CELLS: OBSERVING THE MICROSCOPIC WORLD Lab 7

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1 CELLS: OBSERVING THE MICROSCOPIC WORLD Lab 7 The living cell is to biology what the electron and proton are to physics. Apart from cells and from aggregates of cells there are no biological phenomena. Alfred North Whitehead Science and the Modern World (1925) CAUTIONS AND PITFALLS Please treat the microscopes with care! Use Kimwipes or lens paper to clean lenses. Clean oil from oil immersion lens, stage and slide with alcohol. Return slides to proper storage boxes and click the low power objective into place when finished with your microscope. OBJECTIVES After completing this lab, you should be able to: 1. Identify the parts of a compound light microscope and be proficient with the function and use of each. 2. Describe the procedures used in preparing materials for electron microscopy. 3. Describe the features of specific cells and determine characteristics shared by all. 4. Discuss increasing cell complexity and organization in unicellular and multicellular organisms by observing representative cells and tissues from all five kingdoms. INTRODUCTION According to the cell theory, the cell is the fundamental biological unit, the smallest and simplest structure possessing all of the characteristics of life. In Lab 2 we looked at unity and diversity from an organismal level, in this laboratory activity we will take a cellular approach and focus on similarities and differences between cell types. You will have an opportunity to observe representative cells from each of the different kingdoms. The earliest known cells, found to date, are unicellular prokaryotic cells identified in fossilized sediment about 3.5 to 3.8 billion years old. There is no evidence of eukaryotic cells in the fossil record for another 2 billion years. This suggests that it took 2 billion years for complex eukaryotic cells to evolve from simple prokaryotic cells. The endosymbiotic theory, first proposed by Lynn Margulis, suggests that larger cells became more complex by ingesting smaller cells (see Campbell, Chapter 20). The major evolutionary advantage evident in eukaryotic cells is the elaborate system of membranes and membrane-bound organelles. These endomembranes of eukaryotic cells separate the genetic material from the rest of the cell, compartmentalize specific metabolic functions, and increase the surface-area of these larger, more complex cells.

2 The diversity of form and function in the cellular world parallels the diversity of species in the biosphere. Both free-living and colonial cell types can be observed in the Kingdom Monera and Protista. Many fungal cells are multinucleated and interconnected. Many plant and animal cells are immovably fixed as part of multicellular tissues. The morphology (structure) of tissue cells is highly correlated with their function. In addition to structural and organizational differences, variations in cell size also contribute to the diversity of cell types, to a limit. All cells are destined to be small in order to maintain efficiency. It is essential that each cell be able to effectively take in nutrients and eliminate wastes in order to carry on metabolism. The smaller the cell the greater the surface area/volume ratio and the more efficient the movement of essential molecules into, out of, or within the cell. Therefore, most cells range from micrometers. Exceptions include many bacteria that are closer to 1 micrometer, egg cells that are typically larger than 100 µm, and human nerve cells that may be as long as 3 feet. Cytologists have been studying cells for more than three centuries using one of the most fundamental tools of biology, the microscope. Since its development in the 17th century, modern microscopy has attained sophistication far beyond the wildest dreams of Anton van Leeuwenhoek and Robert Hooke. During this exercise, we will use a compound light microscope (bright-field) to examine cells from all five kingdoms, broadening your familiarity with the diversity of life on earth. MICROSCOPY Over the years several kinds of microscopes have been developed, each adapted for making certain observations. Fill in the following table comparing some of the different kinds of microscopes in use today. Table 7.1 Comparison of Microscope Types and Uses Type Special features Use Phase-contrast Fluorescent microscopy Transmission electron (TEM) Scanning electron (SEM) Scanning tunneling microscope (STM) Contrasts brightness in transparent and unstained cells. Uses flourescent dyes or antibodies to tag specific molecules. Resembles an inverted light microscope. Electrons rather than light pass through the specimen. Beam of electrons scan surface of specially prepared specimen. Computer controlled micro-probe scans the surface of the specimen. Makes structures in living cells clearly visible without stain. Allows visualization of special cell structures especially the cytoskeleton. 200x fold better resolution than a light microscope. Allows visualization of cell ultrastructure. Three dimensional image of cell surface or isolated cell structures. Allows visualization of molecules and atoms.

3 TECHNIQUES IN CYTOLOGY The microscopic study of cells is not without its limitations. Most microscopes require that specimens be dead. In addition, electron microscopy requires that cells be dehydrated due to the vacuum that is necessary to direct beam of electrons. The cells must also be very thin (0.1µm thick or less) since electrons have very low penetrating power through solid material. In contrast, a beam of visible light can penetrate objects 5 to 15 µm thick. In any case, it is necessary to specially prepare an object (specimen) for microscopic examination. Sectioning. A common preparation technique is to section (cut) a specimen with a microtome, an instrument similar to a butcher's meat cutter. Sections are cut with steel knives for light microscopy, but extremely sharp glass or diamond knives are necessary to produce the thin sections required for electron microscopy. Fixing. A specimen is prepared for sectioning by placing it in a solution that kills and preserves it (fixing). The specimen is then dehydrated to remove water and embedded in a material (paraffin or plastic) that can be sectioned easily. Staining. Thin, fixed specimens are fairly transparent and offer little internal contrast for visualization of organelles. Stains are used to distinguish the various structures. Stains used in light microscopy are usually dyes that impart color to specific structures based on the affinity of that stain for macromolecules present in the specimen. Therefore, a different stain would be used depending on if you were interested in studying chromosomes or mitochondria. To enhance contrast in electron microscopy, various metals (usually gold) are used to decrease the ability of electrons to penetrate the specimen. The optical system and type of penetrating beam also influence the degree to which smaller and smaller objects can be magnified and resolved. Human Red Blood Cell 7 µm Chloroplast 5 µm E. coli 2 µm or 2000 nm Adenovirus 300 nm Figure 7.1 Comparison of cell size. Bacteriophage 100 nm

4 ACTIVITY ONE 1. Obtain a compound light microscope. 2. Locate the following parts of your microscope: ocular, revolving nosepiece, objectives, mechanical stage, stage clips, course adjustment knob, fine adjustment knob, condenser, iris diaphragm. 3. Determine the total magnification for each objective lens. Name Ocular Objective Total magnification SCANNING LOW POWER HIGH POWER OIL IMMERSION 4. Discuss and define the following terms before proceeding: field of view- parfocal- working distance- depth of focus - resolution- 5. Make a wet mount of a single Elodea leaf. 6. Place your Elodea slide against the far edge of the slide holder by pinching together the stage clips. If you are unfamiliar with this type of stage clip ask your instructor.

5 HINTS FOR FOCUSING 1. Click the low power(10x) objective into place. 2. Raise the condenser to the highest position. 3. Bring the objective and stage as close together as possible. 4. Center the specimen. 5. Look through ocular, lower the condenser until the graininess disappears. 6. Use the course adjustment knob to slowly focus upward. 7. Sharpen with fine adjustment knob. 8. Adjust diaphragm for proper light. ACTIVITY TWO You will be observing prepared slides and living specimens using the compound light microscope. Cells and tissues representing each of the five kingdoms of living organisms are available. Your lab report should include microscopic drawings and answers to the questions found at the end of the lab. Your drawings should be complete with titles, magnification and labels. Draw the cells you observe at a magnification that best represents the cell or organism. It is always helpful to use your book, lab manual, or other reference material while making observations and writing up your lab report. MICROSCOPIC DRAWINGS Although few of us are artists, drawing the specimens you view will help to focus your observations and reinforce your learning. A few simple rules provide consistency and clarity to microscopic drawings. 1. Use a round object to trace a field of view. 2. Place the title above and magnification below. 3. Use straight lines extending outside the field of view for labels (no crossing lines). 4. Print title and labels using neat, uppercase, block style print.

6 STENTOR Vacuole Cilia 400x Figure 7.7 Drawing from microscope with correct labels. MONERA - Bacteria The Danish bacteriologist Christin Gram developed a diagnostic staining technique which is used to separate bacteria into two groups: Gram-positive (violet) and Gram-negative (pink). The Gram stain, which is based on the different structure and composition of bacterial cell walls, is one of the most important techniques used to classify bacteria. Gram staining is important because it correlates with the sensitivity of a bacterium to antibiotics. Gram-positive bacteria have a thick cell wall that retains a purple dye, whereas Gram-negative bacteria have a much thinner cell wall that does not retain the dye. The identification of thousands of different types of bacteria is based on this diagnostic test in combination with other traits. Obtain a prepared slide of mixed types of bacteria. Observe with high power or oil immersion. (Your instructor will explain how to use an oil immersion objective). The slide should contain both Gram-positive and Gram-negative bacteria and three shapes of bacterial cells. Cocci (spheres), bacilli (rods) and spirilla (comma or corkscrew-shaped bacteria). Because of their small size, it is impossible to see detail inside bacterial cells with the light microscope, consult your textbook for transmission electron micrographs of bacterial and cyanobacterial cells. Draw and label the bacterial types you observe.

7 EXTRA! EXTRA! Yogurt is made by adding Lactobacillus sp. and Streptococcus thermophilous to milk and allowing the bacteria to anaerobically metabolize milk sugar. The lactic acid produced as a by product of this fermentation process lowers the ph of the solution which curdles the milk by denaturing milk proteins. Take a small amount of yogurt and mix it on a slide with a drop of water. Add a coverslip and observe the slide through your microscope. What are the shapes of the bacteria in the yogurt? MONERA - Cyanobacteria (Blue-Green Algae) Like bacteria, cyanobacteria are prokaryotes that grow in many types of environments. They make rocks at the edge of a pond slippery and grow on the water as part of pond scum. Only about half the cyanobacteria are actually blue-green in color - others range in color from brown to olive green. They contain chlorophyll but no chloroplasts. Photosynthesis takes place in extended folds of the plasma membrane. Because cyanobacteria are prokaryotes they are not related to other algae which are eukaryotic. They form colonies and filaments and are often cause many of the disagreeable tastes, colors, and odors in water. Oscillatoria is a filamentous blue-green algae that was named for its tendency to oscillate gently in water. PROTISTA Most of the members of the kingdom Protista are unicellular but differ significantly from the Monerans in size and complexity. These eukaryotic cells contain a nucleus and several membrane bound organelles. Protista is the oldest, most diverse, and most difficult to characterize of the four kingdoms of eukaryotes. For the most part, this kingdom includes all eukaryotes that lack the distinguishing characteristics of plants, animals, and fungi. The kingdom includes simple animal-like organisms such as Amoeba and Paramecium and some unusual multicellular forms such as the brown alga (kelp). The major groups of Kingdom Protista are algae and protozoans, there are also a few fungal-like slime molds. You will be using a dichotomous key to hunt for and identify several different members of this group of organisms. FUNGI Fungi are fairly simple multicellular eukaryotes that differ significantly from plants in several important ways. The cell wall is not composed of cellulose, but is made of chitin, the same polysaccharide in the exoskeleton of insects and crustaceans. Through this chitinous cell wall a fungus secretes enzymes for extracellular digestion. Most fungi obtain nourishment from dead organic matter and are called saprophytes. Other fungi feed on living organisms and are parasites. As you examine prepared slides of fungi you should be able to identify three basic structures: 1) Hyphae - slender filaments of protoplasm enclosed by a cell wall. Hyphae of some species have incomplete crosswalls between cells and are therefore multinucleated. 2) Mycelium - a collective term for a cotton-like mass of hyphae constituting an individual organism. Fungi sexually reproduce via conjugation. Conjugation occurs when hyphae of two strains grow together allowing for fusion of haploid nuclei to form a zygote as seen in Rhizopus (bread mold). 3) Sporangia - reproductive hyphae where spores are produced.

8 PLANTS This kingdom includes a remarkably diverse group of multicellular organisms that are autotrophic, contain chlorophyll and have cell walls of cellulose. In future courses you will study the unique life cycle of various species involving an alternation of generations. The major groups of plants include mosses, ferns, gymnosperms and angiosperms. Significant evolutionary changes include adaptations for survival on land and the development of specialized tissues. Refer to your textbook to identify vasculartissue such as xylem for conducting water and phloem by the plant to carry food. Label and distinguish between the arrangement of this vascular tissue in monocot versus dicot stems of angiosperms (flowering plants). Identify and label the specialized tissues of a leaf. ANIMALS The diversity of organisms in the animal kingdom ranges from multicellular sponges, jellyfish, and corals to birds and mammals. Cells with similar structure and function constitute tissues, such as muscle, skin or blood. The most distinguishing features of animal cells are the lack of cell walls and their high degree of specialization. Refer to your textbook to learn more about how structure fits function within the various cells and tissues you observe. Most well known animals are macroscopic but there are many microscopic species. Hydra is a freshwater cnidarian whose body plan shows radial symmetry. The epidermal cells in the tentacles contain nematocysts capable of harpooning prey. Adults reproduce asexually by budding. Rotifers, sometimes referred to as natures water purifiers are multicellular animals that beat organic matter into their digestive system with tufts of cilia visible at their anterior end.

9 A C E B D F G H I Figure 7.10 Assorted Protists

10 MICROSCOPIC OBSERVATIONS I. KINGDOM MONERA Choose one from each group (A and B) to draw and label. A. Bacteria (oil immersion useful) B. Cyanobacteria (no oil immersion needed) 1. Staphylococci in chains 4. Anabeana (blue-green) 2. Spirillim volutans 5. Oscillatoria 3. Typical mixed bacteria II. KINGDOM PROTISTA Choose one from each group (A and B) to draw and label. Work with a partner to identify and classify all protists found in Carolina Biological Survey Mixture (plant-like protists) and Carolina Biological Protozoan Mixture (animal-like protists). A. Animal-like Protozoans B. Plant-like Protists 1. Paramecium caudatu 7. Euglena 2. Amoeba proteus 8. Closterium (desmid) 3. Chilomonas 9. Synedra (diatom) 4. Stentor 10. Volvox (green algae) 5. Peranema 11. Ulithrix (green algae) 6. Vortecella 12. Pediastrum (green algae) 7. Trypanosoma (prepared slide) 13. Oedoganium (green algae) 14. Scenedesmus (green algae) III. KINGDOM FUNGI Choose one to draw and label 1. Penicillium 2. Rhizopus (conjugation) 3. Punccinia (wheat rust) IV. KINGDOM PLANTAE Choose one to draw and label 1. Zea mays stem, c.s. (monocot stem) 4. Oleander leaf, c.s. 2. Typical dicot stem, c.s. 5. Elodea leaf (wet mount) 3. Tilia stem, c.s. (woody dicot) V. KINGDOM ANIMALIA Choose one tissue and one organism to draw and label A. Tissues B. Multicellular organisms 1. blood smear (Wright stain) 4. rotifer 2. simple squamous epithelium 5. hydra

11 3. muscle tissue (3 types) QUESTIONS TO ACCOMPANY MICROSCOPIC DRAWINGS 1. List the structural differences between prokaryotic and eukaryotic cells. Place a check mark next to the differences you were able to observe using a light microscope. Why couldn t you observe the other structures? 2. Based on your observations and reading, what similarities exist between prokaryotic cells and the organelles of eukaryotic cells? 3. List how algal protists differ from protozoans. 4. List how plant cells differ from fungal cells? 5. What criteria would you use to distinguish colonial or filamentous organisms (such as algae) from truly multicellular organisms? 6. Name the tissues you observed, remember to consider all kingdoms. 7. Name the unicellular organisms you observed, again consider all kingdoms? 8. Would you classify the Oleander leaf as a tissue or an organ? Why? 9. What are the advantages and disadvantages to an individual cell of being part of a multicellular organism? REFERENCES Abamoff P. and Robert Thomson Laboratory Studies in Biology-V. New York: W.H. Freeman and Company. Latta, V.G. et al Principles of Biology: Laboratory Investigations, 3rd Edition. Hunter Textbooks Inc. Bowen, William R Experimental Cell Biology: An Elementary Laboratory Guide. New York: The Macmillan Company.