Bio 110 Lab Manual Spring 2011

Bio 110 Laboratory Manual Spring 2011
Dr. Maureen Knabb and Dr. Win Fairchild Revised, Dr. On R. Pagn Department of Biology, West Chester University
Bio 110 Laboratory Manual - Spring 2011
GENERAL BIOLOGY LABORATORY MANUAL INDEX

PAGE 3 4 14 18 20 28 34 42 49 57 65 81
Laboratory safety Adopt-a-bug Avian evolution Organic molecules Microscopes & cells Diffusion and osmosis Data analysis & statistics Fermentation and metabolism Mitosis, meiosis and life cycles Mendelian genetics Duckweed population biology Daphnia feeding ecology Molecular genetics
LABORATORY EVALUATION: Activity Apply-your-knowledge Lab practical exams Quizzes Total Points 30 60 (2 x 30) 40 130
Bio 110 - Lab Safety & General Rules 1. Read the experimental procedure ahead of lab. 2. Report all accidents to your lab instructor, regardless of how minor. 3. Use goggles and wear lab coats when instructed to do so. It is recommended (but not required) that students wear laboratory coats to protect their clothing. 4. Due to the dangers of broken glass and corrosive liquid spills in the lab, open sandals or bare feet are not permitted in the lab. 5. Learn the location and proper usage of the eyewash fountain, fire extinguisher, safety shower, fire alarm box, evacuation routes, clean-up brush and dust pan, glass/chemical disposal can. 6. Use care whenever samples must be heated. 7. If you get any chemical in your eyes, immediately wash them with the eye-wash fountain and notify the lab instructor. 8. Never look directly into a test tube. View the contents from the side. 9. Food and drink are prohibited in the lab. Avoid hand-to-mouth operations (e.g. chewing pencils, licking labels, mouth pipetting). 10. Return all lab materials and equipment to their proper places after use. 11. Upon completion of work, wash and dry all equipment, your lab bench, and your clean-up area. 12. Wash your hands before you leave the lab. 13. Cell Phones: Not allowed in lab. Please silence them before the lab period starts. If you must take a call, PLEASE take it outside. 14. Grades I will not be e-mailed! 15. Academic Integrity: Be familiar with the University policy on Academic Integrity, whose principles will be adhered to in this course. Note that it is just as dishonest, and unfair to other students, to knowingly allow someone to copy your work as it is to copy someone's work. All instances of academic dishonesty will be dealt with according to University policies.
I have read and will abide by the syllabus and lab safety rules. ______________________________________________________ Print your name here ID#
______________________________________________________ Student signature date
Copies of this form will be available during our first lab. Please print & sign your name and give it to your lab instructor
Lab exercise 1: Adopt-a-Bug! Milkweed Bug Growth Experiment

Large milkweed bug - Oncopletus fasciatus; Order Hemiptera, Family Lygaeidae Congratulations! Youve just adopted a cute and cuddly milkweed bug. The large milkweed bug is used extensively in laboratory work by entomologists because it can be reared easily on a diet of the cracked seeds of sunflower, watermelon, squash and almond. (Note: the seeds must be raw, unsalted and cracked, as the insects cant open the seeds!) Sunflower seeds will be used in this experiment. Host Plant Milkweed bugs in the wild feed on the seeds and tissue of a plant that is common in old fields and along roadways, the milkweed plant (Asclepias sp.). This species is often found in small groups on milkweed leaves. The 1 to 1.5 meter high milkweed plants, which produce a distinctive milky white sap when a leaf is removed, bloom in sprays of small white flowers in the summer. In the fall, seedpods develop which are about 10 mm long and 4 mm wide. When the seedpods ripen they open up releasing seeds that float on fluffy white parasols. Milkweed bugs are often found piercing the wall of the seedpods to feed on seeds inside.
Life History Milkweed bugs are hemimetabolous insects, which mean they exhibit gradual metamorphosis with nymphs (immatures) that look like the adults without full wings. Black wing pads appear early in development and their color pattern also changes gradually with age. Nymphs have bright red abdomens.
Eggs are about 1 mm in diameter and change gradually from yellow to an orange color as they incubate. First instar nymphs are about 1.5 mm long (tip of head to tip of abdomen). As they grow they periodically molt (shed) their old exoskeleton. Milkweed bugs usually molt 5 times which means they have 5 nymphal instars before becoming an adult. Eggs take about 1 week to hatch and a month to become adults at room temperature (75F or 23C). The underside of the abdomen of the female has one black strip and two black dots while the male has two thick black strips.
Aposematic Coloration to Avoid Predation Milkweed bugs are one of a group of insects that are adapted to feed on poisonous members of the milkweed family. The bugs have few predators because they sequester (concentrate in their bodies) bad tasting compounds, cardiac glycosides, found in the sap of milkweed plants. The bugs use the bright (aposematic) coloration to advertise their bad taste. Inexperienced birds that eat their first milkweed bug will vomit from the sequestered glycosides and are unlikely to try to eat another orange and black insect. Some insect species that do not taste bad use similar color patterns to fool birds. These are known as mimics. Interesting Behaviors Milkweed bugs often gather in groups on the milkweed plant. This gregarious behavior probably enhances their warning coloration. In the container you may see groups of bugs forming at night. During the day they are usually found feeding on seeds or walking around the container. Sometimes groups form on the side of the container away from the seeds when some of the bugs are molting. Care You will be provided an appropriate habitat for your bug: a plastic dish with some sunflower seeds (a milkweed substitute) and a water supply, including a plastic tube containing wet cotton. The cotton should be kept moist but not saturated. If the cotton becomes dirty or moldy, get a replacement form your lab instructor. Be sure to keep the water away from the seeds so that they don't become moldy. If you need to handle your bug, a soft camelhair brush can be used to move them. Insects are ectothermic and their growth and development are very sensitive to temperature. Try to find a warm place where the temperature is stable. Check your adopted bug frequently, daily if possible. Examine the habitat for discarded exoskeletons to see if it has molted (they look a little like spiders). Each week, measure the length of your bug (tip of head to tip of abdomen in mm) using a piece or paper with 1 mm gridlines taped to the plastic cup. Record the length and any notes on the datasheet provided. Later in the semester, you will graph and analyze these results in the Data Analysis/ Statistics Lab.
References Price, Peter W. 1977. Insect Ecology. John Wiley & Sons, NY. pp. 514. http://insected.arizona.edu/milkinfo.htm http://www.uwex.edu/ces/cty/milwaukee/urbanag/bugnet/outflowers/milkweedbug/milkweedb ug.html http://www.life.uiuc.edu/entomology/105/milkweedbug.html http://entowww.tamu.edu/extension/youth/bug/bug027.html http://pubpages.unh.edu/~pcj/adoptbug.html http://www.cnr.berkeley.edu/citybugs/Taxa/Heteroptera/Lygaeidae/Oncopeltus/Oncopeltus_fas ciatus/Oncopeltus_fasciatusPage.htm http://everest.ento.vt.edu/Courses/Undergraduate/IHS/distance/html_files/buginacup/journal2.h tml Adopt a Bug! Datasheet Name (your name, not the bugs name): _________________ Variable tested:____________________ Date Size (mm) Notes Date Size (mm) Notes Lab section _______
Lab exercise 2: Avian Evolution

This exercise is intended to demonstrate the processes of natural selection, sexual selection, speciation, and convergent evolution using specimens from the Biology Department's bird collection. Because many of the birds are quite old and valuable, please refrain from handling the specimens as you make your observations. I. Evolutionary Adaptation The physical characteristics and behaviors of birds illustrate a vast assortment of evolutionary adaptations for a particular way of life, often termed the ecological "niche". For example, bill size and shape reflect the diet of most birds. Seed eating species, for example, have short, strong bills for cracking the seed coat. Predators typically have hooked bills for tearing apart their prey. Birds such as swallows have bills which, when opened, provide a wide gape for catching aerial insect prey. Long bills may be used for spearing prey, probing in soft substrates, or hammering in wood. Examine the specimens below and decide on their probable diet. Bird Elf Owl Dowitcher Skimmer Night hawk Chimney swift Hummingbird Evening Grosbeak Woodcock Least Bittern Bill Description Probable Diet
II. The Process of Natural Selection Adaptation typically occurs within populations by the following microevolutionary processes, termed natural selection: a. Individuals in the population differ in their genetic composition (genotypes). b. The different genotypes produce different phenotypes, typically visible as slight variations in height, weight, or the size/shape of particular structures.
c. These phenotypic differences influence survival and reproductive success. d. Differential reproductive success leads to a different mix of genotypes (and thus phenotypes) in the next generation.
Shown is a Crossbill, a bird with an unusual bill that is inserted into pinecones to extract the pine seeds. The Crossbill is thus a dietary specialist. Also shown is a bird of about the same size, a Pine Grosbeak, which is a dietary generalist capable of feeding on a wide variety of seeds. Using the information on the previous page, describe a process of natural selection on a Grosbeaklike bill, which, repeated over enough generations, would cause such a bill to evolve to the Crossbill-like shape.
The theory of natural selection, formally proposed by Charles Darwin, replaced an earlier theory espoused by Lamarck that adaptations acquired during an individuals lifetime were slowly heritable. For example, Lamarck proposed that the giraffe stretched its neck over time to reach leaves on trees and that these adaptations were passed to the offspring. Lamarck's theory slowly lost favor for lack of supporting evidence. For example, the crow shown here survived an apparent accident, but its offspring would not have crossed bills.
III. Phenotypic Variation within Populations In order for natural selection to produce evolutionary change, there must be genetically determined phenotypic variation among individuals within a population. An extreme example of such variation is that of the Eastern Screech Owl Otus asio, which has two distinct color morphs: a grey phase and a red phase. These two phases are heritable and are also associated with behavioral differences. Grey phase owls typically roost (sleep during the day) next to tree trunks, whereas red phase owls more commonly roost in the outer foliage. Why might roosting next to a tree trunk decrease mortality and thereby increase the reproductive success of a grey phase owl? The sparrows shown are all members of the common house sparrow, Passer domesticus, but differ slightly in body size. In a very early, now classic study of the effects of natural selection on sparrow body size, Hermon Bumpus at Brown University collected sparrows incapacitated by a severe ice storm that hit Rhode Island on February 1, 1898 and brought them back to the lab. Of the 136 sparrows collected, 72 survived while 64 died. Bumpus recorded 9 size measurements on each bird. Body weight and total length data for the 59 adult males collected are shown in the graph below. Based on the graph, were smaller or larger birds favored by natural selection in this instance?
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30
Body weight (g)
28
26
24
SURVIVE?
no
22 150
yes
155
160
165
170
T o ta l l en gth (m m )
IV. Sexual Selection Males and females of many species (humans included) lead slightly different lives and have differing roles in courtship. Particularly in polygynous species (where a dominant male may mate with more than one female and some males are left without mates), competition among males for mates is fierce and natural selection favors traits that lead to winning battles (e.g., larger size). Natural selection may also favor traits that "advertise" males (e.g., bright coloration) to females, even if those same traits increase their risk of getting eaten by predators. Selection on the two sexes may thus be different, and lead to sexual dimorphism (males and females look different). Describe the dimorphism in color pattern of the bird species shown.
Male Cardinal Scarlet Tanager Red-bellied Woodpecker Bobolink
Female
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The males of these species molt immediately after the breeding season to less ostentatious winter plumage. Suggest why this may be an advantage: ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ V. Speciation and Divergent Evolution Populations of a species that become isolated from one another often evolve along separate trajectories to the point where, even if the two populations once more came into contact, reproduction is impossible. When the two populations are no longer capable of interbreeding, they are considered distinct species. This separation of one species into two distinct species is called divergent evolution. An example occurs in Chester County, which is situated along a zone where the ranges of the Black-capped Chickadee (Parus atricapillus) and Carolina Chickadee (P. carolinensis) overlap slightly (see the maps). The laptop at this station has pictures and songs of both the Black-capped Chickadee and Carolina Chickadee. Double click on each file to view and hear them. Based on the specimens shown, and the information provided, how do the two species appear to differ in their size, coloration and calls?
______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________
Black-capped Chickadee Size Coloration Calls/ Songs
Carolina Chickadee
VI. Convergent Evolution Most recently evolved species continue to grow more dissimilar over time, showing divergent evolution (see above). Occasionally, however, species of very different ancestry exploit similar niches, and their adaptations show convergent evolution. An example is shown by Grebes and Ducks, both evolved from very different ancestors, but both using aquatic animals and plants as food. What trait do both species share which indicates convergent evolution?
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___________________________________________________________________________________ ___________________________________________________________________________________ ___________________________________________________________________________________
References: http://www.aquatic.uoguelph.ca/birds/morphevol/main.htm http://www.chickadee-web.com/ http://research.amnh.org/ornithology/crossbills/ http://research.amnh.org/ornithology/crossbills/nathist.html
The beaks of Finches and natural Selection

For this part of the lab you will work in pairs. You have been provided with a tool to simulate the beak of a finch. The different tools represent different beak types within a population of a single finch species. Tool Small forceps Medium forceps Long forceps Pliers Wire cutters Wrench At your table there are 4 petri dishes containing different seeds: corn, sunflower, caraway and mixed seeds. Your goal is to determine how many seeds of each type you can successfully pick up with your tool (the finchs beak) in 30 seconds. Success is defined as the transfer of the seeds to a plastic cup (the birds belly). Your professor will provide direction and serve as the timekeeper for this experiment. One of the members of your team will be the bird and the second one will count the seeds. Only 1 seed per transfer!!!! We will collect the data in the tables below.
Which tool are you? _____________________________________ Which tool do you predict will work best for each seed type? Corn: ___________________ Sunflower: __________________ Caraway: ________________
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Seed type = Corn Tool Small forceps Medium forceps Long forceps Pliers Wire cutters Wrench Number of seeds Average
Seed type = Sunflower seeds Tool Small forceps Medium forceps Long forceps Pliers Wire cutters Wrench Number of seeds Average
Seed type = Caraway seeds Tool Small forceps Medium forceps Long forceps Pliers Wire cutters Wrench Number of seeds Average
Which tool (if any) worked better for the:
Corn seeds? _____________ Sunflower seeds?_______________ Caraway seeds?________________ Were your initial predictions correct? ___________ Based on your predictions, which beak type would be an advantage if the environment changed so that the predominant seed type transitioned from corn to caraway seeds? ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________
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In the remaining Petri dish, there is a mixture of seeds. Based on what you learned about the efficiency of each tool for the different sizes of seeds, do you think that any tool would be advantageous for survival (obtaining sufficient seeds in a limited time)? _____________________________________ Seed type = Mixed 10 seconds Tool Small forceps Medium forceps Long forceps Pliers Wire cutters Wrench Number of seeds Average Rate (seeds/second)
Suppose the finches needed to consume seeds at a rate of about 0.5 seeds/sec per feeding to survive long enough to reproduce. Which finches would survive? _______________________________________________________ Suppose the finches needed to consume seeds at a rate of about 1 seed/sec per feeding to survive long enough to reproduce. Which finches would survive? _______________________________________________________ Suppose the finches needed to consume seeds at a rate of about 2 seeds/sec per feeding to survive long enough to reproduce. Which finches would survive? _______________________________________________________ Explain how this activity demonstrates natural selection. ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________
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Lab exercise 3: Organic Molecules

Most organic molecules found in living systems can be classified as carbohydrates, fats, proteins, or nucleotides. Each of these classes of molecules has specific properties that can be identified by simple chemical tests called assays. Assays can be either quantitative or qualitative. A qualitative assay simply shows whether or not a specific molecule is present in a sample at detectable levels. A quantitative assay shows the presence and amount of a molecule in a sample. In this laboratory you will use qualitative assays and learn to detect the presence of the organic molecules: carbohydrates, fats, and proteins.
Exercise 1: Testing for Carbohydrates The basic structural unit of carbohydrates is the monosaccharide (or single sugar). Monosaccharides are classified by the number of carbons they contain: for example, trioses have three carbons, pentoses have five carbons, and hexoses have six carbons. They may contain as few as three or as many as ten carbons. Monosaccharides are also characterized by the presence of a terminal aldehyde group (CHO) or an internal ketone (C=O) group. Both of these groups contain a double-bonded oxygen atom that reacts with Benedict's reagent to form a colored precipitate that will change the color of the original solution. When two monosaccharides are joined together, they form a disaccharide. If the reactive aldehyde or ketone groups are involved in the bond between the monosaccharide units (as in sucrose), the disaccharide will not react with Benedict's reagent. If only one group is involved in the bond (as in maltose), the other is free to react with the reagent. Sugars with free aldehyde or ketone groups, whether monosaccharides or disaccharides, are called reducing sugars.'' In this exercise, you will use Benedict's reagent to test for the presence of reducing sugars.
C C O C C O H
KETONE ALDEHYDE
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Monosaccharides may join together to form long chains (polysaccharides) that may be either straight or branched. Starch is an example of a polysaccharide formed entirely of' glucose units. Starch does not show a reaction with Benedict's reagent because the number of free aldehyde groups (found only at the end of each chain) is small in proportion to the rest of the molecule. Therefore, we will test for the presence of starch with Lugol's reagent (iodine/potassium iodide, I2KI). Part 1: Benedicts test When Benedict's reagent is heated with a reducing sugar, the color of the reagent changes depending on the amount of sugar present. Orange and red indicate the highest proportion of these sugars. Green or yellow indicate lesser amounts of reducing sugar. (Benedict's test will show a positive reaction for starch only if excessive heating has broken down the starch.) Procedure: 1. To each large glass test tube, add approximately 1 ml (about 20 drops) of each solution to be tested. 2. Add approximately 5 drops of Benedict's reagent to each tube. 3. Mix the reagent and the solution by agitating the solution in each tube from side to side. 4. Record the original color of each tube's contents in Table 1. 5. Heat the test tubes in a warm water bath for 10 minutes. Be careful when placing tubes into or removing them from the water bath. Record any color changes. Part 2: Lugol's Test When an iodine solution comes in contact with starch, the yellow-brown color of the iodine changes to dark brown/black. Monosaccharides and disaccharides are too small to interact with iodine. Therefore, the Lugol's test is specific for a polysaccharide, starch.
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Procedure: 1. To each small plastic test tube, add approximately 1 ml of each solution to be tested. 2. Add approximately 2 drops of Lugols reagent to each tube (bottles may be labeled iodine solution). 3. Mix the reagent and the solution by agitating the solution in each tube from side to side. 4. Record any color changes (DO NOT HEAT) in the table below. SUBSTANCE TYPE OF SUGAR MOLECULE control, mono-, di-, polyBENEDICTS TEST before boiling BENEDICT S TEST after boiling LUGOLS TEST after addition
1. Water 2. Glucose 3. Maltose 4. Sucrose 5. Starch
Questions: a. Why did you test water with Benedict's and Lugols reagent? ____________________ b. What color change resulted from a positive Benedicts test? _________________ Lugols test? ____________________
c.
Did maltose cause Benedict's reagent to change color?___________ Lugols reagent? ___________ Why or why not? ____________________ Did glucose cause Benedict's reagent to change color? ___________ Lugols reagent? ___________ Why or why not?_____________________ Did starch cause Benedict's reagent to change color? ___________ Lugols reagent? ___________ Why or why not? _____________________ Did sucrose cause Benedict's reagent to change color? ___________ Lugols reagent? ___________ Why or why not? _____________________
d.
e.
f.
Exercise 2: Testing for Lipids The word lipid refers to any of the members of a rather heterogeneous group of organic molecules that are soluble in nonpolar solvents such as chloroform (CHCl3), but are insoluble in water (polar). Although lipids include fats, steroids, and phospholipids, this exercise will focus primarily on fats.
Triglycerides, a popular topic in discussions of diet and nutrition, are the most common form of fat. They consist of three fatty acids attached to a glycerol molecule. Triglycerides are found predominantly in adipose (fat) tissue and store more energy per gram than any other molecule. At room temperature, some fats are solid (generally those found in animals) and are referred to as fats, while others are liquid (generally those found in plants) and are referred to as "oils." Vegetable oil, a liquid fat, is a mixture of triglycerides. Since both solid and liquid fats are nonpolar, we will test for their presence by using Sudan IV, a nonpolar dye that preferentially dissolves in nonpolar substances like fats and oils, resulting in red droplets on the surface of a liquid containing lipid.
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Procedure 1. Get two small plastic test tubes and add the same amount of water to both of them. 2. Add one small drop of vegetable oil to one tube. 3. Add one drop of Sudan IV to both tubes. What is the result of the water versus water + oil reaction with Sudan IV? ______________________________________________________________________ Why do the droplets float, rather than mix with the water? ______________________________________________________________________ Exercise 3: Testing for proteins and amino acids
Proteins are made up of one or more polypeptides, which are linear polymers of smaller molecules called amino acids. Amino acids derive their name from the amino group and the carboxyl group (acidic) that each possesses. Polypeptides are formed when amino acids are joined together by peptide bonds between the amino group of one amino acid and the carboxyl group of a second amino acid.
The biuret reagent reacts with peptide bonds and will, therefore, react with proteins, like egg albumin, but will not react with free amino acids, like glycine and alanine.
On the other hand, the reagent ninhydrin will react with the amino group of free amino acids, but will not react with polypeptides.
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In this exercise you will distinguish between free amino acids and proteins (polypeptides) on the basis of their ability to react with either biuret reagent or ninhydrin. Part 1: Biuret Test Procedure 1. Obtain two small plastic test tubes and add approximately 1 ml of water to one tube. 2. Add approximately 1 ml of albumin to the second tube. 3. Add about 5 drops of biuret reagent to both tubes. 4. Allow the contents of the tubes to react for about 2 minutes at room temperature. Record your results. ________________________________________________ What is the result of the water versus albumin reaction with Biuret reagent? ________ Part 2: Ninhydrin Test Procedure 1. Obtain a piece of filter paper and divide it into six pieces with a pencil. Label the areas A, B, C, D, E, and F (see picture at right). 2. Place one drop of each solution (labeled A, B, C, D) onto the filter paper in the area with the corresponding letter. Only add a small drop of each solution so that the sample does not spread into the adjacent areas. Allow the spots to dry. Place a drop of water in area E. Press your index finger into quadrant F. 3. Apply one drop of ninhydrin to each spot. Caution: Ninhydrin is poisonous; avoid contact with your skin. Allow the paper to dry at room temperature for 10 to 15 minutes. (The reaction will occur more quickly if the paper is heated using the lamp on your table.) All amino acids, except proline, will turn purple in the presence of ninhydrin. Proline will turn yellow. Record your results in the table below. SAMPLE A B C D E F FINAL COLOR TYPE OF MOLECULE
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Exercise 4: Analyzing unknowns Using reagents and methods from the previous exercises, identify the types of molecules contained in the unknown solutions. Fill out the table below.
SAMPLE
BENEDICTS TEST
LUGOLS TEST
BIURET TEST
NINHYDRIN TEST
SUDAN IV TEST
1.
2.
3.
What are the chemical components in each of the unknowns tested? 1. ____________________________________________________________________________ 2. ____________________________________________________________________________ 3. ____________________________________________________________________________ References: http://www.sidwell.edu/sidwell.resources/bio/VirtualLB/Macro/macro.html http://www.surry.cc.nc.us/ayers_bio/BIO%20111/labexcs/chemistry%20lab/chemistry%20for%20life.htm http://www.science.mcmaster.ca/Biology/1A03/Laboratories/Lab_One/index.html
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Lab exercise 4: The Microscope, Measurement, and Cells

Understanding and properly applying the methods of scientific inquiry require that you become proficient at observing and recording data accurately. To do this, you need to be familiar with the types of instruments used for experimental work and with proper sampling techniques. During this laboratory period you will learn about the use and care of the compound microscope. You will also prepare living materials for observation. In the late 1500s, two Dutch spectacle makers (The Jannsens) developed the compound microscope. Their device had two convex lenses placed at either end of a tube and was capable of magnifying an object to 10 times (10x) its actual size. Today, developments in microscopy provide scientists with a wide selection of instruments with which to view the smallest organisms and even the components of individual cells. These microscopes range in complexity from the relatively simple models you will use in the laboratory today to highly sophisticated scanning and transmission electron microscopes.
Exercise 1: Identifying the parts of the compound microscope To use the microscope properly, you must first be familiar with the care of this expensive and delicate instrument. Keep the following precautions in mind: Always carry the microscope in an upright position. Use one hand to grasp the arm of the microscope; use the other to support the base. The eyepiece (ocular lens) slides into the body tube and could fall out if the microscope is tilted too much. Never place the microscope close to the edge of the table. Keep the electrical cord out of the way. Use only lens paper for cleaning the lenses. Using your fingers, handkerchief, or other materials could smudge or damage the lenses. When you are finished with your observations, turn off the illuminator and rotate the low-power objective into viewing position. Never put a microscope away with the high-power objective in the viewing position. Obtain a compound microscope from your instructor and familiarize yourself with the location and function of each part. Light source: May be built into the base with a lens that focuses light onto the lower condenser lens or may be a separate light that is focused onto the condenser lens by a mirror. Condenser: Contains a system of lenses that focuses light on the object. Locate the knob that raises and lowers the condenser. Iris diaphragm: Used to control contrast. It can be opened or closed using the lever protruding from the lower front edge of the condenser. Greatest contrast is achieved when the iris is fully closed. Closing the iris diaphragm reduces the resolving power of the microscope, the ability to distinguish the smallest structures. As the iris is opened, resolving power increases but contrast decreases. Objective lenses are mounted on a revolving nosepiece or turret (e). Most new microscopes are parfocal, that is, when an object is in focus with one lens, the lenses can be changed without
completely losing focus. Each objective contains a complex lens system. Magnification is indicated on the side of the objective. The nosepiece usually holds the objectives listed below. Check to see which of these are present on your microscope: scanning objective (4 x magnification), low-power objective (10 x magnification), and two high- power objectives (40 x and 60 x magnification).The objective is also responsible for determining the resolving power. Resolving power is the ability to reveal detail, to distinguish two closely spaced objects as being two rather than one. The smaller the distance between two objects that can be distinguished from one another, the greater the resolving power of the instrument used to view the objects. The unaided human eye can distinguish (resolve) two objects when they are at least 0.1 mm apart, whereas with the light microscope, the human eye can distinguish objects as separate when they are up to 1,000 times closer than that! Ocular lens or eyepiece: The lens you look through. It will magnify the image from the objective an additional 10 times (10 x). The body tube (g) holds the ocular lens. Your microscope has two oculars and is said to be binocular. Stage: Holds the slide to be viewed. The stage can be moved vertically by turning the coarse adjustment (i) and the fine adjustment (j) knobs. These are located in different places on different types of microscopes, either separately or together. Coarse adjustment is used for initial focusing of specimens at low power. Fine adjustment makes very slight changes, allowing precise focusing at high power. Mechanical stage: Your microscope is equipped with a mechanical stage. Adjustment knobs are used to move the slide in the horizontal plane, that is, side to side and toward and away from you. Base (l) and arm (m): Important structural parts of the microscope.
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Exercise 2: Using the compound microscope Procedure To get maximum performance from the microscope, you will need to adjust the illumination and focus properly. 1. Place the microscope on the table with the oculars pointing toward you. 2. Revolve the nosepiece until the scanning objective (4x) is in line with the body tube. A click will be heard (or felt) when the objective is properly engaged (otherwise you will see only your eyelashes against a black background). Raise the stage as close to the objective as it will go. You should notice that with this objective there is considerable space between the end of the objective and the stage. The space between the end of the objective and the specimen is referred to as the working distance and it will decrease with the higher power objectives and increased magnification. 3. Turn on the light switch and adjust the illumination. Higher power objectives require more illumination. 4. Adjust the condenser to its highest position. To truly optimize the condenser position, after focusing on the subject, gently place a sliver of paper on the top of the illuminator lens. Now raise or lower the condenser so that the edge of the paper is in sharp focus with the specimen. The iris diaphragm should be adjusted so that the field of view is brightly and evenly lit. 5. Obtain a slide of the letter "e". With the 4x objective in position, place the slide on the microscope stage, making sure that the coverslip is facing upward toward the objective. Hold the slide in place with the mechanical stage. Position the slide so that the specimen is directly over the hole in the stage and confirm that the stage is as close to the objective as possible. 6. Since the stage is as close to the 4x objective as it can get, focus must occur at a lower stage position. Looking through the oculars, slowly lower the stage by turning the coarse adjustment focus knob. As the
specimen comes into view, continue lowering the stage until the image becomes clear. At first continue past focus, then reverse direction and determine the point at which the image is in perfect focus. 7. Once you are sure that the specimen is in sharp focus and in the center of your field of view, rotate the next higher power objective into place. The specimen should appear larger, but perhaps blurred. Turn the fine adjustment knob slowly to sharpen the focus. Your microscope is parfocal, so you should be able to bring the specimen into focus by using only the fine adjustment. If you need to use the coarse adjustment, always return to a lower power objective with a long working distance, check focus, and make sure the specimen is centered. Never turn the coarse adjustment while using an objective with very little working distance.
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8. You can determine how much your specimen is magnified by multiplying the power of the objective lens by the power of the ocular lens. For example, a specimen is magnified 600 times when using the 60x
objective lens and the 10x ocular lens. What is the magnification when using the 4x objective? _______________________
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9. Move the slide to the right. Which way does the letter "e" move (as viewed through the microscope)? _______________________________________. 10. Prepare a slide of Elodea leaf and repeat steps 1-8. Place a drop of water in the center of a clean microscope slide. To the drop, add a torn piece of an Elodea leaf. Place one edge of a coverslip at the edge of the water drop and gently lower it so that the water containing the specimen completely spreads out under the coverslip. Take care not to trap bubbles under or around the specimen. Do not press down on the coverslip. If there is too much water present, draw off the excess by touching the corner of a paper towel to the edge of the coverslip. 11. Rotate the nosepiece so that the 10x objective clicks into place. The working distance (the space between the objective lens and the slide) decreases with higher-power objectives and increased magnification. The size of the field of view (the area you can see) varies inversely with magnification. The greater the magnification, the smaller the field of view. 12. Examine your slide of Elodea. Count the number of cells you can see in one row at 4x and 10x. What happened to your field of view as you changed from the 4x to the 10x objective? ________________________. What is the relationship between magnification and field of view? ____________________________________________________________________ 13. When you can operate the microscope successfully using 10x magnification, change to the 40 x highpower objective. When using high power, the object to be viewed must be at the center of the field because the high-power objective magnifies only a small portion of the field of view observed under low power. You may need to use the fine adjustment knob to focus the specimen. Remember, never use the coarse adjustment knob with high power. What is the total magnification of your specimen with the 40 x objective in place? ________________________. 14. Below, draw what you observe using the 10x and 40x objectives. Drawings should routinely be done in pencil and be labeled to indicate the name of the specimen. A scale bar should be included to indicate the approximate size of the specimen (see exercise 5). A few additional suggestions for using a microscope. a. Be certain that you have the proper amount of light. With an unstained specimen, you can find the right focus by first focusing on the edge of the cover slip. b. Try to keep both eyes open. This will take some practice on your part, but will be less tiring for your eyes. c. Always clean ocular and objective lenses with lens paper before use. d. Always begin by using a low-power objective lens to find the specimen. You can then turn to a higher power to make your observations. e. If you are having difficulty locating the specimen, use a systematic pattern to search the slide. f. If all else fails, ask your instructor for help. Exercise 3: Observing wet mount slides Procedure 1. Examine your wet mount of Elodea under low power and then under high power. Use the 10x objective to focus on the torn edge of the Elodea leaf. Notice that only part of the whole thickness of the leaf is in focus. Using the fine adjustment knob, focus on various planes throughout the thickness of the leaf. Note that the leaf is more than one cell-layer thick. How many cell layers can you discern? _______________________
2. Now switch to your 40x objective. You should notice that even less of the leafs thickness is in focus at any one setting. What you have just accomplished is a demonstration of a principle of magnification that was discussed earlier: the higher the magnification, the shallower the depth of field (depth of the area that is clearly focused). To a microscopist, this means that your DEPTH DISCRIMINATION has been improved. Depth discrimination is the ability to make clear optical sections unblurred by under- and over-lying material. 3. Notice the movement of little green bodies inside each cell. These are chloroplasts, organelles responsible for photosynthesis in plant cells. The movement you observe is called cyclosis, or cytoplasmic streaming. As the cytoplasm moves around the large central vacuole, it carries with it dissolved substances as well as suspended organelles. Does cyclosis occur in the same direction in all cells? __________________________
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Exercise 4: Measuring the size of objects using the compound microscope The microscope can be used as a tool to gather quantitative data in addition to serving as an instrument for making qualitative observations. Procedure The size of objects viewed with the compound microscope can be estimated by first determining the diameter of the field of view for a particular microscope objective. It is then possible to estimate the size of the specimen by comparing it with the total field. 1. Place a transparent mm grid across the field of view under scanning power and record the diameter in millimeters: ________ 2. What is the diameter in micrometers (m)? ________ 3. What is the diameter in cm? ____________ 4. What is the diameter in nm? __________ The diameter of the field of view using the scanning objective (A) can be used to calculate the diameter using any other objective (B): magnification A x diameter A = magnification B x diameter B 5. Calculate the diameters of the fields of view using the other objectives on your microscope. Objective Diameter (m) 10 X 40 X 60 X 6. Using the slide of Elodea, estimate the length of one cell in micrometers. Hint: Use the diameter of the field of view to determine the length of a row of cells, then divide by the number of cells: ___________m
Exercise 5: Comparing prokaryotic and eukaryotic cells Understanding the nature of cell structure and function is important to an understanding of organisms. All organisms are composed of cells whether they exist as single cells, colonies of cells, or in the multicellular form. Cells are usually very small, and for this reason, a thorough understanding of their subcellular structure and function has been possible only through advances in electron microscopy and cell biochemistry.
There are two general types of cells: prokaryotic and eukaryotic. These two words have their root in the Greek word karyon (nut), which refers to a cell's nucleus. The prefix pro- means "before" or "prior to." Thus the word prokaryotic literally means "before having a nucleus." Prokaryotic cells do not have a membrane-bound nucleus and their genetic material (DNA) is only loosely confined to a nuclear area within the cell. Bacteria, including the cyanobacteria (formerly known as "blue-green algae"), are prokaryotes. All other organisms are eukaryotes. The prefix eu- means "true." The cells of eukaryotes have true, membrane-bound nuclei containing their genetic material. There are other distinctions between prokaryotic and eukaryotic cells. Eukaryotic cells are generally larger and contain additional specialized compartments (membrane-bound organelles) in which cell functions such as energy production may occur. Prokaryotic cells lack membrane-bound organelles, their cell functions are carried out in the cytoplasm.
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Observing Prokaryotic cells Bacteria are extremely small (approximately 1 to 2 m). Most are heterotrophic, depending on organic molecules for food, but some are photosynthetic and make their own food. Morphologically, they are either round (cocci), rod-shaped (bacilli), or spiral-shaped (spirilli). To view them with the light microscope, the highest power objective lens (60x) must be used. Even then, not much more than their basic shape will be visible. Both a cell membrane and a thicker polysaccharide cell wall surround the bacterium. Some bacteria have flagella, threadlike structures used in locomotion. Bacterial flagella are composed of the protein, flagellin. We will not observe bacteria in this lab. Cyanobacteria, formerly called blue-green algae, are photosynthetic prokaryotes. However, unlike photosynthetic bacteria, which contain bacteriochlorophyll, cyanobacteria contain chlorophyll a-the same type of chlorophyll found in eukaryotic green algae. The chlorophyll molecules are not located within chloroplasts, as in higher plants, but are found, instead, within photosynthetic thylakoid membranes dispersed throughout the cytoplasm. In addition to chlorophyll a, cyanobacteria contain other accessory pigments including the yellow and orange carotenoids and the phycobilins (reddish phycoerythrins and bluish allophycocyanins). 1. Make a wet mount of the cyanobacteria species, Anabaena, available in the laboratory. 2. Draw your observations at right. The cells of protists, fungi, plants, and animals are eukaryotic, containing both a membrane-bound nucleus and membrane-bound organelles.
Magnification?______ Procedure 1. Prepare a wet-mount slide of the following samples available in the lab and draw a picture of your observations.
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Kingdom Protista Stentor: a ciliated, single-celled eukaryote cells Volvox: a colony of biflagellated green algal (NO COVER SLIP ON VOLVOX)
Magnification?______
Plant cells: All plant cells possess a cell wall, cell membrane, cytoplasm, nucleus, and plastids. Plastids are membrane-bound organelles unique to plants -chloroplasts (containing chlorophyll) and leucoplasts (containing starch). Chromoplasts contain several types of pigment including carotenoids, which give plants an orange or yellow color. Add the dye Lugols reagent to the plant samples in order to visualize starch granules in the plants (if they are present).
Kingdom Plantae Elodea:
Onion skin:
Potato:
How are the Elodea, onion cells, and potato cells similar to each other? ______________ ___________________________
How are they different? ________________________ _______________________________________________________________________
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Animal cells: Unlike plant cells, animal cells do not possess a cell wall or plastids. View the prepared slides of animal cells using the 10 x and 40 x objectives. Kingdom Animalia Nerve cells: Oral epithelial cells
References: http://www.science.mcmaster.ca/Biology/1A03/Laboratories/Lab_One/Microscopy/Microscopy http://www.science.mcmaster.ca/Biology/1A03/Laboratories/Lab_One/Microscopy/microscopy.html http://www.wisc.edu/botit/Botany_130/Microscope/Parts_microscope.html http://www.wisc.edu/botit/Botany_130/Microscope/Microscope.html
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Lab exercise 5: Diffusion and Osmosis

The law of diffusion is of particular importance to our understanding of the movement of molecules into and out of cells. The law of diffusion states that molecules tend to move from areas of high chemical potential (high concentration) to areas of low chemical potential (low concentration). Osmosis, a special case of diffusion with special relevance for cells, is the movement of water molecules from regions of high water potential (low concentration of solutes) to regions of low water potential (high concentration of solutes) across a selectively permeable membrane. The more solute dissolved in water, the lower the water potential. Thus, pure water with no dissolved solutes has the highest water potential.
Exercise 1: Dialysis The process of movement (diffusion) of solutes through a selectively permeable membrane is called dialysis. The cell membrane is a living example of a selectively permeable membrane. Small hydrophobic solute molecules, water, and other very small polar, but uncharged, molecules can move freely through the cell membrane. But, larger molecules may pass more slowly or sometimes not at all. In this experiment, dialysis tubing, made of a material with a specific pore size, will be used to simulate a cell membrane.
1. Work in groups of four. Obtain a piece of dialysis tubing, soak it in water for a minute, and gently rub the untied end to open the bag. Fill the bag with water to make sure that there are no holes in the bag. Empty the water from the bag. 2. Add 4 pipettefuls of 15% glucose to the bag and then add 4 pipettefuls of 1% starch solution to the bag. 3. Hold the bag closed and mix its contents. Carefully rinse off the outside of the bag in tap water. Place the bag in a beaker so that the untied end of the bag hangs over the edge of the beaker. 4. This is important! Fill the beaker with only enough water to cover the liquid within the bag. Add about 1 dropperful of Lugol's reagent (I2KI) to the water in the beaker. Record the color of the solution in the bag and the beaker. 5. Allow the setup to stand until you see a distinct color change in the bag or in the beaker. After about 1 hr, record the final color of both the solution in the bag and the solution in the beaker. 6. Remove about 1 ml of the solution from the beaker, and test with Benedict's reagent (see Organic Molecules lab #2 for the procedure if you have forgotten this test). Record your results in the table below. ORIGINAL CONTENTS Glucose and starch water and Lugols ORIGINAL COLOR FINAL COLOR COLOR AFTER BENEDICTS TEST not performed
Bag Beaker
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How would you explain the results of your experiment? _________________________________________________________________________ _________________________________________________________________________
Which substance or substances entered the bag; which ones left the bag? Give evidence for your answer in terms of the color changes that occurred. _________________________________________________________________________ _________________________________________________________________________ Exercise 2: Osmosis (Diffusion of water) The movement of water molecules through a selectively permeable membrane is a special case of diffusion known as osmosis. Osmosis is a passive process, that is, it requires no metabolic energy. The principle factor driving osmosis in animal cells is osmotic potential. The osmotic potential is a function of solute concentration. The addition of solute to water lowers the osmotic potential of a solution. Other things being equal, if two unlike solutions are separated by a membrane that allows water to pass through but not solutes, water molecules will move across the membrane towards the solution that has more solute. Thus, osmosis results in movement of water from an area of low solute concentration to an area of higher solute concentration. Consider red blood cells that are normally suspended in plasma. Plasma contains salts, proteins and other solutes. The osmotic potential of red blood cells and the surrounding plasma are equal so that they are in equilibrium. If a drop of blood is placed on a slide and pure water is added to it, the blood cells will quickly swell and burst because the solution surrounding the cells has a higher osmotic potential than the cells. Now lets consider a plant cell. When plant cells are submerged in pure water, they will also take up water because the intracellular contents are more concentrated than the surrounding solution. In contrast to animal cells, plant cells do not burst because of the presence of a rigid cells wall located outside the plasma membrane. As water is taken up into the plant cell, pressure builds up within the cell. This pressure is called turgor pressure. Turgor pressure will not increase indefinitely. Eventually, the turgor pressure will prevent any further uptake of water even if the osmotic potentials are different on the two sides of the membrane. Hence the combined effects of osmotic potential and turgor pressure drive osmosis into and out of plant cells. In this experiment, one group at your table will place several dialysis bags containing solutions of different sucrose concentrations into beakers containing distilled water.
The direction of water movement can be determined by weighing the bags before and after placing them in distilled water: the dialysis bags are permeable to water, but not to sucrose, and can gain or lose water. The water potential of the sucrose solutions in the dialysis bags will be negative (recall that the addition of solute to pure water decreases osmotic potential). Can you predict which way the water will move? ___________________________________________________________________
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The other group at your table will investigate the osmotic potential in a living system; the deshelled egg. Share your results with your group when you are done your portion of the osmosis experiment.
Osmosis in an artificial system (for of table) 1) Obtain five presoaked dialysis tubes. Use a pipette to put approximately 5 ml of each of the solutions listed below into separate bags. After adding the solution, remove most of the air from the bag by drawing the unfilled portion between two fingers. Tie a knot near the open end to seal the solution within the tube. You should have 2 times as much empty space in the tube as that taken up by the volume of the solution. This will leave enough unfilled space within the tube to accommodate the possible accumulation of water. Mark the beakers to keep track of which tube contains which solution. a. distilled water d. 60% sucrose b. 20% sucrose e. 80% sucrose c. 40% sucrose
2) Carefully blot the outside of each bag. Tare the balance before weighing each bag, determine the initial mass of each bag, and record the value in the table below. 3) Fill five 250-ml beakers three-quarters full with distilled water. 4) Place each bag in one of the beakers of water and label the beaker to indicate the % of the solution in the dialysis bag. Make sure that all parts of the bag are completely covered by water. 5) Let stand for 1 hour. At the end of the required time, remove the bags from the water and carefully blot them. Tare the balance, determine the mass of each bag, and record your data in the Table below. Calculate % change = [(final mass- initial mass)/ initial mass] x 100 CONTENTS IN BAG distilled water 20 % sucrose 40 % sucrose 60 % sucrose 80 % sucrose INITIAL WEIGHT (g) FINAL WEIGHT (g) % CHANGE IN WEIGHT
a. What is the relationship between the increase in weight and the % sucrose within the dialysis bags? _______________________________________________ b. How does the increase in sucrose concentration affect the water potential of the solutions inside the dialysis bags? _________________________________ c. A solution (A) that contains more solute than another solution (B) is said to be hyper-osmotic (hyper = more than; solution A is hyperosmotic to solution B). Likewise, solution B can be described as being hypo-osmotic (hypo = less than) to solution A. Solutions with equal solute concentration are called what? __________________.
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Osmosis in a living system (for of table) A chicken egg is composed of a large cell surrounded by albumin and encased in a shell membrane and a shell. The shell can be removed by soaking the egg in vinegar or acetic acid. The egg will serve as a living model system for studying osmosis. 1) Place the following solutions in 3- 250 ml beakers: a. distilled water b. 20 % sucrose c. 60 % sucrose 2) Carefully blot the outside of each egg. Tare the balance, determine the initial mass of each egg, and record it in the table on the next page. 3) Place each egg in one of the beakers and label the beaker to indicate the % of the solution in the beaker. Make sure that there is an equal amount of solution in each beaker. 4) Weigh the egg every 15 min for 1 hour. At the end of the required time, remove the eggs from the beakers and carefully blot them. Tare the balance, determine the mass of each egg, and record your data in the table below. 5) Calculate % change = [(final weight- initial weight)/ initial weight] x 100 Calculate rate of osmosis = (final weight- initial weight)/ 60 min
TIME (MIN) 0 15 30 45 60 % change (at t=60 mins) rate of osmosis (% / minute)
EGG IN WATER (g)
EGG IN 20% SUCROSE (g)
EGG IN 60% SUCROSE (g)
Which of the solutions are iso-osmotic, hypo-osmotic, or hyper-osmotic compared to the chicken eggs? _________________________________________________________________________ _________________________________________________________________________
Exercise 3: Diffusion of a liquid in a liquid
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Done by your instructor!

We can make some observations about the rate of diffusion and changes in concentration of a diffusing substance by observing the diffusion of a colored liquid in water.
Procedure 1. Fill a 4000 ml graduated cylinder to the top with water. Position the cylinder so that you can see the milliliter scale. Allow the container to stand undisturbed for a few minutes to be sure that all water convection has ceased. 2. Gently add a dropperful of methylene blue to the surface of the water. Take care to avoid disturbing the surface of the water. Cover the container with a Petri dish lid to prevent disturbance by air currents.
Done by you!
3. Observe the diffusion of dye in the liquid and answer the following questions a. What happens to the leading edge of the dye molecules?
b. What can you say about the chemical potential of the dye molecules at different levels of the cylinder?
c. Do you think that the dye molecules will become evenly distributed with more time? you think that it will take?
How long do
d. What do you think would happen if you used a smaller cylinder?
Exercise 4: Plasmolysis If a plant is immersed in a hyper-osmotic solution, water will leave the cell. Microscopically, increased loss of water and loss of turgor becomes visible as a withdrawal of the protoplast from the cell wall (plasmolysis) and as a decrease in the size of' the vacuole. Obtain a leaf from the tip of an Elodea plant. Place it in a drop of water on a slide, cover it with a coverslip, and examine the material first at low power (10x) and then at higher power (40x). Locate a region of healthy cells and sketch the location of the chloroplasts.
While touching one corner of the coverslip with a torn piece of paper towel to draw off the water, add a drop of 40% sucrose solution to the opposite corner of the coverslip. Be sure that the sugar solution moves under the coverslip. Wait about 5 minutes, and then draw your observations in the space below.
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Before
After
The 40 % sucrose solution is _______(hyper-, iso-, or hypo-) osmotic to the Elodea cells.
References: http://www.umanitoba.ca/faculties/science/biological_sciences/lab3/biolab3_3.html http://biog-101-104.bio.cornell.edu/BioG101_104/tutorials/cell_division.html http://arbl.cvmbs.colostate.edu/hbooks/cmb/cells/pmemb/transport.html http://www.trinity.edu/slibs/OsmosisWeb/default.html Barbara Cocanour and Alease Bruce in "Osmosis and The Marvelous Membrane" JCST November 1985, pp127-130.
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Lab exercise 6: Data* Analysis and Statistics

*Data is the plural form, datum is singular. The Nature of Science A critical part of the methods of learning about the universe that we call science is to gather data. Scientists then analyze the data and try to explain the conditions that caused them. Statistical analysis is simply a collection of methods for evaluating questions or predictions based on data. This lab has two objectives: 1) You should become familiar with the use of Excel spreadsheets to organize and summarize data, and to perform routine mathematical calculations. 2) You will perform a statistical comparison of mean growth rates in milkweed bugs grown under two experimental conditions. Organizing and Summarizing Data Data are usually a collection of either categories or numbers, corresponding to measurements or observations of particular variables. Variables are called variables because the observations for a particular variable usually vary (duh !). Observations are the individual measurements of a variable. Tables usually organize the data into columns of variables and rows of observations under each variable title. Recall from lecture that 1) the species Homo sapiens, to which we belong, varies widely in terms of height and brain size, 2) brain size seems to be generally related to height when comparing humans with some of the species from which we evolved (see figure below), and 3) brain size does not determine intelligence in humans.
Suppose you wished to determine whether head size depends in part on height in humans. We will begin by collecting head lengths (in cm), head widths (in cm) and heights (in cm) in a table on the whiteboard. Following the procedure explained by your lab instructor, use the plastic calipers provided on each lab bench to measure head length (in cm; from the point directly above the nose to the bony bump at the back of the head), head width (in cm; with the caliper positioned directly in front of each ear), and head height (in cm; estimated from right under your ears vertically to the crown of the head). All estimates should be to the nearest half centimeter. Two of the dimensions are shown in the photo above. Finally, estimate your body height (in cm) by multiplying your height in inches by 2.54. Record the class data in Table 1.
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Table 1. Morphometric measurements of head size and body height.
Head Length (cm)
Head Width (cm)
Head Height (cm)
Head Volume (cm3)**
Body Height (cm)
**HV = LWH/6. Calculated as shown below. Turn on your laptop, and download the file Brains and Bugs.xls from the Blackboard website to your desktop (make sure the desktop is in fact the screen and not a directory within the hard drive).
Open the file and note a first spreadsheet marked Brains (see the tab at the lower left) with the same variables you just measured (head length, head width, head height, and body height), and with a fifth variable, head volume, that has yet to be calculated. Enter the data for head length, head width and height under the correct column headings in the Excel worksheet. Note that, once the measurements have been entered, Excel automatically calculates an estimate of head volume. To find out what formula was used, click on the cell E3 in the spreadsheet and read the formula next to the fx button at the top of the worksheet. What is the formula? =____________________ In effect, you have just calculated head volume (HV) as HV = LWH/6, where L, W and H are the head length, head width and head height. This assumes that heads are approximately ovoid, which is only a very crude estimate of head volume, and is approximately twice the brain volume. To determine brain volume, the brain would usually be extracted, and the empty cranial cavity stuffed with metal shot, rice grains or some other non-compressible material. The material could then be poured out into a graduated cylinder to estimate its volume. That procedure is not performed in this lab .
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You now wish to determine the degree to which head size is influenced by body height. Which is the independent variable?__________ Which is the dependent variable? __________________. Graphing and Visualizing data Figures are graphs that provide (with a little practice) a visual summary of relationships among variables. One way to visually evaluate the effect of one variable on another is a scatterplot. The horizontal axis (the X-axis) is used to plot the observations for the independent variable, while the Y-axis is used to plot the observations for the dependent variable. Construct a scatterplot showing the effect of height on head size, using the independent variable as the X-axis and the dependent variable as the Y-axis, in Figure 1 below. To speed up the process, group members can each place a portion of the data in the figure, then copy the remaining data points from other group members. Label both axes and include the units of measurement in parentheses after each axis title (e.g., Height (cm)). Each point in the scatterplot actually represents a pair of observations, one for the independent variable and a corresponding observation for the dependent variable. How many points should there be in the scatterplot?
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To create a scatterplot in Excel, use your mouse to highlight the two columns Head Volume and Body Ht, including the column titles, then pull down under Insert to Chart. In the Chart Wizard Step 1, select XY (Scatter), and click on the sub-type without lines (upper left). Next. In the Chart Wizard Step 2, make sure you indicate that the series are in columns, and click Next. In the Chart Wizard Step 3, add appropriate chart and axis titles (including measurement units in parentheses at the end of each axis title), then click on Legend in the toolbar at the top of the window. Remove the legend. Next. Finally, in Chart Wizard Step 4, include the graph as an object in the current spreadsheet. Finish. If you have been successful, the resulting graph should resemble the hand-drawn plot in Figure 1 above. To obtain a line that expresses the overall effect of your independent variable on the dependent variable, click once on the chart to highlight it, then pull down under Chart to Add Trendline. In the Add Trendline window, make sure the linear option is specified, and click OK. A best fit line summarizing the relationship should now appear. When you are done, Save the file to your desktop where you can locate it easily later. Based on the data points and trendline, does the graph show a very tight relationship, with most of the data points close to the trendline and a clear slope to the trendline, or is the relationship very loose, with lots of scatter and a nearly flat trendline?
Based on your analysis, does height appear to influence head size?________________________ Graphing changes in body length of your milkweed bug over time Here youll use a second kind of scatterplot, in which successive observations are connected by straight lines. Using the data set compiled from your Adopt-a-bug measurements (mm), make a graph below to visualize your bug's length relative to time (days from the beginning of experiment). What is the independent variable in your milkweed bug growth experiment? ______________________What is the dependent variable? ____________________. Label both axes in below, including the units of measurement.
Now create a similar graph in Excel. Reopen the file Brains and Bugs.xls if necessary, and click on the tab for the second worksheet Your Bug at bottom of the screen. Enter your data in the two columns Time (d) and Length (mm) The data in the first column should be the number of days since you started taking data (include all days as integers (0,1,2,3,), even if you missed a measurement on a particular day). Then enter the length data for your bug in a second column (opposite the appropriate days), and a third column with your partners bug lengths further to the right (make sure all three columns are adjacent and have titles). Now highlight all three columns including column titles using your mouse. As before, pull down under Insert to Chart, and specify XY (Scatter). This time, however, choose the subtype with straight lines and markers (lower left option). Next. In Step 3, add axis titles (with units in parentheses). Next. In Step 4, add the Chart as an object in your current Sheet 2. Again save the file. Your bug probably showed a stair-step rather than linear progression of body lengths. Insects, unlike vertebrates, grow by molting. Size remains approximately constant between molts. Shedding the old exoskeleton during molting allows a brief period of rapid increase in size until the new exoskeleton hardens. How many times did your bug molt during the time you observed it? _______ A milkweed bug undergoes 5 molts from the time it hatches from an egg to the time it becomes a reproductive adult. How much does body length change between molts? ____ mm. Was the amount of the increase the same each time? _____________________.
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Variation in Growth Rate within Populations In order to compare the growth of bugs in the class, you need to calculate growth rates for each. Discuss how you would represent growth rate numerically as a class. Growth Rate = The milkweed bugs in your class all have slightly different genetic composition, differed in gender, differed slightly in age when you received them, and were likely subjected to slightly different living conditions. Thus, it is expected that there will be some variation both in the growth rate of growth and the final size. Despite all this variation among individual bugs, you are hoping to be able to discern the effects of one variable that was deliberately manipulated, dividing the bugs into two groups. The (independent) variable, defining the two groups, that was chosen by the class was _______________________________. The two values (treatments) of that variable were __________ and _________. In Table 2 below, list the value (treatment) of the independent variable tested (at the top of each column), and the growth rate for each bug in each treatment in the rows below the treatment titles.
Calculating Descriptive Statistics Using your bug data you will perform two sorts of statistical analyses: (1) the calculation of descriptive statistics and (2) hypothesis testing. Descriptive statistics dont directly test
predictions, but are useful in summarizing groups of data. Two kinds of descriptive statistics are the mean and standard deviation. The mean describes the average value for a group of observations. The standard deviation describes the amount of variation among values within the group of observations. The mean (X) is the average of the data as defined by the formula: X = X/n
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where is the sum of the individual data values (in this case the Xs are growth rates for the bugs). Thus, X represents the combined growth rates of all the bugs. The value for n is the number of observations (in this case the number of bugs). The standard deviation describes how the individual observations within the group of data are spread out around the mean. Large differences among observations (in this case, large differences in growth rates among individual bugs) will result in a larger value for the standard deviation. Leave the mean and standard deviations at the bottom of Table 2 blank for now. Excel will make the necessary calculations for you, but it is important that you know the meaning of both the mean and standard deviation.
Growth rate in two groups of milkweed bugs Treatmentt 1 = Growth Rate (units= ) Treatmentt 2 = Growth Rate (units= )
Mean for trt 1: Std. Dev. for trt 1:
Mean for trt 2: Std. Dev. for trt 2:
You are now going to analyze the milkweed bug growth data in Excel. Reopen the file you saved earlier if necessary, and click on the third worksheet Growth Rates.
Now enter the data for your lab section in columns in the same format as shown in Table 3 above (with 2 columns, including column titles for each treatment and using one row for each bug in the cells below). If you need to, you can widen your columns by clicking on the line between letters (e.g., between A and B) and dragging the column wider. Save the file once more. Now you're ready to analyze your results.
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Computing Descriptive Statistics Click on cell B25 at the bottom of the first column. Click on the Paste Function button on the tool bar, marked fx. Click Statistical under the function category and specify Average for the function name. OK. Click OK again in the next dialog box. The average milkweed bug growth for the bugs in the first treatment should appear in your spreadsheet. To find the standard deviation, click on cell B26. Click on the Paste Function button on the tool bar, marked fx. Click on Statistical under function category as before, and specify Stdev for the function name. OK. Click OK again in the next dialog box. Now compute the mean and standard deviation for the growth rates of bugs in group two at the bottom of the second column. Enter all estimate in Table 2 of your lab book. Hypothesis Testing Hypothesis testing begins by asking a question. In the data involving diet and cancer, for example, the question might have been Is head size influenced by height? What is an appropriate question involving growth rates of the two groups of bugs recorded in Table 2?
Question: The next step is to formulate two hypotheses or possible outcomes related to the question. The first hypothesis is termed the null hypothesis, and in this instance describes the outcome if there is NO effect of treatment. What is an appropriate null hypothesis in this instance?
Null Hypothesis: The other hypothesis, often termed the alternative hypothesis or prediction, is the outcome if there is an effect of treatment. State your alternative hypothesis (prediction) for this experiment. The way you decide among these two possible outcomes is to either accept or reject the null hypothesis. If you reject the null hypothesis, you are really deciding that there is an effect of treatment.
Prediction: A statistical test gives you the necessary information to either accept or reject the null hypothesis. There many types of statistical tests, appropriate for answering different kinds of questions. In this instance you will use a two-sample t-test, which evaluates whether the mean values of observations in two groups of data differ significantly. Now here is the hardest part. A scientists rarely knows with absolute certainty, if the null hypothesis is rejected, whether he or she is right!
As an example, have another look at Figure 3. The null hypothesis for the data is The two means are equal. It sure LOOKS like the two treatments have different means. But suppose the study were repeated, with a different set of observations? How sure are you? Being wrong can be quite embarrassing, so biologists typically limit the likelihood of incorrectly rejecting the null hypothesis. And each statistical test provides a statement of significance that tells the scientist what the probability of being wrong actually is. By convention, most biologists are willing to reject a null hypothesis only if the chance of being wrong (called p or the statistical significance of a test) is less than 1 in 20 (5%), or if p<0.05. Of course, the smaller the actual p value (much less than 0.05), the more confident you are that your decision to reject the null hypothesis is the correct one. Why not instead only reject the null hypothesis if your chance of being wrong were, lets say, less than one chance in a thousand (p<0.001)? The answer is because then the chances of correctly rejecting the null hypothesis when there really is a difference become vanishingly small; in other words, real differences between treatments, or real relationships between variables, would only rarely be uncovered, and discoveries of potential significance to science would be rare. Now its time to either accept or reject your null hypothesis regarding the bug data. Click on cell E17 to the right of the block of data. Then click on the Paste Function button on the tool bar, marked fx. Under the function category click Statistical and t-test for function name. Click OK. The t-test dialog box should appear. Now you need to indicate what data you wish to test. If your cursor isn't already there, click inside the Array 1 box. Now indicate the data in treatment 1 by dragging your cursor over the data in the second column, containing the growth rates of the first treatment. Your data range will automatically appear in the Array 1 box. Now click in the Array 2 box and drag your cursor over the growth rate data for treatment 2. For the number of tails enter 2, and for the type of test enter 2 (dont worry about why). Now click OK. Excel will automatically calculate a (2-tailed 2-sample) t-statistic and enter the resulting statistical significance, or p value, in the cell. What is the chance that you would be wrong if you reject your null hypothesis? ____________________________
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Based on the t-test, should you decide to accept or reject the null hypothesis? ____________________________
What is your conclusion regarding the results of the experiment?
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Lab exercise 7: Fermentation and Metabolism

Lipids, carbohydrates, nucleic acids, and proteins are necessary components to living things yet are very difficult to synthesize in even the best-equipped laboratories. The chemical reactions are hard to start and often produce unwanted side reactions and byproducts. It may seem strange that simple cells can produce macromolecules that highly trained chemists cannot. This is because even the simplest cells have specific tools that are synthesized to facilitate difficult and diverse chemical reactions. These tools are catalytic enzymes. Enzymes are usually proteins (although some RNA molecules also have catalytic properties). These enzymes have a complex three-dimensional shape consisting of one or more polypeptide chains folded to form an active site. The molecules that fit into the enzymes active site are called the substrates. When the substrate binds to the enzyme, it is affected by the specific functional groups of the amino acids of the active site and undergoes a chemical reaction to form a product. The enzyme is not changed by the reaction and is capable of immediately binding to another substrate molecule.
E + S E-S E + P
The conformation of an enzyme is crucial to its activity. Thus, anything that alters the enzyme structure such as changes in pH, temperature, ions, detergents, or inhibitors can influence the enzymes activity. Cells depend on the formation (anabolic reactions) of complex macromolecules, such as DNA, to survive. These types of chemical reactions require chemical energy and complex pathways catalyzed by enzymes. Adenosine triphosphate (ATP) is the energy-rich compound most often used by cells to synthesize polymers from monomers. The major source of ATP for many cells is the oxidation of glucose:
C6H12O6 + 6O2 6CO2 + 6H2O (aerobic respiration)

The oxidation takes place in two major stages. The initial process is called glycolysis and results in the splitting of the glucose molecule into two 3-carbon molecules of pyruvic acid (pyruvate). Glycolysis can proceed in the presence or absence of oxygen. The energy released from this pathway is sufficient to produce a net gain of 2 ATP molecules. During anaerobic (oxygen absent) conditions, some organisms metabolize pyruvic acid to other compounds, such as lactic acid (lactate fermentation) or ethanol and CO2 (alcoholic
fermentation) in order to regenerate cytoplasmic NAD+. For example, in the absence of oxygen, our muscles produce lactic acid but yeast produce alcohol and carbon dioxide (see Figure 1 on the next page).
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C6H12O6 2CO2 + 2C2H5OH (alcoholic fermentation)

When oxygen is available, aerobic respiration proceeds through the Krebs cycle and electron transport. These reactions will yield 36-38 ATP per molecule of glucose.
http://www.accessexcellence.com/AB/GG/ana_Pyruvate.html
Fermentation pathways. (A) When inadequate oxygen is present, for example, in a muscle cell undergoing vigorous contraction, the pyruvate produced by glycolysis is converted to lactate as shown. This reaction restores the NAD+ consumed in step 6 of glycolysis, but the whole pathway yields much less energy overall than complete oxidation.
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(B) In some organisms that can grow anaerobically, such as yeasts, pyruvate is converted via acetaldehyde into carbon dioxide and ethanol. Again, this pathway regenerates NAD+ from NADH, as required to enable glycolysis to continue. In this laboratory, you will perform experiments to observe the processes of fermentation and metabolism.
Exercise 1: Production of carbon dioxide gas by fermentation Yeasts are simple unicellular fungi that are classified as facultative anaerobes- they can live in either anaerobic or aerobic environments. Under anaerobic conditions, yeasts carry out alcoholic fermentation to produce alcohol and carbon dioxide. A capillary tube with a culture of fermenting yeast will be used to measure the displacement of the liquid by gas. Different groups will investigate the effects of different variables on the rate of gas production by yeast. Variables to be tested include 1) different substrates and 2) the addition of a metabolic inhibitor. Procedure: 1) Each student will fill a plastic disposable tube to the bottom etch mark with bakers yeast.
Fill to this mark 2) Add 10 drops of buffer and mix for 3 minutes until the mixture becomes a thick slurry.
3) Add 5 drops of the assigned carbohydrate solution (see table on the next page) or water and mix for an additional 2 minutes. 4) Using a capillary tube open at both ends (1.5-1.8 X 100mm), mark the tube with a Sharpie marker at the midpoint of the tube. Fill the tube to this mark by capillary action by placing the capillary tube into the test tube and holding the tubes so that they are nearly horizontal. Be careful to prevent air from entering the capillary tube, as it should be a continuous column of the yeast solution. 5) After filling, the open end of the half- filled capillary tube should be closed with your index finger to prevent loss of solution. The solution end of the capillary tube is then inserted into the critoseal and gently pushed to the bottom of the container with a slight twisting motion. The sealed tube is then placed in one of the numbered receptacles on either end of tray with the open end downwards (critoseal end is up). The capillary tube should look like this:
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6) Place the tray containing the tube under an incandescent lamp to increase the temperature of the reaction. 7) The mark on the tube will be your 0 mm mark at T = 0. Measure the distance that the fluid level moves downward every 2 minutes until the solution gets to the end of the capillary tube. Remove the capillary tube from the tray and place the capillary tube in a paper towel to catch the solution as it moves out of the bottom of tube. 8) If your solution does not move within 12 minutes, stop your reaction.
Data for exercise 1 TIME (MIN) 0 2 4 6 8 10 12 mm gas/mi n 9) Water Gluc Fruct Galacto Sucr Lact lactose + lactaid Glucose + Na bisulfite
Share your results with everyone at your table and fill in the table above.
Questions to consider: 1) Does the rate of fermentation depend on the carbohydrate fermented? _________ Are there differences between monosaccharides and disaccharides? ___________ 2) Which carbohydrate generates the greatest gas production by the yeast? ______ produces the least amount of gas? ______________ Which carbohydrate
3) What effect does Na bisulfite have on CO2 production? ____________ Sodium bisulfite reacts with acetaldehyde to prevent CO2 formation before it can be reduced to ethanol and, therefore, inhibits fermentation. Graph the results from the glucose, fructose, galactose, sucrose, and lactose experiment on the graph paper and determine the rate of gas production (mm gas/ min). Exercise 2: Examining Oxidation-Reduction Reactions in living cells When cells metabolize food for the production of energy, they oxidize energy-rich (electronrich) substances, resulting in a loss of electrons.
During catabolism, a substrate (such as a carbohydrate) is oxidized initially without the participation of oxygen. Electrons (H atoms in biological reactions) released by enzymatic reactions are accepted by coenzymes (like NAD+ and FAD) that become reduced. The reduced coenzymes (such as NADH + H+ or FADH2) are oxidized by the electron transport chain. The final electron transport carrier is oxygen during aerobic respiration.
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Part 1: Reduction of methylene blue by yeast In the absence of oxygen, electrons may be diverted from the electron transport chain by an oxidation-reduction mediator, such as methylene blue. Methylene blue is normally blue in the oxidized state but becomes colorless when it is reduced. The methylene blue enters the cell, becomes reduced, and leaves the cell in the reduced state. If the cells are metabolically active, the colorless condition will remain indefinitely. The rate of decolorization depends on the metabolic activity of the cells and the presence of oxygen. The equations for the reaction are:
O2 + MBred (colorless) MBox (blue) MBox MBred + R

where MBred is the reduced (colorless) form of methylene blue, MBox is the oxidized (blue) form of methylene blue, and RH represents glucose. Procedure: 1) Label and set up six test tubes with the following reagents: Note: (ADD 1 g YEAST TO 5 ML BUFFER) = ONE PER TABLE. FROM THIS MIXTURE, YOU WILL ADD THE YEAST DROPS INDICATED IN THIS LAST COLUMN
TUBE 1 2 3 4 5 6
BUFFER 2 ml 2 ml 2 ml 2 ml 2 ml 2 ml
DYE 1 drop 1 drop 1 drop 1 drop 1 drop
WATER 15 drops 11 drops 10 drops 5 drops 5 drops
GLUCOSE
NA BISULFITE
YEAST
5 drops 5 drops 5 drops 5 drops
5 drops 5 drops 5 drops 5 drops (Boiled)** 5 drops
Cover the tubes with parafilm and incubate at room temperature for 30 min.
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2) Record your results in the table below. The intensity of color can be graded 0 for no color, +, ++, +++, ++++ indicate increasing intensities of color.
**The boiled yeast will be provided by your instructor.
TUBE 1 2 3 4 5 6
COLOR AT T= 0
COLOR AT T = 30 MIN
EXPLANATION OF RESULTS
4) Which tubes were used as controls?
5) Compare the results in tubes 3 & 4? Is there any difference? If so, can you account for the difference?
What effect did boiling the yeast have on the reaction? Explain.
What effect did adding inhibitor have on the reaction? Explain.
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Part 2: Staining yeast with methylene blue Living cells will reduce methylene blue, resulting in the colorless form. What happens when cells are damaged or cease to metabolize fuels? Prepare a smear (lightly rub the end of the pipette on the slide) of living and boiled yeast from tubes 4 and 5. Draw your results in the space below. Pay particular attention to the color of the yeast.
Does methylene blue get into the cells? How do you know?
Is there a different staining between unboiled and boiled yeast? If so, why?
References http://www.uwrf.edu/biotech/workshop/activity/act1/act1.htm#labfy http://www.oxy.edu/departments/tops/Yeast/yeasthome.htm http://www.biology.arizona.edu/biochemistry/problem_sets/metabolism/metabolism.html http://www.phys.ksu.edu/gene/a1.html http://tidepool.st.usm.edu/crswr/yeastfermmov.html "Fermentation, Respiration, and Enzyme Specificity: A Simple Device and Key Experiments with Yeast," by L. Reinking, J. Reinking, and K. Miller, The American Biology Teacher, Vol. 56, March 1994, pp. 164-168.
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Lab exercise 8: Mitosis, Meiosis, and Life Cycles

One of the basic principles of the cell theory is that all cells arise from pre-existing cells. Cells arise from others by karyokinesis (nuclear division) and cytokinesis (cytoplasmic division). There are two types of nuclear division: mitosis and meiosis. During mitosis, two daughter nuclei are formed that are genetically identical to the original cell: (2n 2n, diploid to diploid). In diploid organisms, chromosomes occur as matched pairs (homologous chromosomes), one paternal and one maternal in origin. Meiosis, however, reduces the number of chromosomes to half the number present in the original cell (2n->n, diploid to haploid). Let us examine the phases the cell cycle when the cell is dividing by mitosis: I) Interphase= preparing for cell division * G1 (first gap) is a period of cell growth and doubling of organelles. * S is the period when new DNA is synthesized. * G2 (second gap) is when preparations are made for cell division, i. e., synthesis of spindle proteins and ATP used during division. In animal cells, a pair of centrioles divides to form two pairs of centrioles. Notable features: DNA is uncoiled and distinct chromosomes are not visible. The chromosomes appear as a diffuse, darkly stained material called chromatin. During the S phase, DNA is replicated and each chromosome is composed of two helices of DNA called sister chromatids. The chromatids are joined at a central region called the centromere, which contains a kinetochorea place where the spindle fibers can bind. A chromosome composed of two chromatids is called a dyad.
II) Mitosis = separation of the replicated chromosomes (4 phases) * Prophase: chromatids become visible, the nuclear membrane and nucleoli disappear, centrioles migrate to opposite sides of the nucleus, and spindle fibers attach to the kinetochore. * Metaphase: the chromosomes line up in the middle of spindle apparatus (the metaphase plate). * Anaphase: the sister chromatids separate into monads and move toward opposite poles. The monads are called daughter chromosomes. * Telophase: sister chromatids have completely separated and begin to uncoil, the spindle apparatus disappears, the nuclear membrane and nucleoli reappear. III) Cytokinesis = the cellular material is divided between the two daughter cells. Daughter cells are usually smaller following cytokinesis compared to other cells in interphase.
Exercise 1: Mitosis in an eukaryote
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Part A: (Allium) root tip Kingdom = Plantae, Phylum = Anthophyta Use this slide to locate the different stages of the cell cycle in plants. Do you find more cells in various stages of mitosis at the root cap or at the apical meristem? ________ What do you suppose is the function of those two regions? ________________________ Focus on the apical meristem. Make drawings and label the nuclear changes that occur during the 5 major stages in the cell cycle (IPMAT).
Mitosis Jumble: Can you determine the correct sequence of events for mitosis? a) Spindle apparatus disappears and nuclei reform. Nucleoli reappear. b) Chromosomes line up at the middle of the spindle apparatus. c) Centrioles replicate and separate. d) DNA replicates. e) Cytoplasmic division. f) Cell grows in size and organelles double. g) Sister chromatids separate and move toward opposite poles. h) Spindle apparatus forms. What is the correct order for mitosis? ___________________________________ Part C: How long does it take for the onion root tip cells to complete the cell cycle? When studying populations of dividing cells, biologists often need to know the length of the cell cycle. If a cell biologist wished to know the effect of an environmental factor such as CO2 level on the cell cycle, it would be necessary to know the length of the cycle under normal and altered conditions. The technique described below only works for randomly dividing populations of cells. For our determination of cell cycle, we will use the onion root tip slide. We will assume that the cells have been treated for 30 min with colchicine, a chemical that stops nuclear division in metaphase by preventing microtubule polymerization. In addition, we will assume that the fraction of the cells in metaphase in an untreated population is 0.01 (1%).
To calculate the cell cycle, fraction of cells in metaphase after colchicine - fraction of cells in metaphase under normal conditions (0.01) = fraction of cells in metaphase in T = 30 min
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Lets say that 0.21 of the colchicine-treated cells are in metaphase (This is a high estimate for these kind of cells, used for simplicity). It means that 0.20 (0.21-0.01) of the cells reached metaphase in 30 min. Thus, 30 min represents 0.20 (20% or 1/5) of the cell cycle:
Cell cycle = T/F

where T = time of the colchicine treatment and = fraction of cells reaching metaphase. For the example above, the cell cycle would be 30 min / 0.2 = 150 minutes Work with a partner where one of you serves as the observer while the other will record the data. The observer scans up and down the vertical rows of cells visible within the field at one time, calling off judgments on the stages of the cell cycle encountered. The recorder marks down the data in the table below. Six such field counts should be made (two slides, as there are 3 root tips per slide), with the observer and recorder changing roles after the third field count. It is suggested that only the three most central rows be counted in each field. Using the 40 x objective, you should see something like this (keep in mind that your field of view will be a circle):
In this example, there are about 144 cells, of which 4 are in metaphase (4/144 = 0.028). Therefore, the fraction of cells in metaphase is will be given by the following relationship (as above): o Fraction of cells in metaphase after colchicine (0.028) - fraction of cells in metaphase under normal conditions (0.010) = 0.018
Assuming a T = 30 minutes, the length of the cell cycle would be: 30/0.018 = 1,667 minutes (~ 27 hours or about 1 day and 3 hours).
Using your own data, determine the length of the cell cycle using the formula on the previous page. Complete the table below. Field Of View 1 2 3 4 5 6 Total Number Of Cells Number Of Cells In Metaphase Fraction of Cells Length Of The Cell In Metaphase Cycle
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Exercise 2: Meiosis and life cycles Meiosis is a form of nuclear division in which the newly formed nuclei contain one half the number of sets of chromosomes as were present in the original nucleus. Meiosis turns diploid nuclei (nuclei with two sets of chromosomes, 2n) into haploid nuclei (nuclei with only one set of chromosomes; one chromosome from each and every "pair" in the original). Any organism that reproduces sexually must have a stage in the life cycle where meiosis occurs to ensure that the total number of chromosomes stays constant over generations. The fusion of haploid (n) nuclei following fertilization produces a zygote (2n). IMPORTANT NUCLEAR EVENTS DURING MEIOSIS Meiosis consists of two nuclear divisions (Meiosis I and Meiosis II) and results in the production of 4 daughter nuclei. The interphase stage of the cell cycle immediately preceding meiosis I is similar to interphase in mitosis (see first page of this lab).
MEIOSIS I
PROPHASE I: Uncondensed homologous dyad chromosomes are brought together to form uncondensed tetrads. Chromosomes condense, the nuclear envelope disappears, and tetrads become attached to the spindle (a tetrad is composed of two homologous dyad chromosomes). Homologous chromosomes synapse (get close together) and may break and rejoin. This process is called crossing over. This is the first major difference between mitosis and meiosis. METAPHASE I: Condensed tetrads are moved to the equator of the spindle. ANAPHASE I: The tetrads are separated as homologous dyad chromosomes move toward opposite poles. This is a second major difference between meiosis and mitosis. In mitosis, each homologous chromosome independently separates into monads.
TELOPHASE I: Condensed dyads are at the poles of the spindle and the spindle subsidesor disappears. By this time the sets of chromosomes have been halved. Each nucleus is haploid, but chromosomes are still in the dyad condition. The condensed dyads may go to the uncondensed state (resulting in a stage called interkinesis.) Interkinesis is a third major difference between mitosis and meiosis. During this time the DNA may again become functional. In some cells, the dyad chromosomes may remain in the condensed state and proceed directly to Prophase II.
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MEIOSIS II
METAPHASE II: The dyad chromosomes are moved to the equator of the spindle. ANAPHASE II: Each dyad chromosome is split into two monads, which move to opposite poles of the spindle. TELOPHASE II: The condensed monad chromosomes become uncondensed monad chromosomes and the nuclear envelope reforms. Meiosis is now complete. In summary: The first meiotic division separates sets of chromosomes, resulting in haploid nuclei, but the chromosomes are still dyads. The second meiotic division reduces dyad chromosomes to monads. THE ROLE OF MEIOSIS IN LIFE CYCLES The objective of this exercise is to illustrate the role of meiosis in the reproductive life cycle. There are three basic life cycle patterns which can be described by the role that meiosis plays. They are: A) Gametic- meiosis is involved in providing nuclei for gametes. This pattern is typical of animals and some protista. B) Zygotic - the zygote nucleus goes through meiosis before the life cycle continues. This pattern is very common among the protista (algae) and the fungi. C) Sporic - meiosis occurs in 2n sporogenous cell nuclei, providing nuclei for spores. This pattern is found in the plants and some protista. The Gametic life cycle = e.g. Homo sapiens (Sorry, no lab experiments for this one)
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Spirogyra is a green filamentous alga commonly found in ponds and water filled ditches. In the vegetative state, the cells contain one or more spiral chloroplasts and a haploid nucleus suspended in the cell vacuole by cytoplasmic bridges. At the time of sexual reproduction, two filaments come to lie parallel to each other. Projections from the cells begin to grow toward each other. These eventually join, forming a conjugation tube. The vegetative cells then go through a morphological change and the protoplast (cell membrane and the cellular contents) functions as a gamete. The protoplast in one cell moves by amoeboid movement through the conjugation tube and fuses with the gamete in the other cell, forming a zygote.
The zygote produces a thick wall around itself and becomes a zygospore. The parent filaments disintegrate, releasing the zygospore into the environment. The zygospore is a resting stage and allows the species to withstand environmental conditions that are unfavorable to the vegetative form. When environmental conditions are favorable, the 2n zygospore nucleus goes through meiosis producing four haploid nuclei. Three of these nuclei disintegrate, leaving a cell with one haploid nucleus. This cell then escapes from the zygospore wall and produces the typical vegetative filament by mitosis.
Part A: Observing the life cycle of Spirogyra Using prepared slides of Spirogyra, observe the organism in its vegetative form and locate a chloroplast and nucleus. Observe the sexual stages and identify - the vegetative form, conjugation tube, gamete, and zygote. Label the diagram below.
The Sporic Life Cycle = e.g. fern The ferns will be used to illustrate the sporic life cycle. In this life cycle there are two generations produced, a sporophyte generation (nuclei diploid) that produce spores, and a gametophyte generation (nuclei haploid) that produce gametes.
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Part B: Observing the life cycle of fern The sporophyte generation is composed of roots, stem, and leaves. The most obvious structures are the leaves or fronds. Observe the frond of the fern on demonstration and note the brownish clusters on the underside of the leaflets. These clusters are called sori (singular = sorus). Each sorus is a collection of sporangia in which meiosis and cytokinesis has occurred to produce spores. Obtain a slide of Fern leaflet and locate the sporangia and spores. Draw a picture of the fern leaflet in the space below. After the spores geminate, they produce a tiny, green flat heart-shaped, plant body called a prothallium. This is the gametophyte generation. The prothallium grows on the surface of the soil. The sex organs are borne on the lower or ventral surface of the prothallium. Study the mature prothallia on demonstration and identify the sex organs, the antheridia (male) and the archegonia (female). The antheridia are usually formed at the point of the heart-shaped prothallium and appear as small bumps. The archegonia are usually formed at the cleft of the heart-shaped prothallium and appear as small finger- like projections. Sperm are released at a time when there is a layer of water on the soil to allow the sperm to move to an egg in an archegonium. The zygote is retained within the archegonium and during the early stages of embryonic development. The prothallium provides nourishment for the developing sporophyte. Study the prothallium with attached sporophyte on demonstration.
Know the structures of the fern on the diagram belowgote, sporophyte, sori, gamete, spore, gametophyte
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References: http://www.umanitoba.ca/faculties/science/biological_sciences/lab3/biolab3_5.html#Mitosis http://www.umanitoba.ca/faculties/science/biological_sciences/lab5/biolab5_2.html#Meiosis http://www.biology.arizona.edu/cell_bio/activities/cell_cycle/cell_cycle.html http://biog-101-104.bio.cornell.edu/BioG101_104/tutorials/cell_division.html
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Lab exercise 9: Mendelian Genetics

Gregor Mendel discovered that hereditable factors come in pairs. We now call these factors genes. In some cases a gene is a piece of DNA in a chromosome that codes for a specific protein that will become a distinct trait, such as the height of a plant, the color of a bird, or part of a chemical pathway. More often, however, a gene codes for a protein that is a part of a complex set of instructions that may include other genes. While diploid cells have two copies of each gene, the copies may not be identical. For example, each of our cells has two genes that control hair color, but one gene may code for light hair and one may code for dark hair. The different versions of a gene are called alleles. The different alleles of a gene both produce a protein. However, these proteins are not expressed equally based on the appearance of an individual (phenotype). For each trait, one allele is usually dominant and the other is recessive. A dominant allele is evident in the phenotype whenever it is present, even if only one copy is present. A recessive allele must be present in two copies to be apparent in the phenotype. Mendel studied pea plants in order to understand hereditable factors. He discovered that when purebred (homozygous, RR = genotype) plants that produce round seeds are crossed with purebred (homozygous, rr = genotype) plants with wrinkled seeds, the first generation (F1) of plants all have round seeds. Thus, the round seed trait is dominant over the wrinkled seed trait (recessive). The offspring from this mating are called hybrids (heterozygous, Rr = genotype). Mendel found that crossing the hybrid offspring led to 75% of the offspring with the round seeds and 25% of the offspring with wrinkled seeds (3:1 ratio). These experiments led Mendel to postulate that each individual carries two factors for each trait.
Exercise 1: Why Do Mendel's Peas Wrinkle? This lab is designed to demonstrate the connections between genotype and phenotype. The sequence of events that influence the production of round and wrinkled seeds is shown on the next page. This complex process involves the synthesis of different forms of an enzyme that lead to the production of starch, accumulation of sugar (sucrose) with the recessive form of the enzyme, differences in the osmotic potential, and water loss with desiccation. Remember that starch is a polysaccharide made up of many glucose monomers bonded together. Specific enzymes are needed to link the glucose onto the growing starch molecule. The R allele produces a starch branching enzyme that, as the name implies, adds branches of glucose units to the growing starch molecule The starch branching enzyme also influences other enzymes (coded by other genes) and their ability to synthesize starch. The r allele codes for a mutant form of starch branching enzyme, leading to an inefficient production of starch.
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You will prepare an extract of both round and wrinkled pea cells to test their: 1) Capacity to form large starch granules whose shape is determined by the type of starch formed, and 2) Difference in osmotic potential that results from sugar accumulation.
Figure 1: Mechanism responsible for round and wrinkled pea shape Procedure: 1) Take a sample of 5 dried peas. Determine the mass of 5 dried round peas and 5 dried wrinkled peas. Record your results in the table below.
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2) Take a sample of 5 presoaked peas. Determine the mass of 5 soaked round peas and 5 soaked wrinkled peas. Record your results in the table below.
PEA TYPE round wrinkled
DRY MASS (G)
WET MASS (G)
CHANGE IN MASS (G)
Which peas gained the most weight? ___________________________ 3) Take 2 presoaked peas of each type from the side counter. Grind the round peas with a small amount of water using a mortar and pestle. Try pushing hard with the pestle while twisting against the mortar until no large pieces remain. Repeat this procedure for the wrinkled peas. MAKE SURE YOU THOROUGHLY WASH THE MORTAR AND PESTLE WITH SEVERAL RINSES OF TAP WATER between each variety. 4) Using a pipet, make a wet mount slide for each variety of peas and cover it with a coverslip. Observe the extract from both the round and wrinkled peas using the 40x objective. 5) Stain the slide with Lugols solution by touching a drop of Lugols to one side of the coverslip and a piece of a paper napkin to the other side of the coverslip. Observe with the high power objective. 6) Compare the types of starch granules. Some grains are compound (see fig. 2 below), i.e., an aggregation of individual granules and some are simple with just one grain.
Figure 2: Diagrammatic representation of compound starch grains found in wrinkled peas (simple grains not shown).
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7) Draw a picture of your observations below. ROUND WRINKLED
Question 1: Did you measure any differences in the osmotic potential of the round and wrinkled peas? Explain.
Question 2: Are there differences in the starch grains of wrinkled and round peas? Explain.
Exercise 2: Human Genetics: what will your children be like? The traits described by classical Mendelian genetics demonstrate simple dominant-recessive characteristics because the trait has only 2 alleles. Polygenic traits, such as skin color or height, depend on several genes and their interaction with each other and with the environment. Traits that have only a few discreet phenotypic states are called meristic traits. Several meristic traits have been identified in humans that illustrate Mendelian genetics quite well. A few are listed below: 1) Acondroplastic dwarfism: The dominant allele causes dwarfed arms and legs; normal proportions are recessive. 2) Attached earlobes: Free earlobes are dominant. 3) Bent little finger: Bent little finger is dominant, a straight little finger is recessive. 4) Pigmented irises: Lack of pigment in the iris results in blue eye color, which is recessive (p). Any other eye color (brown, green, hazel, violet, etc.) is due to the deposition of pigment in the iris and the expression of the dominant allele of the gene (P). 5) Curly hair: Curly hair is dominant over straight hair. Wavy hair is heterozygous. 6) Dimples: Dimples in your cheeks or in your chin are dominant.
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7) Freckles: Freckles on your face are dominant. 8) Interlacing fingers: Relax and casually fold your hands together so that your fingers interlace. If you left thumb is on top of your right thumb, you have the dominant allele. If your right thumb is on top, you are homozygous recessive. 9) Hitchhikers thumb: Place your thumb in a hitchhikers position. If the tip of your thumb curves downward, you have the dominant allele. A straight thumb is the recessive allele. 10) PTC tasting: Touch some test paper impregnated with phenylthiocarbamide to your tongue. If you detect a bitter taste, you have a dominant allele. 11) Tongue rolling: The ability to curl your tongue in a U-shape indicates the dominant allele. 12) Widows peak: A pointed hairline is determined by the dominant allele. Determine your own phenotype for each trait. Can you determine the genotype? Your instructor will tabulate the data for the whole class for these traits. Fill in the chart below with the results. Are the dominant phenotypes always more common than the recessive phenotypes? ______________
DOMINANT TRAIT PRESENT ABSENT Acondroplastic dwarfism Free earlobes Bent little finger Pigmented iris Curly hair Dimples Freckles Interlacing fingers (L/R) Hitchhikers thumb PTC tasting Tongue rolling Widows peak Not all common traits are a matter of simple dominance. Blood types, for example, exhibit a property called codominance. As you probably know, there are 4 major blood groups, A, B, AB and O. These four types are influenced by multiple alleles of a single gene. Blood type describes a particular glycolipid on the surface of red blood cells. The allele IA produces the type-A glycolipid and the allele IB produces a type-B glycolipid. Both of these alleles are dominant over i, which produces neither glycolipid A nor B. The presence of the two dominant alleles is what makes blood type an example of codominance. Thus, six possible genotypes will give the four phenotypes. Type A Type AB IB IB or IBi i i Type B Type O
IA IA or IAi IA IB
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Several traits in humans are located on the X or Y-chromosomes. These are called sex-linked traits. Genes for sex-linked traits are usually carried on the X chromosome and are absent on the Y chromosome. As a result, males only have a single copy of any gene located on the X chromosome and they will express whichever allele is present, even if it is normally a recessive allele. The gene that controls color vision is located on the X chromosome and has two alleles: normal (XB) and colorblind (Xb). The normal condition is dominant over the colorblind condition. Females that have normal color vision, but are heterozygous for colorblindness (XB Xb) are often referred to as carriers. Males that have inherited the gene for color-blindness (XbY) are color blind. Can females be colorblind? ___________________
Why?_____________________________________________________________________________ __________________________________________________________________________________ What will your children be like? For this activity, we will observe patterns of inheritance for three general types of traits: an autosomal trait controlled by two alleles (hair texture), an autosomal trait controlled by multiple alleles (blood type), and a sex-linked trait (color-blindness). To gain experience with genetics, you will work with a partner to determine the combination of traits that would be possible in your offspring. Here is how it works: Step 1: Get a partner: Men will draw a card from the bag marked males and women will draw a card from the bag marked females. If there isnt an equal number of men and women in the class, some people will play the role of the opposite sex. Step 2: Determine your traits. The three traits listed on the card are hair type, blood type, and colorblindness. Write them in the space below:
Step 3: Determine your genotype. Your hair is curly (HH), wavy (Hh), or straight (hh). Your blood type is A, B, AB or O. If your blood type is A or B, you can assume that the genotype is IAi or IBi, respectively. You are a normal-vision (XBY) or color blind male (XbY). Alternatively, you are a normal vision (XB XB ), carrier (XB Xb), or color-blind female (XbXb). Write them in the space below.
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Step 4: Determine all the possible gametes that you could contribute to your offspring. Remember, each gamete will only get one allele from each pair of homologous chromosomes. Write them in the space below.
Step 5: How many possible gametes could your mate have? Write them in the space below.
Step 6: Look at the various combinations of your gametes to answer the following questions. 1. Will any of your children be colorblind? If so, what are the chances that your sons will be color blind? Your daughters?
2. Will any of your children have curly hair? Wavy hair? Straight hair? What are the ratios of the different hair types?
3. What are the possible blood types of your children?
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4. Could you possibly produce a son who was colorblind, had wavy hair, and A type blood?
5. Can you predict the phenotype of your first child based on the most likely combination of these three traits?
References http://esg-www.mit.edu:8001/esgbio/mg/mgdir.html http://biology.clc.uc.edu/courses/bio105/geneprob.htm http://www.blc.arizona.edu/courses/181gh/rick/genetics1/mendel.html http://www.biology.arizona.edu/mendelian_genetics/mendelian_genetics.html http://www.furman.edu/~lthompso/pealab.htm http://www3.ncbi.nlm.nih.gov/Omim/
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Lab exercise 10: Population Growth of Duckweed

In this laboratory you will 1) determine whether the growth of individual duckweed plants is density dependent (involving intraspecific competition), 2) experimentally manipulate nutrients and/or light to determine their effects on growth, and 3) mathematically simulate the effects of varying growth rate and carrying capacity on duckweed growth using a computer model. This is a two-week lab. During the first week you will decide on an experimental design and set up your experiment. During the second week you will analyze the growth response of the plants.
Duckweeds (genus Lemna) are free-floating aquatic plants that undergo continuous growth. Individual plants are stemless and have 1-4 leaves. Roots from the leaves hang down into the water and absorb nutrients needed for growth. Leaves typically reproduce asexually by producing new leaves which, when large enough and equipped with their own roots, break off from the parent to form a new plant.
Fig. 1. Four plants of Lemna minor, each with three leaves
Week 1:
You first wish to determine whether the growth of the duckweed plants is density dependent. To do this you will measure the per individual growth rate (PIG for short). The PIG can be approximated for relatively short time periods using the equation PIG = ln(Nwk2) ln(Nwk1) (1)
where ln(N) is the natural log of the population size. PIG can be computed using most calculators, but in this lab you will use Excel to do your calculations. For example, if a duckweed population increased from 10 leaves to 30 leaves in a week, the PIG = ln(30) ln(10) = 3.4 2.3 = 1.1 individuals/individual/wk. If the population is growing, the PIG will be positive; if it is declining the PIG will be negative. A convenient way to describe the population growth rate is to calculate the number of new leaves produced using the equation
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(2)
PGR = Nwk2 Nwk1
If the PIG remains approximately the same regardless of the initial number of leaves, then growth is presumed to be density independent (with no limiting resources and no competition among individual duckweed plants). If, however, the PIG declines when initial densities are greater, this implies that growth is density dependent, and indicates the presence of intraspecific competition for a limiting resource. In Experiment 1, you will manipulate the number of plants (2 vs. 10 vs. 25 plants) and see whether changes in starting density affect the per-individual growth.
Discuss with your group an appropriate question and null hypothesis for this experiment: State your Question:
What is you Null hypothesis regarding the effect of density on PIG?:
Now obtain 6 glass vials, and fill each with 15 mL of growth medium. Count the number of leaves on 2 healthy duckweed plants, then add the plants to vial #1. Record the number of leaves in column 3 of Table 1. Do the same for vial #2. Place 10 plants in each of vials #3 and #4 (again count the number of leaves for each before adding them to the vials and record in Table 1). Place 25 plants in vials #5 and #6, again after recording the total number of leaves in each.
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Table 1. Growth rates of duckweed (your data). ln(N1) Vial # Initial no. Init. no. Final no. ln(N2) plants leaves leaves (Nwk1) (Nwk2) 1 2 2 2 3 6 4 6 5 10 6 10 7** 2 8 2 9 2 10 2 **Vials 7 to 10 will have your experimental variable.
PIG ln(N2)-ln(N1)
No. new leaves
In Experiment 2, you can experimentally determine whether particular resources control the growth of the duckweed. Vials #7-10 are reserved for your use, and additional materials needed to test for possible growth limitation by light intensity and/or quality, and by nutrient supply, are available in the lab as shown in Table 2.
Table 2. Supplies available for Experiment 2. Potentially Limiting Resource Supplies Available Amount of Light Window screening Quality of light Colored cellophane Nutrients Nutrient stock Discuss with your group how you could test whether a particular resource limits growth. The vials with 2 plants should be used as controls, with which your treatments will be compared. For example, you could ask whether light limits growth by comparing the PIG in your treatment vials with that of vials #1-2. Or you could see whether adding extra nutrients stimulated growth compared to vials #1-2. Vials #7-10 should consist of two treatments, each with two replicates. Suppose you want to know whether either of the two treatments causes the PIG to differ from that of your controls. What is an appropriate null hypothesis appropriate to that question?
Null Hypothesis:
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Now state the experimental design you will use to address the question. Experimental Design:
For Experiment 2, record the total number of leaves in each of vials #7-10 as before in Table 1. Label the entire row of 10 vials with your initials and lab section, and place the vials under one of the fluorescent lights on the side benches. Next week you will again count the total leaves in each vial, entering them in Table 1 as before.
Week 2: Your ability to create, and understand, graphic displays of data is a critical skill in biology. The following three graphs describe relationships among population size, population growth rate (PGR) and per-individual growth rate (PIG). The dashed line describes the expected relationships for exponential growth. In this lab, you are testing to see whether duckweed plants compete for growth-limiting resources, and display logistic growth to carrying capacity. As preparation, draw a solid line corresponding to the prediction for logistic growth in Figure 2b-c below.
Fig. 2a-c. Expected relationships among population size, population growth rate and per-individual growth rate for exponential growth (dashed lines), and logistic growth (solid lines). Add the solid lines to graphs b and c.
Pour out the contents of each vial into a Petri dish and again count the total number of leaves present in each.
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Enter the final leaf counts from week 2 in column 5 of Table 1 (your initial number of leaves from last week should already be entered in column 3. Dont worry about the calculations for the other columns yet. Theyll be computed using Excel. When done, please clean all vials, shake off most of the liquid and place them upside down in the cardboard tray. Once the lab bench is dry, get out your laptop and download the file Logistic Growth Model to your desktop (making sure the desktop is in fact your screen and not a subdirectory of the hard drive). Now open the file. The Excel workbook has two worksheets, one for calculations and one with a simulation of expected duckweed growth. Examine the worksheet with calculations. You should see a copy of Table 1. Enter the initial and final numbers of leaves in the Excel table as you did in your lab manual. Note that Excel automatically calculates the per-individual growth rate (PIG) and the increase in number of leaves. In the first experiment, you want to measure the degree to which the PIG declines as population size (N) increases. To do this you need to know something about simple linear regression. Simple linear regression (called a trendline in Excel) describes the degree to which the value of one variable depends on the value of another variable. For example, suppose you suspected that how you perform on BIO 110 exams is at least partly related to the amount of time spent studying. A mathematical model of this relationship might take the form:
EXAM SCORE = b(STUDY TIME) + a In that simple model, EXAM SCORE and STUDY TIME are the variables, and are related to each other algebraically by two constants, a and b. STUDY TIME is called the independent variable and can be used to predict the dependent variable EXAM SCORE. Stated differently, you want to see how much exam score depends on the amount of time spent studying. A possible relationship between STUDY TIME and EXAM SCORE is shown in Figure 3 below:
Notice that Exam Score was positively related to study time. The actual relationship is shown by an algebraic equation (model) of the form Y = b(X) + a, in this case ES = 3.3(ST) + 66.6, where 66.6 (=a) is the score predicted if you didnt study at all (0 hours) (DONT TRY THIS!) and 3.3 (=b) is the number of additional points above 66.6 that might be expected for each hour spent studying.
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As stated above, both numbers (66.6 and 3.3) are considered constants, and EXAM SCORE and STUDY TIME are dependent and independent variables, respectively. To test your understanding of simple linear regression, answer the following questions:
If a student increased his/her study time by 3 hour, how many additional points might he/she expect on the exam? ____________ If the student studied for 6 hours, what is the expected grade? _________________ In the regression equation youll create for Experiment 1 the per-individual growth rate (PIG) is the dependent variable and the initial number of leaves (N) is the independent variable. You wish to determine whether PIG declines with increasing N. In the Excel worksheet with Table 1, highlight cells D5:D10 (the initial number of leaves in vials #1-6). Then, while holding down the Ctrl key, also highlight cells H5:H10 (the estimates of PIG for those vials). Now pull down under Insert to Chart, specify XY (Scatter), and choose the subtype without lines (upper left). Next. In Chart Wizard Step 3 enter Axis titles for both the X axis (= N) and Y-axis (= PIG). Leave the Chart Title blank. Next. In Chart Wizard Step 4, for chart location, specify the current spreadsheet (Sheet 1). Single click on the resulting graph to highlight it, then move it so you can see both it and the datasheet. To customize your graph, make sure the whole chart area is highlighted, click once to highlight the legend at the right, then delete it. Now double-click on the Y-axis. In the Format Axis Window which should appear, specify Scale, then remove the check from the checkbox for automatic Minimum and set the minimum for the Y-axis at 0 (at a value of 0 there is no growth). OK. Then (with the chart still highlighted) pull down under Chart to Add Trendline. In the Trendline Window, specify Linear. OK. This adds a regression line to the chart. If the regression line doesnt extend all the way to the X axis, double-click on the regression line to open the Format Trendline Window, and click on Options. Click the checkbox for Display Equation on Chart. Then, under Forecast, extend the line by enough units so that it intersects the X axis (how many units are required will depend on your data; overshooting is OK). Rescale the X-axis if necessary as you did the Y-axis so that the line is nicely centered in the graph. Also estimate the number of leaves where the regression line meets the X axis. This is the carrying capacity (=K) (the population size at which the perindividual growth rate has declined to zero)! Sketch the points and regression line in Figure 4 below. In the equation above the figure, remember that in this instance PIG= ___N + ___ is the same as y = b(x) + a. Insert the constants for the slope (b) and intercept (a) in the equation. You can now calculate the carrying capacity based on this equation. What should be the value for PIG at carrying capacity? _____ Enter this value into the equation, then solve for N. Enter your estimate of K next to the equation at the top of Figure 4.
Regression: PIG = ____(N) + ____ K: ______
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Fig. 4. Relationship between per-individual growth and population size in duckweed (Experiment 1) You now need to decide whether to accept or reject your null hypothesis. If the regression line shows a negative slope (the value for b in the regression equation that has been added to your chart is negative) this means that the per-individual growth rate is reduced by intraspecific competition for some limiting resource (in effect, that the PIG is density-dependent).
Decision: Just what resource is limiting is investigated in Experiment 2. Begin by entering the PIG estimates for vials #1-2 and the treatment vials as 3 separate columns in your spreadsheet (see Fig. 5 below as an example). The first column should have the heading control, with the estimates for PIG directly beneath it. In the two adjacent columns insert titles for your two treatments, and place the PIG values underneath them. Then pull down under Insert to Chart, and specify Column. Select the subtype showing a Clustered Bar (upper left option). Next. In Chart Wizard Step 2, click to indicate the data are in rows, then click once in the data range window and highlight the data for Experiment 2, including column titles. Next. In Chart Wizard Step 3 enter appropriate names for the X and Y axes. Next. Make sure the chart will appear on your spreadsheet, then click on Finish. You can again delete the legend, and should again position the graph near the data used to create it.
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control 1 layer 2.3 2.1 2.5 2
2 layers 1.9 1.7
Sketch your chart in Figure 6 below. You will evaluate the null hypothesis of no treatment effect in Experiment 2 qualitatively (without statistical testing) by comparing the heights of the bars. Do the bars for the treatment vials show consistent differences in height from those of the control vials? What can you conclude from the results? Write your decision in the space below.
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Decision:
PIG
Treatment
Fig. 6. Effects of resource manipulation on per-individual growth rate (Experiment 2). Modelling Population Growth using the Logistic Equation: When a population is allowed to grow from a small initial number of individuals in an environment in which resources are limited, it is expected that population size will eventually reach carrying capacity (K) as shown in Figure 7. In Experiment 1 you estimated the carrying capacity by extending the regression of PIG down to the X axis (to the point where PIG = 0 and the population is no longer growing). The carrying capacity can be thought of as the maximum number of leaves that the vials can stably sustain. This is likely determined by the number of leaves that can coexist on the surface of the water.

K MAXPIG
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PIG
Time
Fig. 7a-b. (a) Logistic growth model of the number of duckweed leaves (N) expected over time. (b) the effect of duckweed density (N) on the per-individual growth rate (PIG). You will now create a logistic model of duckweed growth. Switch to the second worksheet in your Excel file. A sheet with the title Logistic Growth Simulation should now appear, with three graphs (of Population trends over time in green, trends in per-individual growth (PIG) in blue, and population growth rate (PGR) in brown. There is also an area in yellow at the right containing equations that produce the three graphs. You will examine the impacts of changing the three values in the magenta box on the three graphs of logistic growth. Begin by setting the carrying capacity (K) in cell N5 to the value you obtained in experiment 1. Similarly, enter the initial number of leaves (the average of the number of leaves in vials #1-2 in week 1 (column 3 in Table 1) in cell N6. Finally insert the value 0.1 for the maximum PIG in N4. Now examine the three graphs. First, the graph of N vs. t (in green) should show an approximately S-shaped curve (or, if the growth rate is rapid, a curve whose slope simply declines with time) beginning at the initial number of leaves and approaching carrying capacity. The second plot describes the predicted effect of population size on the per-individual growth rate (PIG). The third plot describes the population growth rate (estimated as the number of new leaves produced) in relation to population size. Note that the population growth rate is expected to be greatest at a value of roughly of the carrying capacity. Now replace the estimate of MAXPIG in cell N4 of your spreadsheet with the following 5 values: 01, 0.2, 0.4, and 0.6. For each value of MAXPIG, examine the third (brown) graph to estimate the maximum population growth rate (PGR). Enter these data in Table 3. Enter your estimates in Table 3.
Table 3. Effect of changing MAXPIG on PGR MAXPIG Greatest PGR 01 0.2 0.4 0.6
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How does MAXPIG affect the population growth rate?
Similarly, examine the effect of changing the carrying capacity on the maximum population growth rate. Enter the value 0.4 for MAXPIG in cell N4, and enter the following 5 values into cell N5: 80, 100, 120, 140, 160. Then estimate the maximum population growth rate in the brown graph as before, entering the estimates in Table 4.
Table 4. Effects of carrying capacity on maximum population growth rate. K 80 100 120 140 160 Maximum PGR
What is the effect of increasing the carrying capacity on the maximum population growth rate?
Finally, do you think the treatment manipulations you performed in #7-10 affected the MAXPIG or the carrying capacity (or both)? What evidence can you give to support your answer?
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Daphnia Morphology and Feeding Ecology In this portion of the lab you will examine feeding by Daphnia, a common member of the zooplankton in lakes and ponds in this region. It is a filter feeding herbivore that strains phytoplankton algae from the water around it. Your observations should address the following question: Is Daphnia picky about what it eats? If so, what kind(s) of algae does it prefer? Youll examine two kinds of potential algal food first, then observe the response of Daphnia to the food.
Types of Algae There are many kinds of phytoplankton algae, differing in their size, shape and nutritional content. Work with a partner. Begin by looking at two kinds of algae under the microscope. The first is Chlamydomonas, a small (about 10-20 m) eukaryotic, single-celled, and uses flagella to swim through the water. Make a wet mount (with a drop of culture medium and a coverslip), and view it under high power with a compound microscope. Sketch a Chlamydomonas in the space below. The second is Anabaena. Recall from the lab on The Microscope, Measurement and Cell that Anabaena is prokaryotic member of the cyanobacteria, with many very small cells that form long filaments. Again, make a wet mount, view it under high power and sketch it in the space below.
Chlamydomonas
Anabaena
Based on what youve seen so far, which kind of food do you predict will be preferred by Daphnia? Why?
Daphnia Chemotaxis Daphnia are microcrustaceans, very small but related to shrimp and lobsters. Like milkweed bugs, they pass through several immature stages called instars, molting in order to grow. In the culture, youll see different size individuals of various instars. Typically they are all females, and produce eggs that are genetic clones of themselves.
Chemotaxis is movement toward (or away from) a chemical cue. In this part of the lab you want to determine whether Daphnia can chemically detect patches of preferred food. Work as a group of 4. Set up your experiment as follows:
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1) Place a small amount of water (approximately 1-2 pipetfuls) in the center of a Petri dish. 2) Place suspensions of Chlamydomonas and Anabaena in separate capillary tubes. Seal one end of each tube. 3) Place two pieces of florists putty on the rim on opposites of the Petri dish. Place the open end of the capillary tubes at the edge of the puddle of the water in the center of the Petri dish. Mark one end of the Petri dish with a C (for Chlamydomonas) and the other with an A (for Anabaena). Your setup should resemble the diagram below).
4) Add 4-5 Daphnia to the puddle of water, turn on a light and view under the dissecting scope. 5) Observe the swimming behavior of the Daphnia. Is their swimming random or do they stay near one of the two capillary tubes?
Birds-eye view of Petri dish with capillary tubes containing Chlamydomonas (C) and Anabaena (A). Daphnia not shown.
Daphnia Morphology Now its time to get a close-up view of the morphology and feeding behavior of Daphnia. Again, work in groups of four. First fill a well slide with aged tapwater, and place a small piece of florests putty on one side. Second, obtain a short (< 1 cm) bristle, to be used as a tether, and dip on end of it in small amount of petroleum jelly (too much and your Daphnia will be too gooey to perform for you!). Keep your tether ready next to the well slide. Third, you now need to snag a Daphnia. Use a wide-bore plastic pipet (with the tip snipped off to increase the size of the aperture), then chase down a healthy looking specimen from the Daphnia culture. Once the Daphnia is in you pipet, it will typically swim against the current. Carefully evacuate most of the water until just a few drops (including your Daphnia) remain.
Then empty the Daphnia temporarily onto a clean microscope slide (no coverslip!), soak up excess water with a piece of paper towel so the Daphnia cant swim around, and immediately place the slide on the stage of a dissecting microscope. While observing your Daphnia through the microscope and using a forceps, touch the bristle tip with the petroleum jelly to the back or side of the carapace, being careful to avoid the swimming antennae and filtering appendages (see figure below). \ Now insert the other end of the bristle into the putty of the well slide. Adjust it if necessary so the Daphnia is once more under water and approximately in the center of the well. Observe the well slide first under the dissecting microscope to make sure your Daphnia is clearly visible at the end of the bristle, then transfer the well slide to a compound microscope and observe the Daphnia under your scanning objective.
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Tethered Daphnia in the well of a well slide.
Begin by identifying the following structures. 1) The head region has a single compound eye (note the many lenses that together allow the Daphnia to distinguish the general direction of light, but are not adequate to form clear images. Can the eye rotate? (Look for any apparent movement of the lenses). 2) Below the head are the two sides of the carapace, which together are a bit like a trenchcoat that opens toward the front. The carapace encloses the 5 pairs of filtering appendages used to concentrate algae from the water. Do the appendages beat in sequence or in synchrony? 3) The filtered algal food is moved forward between the paired filtering appendages and becomes concentrated next to the mandibles. Look for a pair of moving structures just under the head. Note how they push food into the gut. Does the gut have food in it? (Youll have a chance to compare feeding on various types of food in a bit). 4) Food exits the gut at an anus located at the back of a postabdomen. The front part of the postabdomen has a pair of claws that help keep the feeding area between the legs clean of unwanted algae. 5) At the back of the carapace note the heart, beating rapidly! Daphnia is an ectotherm, and its heartbeat increases with the temperature of its environment.
6) Note the swimming antennae. Your Daphnia may try to use them at times, unaccustomed to such a confined space. Are the antennae branched? Can you see setae on the branches, serving to increase propulsive power? Daphnia swim in a hopping motion, alternating movement with feeding.
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7) Note the brood chamber just below the heart at the back of the carapace. If your specimen is large enough and is healthy, you may be able to see some eggs in the brood chamber. Eggs are deposited all at once in the brood chamber about a half hour after molting, and will be released by the mother (by pulling forward her postabdomen) shortly before her next molt. Anatomy of a female Daphnia Daphnia Feeding Behavior The group at one end of the table should now add a single, very small drop of one of Chlamydomonas to the well slide. The group at the other end of the table should add a drop of Anabaena. You will barely see the algal particles, but can observe several aspects of feeding by the Daphnia. Answer the following questions in the table below. 1) Do the filtering legs continue to beat regularly and successfully concentrate the food? 2) Is there evidence of the food in the gut (does it get increasingly green)? 3) Does the Daphnia appear to be increasingly rejecting the food with frequent upward movements of the postabdomen to clean out the area between the filtering appendages? Make sure you compare notes with the other group to get the responses for both kinds of algae. When done, return your Daphnia to the culture chamber. Feeding behavior of Daphnia on two types of phytoplankton. Chlamydomonas Anabaena Filtering? Food in gut? Rejection movts. by the postabdomen? Based on your observations, which of the phytoplankton types is likely to be the most suitable food for Daphnia?
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Do your observations of food preference based on feeding behavior agree with your observations of chemotaxis? Which type of algae is preferred? Does this agree with your original prediction at the start of the experiment?
Lab exercise 11: Molecular Genetics

Molecular genetics part 1: DNA extraction from wheat germ Introduction: DNA (deoxyribonucleic acid) is the genetic material of all living organisms. A common technique used by molecular biologists is to isolate DNA from cells.
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The DNA can be used for finding and cloning specific genes, for constructing chromosome maps, or for sequencing, to name a few applications. There are a few basic steps in DNA extraction. The cell must be lysed (broken open) to release the nucleus. The nucleus (if present) must also be opened to release the DNA. At this point the DNA must be protected from enzymes (DNAses) that will degrade it. Once the DNA is released, it must then be separated from other cellular components.
The rationale for each step in the procedure is listed below: In order for the cell to be lysed, the cell wall (if present) and lipid membranes must be broken down. Mechanical disruption in a blender and the detergent solution are used for this purpose. Detergent also functions to denature and unfold proteins, making them more susceptible to protease cleavage The meat tenderizer has proteases, enzymes that help digest the contaminating proteins. Protease is added to destroy nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA. Finally, cold alcohol in a high salt environment is used to precipitate the DNA. + ions of NaCl bind to the phosphate groups of DNA molecules, neutralizing the electric Na charge of the DNA molecules. The addition of NaCl allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added. In water, DNA is soluble. When it is in alcohol, it uncoils and comes out of solution. Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands. DNA Extraction from Wheat Germ Protocol (for each table to step 7) For the first 7 steps, it is important to keep the temperature at 60 degrees 1. Add 25 ml distilled water to a beaker and place in water bath at 60o C. 2. Add 0.4 g wheat germ and mix. 3. Add 1.25 ml detergent. Mix and maintain at 50-60o C. 4. Add 0.8 g meat tenderizer 5. Add many drops (approximately 30) of sodium bicarbonate (baking soda) so that the final pH is 8.0. Use the pH paper to check the pH. 6. Maintain 50-60o C and stir for 10 minutes. 7. Remove from the water bath. Each person at the table can perform the next steps.
8. Add 3 ml of the solution to a test tube and cool to room temperature. 9. ***This is the most important step! Pour 3 ml of ice cold ethanol carefully down the side of the tube to form a layer 10. Let the mixture sit undisturbed 2-3 minutes until bubbling stops. 11. The DNA will precipitate at the alcohol interface. Shake the tube gently to increase the precipitation of DNA. Swirl a glass stirring rod at the interface of the two layers to see the precipitated threads of DNA.
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Molecular genetics part 2: DNA Fingerprinting Technicians working in forensic labs are often asked to do DNA profiling or fingerprinting to analyze evidence in law enforcement cases and other applications. DNA fingerprinting may involve polymerase chain reaction (PCR) amplification to analyze minute quantities of DNA or restriction fragment length polymorphism (RFLP) analysis, if large amounts of DNA are recovered. A step in human RFLP analysis requires a comparison of band patterns produced by cleavage of DNA samples when separated on an agarose gel. Two major factors affecting the reliability of DNA fingerprinting technology in forensics are population genetics and genetic statistics. In humans there are thousands of RFLP loci or DNA segments that can be selected and used for fingerprinting analysis. Depending on demographic factors such as ethnicity or geographic isolation, some segments will show more variation than others. If 90% of a given population has the same frequency in its DNA fingerprinting pattern for a certain DNA segment, then very little information will be attained. But if the frequency of a DNA pattern turning up in a population for a particular segment is extremely low, then this segment can serve as a powerful tool to discriminate between individuals in that population. The patterns in this exercise are produced from one sample that represents DNA taken at the crime scene and five samples obtained from suspects in the case. This laboratory exercise models the more elaborate technique that is performed on complex human DNA samples and uses 2 important research tools 1) restriction enzymes and 2) agarose gel electrophoresis.
Restriction Enzymes A restriction enzyme acts like molecular scissors, making cuts at specific sequences of DNA base pairs that it recognizes. A restriction enzyme sits on a DNA molecule and slides along the helix until it recognizes specific sequences of base pairs that signal the enzyme to stop sliding. The enzyme then cuts or chemically separates the DNA molecule at that sitecalled a restriction site. If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme will make a cut at each of those sites, resulting in multiple fragments. Therefore, if a given linear piece of DNA is cut with a restriction enzyme whose specific recognition code is found at two different locations on the DNA molecule, the result will be three fragments of different lengths. If the given piece of DNA is circular and is cut with a restriction enzyme whose specific recognition code is found at two different locations on the DNA molecule, the result will be two fragments of different lengths. The length of each fragment will depend upon the location of restriction sites on the DNA molecule.
Agarose Gel Electrophoresis DNA that has been cut with restriction enzymes can be separated and observed using a process known as agarose gel electrophoresis. The term electrophoresis means to carry with electricity. Agarose gel electrophoresis separates DNA fragments by size. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively charged, they will be drawn toward the positive pole (red electrode = run to red) when placed in an electric field. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is inversely proportional to its size in base pairs. Over a period of time, smaller DNA fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in single bands of DNA. These bands will be seen in the gel after the DNA is stained. Using restriction enzymes Because they cut DNA, restriction enzymes are the "chemical scissors" of the molecular biologist. When a particular restriction enzyme "recognizes" a particular four - or six base pair (bp) recognition sequence on a segment of DNA, it cuts the DNA molecule at that point. The recognition sequences for two commonly-used enzymes, EcoRI and PstI, are shown below. The place on the DNA backbones where the DNA is actually cut is shown with a ( symbol: )
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The line through the base pairs represents the sites where bonds will break if a restriction endonuclease recognizes the site GAATTC.
Using agarose gel electrophoresis The electrophoresis apparatus creates an electrical field with positive and negative poles at the ends of the gel. DNA molecules are negatively charged and will migrate to the positive pole. Smaller fragments move more easily through the gel when the electric field is applied. DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. A loading dye containing two blue dyes is added to the DNA solution. The loading dye does not stain the DNA itself but makes it easier to load the gels and monitor the progress of the DNA electrophoresis. The dye fronts migrate toward the positive end of the gel, just like the DNA fragments. The faster dye comigrates with DNA fragments of approximately 500 bp, while the slower dye comigrates with DNA fragments approximately 5 kb in size. Staining the DNA pinpoints its location on the gel. When the gel is immersed in Fast Blast DNA stain, the stain molecules attach to the DNA trapped in the agarose gel. When the bands are visible, you can compare the DNA restriction patterns of the different samples of DNA.
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For fingerprinting analysis, the following information is important to remember: Each lane has a different sample of DNA Each DNA sample was treated with the same restriction endonucleases. With reference to the numbered lanes, analyze the bands in the gel drawing at right, and then answer the questions. 1. What would be a logical explanation as to why there is more than one band of DNA for each of the samples?
2. Which of the DNA samples have the same number of restriction sites for the restriction endonucleases used? Write the lane numbers.
3. Which sample has the smallest DNA fragment?
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4. Assuming a circular piece of DNA (plasmid) was used as starting material, how many restriction sites were there in lane three?
5. Which DNA samples appear to have been cut into the same number and size of fragments?
6. Based on your analysis of the gel, what is your conclusion about the DNA samples in the drawing?
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Obtain a piece of parafilm and spot the various sample/enzyme mixtures according to the figure below:
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Using an automatic pipetter, load 3 L of distilled water in each well of the flash gel (FG) system (at right). Discard the tip. *Insert cassette into dock *Plug in electrophoresis power supply *Pre-run for 15 seconds (200 V) *Turn off voltage and disconnect cables *Using a new tip, mix and collect the loading dye with the Enzyme/sample from Suspect #1 and transfer it to well #1 of the FG system. Discard the tip. Continue this procedure until you put all the samples, including the crime scene system and the molecular weight standards. USE A FRESH TIP FOR EACH SAMPLE!!! *Plug in and turn on light and voltage *Watch until desired separation is achieved. Record your observations.
Analysis of results 1) Draw the pattern resulting from the gel after staining in the box below:
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2) Compare the fragment sizes of the suspects and the crime scene. Is there a suspect that matches the crime scene?
How sure are you that this is a match?
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Molecular Genetics part 3: Bioinformatics using Internet Resources Introduction to Bioinformatics: In the last couple of decades, there has been a technical revolution in molecular biology, which has made it possible to get enormous amounts of information about the sequences of amino acids in proteins and the sequences of bases in nucleic acids. To construct meaning from the sequence information, the array of computer techniques called "bioinformatics" has been developed. The components of bioinformatics are sequence databases and computer programs that analyze the sequences for patterns and similarities. To understand the basic idea of bioinformatics, one might think of a written language. The text you are reading consists of a series of letters, words, sentences, and paragraphs. If you did not know the meanings of the words and the rules of the language, this page would just be a collection of meaningless symbols. Similarly, the first time scientists saw gene and protein sequences, they saw a string of symbols with no clear meaning in terms of biological function. But now, bioinformatics is showing us many things about what sequences mean. Using bioinformatics, sequences are being used to reveal relationships among different life forms that we could not find out any other way. Bioinformatics is revealing the rules and meaning of a language that is new to human beings but in fact is more than a billion years oldthe Language of Life.
The genetic code The genetic code consists of 64 triplets of nucleotides. These triplets are called codons.With three exceptions, each codon encodes for one of the 20 amino acids used in the synthesis of proteins. That produces some redundancy in the code: most of the amino acids being encoded by more than one codon. One codon, AUG serves two related functions:

it signals the start of translation it codes for the incorporation of the amino acid methionine (Met) into the growing polypeptide chain
The genetic code can be expressed as either RNA codons or DNA codons. RNA codons occur in messenger RNA (mRNA) and are the codons that are actually "read" during the synthesis of polypeptides (the process called translation). But each mRNA molecule acquires its sequence of nucleotides by transcription from the corresponding gene. Note that in the table at right, the left-hand column gives the first nucleotide of the codon, the 4 middle columns give the second
nucleotide, and the last column gives the third nucleotide.
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For example, below is a piece of a sequence for the mRNA strand that codes for a piece of an imaginary short peptide: AUG AAA AAG UCA AGA UUU GGA CCC Using the table in the previous page, determine the amino acid sequence of this short peptide: ______________________________________________________ Show your amino acid sequence to your instructor before continuing to the next page.
Bioinformatics can also be used to compare protein sequences from different organisms. Part 1: Manual Comparison of a few Amino Acid in hemoglobin- refer to Table 1 Table 1: Selected amino acid positions (19 total) in the Hemoglobin of some vertebrates. 1 Primate Primate Primate Primate Primate Human SER 2 3 4 5 6 7 8 9 10 THR THR THR SER SER THR
THR ALA GLY ASP GLU VAL GLU ASP
Chimpanzee SER THR ALA GLY ASP GLU VAL GLU ASP Gorilla Baboon Lemur SER THR ALA GLY ASP GLU VAL GLU ASP ASN THR THR ALA THR SER GLY ASP GLU VAL ASP ASP
GLY GLU LYS VAL GLU ASP ASP ASP
NonPrimate Dog NonPrimate Chicken
SER SER GLY GLY ASP GLU ILU GLN THR GLY GLY ALA GLU ILU ASP SER GLY GLY LYS HIS 11 12 13 14 15 16
ALA ASN SER
NonPrimate Frog
VAL THR ASN SER 17 18 19
Primate Primate Primate Primate Primate
Human
PRO GLY GLY ALA ASN ALA THR ARG HIS
Chimpanzee PRO GLY GLY ALA ASN ALA THR ARG HIS Gorilla Baboon Lemur PRO GLY GLY ALA ASN ALA THR LYS HIS PRO GLY GLY ASN ASN ALA GLN LYS HIS PRO GLY SER PRO SER ASN HIS ASN ALA GLN LYS HIS
NonPrimate Dog
LYS ASN ALA ALA LYS LYS
NonPrimate
Chicken
PRO THR THR ALA HIS
LYS ASN SER GLN ARG ALA
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NonPrimate Frog
ALA LYS ASN ALA LYS ARG ARG
Fill in Table 2 below based on the data provided in Table 1. Table 2: Similarities and differences in the amino acid sequences of hemoglobin ORGANISM NUMBER OF SIMILAR AMINO ACID POSITIONS Human vs. NUMBER OF DIFFERENT AMINO ACID POSITIONS
Chimpanzee Gorilla Baboon Lemur Dog Chicken Frog Based on your results on Table 2 above , prepare a bar graph (below) showing the number of similar amino acid positions (compared with humans) for each type of organism.
Similarities in the amino acid sequences of hemoglobin in selected vertebrates

20 18 16 14 12 10 8 6 4 2 0
Number of Identical positions
um C an hi m pa nz ee G or ill a B ab oo n Le m ur
hi c
Fr og
og D
ke n
Using hemoglobin as a model protein, which primate is most closely related to humans? _______________________
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Which non-primate is most closely related to humans? _____________________ Imagine having to compare hundreds of nucleotides or amino acids in this way (DOH!!!!). There are several analysis tools available on the Internet that will help you compare amino acid (or nucleotide) sequences very quickly. Get a laptop computer. Using internet resources, we will compare mitochondrial DNA sequences. Because mitochondrial DNA mutates more rapidly than nuclear DNA, scientists use it to study the evolution of humans. Mitochondrial DNA Bioinformatics Inquiry Lab Go to the Dolan DNA Learning Center: http://www.bioservers.org/bioserver/ After you enter the Bioservers site, enter the Sequence server.
Click on Bioservers
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Click on Manage groups:
Click here
From the pull down menu on the top right called Sequence sources and briefly look at the wide range of sequence choices. Choose Modern Human mt DNA. Click in all of the boxes to the left of the six choices so that you can see all of different human samples.
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Pull down menu
Click on all six boxes
The goal of this activity is for you to develop a hypothesis about different types of peoples or organisms and compare them using computer Internet resources. Here is an example: This is my hypothesis: The mitochondrial DNA from a Native American is more closely related to the mitochondrial DNA from an individual from Asia than it is to an African American. Here is how I will test my hypothesis: 1) I will compare the sequence of a Native American mtDNA to an Asian mtDNA and count the number of differences in the nucleotide sequence of the mtDNA. I choose Bali #1 as my Asian sample and Blackfoot #1 as my Native American sample. 2) I will compare the sequence of a Native American mtDNA to an African American mtDNA and count the number of differences in the nucleotide sequence of the mtDNA. I choose African American #1 as my sample and Blackfoot #1 as my Native American sample. 3) Click on Compare at the top left.
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Click on Compare after the sequences are selected. I choose to compare these two sequences
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The initial portions of the sequence are shown on the next page. As you scroll down through the sequence, you will see that there are yellow highlighted areas that correspond to the nucleotide sequences that are different. I am going to choose to ignore the beginning part of the sequence where there is no overlap (the first 53 nucleotides) and the end of the sequence (after nucleotide 444). **The computer program will do this for you if you choose to trim the sequence.
Here are my results: Number of nucleotide differences between Asian Bali #1 and Native American Blackfoot #1 = 7 I repeat the same process to determine the differences for an African American and a Native American. Number of nucleotide differences between African American #1 and Native American Blackfoot #1 = 9 Based on these results, I can conclude that my hypothesis is correct (or is it? Do I need to collect more data?)
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Bio 110 Laboratory Manual Spring 2011

Bio 110 Laboratory Manual - Spring 2011

GENERAL BIOLOGY LABORATORY MANUAL INDEX

Bio 110 Laboratory Manual - Spring 2011

______________________________________________________ Student signature date

Bio 110 Laboratory Manual - Spring 2011

Lab exercise 1: Adopt-a-Bug! Milkweed Bug Growth Experiment

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Lab exercise 2: Avian Evolution

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Body weight (g)

Male Cardinal Scarlet Tanager Red-bellied Woodpecker Bobolink

Bio 110 Laboratory Manual - Spring 2011

______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________

Black-capped Chickadee Size Coloration Calls/ Songs

Bio 110 Laboratory Manual - Spring 2011

___________________________________________________________________________________ ___________________________________________________________________________________ ___________________________________________________________________________________

References: http://www.aquatic.uoguelph.ca/birds/morphevol/main.htm http://www.chickadee-web.com/ http://research.amnh.org/ornithology/crossbills/ http://research.amnh.org/ornithology/crossbills/nathist.html

The beaks of Finches and natural Selection

Bio 110 Laboratory Manual - Spring 2011

Which tool (if any) worked better for the:

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Lab exercise 3: Organic Molecules

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

1. Water 2. Glucose 3. Maltose 4. Sucrose 5. Starch

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Lab exercise 4: The Microscope, Measurement, and Cells

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Kingdom Plantae Elodea:

Bio 110 Laboratory Manual - Spring 2011

How are they different? ________________________ _______________________________________________________________________

Bio 110 Laboratory Manual - Spring 2011

Lab exercise 5: Diffusion and Osmosis

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

TIME (MIN) 0 15 30 45 60 % change (at t=60 mins) rate of osmosis (% / minute)

EGG IN WATER (g)

EGG IN 20% SUCROSE (g)

EGG IN 60% SUCROSE (g)

Bio 110 Laboratory Manual - Spring 2011

Exercise 3: Diffusion of a liquid in a liquid

Done by your instructor!

d. What do you think would happen if you used a smaller cylinder?

Bio 110 Laboratory Manual - Spring 2011

Bio 110 Laboratory Manual - Spring 2011

Lab exercise 6: Data* Analysis and Statistics

Bio 110 Laboratory Manual - Spring 2011

Table 1. Morphometric measurements of head size and body height.

Head Length (cm)

__

_________________________________________ _ ___________________________________________

How are they different? _______________________________________________