biology 101 Applications of the Scientif ic Method Laboratory Guide and Workbook Ohio University, Fall 2013
Prepared for the Department of Environmental and Plant Biology Ivan K. Smith & Zachary Rinkes
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Ta B L e O f c O N T e N T S v
Why Study Biology?
laboratory 1 Communicable Diseases and Microorganisms
laboratory 2 Classification of Biological Organisms
laboratory 3 Cell Structure of Biological Organisms
laboratory 4 Membrane Structure and Function
laboratory 5 Enzymes
laboratory 6 Photosynthesis
laboratory 7 Mitosis
ApplicAtions of the scientific Method
Ta b l e o f C o n t e n t s (C ON T I N U E D) 71 Laboratory 8
Mendelian Genetics and Evolution
79 Laboratory 9
Deoxyribonucleic Acid (DNA)
89 Appendix Supplies
Applications of the scientific MEthod
P r e fa c e The subtitle of this laboratory manual is “Applications of the Scientific Method.” Obviously, a two-hour laboratory period cannot embrace all steps in the scientific method. Therefore as you perform the exercises think about which aspect of the scientific method is the primary focus of a particular lab: •
Are you observing and describing organisms, cells, or events?
Are you developing a hypothesis and designing an experiment to test its predictions?
Are you performing an experiment that supports a well-established theory?
Our primary goal is to give you a hands-on experience with some of the methods that are commonly used by biologists to investigate biological problems. Each laboratory exercise begins with the Learning Objectives. Our expectation is that you will meet these objectives as a result of performing the lab. This is followed by a Background section that provides you with the necessary information to understand the lab, even when the subject has not been covered by your lecture instructor. Words or phrases that are bolded in this section and elsewhere are important terms or concepts that may be included on lab quizzes. The Methods section outlines how you are to perform an exercise. However, pay particular attention to your lab instructor’s introduction, because methods evolve as we learn from your experiences with each lab. The Results section includes the outcome of an exercise, which may be a drawing or data presented in a table or graph. Finally, you are asked Questions to determine whether you understood the results. If an experiment did not work out as you expected, this is the place where you can hypothesize as to what conditions may have produced your results. This symbol, , indicates each place where you are expected to record results and answer questions. Your lab instructor will use your responses to grade your work. This laboratory manual is one in a long series used in the Biology 101 course. Many of the experiments in this manual were developed and field-tested by James P. Braselton and Dan L. Moran who authored the previous manual published by Burgess Publishing (1998). We are indebted to them. The first edition of this manual was authored by I. K. Smith and Elizabeth (Betty) Moore to whom this edition is dedicated. The input of the numerous TAs who have taught these labs is gratefully acknowledged. We will make special mention of the contributions of doctoral candidate Aswini Pai and Professor Arthur T. Trese who created some of the illustrations. Although this manual is the synthesis of the ideas and suggestions of many people, we take full responsibility for any errors. Ivan K. Smith and Zachary Rinkes June 2004
Applications of the scientific Method
W h y Study Bi o lo gy? Thomas H. Huxley delivered a lecture on the study of biology on the occasion of an Exposition of Scientific Apparatus, in the South Kensington Museum, London, in 1876. His comments apply to any introductory biology class. (Humboldt Library of Popular Science Literature No. 36. Vol. II, 1882.) “It is my duty tonight to speak about the study of Biology, and while it may be that there are many of my audience who are quite familiar with that study; yet as a lecturer of some standing, it would, I know by experience, be very bad policy on my part to suppose such to be extensively the case. On the contrary, I must imagine that there are many of you who would like to know what Biology is; that there are others who have that amount of information, but would nevertheless gladly hear why it should be worth their while to study Biology; and yet others, again, to whom these two points are clear, but who desire to learn how they had best study it, and, finally, when they should best study it.”
What is Biology? In the seventeenth century all knowledge of facts or “history” was divided into natural history (facts related to minerals, plants and animals) and civil history (facts that resulted from man’s actions). In 1801 Lamark coined the term “Biologie” from the two Greek words which signify a discourse upon life and living things. Why should we study Biology? We should be curious because we are biological organisms ourselves. Huxley says that we are raised by tradition to believe of ourselves as special, but if we consider our physical structure it does not differ markedly from that of a dog. The ultimate conclusion of biologists is that “A fundamental uniformity of structure pervades the animal and vegetable worlds, and that plants and animals differ from one another simply as diverse modifications of the same great general plan.” He cites the theory of infectious diseases and advances in agriculture as additional reasons to be interested in biology. What is the best way to study Biology? Perform experiments in a laboratory. “If you want a man to be a tea merchant, you don’t tell him to read books about China or about tea, but put him into a tea merchant’s office, where he has the handling, the smelling, and the tasting of tea. Without the sort of knowledge which can be gained only in this practical way, his exploits as a tea merchant will soon come to a bankrupt termination.” When should one start to study Biology? As early as possible. In Huxley’s judgment, “The best form of Biology for teaching to very young people is elementary human physiology on the one hand, and elements of botany on the other.”
Applications of the scientific MEthod
L e a r n i n g O b j ecti v e s •
To measure the effect of light quality on the light reactions of photosynthesis.
To measure the effect of detergents on the light reactions of photosynthesis.
Book and CD •
Photosynthesis is discussed in Chapter 10 and Section 10.4 on the CD.
B a c kg r o u n d A. The nature of light Light can be regarded either as consisting of particles or packets of energy called photons or as consisting of electromagnetic waves. White light is composed of a mixture of visible lights (violet, blue, green, yellow, orange and red) each having a characteristic color, wavelength, and energy. For example, blue light has a wavelength of 455–500nm and an energy of 262–239 kJ mol–1, whereas red light has a wavelength of 620–700nm and is of lower energy, <192 kJ mol–1. When light is shone on an object, three things can happen that will determine the appearance of the object. •
When white light is shone on white paper, all wavelengths of light are reflected or transmitted, i.e., passes through the paper, and the paper appears white.
When white light is shone on a black object, all wavelengths of light are absorbed, so no light returns to the eye and the object appears black.
When white light is shone on a green leaf, violet, blue, yellow, orange and red light are absorbed by the photosynthetic pigments, chlorophylls and carotenoids. Green light is either reflected or transmitted, so the leaf appears green. It is important to realize that light is both reflected and transmitted. An analogy is a light bulb colored red. Red light is reflected when the bulb is off and the room lights on. But red light is transmitted when the light bulb is on and the room lights are off.
La b o r a t o r y 6 Photosynthesis
Photosynthesis is the conversion of light energy into chemical energy. The light energy is absorbed by the photosynthetic pigments, mainly chlorophylls, and used to make carbohydrates that are stores of chemical energy. The overall equation is: Carbon dioxide + Water → Oxygen + Glucose + Water 6CO2 + 12H2O → 6O2 + C6H12O6 + 6H2O
Energy from the sun
C6H12O6 + 6 O2 (Glucose & oxygen)
6 CO2 (Carbon dioxide from the air)
6 H 2O (Water from the ground)
Figure 6-1. This illustration is for sample purposes only. Two comments need to be made about this equation. First, it may seem unnecessary to have water on both sides of the equation. The reason is that this formulation correctly indicates that all of the O2 released in photosynthesis comes from water. Second, although glucose is traditionally shown as the product of photosynthesis, free glucose is rarely found in plants. Most of the glucose in a plant is present in starch, sucrose, or glucose phosphates. The above equation is a summary of scores of reactions that can be conveniently divided into two groups, the light-dependent and light-independent reactions. 58
1. Light-Dependent Reactions The light-dependent reactions of photosynthesis involve light-driven electron transport through three isolatable complexes, Photosystem II (PS II), cytochrome b6/f (cyt), and Photosystem I (PS I), with intervening electron carriers. All of the components are embedded in the thylakoid membranes. The two photosystems are special chlorophyll a molecules that receive light energy from light-harvesting chlorophyll a and b.
The original source of electrons is water, the splitting or photolysis of which produces O2 (oxygen gas), H+ (hydrogen ions), and e– (electrons). The final fate of the electrons is to react with NADP+ and H+ to produce NADPH. In the course of electron transport, H+ are consumed on the stromal side of the thylakoid and released into the thylakoid channel. The resultant H+ gradient represents energy, the proton motive force. The H+ pass back across the membrane through an ATPase resulting in the synthesis of ATP from ADP and phosphate. In summary, light energy is converted to chemical energy with the synthesis of ATP and NADPH and O2 is produced as a by-product. The light reactions could be studied by measuring the production of ATP, NADPH or O2, but this is difficult. Fortunately, there is an easier way. The blue dye DCPIP (dichlorophenol indole phenol) is an artificial electron acceptor that receives an e– from Photosystem I and with a H+ becomes reduced to a colorless compound. Some herbicides (compounds that inhibit the growth of or kill plants) have an effect on photosynthetic electron transport. The herbicide, diuron or DCMU (3-(3,4-dichlorophenyl)-1, 1-dimethylurea) inhibits the transfer of electrons from Photosystem II to an electron carrier. This prevents the formation of ATP and NADPH, and thus photosynthesis. PHOTOSYNTHESIS
C6H12O6 + O2 + H2O sugar oxygen water
C6H12O6 + O2 sugar oxygen
+ CO2 carbon dioxide
H 2O water
Figure 6-2. This illustration is for sample purposes only.
Laboratory 6: Photosynthesis
+ CO2 carbon dioxide
2. Light-Independent or CO2-Fixation Reactions In the light-independent reactions the NADPH and ATP produced in the light reactions are used to reduce CO2 to carbohydrate. These reactions occur in the stroma of the chloroplast. Initially, CO2 reacts with a 5-carbon sugar (ribulose bisphosphate) to form two molecules of a 3-carbon sugar, which is variously called triose phosphate, glyceraldehyde phosphate (GAP) or phosphoglyceraldehyde (PGAL). Some of the triose phosphate is used to make ribulose bisphosphate. Some is converted to glucose phosphate, which is converted to starch for temporary storage in the chloroplast. The remainder is exported from the chloroplast and used to make fructose phosphate and glucose phosphate, which react to form sucrose.
Figure 6-3. This illustration is for sample purposes only. Some of the sucrose is used in the cell as an energy source or to make other carbohydrates, such as the cellulose present in cell walls. The remainder is transported out of the leaf to non-photosynthetic parts of the plant, called sinks. In some sinks, sucrose is used to provide energy and raw materials for growth. In other sinks it is stored either as sucrose (sugar beet and many fruits) or as starch (grains and storage organs such as potatoes or carrots).
Part 1: Effect of Different Colors or Wavelengths of Light on the LightDependent Reactions of Photosynthesis
Methods Chloroplast Isolation 1. The TA will homogenize 20g of spinach leaves with 100ml of 0.25M sucrose in a blender on low speed for 10–15sec. 2. The resultant slurry will be squeezed through two layers of cheesecloth and one layer of MiraclothTM (MiraclothTM on the bottom) into a 500ml breaker. 3. Working in groups of 4, place 10ml of the filtered spinach solution in a centrifuge tube. Centrifuge tubes should be balanced and spun for 3min in a Fisher model 282 centrifuge. 4. Note that after centrifugation there is a tightly packed gray pellet below a green layer of chloroplasts. Pour off the liquid. 5. Add 5ml of 0.25M sucrose, and using a disposable plastic pipette, gently suspend the chloroplasts. This chloroplast suspension is the “chloroplast stock solution.” 6. Place 110ml of 0.25M sucrose in a 125ml Erlenmeyer flask, add 2ml of chloroplast stock solution and gently swirl to mix the chloroplasts. This is the “chloroplast working solution.”
Fill one cuvette (test tube) for the Spectronic 20 spectrophotometer with 0.25M sucrose (the blank). Fill another cuvette with the chloroplast working solution.
Place the blank in the Spectronic 20 and adjust the Transmittance to 100% with the wavelength set at 550nm.
Place the cuvette containing the chloroplast working solution in the Spectronic 20 and read Transmittance. If it is between 70% and 80% proceed to the experiment. If the transmittance is less than 70% add some sucrose to the working solution and repeat the above step until you get between 70% and 80%T. Alternatively, if the original transmittance was above 80% add more stock chloroplast solution to the working solution until you reach 70-80% T.
Laboratory 6: Photosynthesis
7. To adjust the concentration of chloroplasts in the working solution do the following:
Measurement of the light-dependent reactions (Work in pairs) 1. Set up 8 glass test tubes inserted in plastic centrifuge tubes as indicated in the following table, and place on the matching cards with the test tube racks. 2. Add ___ drops of DCPIP to tubes 2–7, do not add any to tube 8. Your TA will tell you how many. It will be enough to give 80% T at 480nm in 4ml of sucrose. Place Parafilm over the mouth of the tubes and mix the contents by inverting twice. Place a black cap over each tube. 3. Place the test tube rack in front of the lamps provided, and start timing the reaction. When the color in test tube 2 matches the color in test tube 1 (approximately 10min, but could be 15-20min) the reaction is complete. 4. Take the test tube rack back to your bench. Add DCPIP to tube 8 and mix. Remove the glass tubes from the plastic tubes, and immediately record the relative color. Test tubes 1 and 2 are scored “0” (no dye or complete decolorization), and test tube 8 is scored “5” (no decolorization). Table 6-1.
In this experiment how were the light-dependent reactions of photosynthesis measured?
What quality or color of light was most effective in driving the light reactions? Why is this?
What color of light is not used in photosynthesis? Why is this? 62
Part 2: The Effect of Detergents on the Light-Dependent Reactions
The detergent sodium dodecyl sulfate (SDS) is an amphipathic molecule composed of a chain of 12 carbons with hydrogen attached (hydrophobic) covalently linked to a sulfate group (hydrophilic). It can slip into biological membranes. When its hydrophobic end binds to the hydrophobic ends of the lipids that make up the chloroplast membrane the latter becomes disorganized. ÂŠHayden-McNeil, LLC
Photosynthetic layer Loosely woven hyphae
B. Crustose lichens are compact.
C. Fruticose lichens are shrublike.
D. Foliose lichens are leaflike.
figure 6-4. This illustration is for sample purposes only.
lAborAtory 6: photosynthesis
A. This section of lichen shows the placement of the photosynthetic cells and the fungal hyphae, which encircle and sometimes penetrate them.
Methods 1. Set up 5 tubes as in the following table. The SDS was prepared in 0.25M sucrose, and “From 1” means the number of drops you used in the previous experiment. Table 6-2. Test tube
2. Place a piece of Parafilm on the tubes, and invert them 2 or 3 times to mix the contents of the tubes. Discard the Parafilm. Do not roll it into balls. 3. Place the tubes in front of the lights and wait until test tube 2 has reached the same color as test tube 1.
Compare the color in test tube 3-5.
Was photosynthetic electron transport inhibited in any of the tubes? If so, what is your explanation of the result?
L e a r n i n g O b j ecti v e s
To prepare slides of garlic roots to view stages of mitosis.
To examine mitotic stages in onion.
To review cell structure and function by viewing Voyage Inside the Cell.
Book and CD •
Mitosis is discussed in Chapter 6 of the book, and in 5.1 on the CD, which includes an audio that talks you through mitosis and meiosis.
B a c kg r o u n d At any moment, every cell is at some point in the cell cycle. The cell cycle is composed of interphase and M phase. During M phase the nucleus divides (mitosis) and two cells are created by cytokinesis.
G1 phase This is a period of rapid metabolism, during which the cell synthesizes cellular molecules and structures, replicates cellular organelles such as mitochondria and plastids, and increases in size.
S phase DNA is synthesized and the chromosomes are replicated during this phase.
G2 phase During this phase, the cell synthesizes and begins to assemble structures that will be used later, e.g., the components of the spindle.
La b o r a t o r y 7 Mitosis
M phaseâ€”Mitosis Prophase The chromatin condenses to form chromosomes. Each chromosome was replicated during S phase, and is now represented by two chromatids, which are attached at their centromere. The nuclear envelope and nucleolus become disorganized. The mitotic spindle begins to form. It is composed of bundles of microtubules that form spindle fibers. Metaphase By this stage the spindle has formed completely. Some of the spindle microtubules become attached to the kinetochores at the centromeres. The chromosomes migrate to the equator of the cell. It is important to realize that diagrams of metaphase in text books always show an equatorial view, i.e., the chromosomes are arranged in a row at the equator. When you do a squash, by chance you may be viewing a cell from the pole, in which case the chromosomes will appear spread out. Late in metaphase the centromeres divide. Anaphase During anaphase the sister chromatids, now called daughter chromosomes, move to opposite poles of the cell. Telophase This phase is characterized by the chromosomes reaching the poles, and becoming less condensed. Additionally, new nuclear envelopes and nucleoli form, so the original cell is seen to contain two distinct nuclei.
Interphase Most cells spend most of their time in interphase. During interphase, individual chromosomes Nuclear envelope cannot be distinguished in Chromatin the nucleus of the cell. (duplicated) Chromosomes duplicate as the cell prepares to Nucleolus undergo division. Plasma membrane
Early mitotic spindle Centromere
Telophase and Cytokinesis The chromosomes uncoil and again become indistinct. Nuclear membrane forms around each of the two sets of chromosomes clustered at opposite ends of the cell. Cell division then occurs, resulting in two daughter cells.
The chromosomes condense and become visible in the nucleus. The nuclear membrane disappears and the chromosomes spread Chromosome, out in the cell. A consisting of framework within the two sister cell, called a spindle, chromatids begins to form.
Nucleolus forming Clevage furrow
Nuclear envelope forming
Spindle pole Nuclear envelope fragments
Anaphase Metaphase The chromosomes line up in the middle of the cell. Spindle fibers
The two identical sets of chromosomes migrate along the spindle fibers to opposite ends of the cell.
66 figure 7-1. This illustration is for sample purposes only.
M phase â€” cytokinesis The final step in cell division is cytokinesis, the division of the cytoplasm. In animal cells, this occurs by the formation of a cleavage furrow on the outside of the cell at the equator. The furrow becomes progressively deeper, and eventually pinches the cell and its contents in two. By contrast, in plant cells, a cell plate forms within the cell at the equator, and new plasma membrane and cell wall is formed on either side, thus producing two separate cells.
Comparison of Mitosis and Cytokinesis in Plant and Animal Cells
Laboratory 7: Mitosis
Figure 7-2. This illustration is for sample purposes only.
Figure 7-3. This illustration is for sample purposes only.
Part 1: Preparation of Slides to Observe the Stages of Mitosis Methods Wear protective goggles for this lab
1. Your laboratory instructor will prepare a fixing-hydrolyzing solution by adding 1 part concentrated hydrochloric acid to 2 parts absolute alcohol and mixing. He/she will place drops of this solution in a well in a depression plate. The fixing hydrolyzing solution is very corrosive/caustic. Take care to keep it away from your clothes and body. Keep the depression plate, needle, and tweezers on the absorbent paper on the lab bench in front of you. 2. Place a few drops of distilled water in two of the depressions on your plate 3. Each pair take one clove of garlic (boxed garlic grown for 4–5 days suspended over a test tube of distilled water) and with the aid of a razor blade remove approximately 1 cm from the tips of 3–5 roots and place in the fixing-hydrolyzing solution for 8–10 minutes. 4. With the aid of a toothpick or tweezers, transfer the root tips from the fixing solution to one of the wells containing distilled water and wait for 1–2 min. 5. Again transfer the root tips to the second well containing distilled water, and wait a further 1–2min. 6. Put one root tip into a drop of aceto-orcein stain (2g orcein in 400ml of 45% acetic acid) on a microscope slide and remove and discard all but the very tip (1–2mm) of the root. Make sure you are discarding the right part. 7. Gently tease the root tip apart with a dissecting needle. Allow the root material to take up the stain for 2min. Lower a coverslip over the material, and lightly tap the coverslip with a ball point pen to disperse the cells. 8. Examine to see if the cells are in clumps. If they are, lightly tap the coverslip until the cells are dispersed. 9. Cover the slide with a paper towel or filter paper and evenly press to flatten the cells. 10. Examine the slide for stages of mitosis.
Count the number of prophases, metaphases, anaphases, and telophases you see.
Prophase Metaphase Anaphase Telophase 68
What do you think is the relationship between the number of phases you see and the amount of time a cell spends in that stage?
Cell plate Anaphase
figure 7-4. This illustration is for sample purposes only.
Part 2: View the Video “Voyage Inside the Cell”
Cell membranes are described as “oily films” with protein “trees” extending from the surface. List two functions of membrane proteins.
List three activities that small hormones might instruct a cell to carry out.
lAborAtory 7: Mitosis
The video is an animation of a voyage through a cell. The purpose of viewing the video is to review information you have received about cell structure and function, and realize how various functions are integrated.
What is the function of mitochondria in plant and animal cells?
Where are proteins synthesized in cells?
structures “control traffic” inside cells? (Note: you are not being asked what controls transport into cells)
What two molecules make up chromosomes and how are they organized relative to one another?
Genetic information for making proteins is transcribed to messenger molecules, which move from the nucleus to the cytoplasm. What are these molecules composed of?
What is the name for the array of microtubules that effect chromosome movement during mitosis?
What is the name of the area on a chromosome where microtubules attach?
After nuclear division, the cytoplasm is distributed between the resulting cells. How is this accomplished in an animal cell?