03 microscope lab - santa monica collegehomepage.smc.edu/chen_thomas/bio3 su 18/03 microscope...

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29 MICROSCOPIC LIFE BACKGROUND The purpose of this exercise is to help you appreciate that there is more to life than meets the eye. Because our primary sensory system is visual, we have a natural tendency to be uncertain about things we cannot see (i.e. "I'll believe it when I see it!"). The universe, however, is full of incredible things beyond the range of our natural eyesight, from distant stars to the atoms and molecules of our very own bodies. We have tried to emphasize that the process of science begins with observation. How do you observe the invisible? One answer is to use technology to extend our senses. Our vision was first extended several hundred years ago with the first microscopes and telescopes. Since then, optical technology has steadily improved. In this lab, we'll use two different kinds of microscopes to explore familiar places and things in search of a diversity of microscopic life. What was in that mouthful of ocean you swallowed last weekend? What does your blood look like, magnified four hundred times? Are your fingertips really clean? Do not use the microscopes or other equipment until your instructor has explained their proper use and care. Some of the microscopes you will use cost thousands of dollars, and can be easily damaged by incorrect focusing or cleaning of the lenses. There are also important safety precautions that must be discussed before you begin working on today’s lab. USE OF THE DISSECTING MICROSCOPE Let us first consider the dissecting microscope (illustrated on the following page). This microscope is used to examine relatively large specimens, usually specimens you can see with your unaided eye but need to view in greater detail. Materials appropriate for viewing with dissecting microscopes are frequently opaque (not transparent) and too thick to be mounted on a microscope slide for viewing with a light microscope. The dissecting microscope enables us to see otherwise invisible details of visible objects. Please follow the procedures described below carefully. Again, microscopes can be easily damaged by misuse. 1. Dissecting microscopes can easily be distinguished from light microscopes by observing that they have fewer lenses, knobs, and moving parts. Remove your dissecting microscope from its cabinet by lifting it with both hands, keeping one hand under the instrument. 2. Use the diagram of a compound microscope to locate the following parts on your microscope: ocular lenses, objective lens, magnification zooming knob, focus knobs, and stage. Some dissecting scopes will also have built-in lamps.

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Page 1: 03 Microscope Lab - Santa Monica Collegehomepage.smc.edu/chen_thomas/Bio3 Su 18/03 Microscope Lab...observing that they have fewer lenses, knobs, and moving parts. Remove your dissecting

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MICROSCOPIC LIFE BACKGROUND The purpose of this exercise is to help you appreciate that there is more to life than meets the eye. Because our primary sensory system is visual, we have a natural tendency to be uncertain about things we cannot see (i.e. "I'll believe it when I see it!"). The universe, however, is full of incredible things beyond the range of our natural eyesight, from distant stars to the atoms and molecules of our very own bodies. We have tried to emphasize that the process of science begins with observation. How do you observe the invisible? One answer is to use technology to extend our senses. Our vision was first extended several hundred years ago with the first microscopes and telescopes. Since then, optical technology has steadily improved. In this lab, we'll use two different kinds of microscopes to explore familiar places and things in search of a diversity of microscopic life. What was in that mouthful of ocean you swallowed last weekend? What does your blood look like, magnified four hundred times? Are your fingertips really clean? Do not use the microscopes or other equipment until your instructor has explained their proper use and care. Some of the microscopes you will use cost thousands of dollars, and can be easily damaged by incorrect focusing or cleaning of the lenses. There are also important safety precautions that must be discussed before you begin working on today’s lab.

USE OF THE DISSECTING MICROSCOPE Let us first consider the dissecting microscope (illustrated on the following page). This microscope is used to examine relatively large specimens, usually specimens you can see with your unaided eye but need to view in greater detail. Materials appropriate for viewing with dissecting microscopes are frequently opaque (not transparent) and too thick to be mounted on a microscope slide for viewing with a light microscope. The dissecting microscope enables us to see otherwise invisible details of visible objects. Please follow the procedures described below carefully. Again, microscopes can be easily damaged by misuse. 1. Dissecting microscopes can easily be distinguished from light microscopes by observing that they have fewer lenses, knobs, and moving parts. Remove your dissecting microscope from its cabinet by lifting it with both hands, keeping one hand under the instrument. 2. Use the diagram of a compound microscope to locate the following parts on your microscope: ocular lenses, objective lens, magnification zooming knob, focus knobs, and stage. Some dissecting scopes will also have built-in lamps.

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DISSECTING MICROSCOPE

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3. Turn the magnification knob to its lowest setting (1x). If your ‘scope is equipped with a light, turn it on. 4. Place one of your fingers on the stage, directly under the objective lens. 5. Adjust the distance between the ocular lenses to match the distance between your eyes. Keep both eyes open at all times. When the distance between ocular lenses is properly adjusted you will see a single circular viewing area, not two merging together. 6. Turn the focus knobs on either side of the microscope until your finger comes into focus. Hint: you may need to position the objective lens much higher above your finger than you think to bring it into focus. 7. Turn the zooming knob to a higher power now. Is your fingernail really clean? 8. Use this same procedure to view another object, such as a coin or dollar bill. The designers of the US penny engraved their initials on each side of the coin. Can you find them? Always clean and dry the stage of the dissecting microscope before you put it away, particularly if there has been a snail crawling on it.

USE OF THE LIGHT MICROSCOPE Let’s now consider the more complex light microscope (illustrated on the following page). Light microscopes are used to observe very tiny samples, which are thin and translucent so that light can easily pass through them. 1. Remove your light microscope from its cabinet by lifting it with both hands, one under the scope and the other grasping the main vertical column. Do not grasp the “head” or slide “platform” (stage) of the microscope. 2. Use the diagram of a light microscope to familiarize yourself with the following parts of your microscope: ocular lenses, lens turret, objective lenses, coarse and fine focus knobs, mechanical stage, slide clip, slide positioning knobs, iris diaphragm, light switch and lamp power adjustment. 3. Plug the power cord into an electrical socket and turn on the light switch. Notify the instructor if the light does not come on. The bulb may need replacing. 4. Adjust the distance between the ocular lenses to match the distance between your eyes. Keep both eyes open at all times. If the distance between ocular lenses is properly adjusted, you will see only one circular viewing area. Rotate the objective lenses until the lowest power (shortest) lens is pointed downward. 5. Your instructor will now provide you with a prepared microscope slide, preferably one that has a relatively large object in it.

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LIGHT MICROSCOPE

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6. Position the slide in the slide holding mechanism on the stage of the microscope. 7. Looking at the microscope from the side (not through the ocular lenses), you will see a beam or circle of light striking the slide from the lamp below. If you do not see the beam, you will need to open up the iris diaphragm. Twist the slide positioning knobs until a sample of material on the slide is centered in the circle of light coming through the stage. 8. Still looking from the side, turn the coarse focus adjustment knob to raise the stage closer to the objective lens. Continue moving the stage upward until the slide nearly touches the objective lens or until you reach the upper adjustment limit. 9. Now you may look through the ocular lenses and begin bringing the subject into focus. Use the coarse focus knob to slowly move the stage down and away from the objective lens.

TIPS FOR FOCUSING LIGHT MICROSCOPES

Always focus on low power initially unless you know you are looking for very small objects such as individual cells. Always start with steps 8 and 9 above. Be aware that as you move the slide into focus the first objects you are likely to see are lint particles, salt deposits, or fingerprints on the surface of the slide. These appear black or gray and are irregularly shaped. If you detect these on your slide, you can clean the slide with water vapor from your breath and lens tissue. A little lint can be helpful, however. If you succeed in binging lint into focus you can anticipate the samples will lie just above or below it (lint occurs on both the top and bottom surfaces of slides). As you move the slide into focus, anticipate the appearance of a blur of color if you are looking at a prepared slide. The material samples in prepared slides are usually stained blue or red. The appearance of color while you are focusing indicates you are nearing the focus plane of the sample. Switch to a fine focus knob, if you haven't already, and bring the color into sharper focus. If you are lucky, the outline of your subject will also emerge with further, very slight, up or down focus adjustments. If light coming from the lamp is too bright, it can "wash out" the objects on your slide, making them impossible to see. Likewise, if too little light is reaching your slide, you will be straining to see anything on it. Try adjusting the lamp power adjustment until the viewing area appears bright gray, not bright white or darkened. If you find that you can only bring a small region of your subject into focus while the rest is blurred, try closing the iris diaphragm a bit. This will increase the depth of the region in focus (but also decrease the amount of light striking the slide). If your subject does not appear after bringing the stain color into sharp focus, you will need to see the tips below for finding objects on a slide.

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10. After bringing your sample into focus, you may want to increase the magnification for a closer look. Turn the lens turret until the next higher power objective lens clicks into position. Because your light microscope is "parfocal," a sample that was in focus on low power will also be roughly in focus when you change objective lens. If you experience difficulty re-focusing at higher power, it may be necessary to ask the instructor to clean the lenses of your microscope. Never attempt to clean microscope lenses yourself.

POWER OF MAGNIFICATION It is easy to determine how many times larger than life a subject appears in a microscope. The total magnification power of a microscope is the product of the power of the ocular lens(es) times the power of the objective lens. The magnification power of each lens is printed in small letters on the side of the lens. Typical powers include 1x, 2x, 4x, 10x, and 40x. If a 10x ocular lens is matched with a 4x objective lens, the total magnification power will be 10 x 4 = 40x. Thus the subject will appear 40 times larger than life. It is a good idea to record the power of magnification used alongside each sketch you make. PROCEDURES There are more materials available for this lab than you will have time to observe. Your instructor will explain which activities you should and should not do. Whichever materials you use, please return them to where you got them, and please help keep the side counters clean and tidy. 1. PLANT VS. ANIMAL CELLS. Compare the major cellular structures of a typical plant cell to those of an animal cell. Draw examples of each cell type in the appropriate box in your worksheet. Onion Skin: The plant cells you will use are from the transparent, cellophane-like membrane found between each layer of an onion. Isolate a layer of onion and gently peel this delicate layer from what was the inner surface of the layer. Remove a small piece of the tissue – about 2mm x 2mm -- and place it on your slide. Make sure the tissue lies down in a single layer without folds. If your sample is too thick, it will not fit under the microscope when the high power objective is in place. Prepare the onion tissue as a stained wet mount, substituting a drop of methylene blue for water. Elodea Cells: See “2. MOVEMENT WITHIN PLANT CELLS” below. Cheek Cells: Observe cheek cells that your instructor bravely extracted from the inside of his or her cheek. Using 400x magnification, compare the onion cells you have prepared to the cheek cells prepared by your instructor. You’ll answer questions about this comparison in the QUESTIONS section of the lab worksheet. Adipose Cells: Observe adipose cells from a prepared slide using 400x magnification.

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Allium Root Tip: Observe Allium root tip cells from a prepared slide using 400x magnification. Sperm Cells: Observe sperm cells from a prepared slide using 400x magnification. A word of warning: the ever-popular human sperm is difficult to find (even at 400x magnification) because of the extremely small size of these cells.

TIPS FOR FINDING OBJECTS ON A SLIDE

To get started, you must follow the Tips for Focusing to bring something into focus. If you are looking at a prepared slide, you will generally be looking for something brightly stained. If you are looking at a wet mount, you will be looking for something floating or moving. Avoid spending your time in today's biology lab studying lifeless, gray, lint or salt deposits on the surface of a slide! Always begin searching on low power unless you know ahead of time that the sample materials are very small and widely dispersed, such as blood or sperm cells. Why? Because searching a microscope slide with high power is the equivalent of looking through a drinking straw to find a friend seated in the Staples Center. Low power provides a much wider area of view. If you don't spot anything in the area of view, you will need to begin systematically searching the entire slide. Use the slide positioning knobs to bring a corner of the cover slip into view. The cover slip is the small, thin, sheet of glass covering the sample. On low power, rotate a single slide positioning knob so that the slide moves in a straight line, either up, down, or sideways, along the top or side of the cover slip. Note: if your slide or microscope stage is wet, the slide will not move properly. Glance at your slide to check that it is not adhering to the stage. As you move the slide in a straight line, look for objects on the slide. Eventually, the far edge of the cover slip will come into view. At this point, rotate the other slide positioning knob so that the area of view begins moving along the edge of the cover slip you just encountered. Move in this direction until the area of view encompasses entirely new "terrain" on the slide. At this point return to the first slide positioning knob you used and twist it so the area of view moves alongside and parallel to the first long swath you traversed across the slide. Continue this process as indicated in the figure below, or until you find something on the slide.

Once you’ve spotted something on low power, use the slide positioning knobs to center the object in the area of view and then switch to a more powerful objective lens. Alas, if you’re looking at a wet mount and you don’t find anything after a thorough search, it is possible that your sample was a dud. Look below for tips on making wet mounts, and try a new sample.

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2. MOVEMENT WITHIN PLANT CELLS. Make an unstained wet mount containing a single leaf picked from the water plant, Elodea (=Anacharis). Select a young, thin leaf from near the tip of the stem, if possible. Make sure the leaf lies down in a single layer without folds. Observe the leaf on high power (400x). Notice the numerous, tiny green disks within boxlike cells. These disks are chloroplasts, the structures that do photosynthesis within a plant cell. If you watch closely, you will notice that the chloroplasts of some of the cells move within the cell. Try to determine the average number of chloroplasts per cell. How long does it take for a chloroplast to circumnavigate a cell? Is the leaf more than one cell layer in thickness? Respond to these and other questions in the QUESTIONS section of the lab worksheet.

TIPS FOR OBTAINING MICROORGANISMS FROM A CULTURE

In most cases, your source of microorganisms will be a small bottled culture or a small dish of pond water. Note that there is usually some fine particulate matter at the bottom of the source container. Consider this to be the food and habitat of the organisms. You want to transfer a small amount of this material onto a glass microscope slide. Do not collect any of the larger stringy, leafy, or sandy material that might be present in the source container. Such items are too large to fit under the cover slip of the wet mount you will prepare. Do not stir or agitate the source cultures. This will disperse the organisms throughout the water column and make them harder to find. Certain kinds of organisms will be large enough for you to spot with the unaided eye. Bring the source container to within a few centimeters of your eye and look for small organisms swimming around. Others may be attached to the sides of the container. If you spot an organism, try to suck it into the tip of a dropper. If you do not see any 'large' organisms, suck up a small amount of the fine particulate matter ("muck") from the bottom of the container. Do not go after it with the bulb of the dropper fully depressed between your fingers. This will result in too much fluid being sucked into the dropper. Before submerging the tip of the dropper, gently squeeze the dropper bulb between your thumb and pointing finger until it is about half compressed. Position the tip of the dropper near your target and allow the bulb to expand quickly to suck in your target. Before you transfer the sample to a glass slide, make sure you read the tips below on Making a Wet Mount. 3. POND WATER. Using the illustrations provided in the Bio 3 Guide to Microscopic Life, sketch and attempt to identify four different organisms from pond water. Prepare unstained wet mounts for this purpose. See “Tips for Obtaining Microorganisms From a Culture” and “Making a Wet Mount” for instructions. Search initially on low power, and increase power after you have found something. Make your sketches where indicated in the lab worksheet. Satisfy yourself that you are observing a living organism and not a speck of detritus or muck before you begin your sketch. Rinse and reuse the same slide and cover slip after each use.

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HOW TO MAKE A WET MOUNT

Water samples viewed under a light microscope must ALWAYS be covered with a cover slip. In the absence of a cover slip water could touch and damage an objective lens. 1. Obtain a clean microscope slide, a cover slip, and a sample of the material you want to observe. See the tips above for collecting protist samples. 2. If the material of interest is a protist sample in an eye-dropper, position the tip of the dropper about a half-centimeter above the center of your microscope slide. Steady the dropper with both hands if necessary. Slowly squeeze the bulb until a single drop emerges, dangling from the tip of the dropper. Allow the drop to dangle on the tip of the dropper if you can. This will often draw additional protists from the dropper into the drop. If the material of interest is not a fluid (e.g., plant tissue), place a small, paper-thin, sample on the slide and then place one drop of water on it. If the procedure calls for stain, substitute a drop of stain for the drop of water. 3. Place a cover slip on edge next to the drop of water. Let adhesion draw the water across the edge of the slide, then gently lower the slide down onto the drop of water. 4. Dab any excess fluid from the bottom and sides of the slide with a paper towel before placing the slide on the microscope stage.

Caution: cover slips are sharp-edged and can easily shatter into shards of glass. Handle them with care. Do not leave intact or broken cover slips on counter tops or the floor, where someone could inadvertently cut himself. Dispose of all broken glass (slides, cover slips, or otherwise) in the receptacles provided specifically for broken glass. 4. SAMPLE CREATURES. Mixed cultures of several different kinds of microscopic creatures are provided on the side counter. Make an unstained wet mount of each culture. Identify the organisms by comparing what you find to the illustrations in the Bio 3 Guide to Microscopic Life. Rinse and reuse your slide and cover slip after each sample. Sketch and identify four organisms as indicated in your worksheet.

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Important: a. Don't cross-contaminate cultures by using the same dropper in two different cultures. b. Don't shake or stir the cultures. c. A drop of "Protoslo" (protozoan 'downer') will slow fast-moving organisms. d. Cultures may contain small, fast-moving protozoans that have been put there as food

for the bigger "unknown" organism. 5. HAWAIIAN SAND. What is there to see, except a bunch of little rocks, you say? Once again, the microscope gives new meaning to a familiar substance. This sample was taken from a coral-reef-enclosed bay (Hana Bay, Maui). Many of the "little rocks" are actually bits and pieces of tiny organisms! Transfer some into a watch glass and have a look. Some of the things you might see are shown on the following chart. Using your thumb and forefinger, place a pinch of sand directly onto the stage of your dissecting microscope. Now use a needle (or pencil point) to randomly isolate 50 pieces of sand. Next, from this sample of 50 pieces, isolate the pieces with organic origins (biological) from those with inorganic (geological) origins. Determine the percent composition of various sand ingredients with the following equation: (Number of pieces ÷ 50) x 100 = percent of total

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HAWAIIAN SAND

Ordinary beach sand can be quite extraordinary on closer inspection. The coral reef is the most diverse and complex ecosystem in the ocean. Sand taken from beaches enclosed by a coral reef is often largely composed of materials made by living organisms. Biologists consider any material made by living organisms to be “organic”. The biological definition of organic is different from the chemical definition (“organic” chemicals contain carbon atoms). It is also different from “organic” food (food plants grown without using pesticides or artificial fertilizers).

The sand you have in lab is from the island of Maui. This sand contains both a variety of animal parts and a large quantity of material from Hawaii’s famous volcanic activities. It’s quite a bit more interesting than Santa Monica beach sand!

You should be able to find things not shown below. Your instructor may be able to identify some of these “unknowns”. Students have found dried shrimp, fish scales, fish bones, sponge spicules, and tiny “Pele’s Tears”. What can you discover?

The pitted black grains are bits of lava. (Inorganic)

The polished, shiny black “broken glass” and “melted glass” is obsidian. (Inorganic)

The pitted, brick red grains are cinders. (Inorganic)

The dark green glass-like pebbles are olivine. (Inorganic)

Purple or yellow cylinders or wedges are bits of sea urchin spines. (Organic)

The irregular shaped yellow or orange fragments are coral. This is the most common material in the sand. (Organic)

White tusk-like structures are wave worn sea urchin spines. (Organic)

The tiny snail shells usually have bright colors and patterns. These are gastropod molluscs. (Organic)

Yellowish, concave fragments are parts of sea urchin tests (“shells”). (Organic)

Bivalve mollusc (clam) shell fragments are relatively rare. (Organic)

Very tiny and translucent flakes are salt crystals. (Inorganic)

The shiny, pill like discs are the shells of foraminiferans, relatives of Amoeba. (Organic)

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BEFORE RETURNING MICROSCOPES TO THEIR CABINETS