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9-1 Biology 1001 Laboratory 9 PROKARYOTES AND EUKARYOTES PREPARATION Read this exercise before you come to the laboratory. TEXTBOOK READING - Biology (Campbell et al.)- Chap. 27 (Bacteria), 28 (Protists) and 31 (Fungi) - Biology 1001 Laboratory Manual: Appendix IV – Tables on page A-12 OBJECTIVES - to correctly use the oil immersion objective - to observe bacteria and understand cell types and Gram’s stain - to prepare and stain wet mounts of yeast, mold and algae - to discover and understand the differences between prokaryotes and eukaryotes PREPARATION Be able to answer the following questions: 1. What are the differences between prokaryotic and eukaryotic cells? 2. Why is immersion oil used? 3. Why are bacteria heat fixed to the slide? 4. Describe the differences between the cell walls of Gram positive and Gram negative bacteria? 5. Check the internet for images of bacteria (various forms of cocci, bacilli, spirilli) as well as Anabaena, Zygnema, Rhizopus and Yeast. LABORATORY ASSIGNMENTS 1. After examining a slide of stained bacteria under oil immersion, prepare a proper table indicating the cell types observed, cell sizes and their Gram staining properties. Pass this in before you leave today. 2. Prepare labeled drawings of fungal cells from wet mounts of Saccharomyces and Rhizopus. 3. Prepare a labeled drawing of the prokaryotic Blue-green algae, Anabaena. 4. Prepare a labeled drawing of the eukaryotic Green algae, Zygnema. 5. Answer the questions on pages 9-9 and 9-10. 9-2 Prokaryotes and Eukaryotes - General Introduction The major differences between prokaryotes and eukaryotes are listed below. Some, but not all of these differences can be observed with light microscopy. Prokaryotic Cells Eukaryotic Cells ___________________________________________________________________________ Nucleus not bounded by a nuclear Membrane DNA single circular molecule bounded by a double unit Membrane organized to form chromosomes Complex organelles Absent Present Microtubules Absent Present Flagella Simple Complex (9+ 2) Sexual reproduction Absent Present ___________________________________________________________________________ The basic living unit of all organisms is the cell. Although different cell types vary in size and shape and although they perform widely different functions (e.g. muscle cells contract, red blood cells carry oxygen, epidermal cells protect, etc...) their basic components are quite similar. It is, however, possible to divide living organisms into two broad cellular groups: the EUCARYOTES and the PROCARYOTES. Prokaryotic cells belong to the smallest and simplest of all living organisms. Mycoplasms, bacteria and blue-green algae are all prokaryotic cells and are considered primitive in evolutionary terms. However, in terms of biomass and variety you should not think of them as unsuccessful organisms. The oldest known fossils, approximately 3 billion years old, are of prokaryotes. Prokaryotic cells do not have their chromosomal material organized into a membrane-bound nucleus, they lack a nucleolus and membranous organelles such as mitochondria, Golgi, chloroplasts, etc... A. Bacteria Introduction Despite their minute size and simplicity of organization, bacteria are highly successful organisms. As a group, they are adapted to occupy an extremely wide range of environments, and in fact, they are more widely distributed than any other form of life. In soil, where bacteria are especially abundant, some species play an important role in the nitrogen cycle converting the inorganic nitrogen gas of the atmosphere into nitrogenous salts which become available for plant growth. Bacteria are also responsible for much of the degradation of dead 9-3 organic material which releases carbon dioxide for reutilization in photosynthesis (the original recycling factory). Bacteria are important primary producers (many small organisms eat them) so they occupy an vital place in world food chains. But, on the other hand, we have also been subjected to innumerable diseases which can be traced to bacteria as causative agents: gonorrhea, pneumonia, scarlet fever, tuberculosis, diphtheria and many more. Bacterial cells, as we have already mentioned, are very small: we are talking about organisms which are about one micron in diameter. If you consider that the limit of resolution of the light microscope is 0.22 microns, then you can appreciate that until the development of the electron microscope, not much was known about the internal structure of these organisms. In fact during today’s lab session you should be able to get a first-hand appreciation of what technical limitations mean in terms of the quality of information one can derive from light microscopy. Bacteria occur in three typical shapes: round (the cocci, coccus, singular); rod-shaped (bacilli; bacillus, singular) and curved-shaped (spirilli; spirillus, singular). See Figure 27.2 in your text book. All of these can occur singly or in groups known as bacterial colonies. Beyond the actual shape differences, bacteriologists observe certain clues which enable them to further classify bacterial types. (a) Clustering – bacteria may exist as individual cells or as groups of two cells, clusters or chains of individual cells. These characteristics are observable microscopically and these characteristics are useful in identifying bacteria. (b) Colony Characteristics - bacteria organize themselves in large distinct groupings presenting characteristic colours, shapes, margins and profiles. These characteristics are observable macroscopically and used with microscopic evidence to identify bacteria. (c) Motility - whether a particular species of bacteria is capable or not of movement (i.e., is motile) may aid in its identification. (d) Staining Reaction - The reaction of different species of bacteria to certain stains may be a useful identifying feature. Over the years, the Gram stain (named for its discoverer, Christian Gram) has proved to be a very useful diagnostic tool. The reaction to the Gram stain correlates with differences in the cell wall composition of bacteria. It is called a differential staining procedure because it can be used to divide bacteria into two groups, called Gram positive (G+) and Gram negative (G-). After Gram staining, the positive bacteria appear blue and the negative bacteria, appear red. Not all bacteria are either Gram positive or Gram negative; a few display both staining reactions and are referred to as “intermediate” or Gram variable. See Figure 27.3 in your text book. Demonstration: Colony Types On the demonstration bench, observe a number of Petri dishes containing bacterial cultures grown on an agar medium. Note the variety of colours, colony shapes, surface characteristics and margins. 9-4 Macroscopic characteristics such as these are used to help identify what kind of bacteria constitute these colonies. What are the most common colony shapes, colony margins and colony surface characteristics found in the species you observed on the demonstration bench? Microscopic observation of bacteria: Examine slide 108 from your slide box. This is a bacteria smear showing a variety of bacterial shapes and gram-staining properties. You will need to examine the slide using the 100X objective lens. You must use the oil immersion technique to do this. Before you begin, thoroughly clean the slide by breathing on it and polishing with a piece of lens tissue. This will remove dust and fingerprints. This is a good time to clean the 40x and 100x lens on your microscope. Use lens cleaner from the stain rack and lens tissue to do this. Smudgy lenses will make it hard to bring microscopic organisms into sharp focus. Procedure for using Oil Immersion 1. Centre the slide using the scanning (4X) lens. Bring the smear into focus. 2. Change to the low power (10X) lens and then the high power (40X) lens, centering and focusing the slide each time. Be sure to use only the FINE FOCUS knob with the 40X lens. 3. While watching carefully from the side, swing the 40X lens out of the way and the oil immersion (100X) lens toward the functional position. Before snapping it into place (aligned with the body tube), place a small drop of immersion oil (found in your stain racks) on top of the circle of light from the condenser, showing through the slide. 4. Still watching carefully from the side, snap the 100X lens into position and allow it to contact the oil droplet. 5. Look through the ocular lens and carefully focus the microscope, using the FINE FOCUS KNOB ONLY. NOTE: you cannot go back to high power (40X) without removing the immersion oil from the 100X lens and the slide. 6. You may need to increase the light intensity if the field of view seems too dark. 7. Examine the slide, using the mechanical stage to move the slide as you require. Use the ocular scale to measure each cell type you observe. Refer to the calibration information from lab 4 to calculate the length on one ocular unit at 1000X. 8. When you are finished examining your slide, you must clean all the immersion oil off your lens and slide while being very careful not to get oil on the high power lens while doing so. While watching carefully from the side, swing the 100X lens out of the way and the scanning (4X) lens into place. Using lens tissue, clean the 100X lens of immersion oil. Repeat using a new piece of lens tissue moistened with lens cleaner. Remove the slide and clean it thoroughly with tissue and lens cleaner before replacing it in your slide box. 9-5 Assignment 1: Carefully survey slide 108, locate and measure as many different kinds of bacteria as possible. Note the size and Gram staining property of each. (Figure 27.2 and 27.3 in your text provides information on Gram stain and bacterial cell shapes) Be sure to only measure individual cell size, NOT the size of a cluster of cells. Organize your notes and construct a table to summarize: a) the variety of cell types (cocci, bacilli, spirilli), b) the length of each cell type and c) the Gram-staining properties of each type. Be sure to refer to your lab manual, Appendix IV, page A-12 for instructions on preparing proper tables. Pass this table in before you leave today. B. Fungi Introduction Say “Fungi” and people immediately think of awful, unpleasant things. Fungi are often thought of as sprawling blobs which occupy a very low place in the evolutionary order of things. In fact, the truth is nearly at the opposite extreme. Many fungi are exquisitely constructed and their life cycles are among the most complex to be found any where in nature. There is great diversity in anatomy and life cycles and for this reason the Kingdom Fungi is difficult to define as a taxonomic unit. The current view is that the Kingdom encompasses all heterotrophic organisms with absorptive nutrition. They are saprophytes (living on dead matter) or parasites. The fungi are placed in division Eumycophyta. This division can be divided into four phyla, we will only examine two, the Ascomycetes and the Zygomycetes. The Ascomycetes, the sac fungi, have hyphae (singular: hypha) with crosswalls and each resulting cell has a single nucleus. We will look at Saccharomyces (yeast, a unicellular variety) from this class. The Zygomycetes, the algal fungi, have multinucleated hyphae without crosswalls. From this class we will examine Rhizopus, the black bread mold. Procedure YEAST: 1. Obtain a clean dry slide. Use only a small drop of culture and a VERY SMALL drop of methylene blue. If you put the stain on the slide first, you can wipe away any excess before you add the yeast. Add a small drop of yeast culture and a coverslip. 2. Observe and draw a few typical cells. These cells are alive. They reproduce vegetatively by budding. You will see buds of all sizes, some as big as the parent cell. (Usually only one bud per cell). Dead cells will stain dark blue. Live cells will remain clear. Find an example of budding and include it in your diagram. These cells have organelles inside, but you will not be able to identify them. Refer to Figure 31.7 in your text. 3. When you have completed the drawing of a few yeast cells, put the slide aside while you go on with the exercise. After 20 or 30 minutes, recheck the slide. Do you notice air bubbles that were not there before? Where did they come from? 9-6 Assignment 2: Prepare a labeled drawing of Saccharomyces. Don’t forget to include title, magnification and scale bar. You may wish to refer to Appendix III to review the instructions for preparing biological drawings. Procedure 1. you Rhizopus has been grown on agar in Petri dishes. Examine the colony in the petri dish under the dissecting scope on the demonstration bench. Observe patterns of growth and formations. The hyphae are the network of clear tubular strands. Occasionally will see large upright round expanded ends on some hyphal strands. These are sporangia (sporangium = singular), the spore producing structures. Refer to Figure 31.12 in your text. 2. Prepare a stained whole mount of Rhizopus. Use a clean dry slide. Bread mold is not easy to spread on the slide. Put a SMALL DROP of methylene blue and a drop of water on the slide. Then, using two dissecting needles or toothpicks, remove some of the bread mold from the petri dish and spread the strands apart in the drop of liquid on the slide. Carefully tease the clump in the liquid to immerse it and separate the hyphae. Add cover slip. 3. View your slide under your microscope. Locate an intact hyphal tip and watch for 3-5 minutes, you may see the hyphae growing. 4. Draw a portion of your Rhizopus slide, showing branching pattern and as many structural Assignment 3: Prepare a labeled drawing of Rhizopus. Don’t forget to include title, magnification and scale bar. You may wish to refer to Appendix III to review the instructions for preparing biological drawings. C. ALGAE Introduction Although an extremely diverse and plentiful group, the algae are often overlooked or ignored. They range from small unicellular and simple, to large, multicellular and complex. They come in a variety of colours and can be found living alone, in colonies or in association with others (Paramecium, seaweeds, lichens). Algae are food suppliers, oxygen producers and some are nitrogen fixers. These interesting organisms can stabilize ice cream and toothpaste, kill fish or poison cattle. Algae can be subdivided into several divisions, we will examine two, Cyanobacteria (the called the blue-green algae) and Chlorophyta (green algae). The prokaryotic Cyanobacteria (blue-green algae) lack distinct chloroplasts and the photosynthetic apparatus consists of a system of cytoplasmic membranes. The pigments of phytosynthesis are chlorophyll a, four different carotenoids, several kinds of phycocyanin (blue pigment) and phycoerythrins (red pigment). The range of colours of these algae are due to the different concentration of these pigments. The eukaryotic Chlorophyta (green algae) are the only group of algae which contain the full complement of photosynthetic pigment characteristic of the terrestrial plants. Other algae groups features as yo 9-7 include simpler algal groups such as Golden, Brown, Red Algae and Diatoms. There is little doubt that the green algae and land plants are related. Members of the Chlorophyta exhibit a wide variety of shape, anatomical structures and life cycles. i) Cyanobacteria (Blue-green Algae) Procedure 1. Prepare a wet mount of the cyanobacteria, Anabaena. It is a very narrow uniseriate (one cell wide) filament. It is important to note the colour to facilitate comparisons to the other algal groups that you will examine. The filament is composed of round vegetative cells surrounded by a gelatinous sheath. In cyanobacteria there are no membrane bound organelles. The photosynthetic lamellae float free in the cytoplasm (they are not bound onto chloroplasts) and the DNA is in the form of fine fibrils. Examine Figure 27.10 in the text. 2. Put only one SMALL drop of culture on a dry slide and cover with a cover slip. Too much liquid will cause the cover slip to float and the whole thing will jiggle with every vibration. 3. Use the 4x lens on your microscope and shut the light way down (use the iris diaphragm lever). Search for uniformly thin, dark, fairly straight, hair-like filaments. Center a specimen in the field of view and go to 10x. Increase the light. You should be able to see the outline of the cells and the blue-green colour. 4. Center the specimen in the field of view and go to 40x. Focus carefully through the cells. Use the iris diaphragm lever to vary the light. At low light you should be able to see the transparent gelatinous sheath (it looks like an aura or halo when you adjust the focus). With brighter light, and careful focusing, you can see granular looking inclusions inside the cells, these are gas vacuoles. With brighter light the blue-green pigment is more obvious. Almost all the cells you will see are the photosynthetic small, bead-like vegetative cells. Larger heterocytes (lighter cells with thick cell walls, they are nitrogen fixing cells with very little chlorophyll) and granular oval cells the akinetes (resting cells which contain energy reserves for up to 30 cell divisions and functions to ensure survival through unfavourable conditions) are rare in these cultures. Draw a segment of an Anabaena filament at 400x on your microscope. Do not use oil immersion. (Any movement of the cover slip, such as when the 100x lens contacts the oil droplet, will cause the specimen to move out of the field on view). 5. Assignment 4: Draw a section of an Anabaena filament and label the underlined words as well as the cell wall and cytoplasm. 9-8 ii) Chlorophyta (Green algae) Procedure 1. Prepare a wet mount of Zygnema, a filamentous chlorophyte. Unlike Anabaena, there are membrane bound organelles. Again, note the colour of the cells. This is also a uniseriate filament. 2. Increase the power to 400x and examine one cell. The cells of the filament are short and cylindrical. At either end of the cell there is a large stellate (branching) chloroplast. The nucleus is in the center but not easy to see. Like Anabaena, the filament is surrounded by a gelatinous sheath, which is easiest to see in reduced light. Assignment 5: Draw a section of a Zygnema filament and label the underlined words as well as the cell wall and cytoplasm. Assignment 6: Complete the Assignment Questions that follow. Assignment Questions: 1. Gram-positive and Gram-negative bacteria differ in the nature of their cell walls. Explain why Gram-positive bacteria trap crystal violet stain and appear purple and why Gram-negative bacteria do not and thus appear red. _______________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 2. What physical features of bacteria (macroscopic and microscopic) are used to identify different kinds of bacteria? _____________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 3. What cellular detail is apparent in the yeast? ____________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 9-9 4. What cellular detail is apparent in the mold? _____________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 5. How do the two types of fungi differ? __________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 6. How do the two algae types differ? ____________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 7. Divide the organisms you observed into prokaryotes and eukaryotes and explain your division. Prokaryotes: _________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ Eukaryotes: _________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________