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I n this exercise you will learn about the principles of optical microscopy and become familiar with the use of the microscope. Microscopes are delicate.

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Presentation on theme: "I n this exercise you will learn about the principles of optical microscopy and become familiar with the use of the microscope. Microscopes are delicate."— Presentation transcript:

1 I n this exercise you will learn about the principles of optical microscopy and become familiar 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 thoroughly 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 microscopes: light and electron. Light microscopes have glass lenses which magnify objects, and light is necessary to illuminate the objects being examined. You will be using two different 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 individual 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 organisms 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. While the magnification and depth of field are smaller in the dissect- ing scope, the field of view is much larger. As its name implies, the dissecting scope is often used to dissect plants, 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 The Biology Department has an electron microscope suite, used for teaching graduate-level students and for research. In these microscopes a beam of electrons (in place of light) and circular magnets (in place of glass lenses) permit the resolution of structures in much finer detail than in an optical microscope. We have two electron microscopes. The first is a "traditional" transmission electron microscope (TEM) in which an electron beam passes through the specimen. The second is the more recently-developed 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 cathode tube, or more critically by exposing photographic film. Many of the photographs of cell structure used in your text were taken with an electron microscope. THE MICROSCOPE In this exercise you will learn about the principles of optical microscopy and become familiar 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 thoroughly 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 microscopes: light and electron. Light microscopes have glass lenses which magnify objects, and light is necessary to illuminate the objects being examined. You will be using two different 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 individual 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 organisms 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. While the magnification and depth of field are smaller in the dissect- ing scope, the field of view is much larger. As its name implies, the dissecting scope is often used to dissect plants, 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 The Biology Department has an electron microscope suite, used for teaching graduate-level students and for research. In these microscopes a beam of electrons (in place of light) and circular magnets (in place of glass lenses) permit the resolution of structures in much finer detail than in an optical microscope. We have two electron microscopes. The first is a "traditional" transmission electron microscope (TEM) in which an electron beam passes through the specimen. The second is the more recently- developed 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 cathode tube, or more critically by exposing photographic film. Many of the photographs of cell structure used in your text were taken with an electron microscope. THE MICROSCOPE In this exercise you will learn about the principles of optical microscopy and become familiar 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 thoroughly 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 microscopes: light and electron. Light microscopes have glass lenses which magnify objects, and light is necessary to illuminate the objects being examined. You will be using two different 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 individual 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 organisms 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. While the magnification and depth of field are smaller in the dissect- ing scope, the field of view is much larger. As its name implies, the dissecting scope is often used to dissect plants, 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 The Biology Department has an electron microscope suite, used for teaching graduate-level students and for research. In these microscopes a beam of electrons (in place of light) and circular magnets (in place of glass lenses) permit the resolution of structures in much finer detail than in an optical microscope. We have two electron microscopes. The first is a "traditional" transmission electron microscope (TEM) in which an electron beam passes through the specimen. The second is the more recently- developed 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 cathode tube, or more critically by exposing photographic film. Many of the photographs of cell structure used in your text were taken with an electron microscope. The Microscope

2 Rules for using 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 microscope, 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 protects 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 instructor. 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 either 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. 10. Never leave any microscope slides on the microscope. Always remove them and return them to their proper place before leaving the room.

3 THE COMPOUND MICROSCOPE

4 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 magnification observed is the product of the power of magnification of the eyepiece and the objective. 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 without 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 mechanical stage to move the slide where you want it.

5 Using the Compund Microscope 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. If necessary, clean the lenses with lens paper only. Do not use anything else, like KimWipes 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 always 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 reflection 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 The field of view is the area visible when you look through the microscope. Knowing the field of view will enable you to determine the size of the object you are observing. To calculate the field of view, multiply the ojbective power by the ocular power (which is always 10 in our microspcopes). There are also special rulers are used to determine the field of view and measure objects under the microscope. Accurate measuring can be very important when identifying plants or plant structures. 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: 4X x 10 = 4.6 mm, 4600 um 10X x10 = 1.8 mm, 1800 um 40X x 10 = 0.46 mm, 460 um where μm = one micron, or one millionth of a meter

6 The Biology of Periphytons 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 disgusting 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 under 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 is also a good exercise for you to learn how to use a microscope. Periphyton is ecologically important in the Everglades because it is the source of much of the carbon fixed in photosynthesis, and this is passed to other organisms, particularly apple snails and small fish, in food webs. The snails and fish are eaten directly by birds, or often by larger fish, that are then eaten by birds and alligators. So this is the stuff on which the Everglades runs. A number of scientists at FIU are studying 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 to break apart. This may have unknown consequences for the function of this ecosystem, and we are trying to figure this out.

7 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 enjoyable 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 Microorganisms Take a piece of the periphyton mat without squeezing it and place it in a petri dish. (Sometimes there are flies in the periphyton that will bite if you squeeze the periphyton too hard.) 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 Clam or Mussel Leech Mayfly larva Rotifer Water Mite Gammarus (Scud) Copepod Midge larva Stonefly Larva Hemiptera (Water Bug) Snail

8 Examining Microorganisms 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 microscope. This has already been completed for you. Place one drop of homogenized periphyton 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 excess 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 Volvox Euglena Peridinium GREEN ALGAE Bulbochaete Mougeotia Chlamydomonas Oedogonium Spirogyra Ulothrix

9 DIATOMS Fragilaria Frustulia Gomphonema Navicula Nitzchia CYANOBACTERIA Nostoc Oscillatoria DESMIDS Desmidium Experimenting on Periphytons 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 hypotheses 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 periphyton. 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 organisms 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 organisms should change in abundance at different times of the year. Again, you could count organisms in mats at different times of the year.

10 Fossils AMMONITES These animal fossils were formed because of the hard calcareous shell secreted by the organisms. These are members of the Cephalopoda. What living organisms do you think these organisms were related to? These organisms lived during the Mesozoic era. TRILOBITES These are the most ancient of the organisms displayed among these fossils, dating back to the Cambrian era. These fossils were formed because of a hard exoskeleton, so durable that the shape of the fossil gives a good idea of the three dimensional shape of the organism. The exoskeleton consists of segments, with many small appendages. What living organisms do you think these organisms were related to? Fossils are remnants of once living organisms. Fossils are direct evidence for organisms that lived in the distant past. Fossils were produced under optimal conditions and only by organisms with hard body parts that allowed such formation. These were places on the earth where dead organisms were covered by fine sediments, oxygen was excluded from oxidizing the structures, and the organisms were thus preserved. Fossils are only found in sedimentary rocks. Fossils may be impressions or compressions of once-living organisms. Hard structures, as shells or skeletons may be fossilized directly. Other fossils are formed with minerals gradually replace the once-living tissues in a process termed petrifaction, like petrified wood. Fossils are the best evidence available on the organisms that were present in the distant past. In this display, observe the fossils on display. For each fossil determine its age (approximately in millions of years before present) by matching the eras during which the organisms lived with the time scale on the poster. Determine the process by which each fossil was formed. Finally, determine the phylum and kingdom to which each fossil belonged. LEPIDODENDRON This is a plant fossil, common in the coal deposits of Pennsylvania and West Virginia, dating back to the Carboniferous. What you are looking at is the surface of the trunk of a small tree, and the diamond shaped structures are the scars where leaves were once attached.

11 More Fossils CALAMITES This is a plant fossil, common in the coal deposits of Pennsylvania and West Virginia, dating back to the Carboniferous. You are looking at the surface of a stem whose branches all originate at a single node, all the way around the stem. On the small branches, leaves also appear at the nodes. These plants produced spores, on cone-like structures, and did not produce seeds. These fossils are of plants that were related to the living genus Equisetum, or the scouring rush. PETRIFIED WOOD These are various samples of wood, deposited in sandstones in deserts of the southwest. These are the youngest of fossils on display, produced during the Tertiary. Wood anatomists can trace the arrangements of cells in these fossils to genera of conifers living today. Thus the names on the fossils correspond to plants you may be familiar with, such as Taxodium (cypress). What are desert localities today once supported coniferous forests back then. If you look carefully at parts of the wood you can see the annual growth rings of the wood. The thickness of these rings can also analyzed to estimate rates of growth and the types of climates in which these trees lived. LEPIDOSTROBUS This is also a plant fossil, common in the coal deposits of Pennsylvania and West Virginia, dating back to the Carboniferous. You are looking at the surface of a cone-like structure of a plant that did not produce seeds. This name is the example of a form genus. It was given its name as a structure. Later on other fossils were found that connected it to the stem, Lepidodendron. Later on another form genus was established for the roots. Yet these are all fossils of a single plant species. These fossils are of plants that were related to the living genus Lycopodium. This genus is often given the name of a “living fossil” because of its ancient origins.

12 Kingdom Archaebacteria Archaebacteria of domain Archaea may be the oldest form or life an earth, an domains Bacteria and Eukarya probably diverged from Archaebacteria independently. Archaebacteria are diverse prokaryotes that share ribosomal RNA sequences as well as several important biochemical characteristics that are quite distinctive from those of all other kinds of organisms. Archaebacteria have distinctive membranes, unusual cell walls, and unique metabolic cofactors. Today’s Archaebacteria are probably survivors on ancient lines that have persistent in habitats similar to habitats found throughout the world when bacteria first evolved. These environments are often extremely acidic, hot, or salty. Thus, many Archaebacteria are called extremophiles. Many Archaebacteria can live in an anaerobic atmosphere rich in carbon dioxide and hydrogen as well as the more benign environments. Kingdom Bacteria Bacteria of the kingdom Bacteria are distributed more widely than any other group of organisms. Individual bacteria cells are microscopic (1  m or less in diameter); a single gram of soil may contain over a billion bacteria. Bacteria have cell walls, which give them three characteristic shapes. (See figure on next page for example of the different bacteria shapes.) Bacillus (rod-shaped) Coccus (sphirical) Spirillum (spiral) Most bacteria are heterotrophic, meaning that they derive their energy from organic molecules made by other organisms. Heterotrophic bacteria are decomposers because they feed on dead organic matter and release nutrients locked in dead tissue. Bacteria that derive their energy from photosynthesis or the oxidation of inorganic molecules are called autotrophic. However, photosynthesis in bacteria is often different from that in eukaryotes, because sulfur rather than oxygen is sometimes produced as a by-product. Survey of Bacteria Cellular organisms have evolved along two lines. Species with cells lacking membrane- Bound organelles are prokaryotes. Those with membrane-bound organelles are eukaryotes and include plants, animals, fungi, and protists. About 5000 species of prokaryotes have been described, and many more await identification. Prokaryotes were long thought to be a unified group commonly called bacteria. However, genetic analysis as recently as 1996 of the DNA of prokaryotes has revealed two groups with surprisingly different DNA sequences, both of which are strikingly different from the DNA sequences of eukaryotes. This has led to recognition of three domains of organisms. Domain Archaea include kingdom Archaebacteria, all species of which are prokaryotes. Archaebacteria often inhabit but are not restricted to extreme and stressful environments on earth. Domain Bacteria includes kingdom Bacteria, all species of which are prokaryotes and are the most abundant organisms on earth. Domaine Eukarya includes kingdoms Protista, Fungi, Plantae, and Animalia. These kingdoms are all eukaryotes. This classification of living organisms into three domains and six kingdoms is widely accepted, but much phylogenetic information remains to be revealed. Classification is an exciting and ongoing process.

13 Here are some examples of the 3 shapes of bacteria. Bacillus = rod-shaped Streptococcus = coccus (round) Spirillum = spiral Although bacteria can reproduce sexually through a process called conjugation, their mode of reproduction is almost always asexual, by simple binary fission. Nitrogen fixing bacteria- Certain bacteria and cyanobacteria transform atmospheric nitrogen (N 2 ) into other nitrogenous compounds that can be used as nutrients by plants. This process in called nitrogen fixation. All organisms need nitrogen as a component of their nucleic acids, proteins, and amino acids. However, only certain bacteria and cyanobacteria have the capability to break the bonds between atmospheric nitrogen and convert it to ammonia (NH 3 ) which can be used by plants. These bacteria form nodules on the plant roots where they convert the nitrogen in exchange for sugar and other nutrients provided by the plant. Such an association, where both organisms benefit, is known as symbiosis. The prokaryotes: Bacteria Root nodules containing nitrogen-fixing bacteria

14 Some Important Prokaryotes Cyanobacteria A member of the kingdom Bacteria, 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 bacterium, as you will discover in one of your exercises. Cyanobacteria

15 Fern Spore Germination Ferns are a part of the plant kingdom, but do not produce flowers, fruits, or seeds like many other plants. Instead, ferns produce spores on the underside of leaves. These spores are dispersed by wind and will germinate if they land on moist soil. Eventually each spore develops into a flat, heart-shaped structure called a thallus. Once fertilization occurs, a new fern starts to grow from the thallus. Over time, this new fern becomes much larger than the thallus and takes on the fern form that we are familiar with. In this procedure, you will be sowing spores of the fern Ceratopteris, a very rapidly growing fern. In two or three days, the spores will germinate and after days, thalli should be apparent. Throughout the semester, you will observe the development of the fern spores and record your observations. The following procedures are based on those provided by the C-Fern company and are protected by copyright laws. Procedure: 1. Use small Styrofoam containers, such as those used for soup take out. There should be one container per table, with a total of 6 per lab. Fill it with sterilized potting soil to a depth of about 2.5 cm (or 1 inch). 2. Gently moisten the soil with the small spray bottle. 3. Using a cotton swab (or q-tip), place the cotton tip into the vial of fern spores. 4. Place the cotton tip above the soil, tap the shaft of the q-tip as you move the cotton tip around on the soil of the dish. 5. Puncture two small holes in the lid of the dish, and label it using the label tape and marker. Indicate your lab group, the lab section and the date. 6. Place the plastic dish on the shelf indicated by your instructor, under the fluorescent lights. 7. Observe the changes in the development of the spores each week during the term. Note the numbers of cells and their arrangement. Document with simple sketches.


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