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Exercise 1 Microscopy Introduction: Today’s lab is one of the most important as we cover both safety (see separate section) and microscopy. The microscope is one of the most important tools in all of biology and it has led to major discoveries for over 400 years. Robert Hooke was the first person to see cells when he examined the bark of an oak tree with his microscope in 1665. Antoni van Leeuwenhoek developed a better microscope and was able to see living cells, including bacteria, in 1674. We use light microscopes in this lab as they use light to magnify and focus the specimen. (Electron microscopes use a beam of electrons to perform the same functions.) Two important features of microscopes are magnification and resolution. Magnification is simply the enlargement of an image—we will cover that in more detail later in lab. Resolution is the ability of a microscope to reveal fine detail. Technically, it is the ability of a microscope to distinguish the distance between two small objects. Resolution and magnification are not the same! If you saw Harry Potter 2, you saw an example of resolution. You may remember that Uncle Dersley screwed bars over Harry’s bedroom window, and the Wealeys flew their car to Harry’s rescue. When Harry first sees the car, it is so far away that the two headlights appear as one—human eyes cannot resolve the two headlights as separate from so far away. However, as the car gets closer, the single light resolves itself into the two headlights. So even though two objects may in fact be separate, the microscope may not be able to reveal that fact. And if the microscope cannot resolve two images, magnifying the image will just result in the same blur—only bigger. The microscopes at Solano are quite good and are an important tool for you to master the lab material. We will use the scopes almost every lab, so you want to be comfortable using them. You’ll also be introduced to the major groups of organisms that we’ll be discussing for the rest of the semester. These include the eubacteria, protists, fungi, plants, and animals. Goals/Student Learning Outcomes: Identify the parts of the microscope and briefly explain the function of each. Using prepared slides or wet mounts that you’ve made yourself, focus a specimen clearly and with appropriate illumination. Determine the total magnification using any of the objective lenses. List the kingdoms that we’ll be discussing throughout the semester. Activities: The compound microscopes are located in the tall cabinets in the middle of the tables (do not confuse them with the dissecting scopes). There should be one for each of you. Grab the microscope by the arm with one hand and down. Turn the dimmer up and light should now appear.) support the base with your other hand. The power cords are in the drawers above the cabinets; plug the cord into the microscope and the electrical outlet, some of which are located on top of the table while others are located just beneath the table tops on the sides. Obtain a prepared slide (one that has already been made for you) with the letter “e.” Hold it up to the light or against white paper so it’s oriented the way you would read it. Open the slide clip and place the “e” so that the corners of the slide fit into the corners of the slide holder on the stage as demonstrated in this photograph: See the photograph below for parts of the microscope: on/off switch, dimmer/rheostat, light source, iris diaphragm lever, condenser, stage, stage adjustment knobs, slide clip, objective lens, ocular lens, course focus adjustment, and fine focus adjustment. Ocular Lenses Slide clip Stage Now center the “e” over the hole in the stage by using the stage adjustment knobs; these are located to the right and below the stage. Objective Lenses Stage adjustments Iris diaphragm lever Fine Focus Dimmer Course Focus Light Source On/off switch The objective lenses (as you can see there are four with our microscopes) are attached to a rotating ring known as the “turret.” To change objective lenses, please grab and turn the turret, not the lenses themselves. Turn the turret so that the 5X objective lens is centered over the “e”; it should make a small “click” when centered properly. You now want to focus the “e.” You do so by rotating the coarse focus knob (the larger one on either the right or left side of the scope) toward or away from you. Note that the stage moves when you focus; the objective lens stays in one place. If you don’t have the “e” in focus, raise your hand to get help from your instructor. At this point, you may need to better Turn the microscope on with the switch on the right side in the back. These scopes also come with a dimmer (or rheostat), located above the on/off switch. (If you turn the scope on and no light appears, the dimmer is probably turned Page 2 from side to side as you look at the light emerging from the hole in the stage—you should see it brighten and dim. You can also control the light by adjusting the dimmer switch; again, turn the dimmer forward and backward while you look at the light emerging from the hole in the stage—you should see the light brighten and dim. After the light leaves the specimen, it travels through the objective lens; it then moves through a series of prisms and is reflected by mirrors so that it emerges from the ocular lens. center the “e” using the stage adjustment knobs. Also at this point, you’ll probably want to make two adjustments to the oculars. The first is to adjust them to match your interpupilary distance—the distance between your two eyes. So looking at the “e”, move the oculars closer together or further away so that you see just one image. (When you’re new to microscopy, your head may move closer to or further away from the oculars, which complicates this step.) You may also need to adjust the oculars if your eyes do not have the same power. If you have contacts or glasses, your optometrist has compensated for the fact that your two eyes may be different. If this is the case, you’ll want the oculars to be in the same position so turn both oculars so the “0” aligns with the white dot of the housing. What is the orientation of the “e”? Right side up? Readable orientation? As you observed, the lenses of the microscope invert the image of the “e”; why will this flipflopping NOT be a problem for us in lab? If your two eyes are significantly different from one another and you don’t have corrective lenses, close your left eye and focus the “e” with your right eye. Now close the right eye and open the left. Rotate the left ocular until the “e” is in good focus. You should now be able to use both eyes to see the specimen in good focus. Return the “e” to its proper tray. (All the prepared slides that you will use will come in shallow trays, which are labeled in the front. Please return all slides to their correct trays.) Next, obtain a “cross thread” or “cross fiber” slide. Three different colored threads—blue, red, and yellow—were placed on top of one another; however, the arrangement will vary (they are not all the same). Again, examine the slide against the classroom lights or white paper so you see the intersection of the threads; open the clip to place the slide into the corners of the slide holder; and then center the intersection of the threads by using the slide adjustment knobs. It is important to make these adjustments so that you can complete your lab work without eye strain. Note that light is produced in the bulb controlled by the dimmer; it travels through the iris diaphragm then the condenser and then the hole in the stage through the specimen. You can control the amount of light that comes through the condenser by closing down or opening up the iris diaphragm; do so by moving the lever from side to side. Move the lever Using the 5X objective, focus the slide with the coarse focus knob. Now rotate the turret to swivel the 10X objective into place. Our Page 3 how much of the specimen you can see. The fields-of-view with the other objectives become smaller with the 63X objective having a miniscule one. WHEN USING THE MICROSCOPE, ALWAYS START WITH THE 5X OBJECTIVE AS THAT GIVES YOU THE BIGGEST FIELD OF VIEW AND THE GREATEST DEPTH OF FOCUS TO FIND THE SPECIMEN. And once you focus a specimen with the 5X objective, always be sure to center the specimen as you move to the 10X objective, etc. (It is easy to lose the specimen as you move up in magnification as the fields-of-view become smaller and smaller.) microscopes were constructed to be “parfocal,” that is, once you focus a specimen with one lens, the other lenses should be close to focus as well—of course they will be off just a bit. Here is where you use the fine focus (the small focusing knob) to get a crisp image of the threads. Starting at the top of the intersection or at the bottom, slowly move the fine focus to determine which thread is on top, which one is in the middle, and which one is on the bottom. The top thread is ____________, the middle thread is _____________, and the bottom thread is _____________. For the rest of the lab period we’ll examine the major groups—known as kingdoms in biology— of organisms that we’ll be discussing throughout the semester. You’ll be practicing your microscopy techniques as you go. Return this slide to its correct tray. When you use the 5X objective, you are actually magnifying the specimen a total of 50 times as the ocular lens magnifies the specimen by 10X. Note that total magnification is a PRODUCT of the objective and ocular lenses, that is, you MULTIPLY the individual magnification powers of the two lenses to get the total. We’ll start off with the Kingdom Eubacteria. These small organisms, generally single-celled, are prokaryotic because they have no membrane organelles such as nuclei or chloroplasts (“prokaryotic” means “before having a nucleus” and refers to organisms that evolved before “eukaryotic” organisms that have them). Our objectives magnify 5X, 10X, 40X, and 63X; remember that the ocular always magnify 10X. So what is the total magnification when using the 10X objective? ______ The 40X objective? ______ The 63X objective? _____ Examine the prepared slides of two cyanobacteria: Anabaena and Stigonema. (The art majors in class may recognize “cyan” as a blue-green color; and the cyanobacteria were once called the blue-green algae.) We start with these bacteria as they are very large for prokaryotic organisms and should be fairly easy to see. Both of these organisms occur in chains, though they are still considered single-celled. Stigonema grows in branched chains, which is uncommon in bacteria. These organisms respire as you do, but they also photosynthesize and fix nitrogen—the latter two topics will be covered in detail later in the semester. But for The different objective lenses also have different depths-of-focus, that is, how much of the specimen you can see in focus from top to bottom. The 5X has the deepest depth-of-focus while the 63X has almost none. Consequently, when you use any of the lenses, but especially the 40X and 63X, you’ll need to make fine focusing adjustments if you want to see the entire specimen from top to bottom. The other nice thing about the 5X objective is that it also has the biggest field of view, that is, Page 4 now, organisms that resembled present-day cyanobacteria were the first organisms to develop photosynthesis and they started adding oxygen to the atmosphere; without cyanobacteria (and algae and plants) there would be no oxygen for us to breath. Solids/liquids of yogurt Air bubbles Much smaller bacteria can be found in yogurt. Humans all over the planet have figured out that fermenting milk is one way to preserve it. Milk is very nutritious as it contains proteins, lipids, and carbohydrates; but you’re probably aware that it sours/rots very quickly, especially if kept at room temperature (many countries still lack refrigeration). The bacteria that ferment milk produce acids that curdle the milk, that is, cause the proteins to clump together into curds as well known in the nursery line: “Little Miss Muffet sat on her tuffet eating her curds [the clumped proteins] and whey [the liquid portion].” These acids also protect the milk from different bacteria that would decompose it. In the U.S., we eat mostly cow yogurt, but you can also find goat yogurt. In Tibet, they make yak yogurt, in Russia horse yogurt, and in the Middle East camel yogurt. refracts light as you focus up and down, which produces something of a rainbow effect as the colors change. However, air bubbles are not of interest in our wet mounts and we’ll ignore them.) Place the yogurt slide on the stage and try to center an area on the slide that is more transparent as you want to avoid the coagulated proteins—the clumps or curds—in the slide. Be aware that bacteria are composed mostly of water and you’ve mounted them in water, so turn the light intensity down. (Remember the two ways to reduce light?) As you fine focus from the 5X to the 10X to the 40X to the 63X, you should see very small particles floating in the water—remember to recenter the slide as necessary. These are the acidproducing bacteria of yogurt, which are teeny compared to the cyanobacteria. Using a toothpick place a very small dab of yogurt onto a slide and add a drop of water (remember for safety reasons there can never be any eating or drinking in the lab). Add a coverslip by placing one end down and then lowering the rest of it to reduce air bubbles that might get trapped. (When students are first making wet mounts, it is very common to get air bubbles, and they are huge compared to bacteria—see the photograph in the next column.) Coverslips flatten the specimen and provide some protection for the objective lens. After about two billion years of evolution from prokaryotic ancestors, protists appeared on the planet—this occurred about 1.5 billion years ago. These organisms are eukaryotic as they have membrane-bound organelles such as nuclei, chloroplasts, and mitochondria. Many are single-celled while others are colonial (aggregates of cells that perform the same functions) while others are multicellular (composed of many cells that do different So it is very common for students to get excited about their “discoveries,” especially as the air Page 5 Around 500 million years ago, fungi—our next kingdom—evolved from protists. They come in two main forms: filamentous molds and unicellular yeasts. Examine the slide labeled “Cyathus,” more commonly known as the bird’s nest fungus. When Cyathus produces its spores, it packages them up (the eggs) and then protects them with the “nest.” Cyathus is a decomposer; it is uses the energy of dead wood to grow and reproduce. functions, e.g., most cells photosynthesize in algae while a smaller number of them are specialized for reproduction). Make a wet mount of amoeba, which are always found at the bottom of the jar—never on the sides or floating above. To obtain a few, expel the air out of a dropper BEFORE you place the tip of the dropper at the bottom of the jar. Suck up a small amount of fluid and transfer it to your slide; remember to gently lower a coverslip. Yeasts are one of the most important fungi economically—they are used for brewing alcohol (Budweiser is just across the highway and the Napa Valley is just a few miles away), baking, and genetic engineering. Place just a small drop of the living yeast culture on a slide. As water is the largest component of yeast cells and water surrounds them, they are difficult to see. Biologists overcome this problem by using dyes that the organism or parts of the organism absorb. To your small drop of yeast cells, add a small drop of methylene blue; then add the coverslip. (There should be dropper bottles of methylene blue on the cart.) Amoebae are single-celled organisms that move and capture food by pseudopods—temporary extensions of their cytoplasm. Most amoeba are free-living as they capture and digest bacteria, other protists, and bits of organic matter (only a few amoebae are parasites and you may have heard of amoebic dysentery). Amoebae are delicate creatures and they get beat up as they are shipped through the mail from Sacramento; so there will be quite a few dead ones. A little searching should reveal the living ones. For comparison, examine the prepared amoeba slide labeled “Chaos carolinensis” or “Pelomyxa carolinensis.” (“-ensis” means found in or located in, so “carolinensis” indicated that these amoebae were first discovered in Carolina.) If you search a bit, you should find cells that are reproducing asexually—a process known as budding where a larger cell produces a small one that begins to grow and then detaches. A photosynthetic protist is Volvox. This is a colonial organism in the shape of a sphere. Examine the prepared slide of Volvox (living Volvox is green; this prepared slide has been stained so the organisms are unnaturally colored red or purple). It can reproduce asexually; when it does so, daughter colonies develop inside the parent organism. When the parent splits open, the daughter colonies are released. Also about 500 million years ago, a green alga gave rise to plants—our fourth kingdom. Make a wet mount of Elodea, an aquatic plant. Remove one leaf, place on a slide, add a drop or two of water (there are dropper bottles of water near the sink), and then add a coverslip. You should see chloroplasts—green organelles that are the site of photosynthesis—moving around the cell. (The rhythmic cycling of the chloroplasts ensures that each one receives the same amount of sunlight as its neighbors.) Page 6 As the final slide for today, examine the flea. Note the piercing/sucking mouthparts at the front. Fleas are parasites and as they feed from one host to another, they transmit pathogens. For the next plant—the potato—make the thinnest section of potato tuber that you can— a quarter of an inch is more than enough; place it on a slide. Now add a drop of iodine (labeled IKI) then the coverslip. You may know that potato tubers store starch, which turns purple with the addition of iodine. Look for the amethyst-like starch granules. Historically, one of the most devastating was the Black Plague of the 1300s (though there were plague epidemics prior to and after this one). Fleas transmitted the bacterial pathogen Yersinia pestis from rats and other rodents to humans. As the bacterium kills human cells they turn black (hence “Black Death”) as other bacteria decompose the dead tissue. The animal kingdom is our last one for today. Animals are multicellular organisms that have no cells walls. They are also the only organisms that have muscle and nerve tissues. This disease is also known as “bubonic plague” for the production of buboes—swollen lymph nodes of the groin, arm pit, or neck. Plague killed one-quarter to one-third of Europeans in the 14th century. Obtain a sterilized toothpick and GENTLY scrape (do not pierce!) the inside of your cheek. Transfer your cheek cells to a slide; add a drop of methylene blue and a coverslip. You should see clumps of flattened cells—try to find one that is separated from the others. Your nuclei, in the middle of the cells, are stained blue— you’re looking at your DNA! You have 46 chromosomes in your check cells and if you placed them end-to-end, they would extend for over six feet! If you’re lucky, some of the bacteria that reside on the surface of your cheek cells will also stain darkly blue. They are generally spherical or rodshaped and much, much smaller than the cheek cell. (The human body is made up of 3 to 200 trillion cells, yet we have 10 times that number of bacteria on and in our bodies! These bacteria are generally beneficial to our health as they synthesize vitamins and protect us from potential pathogens.) As humans can carry bacterial, viral, or fungal pathogens, please discard your cheek cell slide with coverslip and toothpick into the beaker labeled “Cheek Cell Slides.” We will add bleach to kill any potential pathogens before we dispose of the slides. Page 7 Other than magnification, how does the microscope process the image of a specimen? For review: Label the parts of the microscope. When viewing a slide for the first time, why do you always start with the 5X objective? Give at least two reasons. Explain why dyes are used in lab. List the five kingdoms that we saw in today’s lab. If the total magnification of an image is 400X, what objective lens was used? __________ If it is 100X? _____ Briefly describe the two ways to add or reduce the amount of light when using the microscope. Page 8