor will

demonstrate a simple method to remove the epidermis. 3. Place the piece of epidermis in a drop of iodine solution (I2KI) on a clean slide. Be careful not to stain

your skin or clothes. Try to keep the epidermis in a flat sheet. The iodine solution should provide some needed contrast.

4. Add a cover slip and find the specimen under scanning power. 5. Observe the onion skin under low and high power. 6. Observe the arrangement of cells and the location of the nucleus within each cell. QUESTION 1-11: Draw and label the major parts of some onion epidermal cells under high power. 7. How do the onion skin cells differ from the Elodea cells? Estimate and record the size of a typical

epidermal cell next to your drawing.



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Wet Mount of Human Epithelial Cells: The cells lining the inside of the mouth are easy to obtain for investigation. These epithelial cells exhibit characteristics of typical animal cells. 1. Obtain a clean slide and cover slip from the supply area. 2. Place a drop of 0.9% NaCl (sodium chloride) on your slide. 3. With the broad end of a clean toothpick gently scrape the inside of your cheek. 4. Stir the cells into the drop of salt solution on the slide. 5. Gently lower a cover slip onto the slide. 6. Locate the cells under low power. You will probably have to adjust the light intensity and the diaphragm in

order to see them. 7. Observe the cells under high power. QUESTION 1-12: How do these cells compare with the plant cells? What structures can you find? 8. Add a drop of methylene blue to the edge of the cover slip and draw it under by touching a piece of towel to

the opposite side. QUESTION 1-13: What structure has been stained with the methylene blue? QUESTION 1-14: Draw and label the human epithelial cell and its parts under high power. Estimate and record the size of a typical epithelial cell.

Clean-Up All glass cover slips should be placed in the container marked “sharps”.

Slides should be wasted in soap and water, dried, and returned to the supply table.

Make sure the lenses and the stage of your microscope are clean.

Rotate the nosepiece until the scanning lens is in place.

Turn the coarse adjustment knob until the stage is in its uppermost position.

Center the mechanical stage so that its arm is even with the side of the stage.

Wrap the power cord around itself as demonstrated by your instructor. Do not wrap it around the base of the

microscope.

Put the microscope back into the cupboard.

Clean up your work area.

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