lab 5.pdf

Lab 4: Cells Learning Outcomes Addressed in this Lab: LO-1: Describe the characteristics of life. LO-4: Compare and contrast the structures, reproduction, and characteristics of viruses, prokaryotic cells, and eukaryotic cells. LO-9: Describe the unity and diversity of life and the evidence for evolution through natural selection. LO-11: Apply scientific reasoning to investigate questions and utilize scientific tools such as microscopes and laboratory equipment to collect and analyze data. Objectives  Differentiate between structures and appearance of prokaryotic versus eukaryotic cells.  Compare and contrast structural characteristics in a variety of cells types through visual observation using light microscopes, including bacteria, plants and animals.  Estimate size of a variety of cell types and identify key differences. REMINDER: Don’t forget to read this entire lab and complete the pre-lab assignment BEFORE the start of lab (as directed by your instructor either on paper or online). Background Information Cells are considered the basic unit of living organisms because all living organisms are composed of cells, and cells perform all of the processes we collectively call “life”. Cell theory incorporates three basic principles: A) All organisms are composed of one or more cells; B) the cell is the basic living unit of organization; and C) all cells arise from preexisting cells. Although most individual cells are visible only with the aid of a microscope, some may be a meter long (e.g. nerve cells) or as large as a small orange (e.g. due to the yolk of an ostrich egg). Despite these differences, all cells are designed similarly and share fundamental features. All cells share four fundamental structural features: 1) All cells have a plasma membrane defining the boundary of the living material 2) All cells contain DNA (deoxyribonucleic acid), which stores genetic information 3) All cells contain cytosol (the thick fluid that is contained by the plasma membrane as part of their cytoplasm (which refers to everything inside the plasma membrane that is not part of the DNA region) 4) All cells contain ribosomes, the protein and RNA-based complexes that produce proteins that allow the cell to function and maintain life. With respect to internal organization, there are two basic types of cells, prokaryotic and eukaryotic. The Greek word karyon means “kernel” which refers to the nucleus. Thus, prokaryotic means before a nucleus and, eukaryotic indicates the presence of a true nucleus. Organisms with prokaryotic cells fall under two domains, the Archaeans and Bacteria. A subgroup of bacteria called cyanobacteria (also called blue-green algae) are capable of photosynthesis despite not having a nucleus or organelles like chloroplasts. Prokaryotic cells are believed to be most similar to the first cells, which arose on Earth 3.5 billion years ago. Organisms with eukaryotic cells include the protists, fungi, plants and animals. All of these cells contain a true nucleus to protect their DNA by enclosing it inside a phospholipid membrane barrier, as well as a variety of other organelles. It is believed that eukaryotic organisms arose from prokaryotic ancestors. BIOL-1406 Lab Manual Page 34 of 118 Cytology is the study of cellular structure and function. The major tools of cytologists are light microscopy, electron microscopy, and cell chemistry. By studying the anatomy of a cell, we can find clues to how the cell works. In today’s lab, you will study some of the features and variations among living cells to understand life processes of organisms. Activity 4-1 Preparing a wet mount slide In many instances you will be asked to prepare a fresh slide of a biological specimen. Some of these will be liquid (e.g. a slide of a blood sample), some may be solid and liquid (e.g. a piece of onion skin with a few drops of a dye). This first activity will guide you through preparing a wet mount using the first cell type you will view (so the prepared wet mount slide from this activity will be used in Activity 4-2). Figure 4-1. Preparing a wet-mount slide 1) To prepare a wet mount, start by obtaining a clean and dry microscope slide. 2) Place a droplet of sample directly onto the center of the slide. In this case, obtain a drop of Gleocapsa (focus on capturing at least some visible green stuff from the liquid in your drop). 3) Open the coverslip box and carefully separate out ONE clean coverslip. IMPORTANT: Coverslips stick together easily and more than one will make focusing difficult, so check to ensure you do not see a “rainbow” when you look at the coverslip as you angle it back and forth – this indicates 2 are stuck together. Slide the two slips to separate them. 4) Place the edge of the coverslip at an angle just next to the drop of liquid on your slide (see Fig. 4-1). Slowly lower the coverslip until it falls onto the drop. Try to trap as few air bubbles as possible. 5) If your coverslip seems “loose” or wobbly, there may be too much liquid. Remove excess liquid by touching a “Kimwipe” to the edge of the coverslip – water will quickly wick out, so do not touch for too long or you will dry out your sample. 6) For every slide you make, always follow the procedures learned back in Lab 2 to focus and view a sample with your microscope. For this sample, read on and follow the instructions in Activity 4-2 for what to look for on this slide. BIOL-1406 Lab Manual Page 35 of 118 Prokaryotic Cells Domain Archaea: Archaeans are prokaryotes found in extreme environments (e.g. near volcanoes, sea- vents, and salty seashores). They will not be studied in this lab. Domain Bacteria: Consists of the common bacteria and the cyanobacteria (also called blue-green algae). Review the generalized structure of bacteria by identifying the structures in the cell shown below. A basic diagram on the elements conforming a prokaryote cell. in this case a bacteria cell. The image is in public domain. Prokaryotes do not contain a membrane-bound nucleus or any other membrane-bound organelles. Organelles are organized structures of macromolecules having a specialized function and are suspended in the cytoplasm. The cytoplasm of prokaryotes is enclosed in a plasma membrane (cellular membrane) and is surrounded by a supporting cell wall covered by a gelatinous capsule. Flagella and hair-like outgrowths call pili are common in prokaryotes. Within the cytoplasm of prokaryotes are ribosomes (small particles involved in protein synthesis), mesosomes (internal extensions of the plasma membrane), and chromatin bodies (concentrations of DNA). Prokaryotes do not reproduce sexually, but they have mechanisms for genetic recombination. Cyanobacteria The largest prokaryotes are cyanobacteria, also called blue-green algae. They contain chlorophyll a and accessory pigments for photosynthesis, but these pigments are not contained in membrane-bound chloroplasts. Instead, the pigments are held in photosynthetic membranes called thylakoids. Cyanobacteria are often surrounded by a mucilaginous sheath. Activity 4-2: Cyanobacteria 1. Place the wet-mount of Gleocapsa that you prepared in Activity 4-1 onto the stage. Gleocapsa are cyanobacteria that live in colonies (loosely attached clusters). 2. Focus beginning with the 4X scanning objective as usual, looking for something green to know you’re seeing the actual Gleocapsa cells. When you see something green, adjust the fine focus up and down until you feel confident you are seeing tiny clumps of rounded green cells. 3. Work your way up in magnification from 4X Scanning to 10X objective and then to 40X – focusing one or more clusters of cells and centering them each time before you change objectives. Remember to use the iris diaphragm slider to adjust the light as you change magnification. BIOL-1406 Lab Manual Page 36 of 118 4. Observe the cellular structures and draw the cellular shapes and relative sizes of cells as you see them in the FOV (field of view) with the 40X Objective. Concentration on making the cells appear below at the same size they appear relative to the entire FOV (don’t make them larger than they look in the microscope). 5. Use your drawings and what you see in the microscope to estimate the size of a single Gleocapsa cell and mark the approximate size in response to the question below (using the FOV diameter comparison technique learned in Lab 2), then clean your slide and dry it with a Kim Wipe to prepare for the next activity. Gleocapsa Cells under 40X Objective: QUESTIONS 1. About how large is one Gleocapsa cell? a. 5 to 50 micrometers b. 50-250 micrometers c. 250-500 micrometers d. 500-1000 micrometers (which is 0.5 to 1.0 millimeters) 2. How do cyanobacteria do photosynthesis without any chloroplasts? (hint: look back at the introduction!) Eukaryotic Cells Domain Eukarya: Organisms that do contain nuclei. Includes plants, animals, fungi and protists. Eukaryotic cells contain membrane-bound nuclei (plural, nucleus = singular) and other organelles. Nuclei contain genetic material of a cell and control metabolism. Cytoplasm forms the matrix of the cell and is contained by the plasma membrane. Within the cytoplasm are a variety of organelles. Chloroplasts are elliptical green organelles in plant cells. Chloroplasts are the site of photosynthesis in plant cells and are green because they contain chlorophyll, a photosynthetic pigment capable of capturing light energy. Mitochondria are organelles found in plant and animal cells. These organelles are where aerobic respiration occurs. When viewed with a conventional light microscope, mitochondria are small, dark, and often difficult to see. Eukaryotic cells are structurally more complex than prokaryotic cells. Although some features of prokaryotic cells are in eukaryotic cells (e.g. ribosomes, cell membrane), eukaryotic cells also contain several organelles not found in prokaryotic cells. In the following exercise, you will investigate some of these organelles. BIOL-1406 Lab Manual Page 37 of 118 Activity 4-3: Structure of Plant Cells Examine living Elodea cells and chloroplasts. Elodea is a common pond weed used frequently in studies of photosynthesis, cellular structure, and cytoplasmic streaming. 1. Remove a young leaf from the tip of a sprig of Elodea. Try to avoid any stem or thick veins. 2. Place a fresh drop of water on a microscope slide and place your leaf into the water with the top surface facing up. The cells on the upper surface are larger and more easily examined. 3. Add a coverslip following wet mount procedure. If you need to remove a bit of water, be careful to avoid letting the leaf dry out. If your leaf begins to dry out, ask the instructor for help rehydrating it. 4. Examine the leaf with your compound microscope following normal procedure. You may notice that the leaf is composed of several layers, so choose the layer with the largest number of chloroplasts (the small, green ovals inside the boxy cells). 5. When you reach the 40X Objective, adjust the stage until you are looking at an area where you can easily discern the individual cells of one layer of the leaf. Take time to observe the following structures: a. Cell Walls: Each of the small, regularly shaped units you see are cells surrounded by cell walls - a feature that distinguishes plant cells from animal cells. Cell walls are made primarily of cellulose. Cellulose is a complex carbohydrate made of glucose molecules attached end- to-end. The plasma membrane lies just inside the cell wall and may not be visible. b. Chloroplasts: Small, green ovals floating in the cytoplasm are chloroplasts. There should be at least 5-10 in each of the cells, often as many as 50. c. Central Vacuole: The cells should have an area in the middle where none of the chloroplasts seem to be going – this is usually the large, central vacuole. d. Nucleus: You may not be able to see the nucleus itself because we have not stained it, but try to look for evidence of another, smaller area near one side where the chloroplasts are not streaming into as they move around. 6. Draw everything you see in the current FOV (feel free to move around to ensure you can draw less, but don’t ignore things you see in your drawing), then again estimate the size of these cells using the technique learned in Lab 2. Elodea Cells under 40X Objective: QUESTIONS 1. About how large is one Elodea cell? a. 5 to 50 micrometers b. 50-250 micrometers c. 250-500 micrometers d. 500-1000 micrometers (which is 0.5 to 1.0 millimeters) BIOL-1406 Lab Manual Page 38 of 118 2. How do the Gleocapsa compare to the chloroplasts in the Elodea cells? How does their appearance relate to the Endosymbiotic Theory discussed in your textbook? Activity 4-4: Plastids Plastids are specialized organelles in plants and other photosynthetic eukaryotes where food is made and stored. You have already examined chloroplasts, a type of plastid in which photosynthesis occurs. Other plastids have different functions. Next, we will examine a type of plastid called an amyloplast, which stores starch. In many plants, amyloplasts take up the vast majority of the cell volume in the food storage sections of their root system, such as in potatoes or carrots. As we learned in the chemistry and life lab, starch stains a dark blue-black when mixed with iodine (IKI solution). In today’s lab, we’ll discover that blue-black is actually a dark purple! 1. Prepare a wet mount of a thin section of potato: a. Use a razor blade to slice as thin a section of potato as you can. b. Now, make this slice even smaller and thinner by cutting away 90% of what you thought was a small, thin slide. Remember that we’re using a microscope to view the cells, not trying to observe the potato with our eyes, so we need very, very little on our slide. Ask your instructor to check if your slice is thin enough before moving on. c. Place a drop of IKI solution on the slide and carefully place your tiny potato slice into the liquid. d. Add a coverslip carefully – wick away any excess liquid only if needed (do not dry it out!) 2. Follow the standard focusing process to work your way up in magnification. When you have the cells in focus with the 10x objective, try to locate the small, clam-shaped amyloplasts within the cells. a. Cell Walls: in these cells, the cell walls will be grayish lines and the cells may be more polygon shaped rather than rectangles or squares b. Amyloplasts: these will make up most of the cell volume and ask the iodine enters the cells, they will turn bright purple. 3. Draw everything you see in the current FOV (feel free to move around to ensure you can draw less, but don’t ignore things you see in your drawing), then again estimate the size of these cells using the technique learned in Lab 2. Stained Potato Cells under 40X Objective: QUESTIONS 1. About how large is one amyloplast inside the potato cell? a. 5 to 50 micrometers b. 50-250 micrometers c. 250-500 micrometers d. 500-1000 micrometers (which is 0.5 to 1.0 millimeters) BIOL-1406 Lab Manual Page 39 of 118 2. About how large is one whole potato cell? a. 5 to 50 micrometers b. 50-250 micrometers c. 250-500 micrometers d. 500-1000 micrometers (which is 0.5 to 1.0 millimeters) 3. Do you see any chloroplasts in these potato cells? Why do you think that is so? Activity 4-5: Human Epithelial Cells Human epithelial cells are regularly sloughed off of the inner surface of your mouth. They are flat cells with a readily visible nucleus, especially after staining with a dye that sticks to nucleic acids. NOTE: After viewing the human epithelial cell slides, dispose of them in the bleach solution on the side counter. Do NOT remove the cover slip or attempt to wash this slide – place the entire slide into the bleach solution whole. *This is the ONLY slide that should be disposed in the bleach solution today! Examine human epithelial cells by following the directions below: 1. Obtain a clean, dry slide and place a very small drop of methylene blue in the center. If you think the drop is too big, wick away some with a Kimwipe now, before it is mixed with human tissue and becomes biohazard waste. 2. Now, wash your hands thoroughly, then obtain a clean toothpick and gently scrape the inside of your cheek with the broad, flat end of a toothpick. You should feel it, but it should NOT hurt. 3. Stir the scrapings into the very small drop of blue dye on the microscope slide and then dispose of the toothpick in the provided bleach solution. 4. Carefully add a coverslip using wet mount techniques and verify that you don’t have too much liquid under your coverslip before continuing. If you do, wick away a tiny amount carefully, but dispose of this Kimwipe in the biohazard trash because human tissue could be on it. 5. Now, follow standard techniques to focus on the cheek cells with your compound light microscope. Your instructor will provide guidance on what to look for as you begin. Here are the structures to locate: a. Plasma Membrane: animal cells do not have cell walls, so notice the varying shapes and rounded edges of these human epithelial cells and see if you can discern the phospholipid barrier separating cell from outside. b. Nucleus: the nucleus will absorb most of the methylene blue stain that sticks to nucleic acids (DNA and RNA), so you should see one in each cheek cell as a round, relatively dark staining center. 4. On the next page, draw everything you see in the current FOV (feel free to move around to ensure you can draw less, but don’t ignore things you see in your drawing), then again estimate the size of these cells using the technique learned in Lab 2. BIOL-1406 Lab Manual Page 40 of 118 Epithelial Cells under 40X Objective: QUESTIONS 1. About how large is one epithelial cheek cell? a. 5 to 50 micrometers b. 50-250 micrometers c. 250-500 micrometers d. 500-1000 micrometers (which is 0.5 to 1.0 millimeters) 2. How do the size and shape of human epithelial cells differ from those of the Elodea and onion cells that you examined earlier? Activity 4-6: Protists Amoeba and Paramecium are members of the multi-kingdom group called protists. This group has a wide variety of primarily microscopic organisms that includes algae and sporozoans. Today you will examine Amoeba and Paramecia, both of which are single-celled (or “unicellular”) organisms. Amoeba are irregularly shaped protists with many internal organelles. Amoeba move via amoeboid movement. Amoeboid movement occurs by means of pseudopodia, which are temporary protrusions of the cell. Pseudopodia also surround food particles and create food vacuoles, where food is digested. Another important structure in Amoeba is the contractile vacuole that accumulates and expels water and waste products. 1. Examine an Amoeba by obtaining and viewing one of the prepared slides. Choose the best objective for this organism as it may be larger than the cells you have previously viewed. 2. Draw everything you see in the current FOV (feel free to move around to ensure you can draw less, but don’t ignore things you see in your drawing), then again estimate the size of these cells using the technique learned in Lab 2. Amoeba Cells under _______ Objective: BIOL-1406 Lab Manual Page 41 of 118 QUESTIONS 1. About how large is one Amoeba cell? a. 5 to 50 micrometers b. 50-250 micrometers c. 250-500 micrometers d. 500-1000 micrometers (which is 0.5 to 1.0 millimeters) 2. Does an Amoeba have a cell wall? How can you tell? Like Amoeba, Paramecia are also single-celled protists (Paramecia = plural, Paramecium = singular). On the surface of each Paramecium are cilia, which are short hair-like structures used for locomotion. Examine Paramecia by looking at the live cultures set up for you on a light microscope. 1. How does movement of Paramecium compare to that of Amoeba? Are they faster or slower moving? Why do you think that is so? Activity 4-7: Fungi (time permitting) Fungi are among the most common and most important groups of organisms. Fungi are basically filamentous strands of cells that secrete enzymes and feed on the organic material on which they are growing. That organic material may be humus in the soil where mushrooms grow or a stale loaf of bread where mold thrives. It may be the skin between your toes inhabited by athlete’s foot fungus or decaying animal on the forest floor being decomposed by fungi digesting the animal’s dead tissue. Fungi not only cause disease; they are also important decomposers that recycle nutrients from dead organisms. The basic structure of most fungal cells is the hypha (pl., hyphae) – a slender filament of cytoplasm and nuclei enclosed by a cell wall. A mass of these hyphae make up an individual organism and is collectively called a mycelium. A mycelium can permeate soil, water, or living tissue; fungi certainly seem to grow everywhere. In all cases, the hyphae of a fungus secrete enzymes for extracellular digestion of the organic substrate. Then the mycelium and its hyphae absorb the digested nutrients. For this reason, fungi are called absorptive heterotrophs. Examining Fungal Hyphae Examine the provided slide of the mold Penicillium, which is a mold that also served an important role in helping scientists develop the first antibiotics. What unusual structures do you see? BIOL-1406 Lab Manual Page 42 of 118 Examining Yeast, a single-celled fungus Now prepare a wet mount of another kind of fungus, yeast. These are single-celled organisms that reproduce by budding and are commonly used in production of ethanol and as a rising agent in bread. 1. Place a drop of the yeast suspension on a clean, dry slide 2. Use the standard techniques to focus and observe the yeast cells under the compound microscope. 3. Look for nuclei and small glistening food granules. Do any of your yeast cells exhibit small, rounded projections called buds? Your instructor may have you stain the yeast sample with Congo Red. QUESTION: What differences do you notice in the shape and size of the Penicillium and yeast cells? END OF LAB REVIEW 1. Complete Table 4-1 to review the structures in different cell types. Table 4-1 Comparison of prokaryotic and eukaryotic cells. Complete the table to identify the presence/absence of the structures listed. Write YES, NO or SOMETIMES in each box. Cellular Structure Prokaryote: Eukaryote: Eukaryote: Eukaryote: Eukaryote: Bacteria Animals Plants Fungi Protists Cell Wall (Exterior Structure) Plasma Membrane (Exterior Border) Endoplasmic Reticulum Ribosomes Golgi Bodies Nucleus Mitochondria Chloroplasts DNA BIOL-1406 Lab Manual Page 43 of 118 2. Four different cell types (Cell X, Cell Y, Cell Z and Cell W) were analyzed under a microscope to determine which structures were present. Table 4-2 summarizes the findings. Use the information provided in the table to answer the questions and identify the cell types. Table 4-2. (Yes = the structure was present), (No = the structure was absent) Structural Feature Cell X Cell Y Cell Z Cell W Cell Wall Yes Yes No No Nucleus No Yes Yes Yes Chloroplasts No Yes No No Mitochondria No Yes Yes Yes Unicellular Yes No No Yes 1. Which of the cells does the data indicate could be a bacterium? How do you know? 2. Which of the cells does the data indicate could be an Amoeba? How do you know? 3. Which of the cells does the data indicate could be a plant cell? How do you know? 4. Which of the cells does the data indicate could be an animal cell? How do you know? References: 1. Perry et. al.: Laboratory Manual for Starr and Taggart’s Biology: The Unity and Diversity of Life: 2002, Brooks/Cole 2. Vodopich & Moore: Biology Laboratory Manual, 6th edition, 2002, McGraw Hill BIOL-1406 Lab Manual Page 44 of 118

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