Microscope 16

THE MICROSCOPE In this exercise you will learn about the principles of optical microscopy and become fa-miliar with the use of the microscope. Microscopes are delicate and expensive instruments; they should be handled with utmost care! Before you use the microscope, your instructor will explain its proper use. Following are rules that will protect the microscope and insure that you can make maximum use of it. Microscope safety rules are explained more thor-oughly in the Biology Department's Lab Safety Rules which you signed at the beginning of the semester.

TYPES OF MICROSCOPES

There are two different types of mi-croscopes: light and electron. Light micro-scopes have glass lenses which magnify ob-jects, and use light to illuminate the objects being examined. You will be using two dif-ferent kinds of light microscopes in this lab, the compound microscope and the dissecting microscope. Electron microscopes use beams of electrons to examine incredibly small objects (like the components of an indi-vidual cell) that have been specially prepared. The different microscopes are explained in more detail below. The Compound Microscope Compound microscopes are used to examine objects in two dimensions. Very small organisms or cross-sections of organ-isms are placed on clear glass slides; these objects are viewed as light passes through them. The parts of the compound microscope are reviewed below. The Dissecting Microscope Dissecting microscopes are used to observe material that is either too thick or too large to be viewed with the compound light microscope. With these microscopes, you see the surface of things that reflect the light. While the magnification and depth of field

are smaller in the dissecting scope, the field of view is much larger. As its name implies, the dissecting scope is often used to look at plants as you dissect them, since it allows for manipulation of material. Since most of the parts of the dissecting microscope are the same as the compound microscope, they will not be reviewed here. Electron Microscopy In these microscopes a beam of elec-trons (in place of light) and circular magnets (in place of glass lenses) permit the resolu-tion of structures in much finer detail than in an optical microscope. There are two elec-tron microscopes. The first is a "traditional" transmission electron microscope (TEM) in which an electron beam passes through the specimen. The second is the scanning electron microscope (SEM) in which a beam of electrons scans the surface of an opaque object and produces an image of that surface. The images are viewed on a cath-ode tube, or in pictures taken with the micro-scope. Many of the photographs of cell structure used in your text were taken with an electron microscope. FIU hosts the Flor-ida Center for Analytical Electron Micros-copy, which has an SEM.

Microscope 17

RULES FOR USE OF THE MICROSCOPE -- THE TEN COMMANDMENTS

1. Always carry the microscope in a straight upright position with one hand around the arm and the other hand under the base. The eyepieces are not attached and will fall out if the microscope is carried at an angle or upside down. 2. Check out the microscope to make sure all the lenses are clean and the mechanical parts are in working order. Report any malfunction to the instructor so that it may be remedied. 3. Keep the microscope clean. When anything is spilled or otherwise gets on the micro-scope, clean it up immediately. 4. When using the microscope start with the low power lens and work up to the desired magnification. These microscopes are parfocal, which means that all powers should be in focus when the turret is rotated. 5. Never move the stage upwards with the coarse adjustment while viewing through the eyepieces. Get the lens close to the slide while viewing from the side to make sure that they never touch. Then move the stage downward with the coarse adjustment while viewing through the lense. This will prevent the possibility of ramming the lens into the slide, thereby ruining a slide you have just made and, quite possibly, damaging the lens. 6. Moist, living or preserved materials must be observed through a coverslip. This pro-tects the lens as well as tends to make the object under view optically flat. Be sure to maintain a safe distance between the coverslip and the objective lenses. 7. Clean the lenses with lens paper only. DO NOT CLEAN THE LENSES WITH HANDKERCHIEFS, FACIAL TISSUES, PAPER TOWELS, ETC.--they will scratch the lenses. If your lenses are very dirty, obtain some lens cleaning solvent from the in-structor. 8. If you cannot obtain clear focus or good lighting, or if your microscope seems not to be working properly, IMMEDIATELY CALL YOUR INSTRUCTOR. He/she can ei-ther assist you or see that the microscope is repaired. 9. Return your scope to the cabinet with light cord wrapped around its base and with the lowest power objective lens in position.

Microscope 18

THE COMPOUND MICROSCOPE

Microscope 19

THE PARTS OF A COMPOUND MICROSCOPE 1. The microscope has two magnifying lenses: the eyepiece or ocular lens and the objective lenses on a turret which revolves above the stage. The eyepiece lenses are usually 10X and are moveable so that they can be adjusted to the distance between the pupils of each viewer. The objective lenses (there are four: 4X, 10X, 40X and 100X) rotate on the nosepiece. By changing the objectives the effective power of magnification is changed. The total magnifi-cation observed is the product of the power of magnification of the eyepiece and the objec-tive. Only the 100X objective is used immersed in a drop of special oil (between the lens and the slide; all others are designed to be used with air between the object and lens surface. The 100X objective will not be used in this course. The power of magnification is clearly indicated on each lens along with the numerical aperture of each lens. Depending upon their design and quality, different objectives have different resolving distances. The latter is the smallest distance between two points that allows both points to be viewed as separate. This resolving distance is dependent upon the wavelength of light used as well as the construction of the lens. 2. Microscopes contain elements designed to project parallel beams of light through the specimen and into the objective. These include the projection lens which focuses light onto the condenser lens. The condenser lens focuses light onto the object. To get the condenser lens in focus, place a slide containing a wax pencil mark on the stage. Focus on it with the lowest power objective lens and turn the iris diaphragm to the smallest opening. Then focus the condenser up and down until the edges of the iris diaphragm come into sharp focus with-out using the objective focusing adjustments. The condenser is now in focus. 3. The focusing knobs move the lens assembly up and down to bring the object in focus. The coarse adjustment should only be used with the shortest, low power objective lens. The fine adjustment (smaller knob) brings the object into critical focus. Notice that all objects are projected upside down in the microscope field. It takes a little practice in using the me-chanical stage to move the slide where you want it.

Microscope 20

USING THE COMPOUND MICROSCOPE 1.Use both hands to carry the microscope to your seat. Place the microscope on the table in front of you and position yourself so that you are comfortably seated while looking through the microscope. 2.If necessary, clean the lenses with lens paper only. Do not use anything else, like Kim-Wipes or your shirt to clean the lenses--this will damage the microscope. 3. Place a slide of the 'letter e' on the stage. If your microscope has a built in light, plug in the scope and turn the light on. If not, bring a lamp to your table and position it so that the light shines above the object being viewed. 4. Turn the nosepiece so that you are using the lowest power objective lens. You should al-ways use the lowest power objective when you begin viewing an object. While looking through the ocular lenses with both eyes, begin to focus on the object by turning the focus adjustment on the side of the microscope arm. If you see two images of the object or the re-flection of your own eye/eyelashes, you probably need to adjust the ocular lenses. These lenses can be moved together or apart to better match the distance between your eyes. 5. Once the object is in focus, increase the magnification by rotating the nosepiece. Adjust the focus by using the fine adjustment knob only. Make sure that the objective lens does not come in contact with the slide. 6. Examine different parts of the object by moving it around the stage. Notice the direction that the image moves when the object is moved from left to right. Change the light level and observe differences in the way the image appears.

ADDITIONAL CONCEPTS 1. The field of view is the area visible when you look through the microscope. Knowing the size of the field of view will enable you to determine the size of the object you are observ-ing. Special rulers are used to determine the field of view and measure objects under the mi-croscope. Accurate measuring can be very important when identifying plants or plant struc-tures. 2.Drawing Objects To Scale: In drawing objects that you have seen with the microscope it is important to describe how large they actually are. The actual magnification will depend upon whether you have drawn "little" or "big" (you should draw "big"). The way to estimate the actual size of the object is by knowing how wide the microscope's field of view is. This can be estimated by using a scale that has been etched on a microscope slide. Using this scale we have measured the width of each field for your microscopes: 44X = 4.6 mm, 4600 um 100X = 1.8 mm, 1800 um 440X = 0.46 mm, 460 um

Prokaryotes 25

Prokaryotes are composed of organisms with very simple cell struc-ture: no nucleus or organelles. These cells are usually very small, no more than 2 microns in diameter. Their mode of reproduction is almost always asex-ual, by simple binary fission. The Pro-karyotes include the Archaebacteria, the Eubacteria (or true bacteria) and the Cyanobacteria (or blue-green algae). Most of the prokaryotes dont look very different from each other, partly be-cause they are so small. Most bacteria can be split into groups based on their cell shapes: as rods, spheres, spirals and filaments.

They also can be distinguished because of differences in their cell walls, most nota-bly as Gram + and Gram bacteria. They can also be distinguished because of the dramatic differences in their metabolism.

Prokaryotes are important to us in a variety of ways. Metabolically, bacteria perform the work, and form the chemical links, that make different ecosystems operate. Many bacte-ria (but a tiny proportion of the total number of species) cause diseases in plants and animals, including humans. These diseases include many of the great killers in history, like the plague. Several sexually transmitted diseases, like syphilis, chlamydia and gonorrhea, are caused by bacteria. Anthrax, of danger as a biological weapon, is a disease of animals that can infect humans, but not be transmitted by us. Prokaryotes also perform many economi-cally important tasks. They are used in food processing, and bio-engineered bacteria pro-duce many valuable medicines and other substances.

Identify the bacteria on the plates that you prepared the first week of lab. The bacte-ria form shiny colonies, whereas fungi usually form fussy colonies. Are there differences in among the cultures? These indicate different types of bacteria.

THE PROKARYOTES

Prokaryotes 26

SOME IMPORTANT PROKARYOTES

CYANOBACTERIA These photosynthetic prokaryotes, formerly called blue-green algae, were the first organisms to fix carbon dioxide and produce oxygen in photosynthesis. They transformed the early atmosphere from reducing to oxygen-rich. They continue to be important today in many ecosystems. Cyanobacteria, both filamentous and single to multiple cell organisms, are an important portion of the periphyton communities in the Everglades.

LACTOBACILLUS These bacteria modify milk in the production of yogurt. Some people also take them as a dietary supplement to improve their digestion, particularly after having received a dose of antibiotics. They can easily be observed in yogurt that contains live cultures of this bacte-rium.

CYANOBACTERIA

Algae 31

THE ALGAE The algae are organisms with eukaryotic cells (with organelles and a nucleus) that are surrounded by a cell well. The cells are photosynthetic. Here we examine the major groups of algae, using examples that you will see in South Florida, either in ponds or on the coast. These groups vary in their photosynthetic pigments (although all have chlorophyll a), in their cell walls, their storage of sugars, and other details.

CHRYSOPHYTA (Golden Algae) These are the diatoms, single-cell photosynthetic organisms of both fresh and marine waters. They are most closely related to the brown algae. These organisms cover rocks and trunks, making them slippery. Diatoms have glass (silica) coverings. They divide repeat-edly by cell fission, but in each generation the cells become smaller and smaller. Then they produce gametes, reproduce sexually, and re-establish the original cell size. Their chloro-plasts have both chlorophylls a and c. Here some typical diatoms are illustrated, and exam-ples can also be seen in the periphyton exercise.

ALGAE, SEAWEEDS, AND SEAGRASSES

Algae 32

PHAEOPHYTA (Brown Algae) The brown algae are almost exclusively marine and are very common in the coastal waters of Florida. Many are very large in size, as the kelps of the Pacific coast. Brown algae have walls containing cellulose and chloroplasts with chlorophylls a and c. They often store their sugars as laminarin. The most common brown alga in South Florida is Sargasso (Sargassum sp.), which is commonly left on our beaches after high tide. Sargasso is very similar to the related Fucus, which is common to New England coasts and is depicted in your botany text. Here is a diagram of Sargasso, showing the blades and flotation bladders. The life cycle of Fu-cus, very similar to ours in that the only haploid cells are the sexual gametes, is almost identical to that of Sargasso. It is diagrammed on p. 353 of your textbook.

Sargasso (Sargassum filipendula)

Here are some other brown algae, quite commonly seen on rock reefs and mangrove areas in south Florida.

Ectocarpus

Stypopodium Turbinaria

Algae 33

RHODOPHYTA (Red Algae)

The red algae are characterized by chlorophyll a and red pigments, called phyco-bilins, in their chloroplasts. These multicellular algae appear reddish in appearance and are extremely common in marine waters in south Florida. Their walls contain cellulose and quite often accumulate calcium carbonate. They store sugars as Floridean starch. Red algae take on a variety of forms, often as flat blades or as highly branched trees. Here are some examples of red algae often seen in south Florida.

Grateloupia Spyridia

Cryptarachne

Dasya

Porphyra

Algae 34

CHLOROPHYTA (Green Algae) The green algae are the ancestors of terrestrial plants. They have chloroplasts with chlorophylls a and b, the same as in land plants. They also have walls of cellulose, and they store sugars as starch. They vary dramatically in size, from single and motile cells, to fila-ments, to much larger blades and branched structures. Some accumulate calcium carbonate in their walls and are quite tough. Others, as Ulva the sea lettuce, are very fragile. Single celled and filamentous algae will be common in ponds and periphyton. The larger marine algae will be encountered in coastal waters throughout south Florida. For examples of the single-celled and filamentous algae look at the illustrations in the description of periphyton. Here we give some examples of algae commonly seen in coastal marine waters.

Halimeda

Ulva

Caulerpa

Codium

Udotea

Algae 35

PYRROPHYTA (Dinoflagellates)

These single celled algae are commonly known as the dinoflagellates. They are im-portant in food webs in tropical marine waters. These algae are distinguished by their two flagellae and the plates in their cell walls; also, they generally are motile. The toxic red tides that occur on the Gulf Coast are blooms of dinoflagellates. Ciguatera, a toxin in some reef fish, is due to the passage of a product of a dinoflagellate in the food web. Some dinoflagel-lates are illustrated below.

Algae 36

SEA GRASSES Sea grasses are specialized flower-ing plants that grow in shallow coastal ar-eas, particularly in the tropics. They are ecologically important because they pro-vide habitats for fish and crustaceans, par-ticularly during reproduction and early de-velopment. Thus, they are nurseries for many economically important species. You often see parts of sea grasses along with various algae, after high tide at an ocean beach or at locations around Bis-cayne and Florida Bay. Here we illustrate two of the most common sea grasses in South Florida.

Turtle Grass (Thalassia testudinum)

Halodule

Periphyton 21

Periphyton

Dead material

Prawns

Crayfish

RotifersCopepodsInsect Larvae

Shellfish

Alligators

Wading Birds

Gar

Bass

THE BIOLOGY OF PERIPHYTON You may have noticed that the ponds in the Miami area are frequently covered with clumps of light-colored slime, what some might call pond scum. It might look pretty dis-gusting to the average person, but to those of us who study the Everglades, it is very special stuff. We call it periphyton. It is a community of micro and macro-organisms that lives un-der the water surface in the Everglades, or floats if it accumulates enough bubbles of oxygen. Periphyton forms on the skeletons of flowering aquatic plants, particularly the bladderworts (genus Utricularia). As a community, periphyton consists of a variety of organisms that live in the matrix of dead organic matter: bacteria, protozoans, green algae, diatoms, rotifers, insect larvae, and much more. We have added several pages of illustrations of organisms that you can easily find when you observe preparations with a microscope. This exercise also helps you learn how to use a microscope. Periphyton is ecologically important in the Everglades because it photosynthesizes, taking carbon dioxide from the air and transforming it into organic carbon, that can serve as an energy source for other organisms. Much of this organic carbon is passed to other organ-isms, particularly apple snails and small fish, in food webs. The snails and fish are eaten di-rectly by birds, or often by larger fish, that are then eaten by birds and alligators. So pe-riphyton is the stuff on which the Everglades runs. A number of scientists at FIU are study-ing the effects of adding phosphorus (a key ingredient in the water from the sugar cane farms to the north) on the function of the wetlands ecosystems. We are finding that even modest additions of phosphorus cause the periphyton mat to break apart. This alters the Everglades ecosystem.

Periphyton 22

EXAMINING PERIPHYTON UNDER THE MICROSCOPE In this laboratory exercise you will observe and identify organisms in the periphyton community, taken from an Everglades pond, and supplied to the classroom. This is an en-joyable process, because you may see amazingly bizarre living organisms swimming around in the water, or non-moving green and photosynthetic algae. Try to match what you see to the organisms illustrated here. Make a list of the organisms you have seen. If it is unusually interesting, share the view with your table partners.

EXAMINING MACRO-ORGANISMS

Take a piece of the periphyton mat without squeezing it and place it in a petri dish. Place the dish on a dissecting microscope and examine the periphyton for macro-organisms. Use the following diagrams to help you identify what you are observing. If possible, try to isolate a few of the more interesting organisms by using tweezers.

Adult Beetle

Hemiptera (Water Bug)

Clam or Mussel

Gammarus (Scud)

Leech

Midge larva

Mayfly larva

Snail

Water Mite

Stonefly Larva

Rotifer

Copepod

Periphyton 23

EXAMINING MICRO-ORGANISMS

In order to see micro-organisms present in the periphyton, it needs to be homogenized (ground up or pureed), diluted with water, and examined under a compound light micro-scope. This has already been done for you. Place one drop of homogenized periphyton (periphyton pure) on a microscope slide. Gently add a cover slip, by placing one edge against the slide and allowing it to fall over the tissue. This helps force out the air bubbles that tend to be trapped under the coverslip. You can remove excess water by twisting an end of a kleenex (or kimwipe) and placing it on the edge of the coverslip. It will absorb the ex-cess water. Then place the slide on the microscope stage and begin your observations under low power (10X). Look for a variety of micro-organisms, as illustrated in the following pages, in the periphyton. You can boost the power by turning the nosepiece to a higher power objective (watch your instructor demonstrate this). You can estimate the size of the organism by comparing its length to the diameter of the field at any given magnification. If you see absolutely nothing, then try preparing another slide of periphyton, then look again.

PROTOZOANS

Bulbochaete

Euglena

GREEN ALGAE

Peridinium

Chlamydomonas

Volvox

Mougeotia Oedogonium Spirogyra Ulothrix

Periphyton 24

DIATOMS

Fragilaria Frustulia

Gomphonema

Navicula Nitzchia

CYANOBACTERIA

Nostoc

Oscillatoria

DESMIDS

Desmidium

EXPERIMENTING ON PERIPHYTON Using the techniques you have learned in the lab you could ask some interesting questions about periphyton, and collect observations consistent or inconsistent with the hy-potheses stemming from these questions. Here are some sample questions.

1. Light levels at the top should be much higher than at the bottom of a floating mat of pe-

riphyton. Organisms adapted to different light intensities should be found at different levels in the mat. You could simply sample different levels of the mat and count the or-ganisms you have observed.

2. Organisms adapted to specific light levels may move vertically up and down in the mat during the day. You could count organisms at different levels and at different times of the day.

3. Conditions, as water temperature and sunlight, change during the year. Periphyton or-ganisms should change in abundance at different times of the year. Again, you could count organisms in mats at different times of the year.

Fungi 27

FUNGI AND LICHENS Traditionally, fungi had been lumped with plants and always studied in courses of botany. They do have cell walls and often grow in soil or are associated with plants. How-ever, their cell walls typically have quite a different chemical basis than do those of plants, consisting primarily of chitin (the same compound as in insect exoskeletons) rather than cel-lulose. Furthermore, fungi are not photosynthetic. They obtain their carbon compounds from other organisms, or from organic compounds in the soil. The distinctness of the fungi from both plants and animals has led to their being classified as a separate kingdom among all organisms, and they are now recognized as being more closely related to animals than to plants. The fundamental organization of all fungi is a tube consisting of a series of cells with one or two nuclei (or sometimes with no cell walls partitioning the tube), which is called a hypha (or plural, hyphae). They typically grow together as a mycelium, sometimes form-ing large, complex body like a mushroom, .

The fungi are typically classified into phyla based on the organization of their myce-lia and their modes of reproduction. We will observe fungi in four of the more common phyla. The classification of the fungi is based on the structures that are unique to each phy-lum and associated with sexual reproduction. However, many fungi do not reproduce sexu-ally and are placed in a separate phylum, the Deuteromycota.

Fungi 28

THE ZYGOMYCOTA These are molding fungi, that primarily attack plants and food products. The most common of these is the black bread mold, Rhizopus stolonifer. This phylum has a distinct life cycle, that includes a brief diploid stage, the zygospore. This structure makes the bread mold look black. This mold also attacks strawberries during their storage. Look at the mold on the bread that you started the first week of class.

THE ASCOMYCOTA These are the cup fungi, named for the fruiting structure that is characteristic of this phylum. The fundamental reproductive structure of this group is a sac of 8 haploid spores, called the ascus. The spores are called ascospores. Although yeasts can be either ascomycetes or basidiomycetes, Bakers yeast is an ascomycete. It is unusual, however, in not producing an ascus, an it most often reproduces asexually by budding. Look at the bakers yeast that has been growing in the lab.

Fungi 29

THE BASIDIOMYCOTA These are the club fungi, named for the fruiting structure, the basidium, character-istic of this phylum. Each basidium produces four haploid basidiospores. The basidiomy-cetes vary dramatically in their appearances, from parasitic fungi like rusts to soil and wood-dwelling fungi that produce large fruiting bodies, like mushrooms.

The basidiomycetes have fairly complex life cycles. These include (1) phases where the hyphae contain a single nucleus, (2) then two nuclei per cell, (3) a mechanism for transfer-ring nuclei to different hyphae, (4) then a fusion of nuclei, and (4) finally a meioitic division that results in the formation of the basidiospores.

Such a life cycle produces the fruiting bodies of the most commercially important mushroom species, Agaricus campestris, as well as the shitake mushroom, Lentinula edodes. Both of these will be observed in the laboratory. The surfaces of the gills of these mushrooms are covered with basidia and basidiospores. When these are ripe, tapping the mushrooms releases the smoke of the mature spores. These will germinate to repeat the life cycle.

Fungi 30

THE DEUTEROMYCOTA These fungi do not reproduce sexually. They also typically have an asexual spore-producing stage called a conidium. The Conidia produce conidiospores. These are seen very commonly in mold fungi. These fungi are often seen in moldy bread, Aspergillus in whites and blacks and Penicillium in grays. You may particularly note the conidiospores of these fungi, when viewed under a dissecting microscope. Penicillium is a particularly eco-nomically important member of this phylum. It and its relatives produce a variety of impor-tant antibiotics (penicillin, amoxocillin, etc.). It is the fungus that produces the distinct fla-vor of the blue cheeses and Camembert, the most famous being the French goat cheese, Roquefort.

LICHENS Fungi form important partnerships with other organisms. For example, fungi live on or in the roots of many plants, as mycorrhizae. These fungi receive energy as carbohydrates from the roots, and supply nutrients (particularly phosphorus) and water in return. However, the most visible partnership is that with algae. Such lichenized fungi, or lichens, are found throughout the world. They are among the toughest organisms, particularly abundant in ex-treme environments. They are common on high mountains and in the polar regions. Lichens can be seen in Miami on old trees and palm trunks, and can be distinguished from the large structures they develop. These include crusts (crustose) that hug the surface where they grow, such as rocks or palm trunks, thalli (foliose), which are flat, leaf-like sheets, or branches (fruticose), which stand erect. We mainly have crustose lichens in Miami. Look at the examples of lichens on display.

Microscope.pdfProkaryotesAlgaePeriphytonFungi and Lichens