CAMP’S AP BIOLOGY 2013-2014 LABS

LAB 1 Enzyme KineticsLAB 2 Passive Transport & Cell BiologyLAB 3 Cellular RespirationLAB 4 PhotosynthesisLAB 5 Mitosis & MeiosisLAB 6 Mendelian Genetics of DrosophilaLAB 7 Bacterial CloningLAB 8 DNA FingerprintingLAB 9 Hardy-Weinberg EquilibriumLAB 10 Artificial SelectionLAB 11 Comparative Anatomy Part ILAB 12 Comparative Anatomy Part IILAB 13 TranspirationLAB 14 Food Web EnergeticsLAB 15 Primary Productivity

LAB 1: ENZYME CATALYSIS1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Enzyme Basics1) Sketch an enzyme and describe how the primary, secondary, tertiary, and quaternary functions

contribute to its correct function.2) With regard to #1 above, explain how extreme temperatures inactivate or denature enzymes.3) With regard to #2 above, explain how pH or extreme concentrations of salt ions can also

inactivate or denature enzymes.4) List the substrate and products of the catalase enzyme.5) How can you prove the identity of the gaseous product?6) Describe why catalase is ubiquitous in all living cells and particularly abundant in tissues like

roots, muscles, and livers.7) Describe the relative activity (0-10) of the catalase enzyme in each of the following:

Raw Potato Cooked Potato Raw Liver Cooked Liver

QUESTIONS FOR SECTION 2: Enzyme Catalysis of Peroxide by Catalase Enzyme

1) Fill out the table for the baseline assayFinal burette reading (mL)Initial burette reading (mL)Base Line (Final – Initial)

2) Figure out the molarity of the hydrogen peroxide solution from your baseline. The reaction is as follows:

Remember that the KmNO4 is at a concentration of 2% (20 g/L) and that you can use the formula weight and the ratio in the reaction above to figure out the molarity of H2O2.

3) Now figure out the rate of UNCATALYZED peroxide decay (SIT OUT OVERNIGHT)Final burette reading (mL)Initial burette reading (mL)Amount of KmNO4 used upAmount of H2O2 decomposed(mL baseline – mL KmNO4)Percentage of H2O2 decomposed in 24 hours(Baseline – 24hr)/Baseline X 100%

4) Why is sulfuric acid added to stop the reaction in the timed trial? How does it work?5) What factor causes the endpoint of the reaction (titration has stopped)?

2KMnO4 + 5H2O2 + 3H2SO4 --> 2MnSO4 + K2SO4 + 5O2 + 8H2O

6) Fill out the table for the catalyzed rate of decomposition10 30 60 90 120 180 360

A) Base line

B) Final

C) Initial

D) Amount KmNO4

E) Amount H2O2

7) What is the independent variable in this experiment?8) What is the dependent variable in this experiment?9) What are some control variables in this experiment?10) Discuss at least 4 sources of potential experimental error.11) Graph peroxide consumption over time in Excel and attach the graph. Make sure you

remember TIXDYLU and that you label everything and get the graph correct!12) Calculate reaction rates between each data point on your graph. This is simply dy/dx (slope).

0-10 sec 10-30 sec 30-60 sec 60-90 sec 90-120 sec 120-180 sec 180-360 secRx Rate

13) What are the units for reaction rate in this experiment?14) When is the rate highest? Explain.15) When is the rate the lowest? Explain.16) Sketch hypothetical graphs of this experiment if it were run under the following conditions

(Note: It is not necessary to draw 5 separate graphs. Just make different lines!)a. Temperature = 5 Cb. Temperature = 37 Cc. In the presence of a reversible competitive inhibitord. In the presence of an irreversible competitive inhibitore. In the presence of an allosteric inhibitor

QUESTIONS FOR SECTION 3: Turnip Peroxidase Enzyme1) How is the function of peroxidase similar to catalase?2) What organelle generates toxic peroxides and why?3) What organelle is responsible for removing peroxides?4) How is absorbance a useful measure of enzyme activity?

5) Fill out the data table for absorbances at different pH levels.pH 3 pH 4 pH 5 pH 6 pH 7 pH 8

6) Fill out the data table for absorbances at different temperatures.Ice Bath (0 C) Room Temp (24 C) Incubator (37 C) Lukewarm (50 C)

7) Graph and plot absorbance curves for turnip peroxidase enzyme at various temperatures & pHs.8) Peroxidase activity would invariably depend on plant species. Predict the optimum range of

peroxidase activity in the following examples…..a. A bog plant, such as a Venus flytrapb. A banana root c. A sagebrush from New Mexico

LAB 2: DIFFUSION AND OSMOSIS1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Diffusion of Materials into a Model Cell9) How does shape correlate with the rate of diffusion and osmosis in cells?10) How does size correlate with the rate of diffusion and osmosis in cells? 11) Give 3 examples of cell types where form follows function, with regard to diffusion and osmosis.

Explain how their shapes and size correlate with the rate of transport across their membranes.12) What color is phenolphthalein in the presence of acid? Base?13) Briefly explain the experiment you will run to test the rate of diffusion in blocks of 3 different

sizes, given that you have blocks impregnated with phenolphthalein and acid/base solutions.14) State your hypothesis for what you think will happen.15) Fill out the following table for your 3 agar blocks (3 points):

Block Surface Area Volume Diffusion TimeSmall

Medium

Large

16) Based on your data, was your hypothesis correct? Explain in several sentences. QUESTIONS FOR SECTION 2: Osmosis Across a Cell MembranePART A

1) How is dialysis tubing a good model for a cell membrane?2) Based on your chemical tests, which molecules could cross the cell membrane?3) Based on your chemical tests, which molecules were too large?

PART B1) Fill out the table for your dialysis tubing bags below (5 points)

BAG 0.0 M 0.25 M 0.50 M 0.75 M 1.0 MInitial Mass (g)

Final Mass (g)

Change in Mass (g)

Percent Change in Mass (%)Classification of Solution(Hyper, Iso, Hypo)

2) Graph the results of your osmosis experiment on EXCEL (10 points).3) Using the X-intercept of the graph of your results, estimate the molarity of the sucrose solution

you placed in the bag. I’m not telling you until the end (2 points).

4) What happened in the bags that lost mass? Why did this happen?5) What happened in the bags that gained mass? Why did this happen?6) How might this result have been different if glucose solutions had been used instead of sucrose?

QUESTIONS FOR SECTION 3: Osmosis in Living CellsPART A1) Make a sketch of an elodea leaf under the microscope in the two different solutions (3 points).

Elodea Leaf in Freshwater (Hypotonic) Elodea Leaf in Saltwater (Hypertonic)

2) What is crenation? How does it apply here?3) Which organelle changes the most dramatically, based on your observations?

PART B1) Re-write the water potential equation and identify what each of the variables means.2) What two factors can affect water potential?3) Why does air pressure have no effect under normal circumstances?You have a U-shaped tube with 0.2 M sucrose on the left, a dialysis tubing membrane in the middle, and 0.7 M sucrose on the right. 4) On which side is water potential negative?5) Based on the water potential equation, what direction will the water move?6) What eventually stops the solution from continuing to move? 7) Based on the experiment you did in Section 3, how could you determine the molarity of the cells

of the vegetable that was assigned to you?8) Fill out the following chart of your experiment (15 points).0.0 Molar Vegetable Core 1 Vegetable Core 2 Vegetable Core 3 AverageInitial MassFinal MassMass ChangePercent Change

0.25 Molar Vegetable Core 1 Vegetable Core 2 Vegetable Core 3 AverageInitial MassFinal MassMass ChangePercent Change0.50 Molar Vegetable Core 1 Vegetable Core 2 Vegetable Core 3 AverageInitial MassFinal MassMass ChangePercent Change

0.75 Molar Vegetable Core 1 Vegetable Core 2 Vegetable Core 3 AverageInitial MassFinal MassMass ChangePercent Change

1.0 Molar Vegetable Core 1 Vegetable Core 2 Vegetable Core 3 AverageInitial MassFinal MassMass ChangePercent Change

9) Graph the AVERAGE PERCENT CHANGE (10 points).10) According to your X-intercept, what is the molarity of the sugars in the vegetable? 11) Calculate the water potential of your vegetable (5 points)12) Which has a higher concentration of water molecules, a red blood cell or water?13) Which of the two would have a higher water potential?14) What would happen to red blood cells placed in water?15) Why will plants die if they are over-fertilized, based on what you saw here?

LAB 3: Cellular Respiration1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Cellular Respiration of Peas1) Identify the hypotheses investigated in this activity with regard to the following criteria. Justify

each hypothesis with a BECAUSE statement, explaining the rationale for the hypothesis.a. Temperatureb. Developmental stagec. Type of organism

2) Write the ideal gas law and state what each component of the equation means.3) Which factors in the ideal gas law will you manipulate in this experiment? Consequently, what

other components of the equation will change in response?4) State the basic formula for cellular respiration. INCLUDE energy molecules (ATP, NADH, etc.)5) Explain how you can know how much ATP an organism is producing or how much glucose it is

consuming, based on stoichiometry.6) Which factor will we measure in the cellular respiration equation?7) Based on this measurement, what molar ratio (vs. oxygen) of each of the following would we

expect? Provide a numerical factor.a. ATP producedb. NADH producedc. Glucose consumed

8) What are the control variables in this experiment?9) Why is sealing the chamber and minimizing movement a critical part of the procedure?10) Fill out the data table below11) Identify the independent and dependent variables in the experiment.

12) Graph your results on Excel, remembering to follow all graphing rules.13) Calculate the rate of oxygen consumption for each condition, based on the slope of your graph.

14) Why is it necessary to correct the readings from the peas with the readings from the beads?15) Explain the effect of germination on pea seed respiration.16) Suppose that you ran a further experiment with young pea plants that had grown foliage. How

might your results be different? Why?17) The graph below is a sample graph of possible data for oxygen consumption by germinating

seeds up to about 8 C. Draw the predicted results through 50 C and explain your results.

18) Why was KOH critical to the accuracy of the experiment?19) Why was it critical to completely seal the stopper? What part of the ideal gas law equation did

this control? What factors could be altered if this was not done correctly?20) Explain why water moved into the respirometer pipettes.21) IMPORTANT: 10% of a future lab grade will be determined by farming. That’s right. Farming.

Go to the greenhouse and plant your germinated peas. You are responsible for tending to these plants and raising them for use in our later transpiration lab. Keep ‘em alive!

QUESTIONS FOR SECTION 1: Cellular Respiration of Toads

1) Record the mass of your peas (from prior experiment) and the mass of the toad below.Dry Peas Germinated Peas Toad

2) Record your respirometry data for your toad below. Indicate whether you measured the toad’s respiration at 0 C or at room temperature (24 C).

TEMPERATURE

Time Beads Bead Difference Toad Toad Diff Toad Corrected Diff0

5

10

15

20

3) Graph your data.4) Calculate the slope of your graph to determine oxygen consumption in mL/min.5) Now convert oxygen consumption to mass for each organism. Report your results as mL/min.g.6) Compare your results for the toad to another group that ran the experiment at the other

temperature. What differences do you note?Organism Dry Peas Germinated Peas ToadOxygen Consumption (mL/min.g)

7) How might this experiment have differed if you had used a mouse instead of a toad?

LAB 4: PhotosynthesisQUESTIONS FOR SECTION 1: Plant Pigments

1) Are plant pigments primarily polar or non-polar? Why?2) How does changing a solvent affect chromatography?3) Identify the major pigments found in plants and tell what their optimal absorbance range is.4) Attach a sketch of your chromatogram or the actual chromatogram to your lab. 5) Record your pigment migration below and calculate the Rf for each pigment.

QUESTIONS FOR SECTION 2: The Light Reactions6) Provide a short synopsis of the inputs and outputs of the light reactions of photosynthesis.

Break this explanation down into…..a. Photosystem IIb. Photosystem I

7) Explain what happens to the components of a water molecule after it is split.8) Why are pigments necessary to this process? What part of the water molecule do they pass on?9) Why are DIFFERENT pigments necessary for the maximum efficiency of photosynthesis?10) What component of the water molecule ends up on the NADPH molecule?11) Why is DPIP necessary for this experiment?12) What is the source of electrons for DPIP?13) Explain what it is that the spectrophotometer is measuring. Provide a solid paragraph

explanation for the rationale of the experiment and what is going on here.14) Why is it necessary to have phosphate buffer in the photosynthesis cocktail. What molecule

would not be made without it?15) Record your spectrophotometer data in the space below

16) Determine your independent and dependent variables in this experiment.

17) Graph your data in Excel, remembering to include all components.18) What was the effect of incubating the vial of chloroplasts in the dark? 19) Explain what part of the light reactions will fail to function without incoming photons.20) What was the effect of boiling the chloroplasts?21) Identify at least 2 photosynthetic proteins that may have been affected by boiling the

chloroplasts. Explain why they might fail to function.22) How might this experiment have differed if you had done the following? Explain your answers.

a. Used a chloroplast extraction from a C4 plant like pineappleb. Added sodium bicarbonate to the solution (generating more CO2)c. Carried out the experiment at 5 C.d. Carried out the experiment at 40 C.e. Done the procedure under a bank of intense greenhouse lights.f. Done the experiment with a diatom culture (primary pigment = beta carotene)

LAB 5: Mitosis & MeiosisQUESTIONS FOR SECTION 1: Stages of Mitosis in Onion Root Tips

1) Use the prepared onion root tip slides to fill out the table below.Stage Sketch of Cell Summary of Major Events Number Percent TimeInterphase

Prophase

Metaphase

Anaphase

Telophase

2) What is a meristem? 3) What type of meristem is the onion root tip?4) If you had not restricted your view to the root tip, how might your results have differed?5) What other location on the onion plant might one find a high level of mitotic division?6) Complete a pie graph chart of onion mitosis cells, using time in minutes, as your units.7) Which stage is the longest? What events in this stage would explain this?8) Which stage(s) are the shortest?

QUESTIONS FOR SECTION 2: Growing Your Own Onions

1) Calculate the approximate number of cell divisions that it took to become an organism with around 300 trillion cells, like you are now.

2) Why do single celled organisms still need to divide?3) What advantage is there for an onion to reproduce asexually by putting out sets?4) Why is it fundamentally necessary for DNA to replicate before cell division?5) How are the following checkpoints of mitosis controlled?

a. Passage from telophase into interphase and DNA replication in S phaseb. Passage of cell from G2 of interphase to metaphase and anaphasec. Return to day-to-day activity in normally functional non-growing cells.

6) What effect do lectins have on cell division in plants?7) How might secretion of lectins by a fungus weaken and kill a plant? Why would this ultimately

benefit the fungus?8) Fill out the data tables comparing onion sets growing in the presence or absence of lectins.

Growth Conditions Interphase Mitosis TOTALSControl

Lectins

TOTAL

9) What effect did the lectin solution seem to have on the cells in the onion root tip?10) Fill out the table of expected values below.

Growth Conditions Interphase MitosisControl

Lectins

11) What would the null hypothesis be in this experiment?12) Compute Chi squared for your experiment.Conditions Observed Expected (O-E) (O-E)2 (O-E) 2

EControl InterphaseControl MitosisLectin InterphaseLectin Mitosis

DEGREES OF FREEDOM VALUE FOR CHI SQUARED WAS YOUR NULL HYPOTHESIS CONFIRMED OR REJECTED?

QUESTIONS FOR SECTION 3: Meiosis in Sordaria Fungus

1) List five major differences between mitosis and meiosis.2) How is the life cycle of fungi different from that of people and other diploid organisms?3) What is the difference between sister chromatids and homologous chromosomes?4) What is the difference between meiosis I and meiosis II?5) Sketch (or draw a flow chart for the artistically challenged) the life cycle of Sordaria mold.

Designate each stage as haploid, diploid, or dikaryotic.6) Explain the significance of the following in meiosis

a. Crossing-over in Prophase Ib. Independent Assortment of chromosomes during Metaphase & Anaphase Ic. Independent Segregation of chromosomes into separate gametes in Meiosis II

7) Using the Sordaria plate or a prepared slide, collect the following data:

8) Why did you divide the number of crossed-over asci by two?9) How did your data compare to the published map-unit distance of 26 units?10) How might you account for any disparity between your results and the published value?11) Draw a step-by-step diagram of how Sordaria spore sacs form when there is no crossover.12) Draw a step-by-step diagram of how Sordaria spore sacs form when there is crossover.13) Prepare a complete movie manuscript, film, and produce a full length 2 hour film about a lonely

Sordaria researcher who finds love in the arms of an eccentric onion farmer. Not really.

LAB 6: GENETICS OF ORGANISMS1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Indian Corn Genetics1) What is a mutation? How do they change phenotypes?2) What pigment is mutated in some of the corn? Why would this result in yellow kernels?3) What mutation leads to some kernels being sweet?4) Solve the Punnett Square below predicting the ratio of phenotypes given by an RrSs x RrSs cross.

5) Give the predicted ratios of each corn phenotype.

PHENOTYPE Starchy SweetRed

Yellow

6) Count 10 rows of an ear of corn and determine the number of kernels of each phenotypePHENOTYPE Starchy SweetRed

Yellow

TOTAL NUMBER OF KERNELS

7) Based on your percentages and counts above, find the expected number of kernels for each phenotype. Then use this to calculate Chi-Squared.Phenotype Observed Expected (O-E) (O-E)2 (O-E) 2

ERed StarchyRed SweetYellow StarchyYellow Sweet

VALUE OF CHI-SQUARED FOR YOUR EAR OF CORN

8) How many degrees of freedom did you choose for Chi squared and why?

QUESTIONS FOR SECTION 2: Fruit Fly Dihybrid Cross

1) How do you tell the difference between male and female fruit flies? Provide labeled drawings.2) Why is Drosophila an ideal test organism for genetic crosses? Give 3 reasons.3) Research Drosophila mutations. List 10 such mutations, their resultant phenotype, and provide

the chromosome location for such mutations.4) What would you expect the F1 phenotypes to be from a parental cross of NNww x nnWW?5) Provide a count of the flies in your F1 generation.

Phenotype Females Males TOTAL

6) Why is it important to NEVER allow flies from the P generation to mix? Why is it necessary to kill off all of the parental flies before the larvae mature?

7) Why don’t you have to show similar care with the F1 generation?8) Provide a count of the flies in your F2 generation

Phenotype Females Males TOTAL

9) Solve a Punnett Square for your F1 x F1 cross and show the expected phenotypes.

9) Give the predicted ratios of each fly phenotype.PHENOTYPE Winged VestigialNormal Coloration

Sepia Coloration

10) Based on your percentages and counts above, find the expected number of kernels for each phenotype. Then use this to calculate Chi-Squared.

Phenotype Observed Expected (O-E) (O-E)2 (O-E) 2 E

Normal WingedNormal VestigialSepia WingedSepia Vestigial

VALUE OF CHI-SQUARED FOR YOUR DROSOPHILA

11) What is the null hypothesis for this experiment?12) How many degrees of freedom did you use for Chi-squared and WHY?13) What is the probability value for this data?14) According to the p-value, can you accept or reject the null hypothesis?15) Do the actual results deviate from the expected results?

a. If NO, explain how this fits with Mendel’s Lawsb. If YES, explain at least 4 sources of experimental error that could have caused the

erroneous results.

LAB 7: Genetic Transformation of E. Coli with pGLO1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Transformation of E. Coli with pGLO 1) Why do bacteria normally use plasmids?2) Explain the difference between conjugation and transformation in nature.3) What is the role of restriction enzymes in genetic engineering procedures? 4) How were restriction enzymes and DNA ligase used to create pGLO?5) Draw a step-wise diagram showing how the pGLO plasmid was constructed from

jellyfish DNA and from purified bacterial plasmids.6) What does transformation competence mean? You may have to look this one up.7) In order for transformation to occur, the plasmid must be moved into a host cell.

Explain the mechanism we used to get the plasmid into the cell, noting the physiology of the cell membrane.

8) How do antibiotics normally kill bacteria?9) Why does ampicillin have no effect on bacteria that have taken up the plasmid?10) Count the number of colonies on each of your bacterial plates and record here:

LB and Plasmid LB/Amp & Plasmid LB (-) LB/Amp (-)

11) Explain the pattern of growth on each of the plates above. Why were there colonies or why weren’t there colonies?

12) Calculate transformation efficiency below.

LAB 8: DNA Fingerprinting & Gel Electrophoresis1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Transformation of E. Coli with pGLO 1) Why do bacteria have restriction enzymes in nature?2) Explain how restriction enzymes work. 3) Give examples of 3 specific restriction enzymes and the sequences that they cut.4) Why are restriction enzymes that produce staggered cuts, generally, more useful for

genetic applications? In the case of DNA fingerprints, does this really matter?5) Why are exons with functional genes usually NOT used for DNA fingerprints? 6) Why are sequences within exons so highly conserved? What happens if mutations

change them, rendering sequences different?7) Why are introns much more prone to mutations and differences between individuals?8) Calculate the probability of sharing the following with your neighbor (provided that they

aren’t your identical twin or a sibling or relative)…….a. A stretch of 3 DNA basesb. An identical run of 10 DNA basesc. A stretch of 200 identical DNA bases (standard procedure in DNA fingerprint)

9) List at least 4 commercial applications of DNA fingerprints.10) Why is the use of restriction sites to make RFLPs somewhat obsolete in biotechnology?

What techniques have replaced them? 11) What is a microsatellite? Why are they potentially useful in fingerprints?12) What is a transposable element? Again, why are they potentially useful in fingerprints?13) What is a haplotype? How are DNA fingerprints used in their construction?14) Explain how gel electrophoresis separates DNA fragments. Be sure to describe the role

of the gel, the electrical current, and the properties of the DNA, itself.15) How does ethidium bromide work to visualize DNA?16) What other technique, besides EtBr, is used in more technologically equipped labs?17) What is a DNA ladder? Why are they useful in molecular biology?18) For your gel, fill out the following tables & then use the log paper on the page that

follows to graph the migration of your fragments.

19) How does each of the following factors affect the results of a gel?a. Voltageb. Running timec. Amount of DNA usedd. Reversal of polaritye. Concentration of gel used

20) Suppose that you had two DNA fragments of extremely similar size that tended to run together in one band on a gel. How could you successfully separate the two fragments?

21) Use the graph you prepared from the lab to predict the distance that a 1000 bp fragment would migrate down the gel.

22) How can gel electrophoresis be used to show that a mutation has occurred?23) Give an example of an application of #22, where a change in the appearance of the gel

indicates a mutation. Attach a picture or article as evidence.

LAB 9: Hardy-Weinberg Equilibrium Simulation1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Taste the PTC Paper. 1) Discuss why taste perception is genetic. What is different between a taster and a non-taster?2) Record your taste data in the space below:

3) Why might the distribution in this class NOT reflect the distribution of genes in the USA as a whole? In what type of location might you find the closest statistical comparison?

4) What is the percentage of heterozygote tasters in the USA?5) What is the percentage of heterozygote tasters in the class?6) Why is it not possible (easily) to know WHO is a heterozygote?

QUESTIONS FOR SECTION 2: Case Study I: An Ideal HW Population 1) Record your initial data here and make your calculations.

2) What does the Hardy Weinberg equation predict for the new p & q?3) Do the results you obtained in your simulation agree?4) What major assumptions of HW were not strictly enforced here?5) What might you expect, had you run this simulation with the entire school?

DATA CHART FOR CASE STUDIES 1-4 (Use with all four simulations)

QUESTIONS FOR SECTION 3: Case Study 2 (Natural Selection) 1) How do the new frequencies of p & q compare to the original frequencies in case I?2) What major assumptions of HW were violated during this simulation?3) Predict what would happen if the simulation continued for 5 more generations.4) In a large population, would it be possible to completely eliminate a deleterious mutation?

Explain why or why not?5) Give an example of a real-life genetic disorder that follows the inheritance pattern of this

simulation. Explain why it has persisted, in spite of being lethal in its homozygote form.

QUESTIONS FOR SECTION 3: Case Study 3 (Heterozygote Advantage) 1) Explain how the final p and q frequencies compare with cases I and II.2) Do you think the recessive allele will be completely eliminated in this case?3) What is the importance of heterozygotes in maintaining genetic variation?4) Give an example of a real-life genetic disorder that follows the inheritance pattern of this

simulation. Explain the circumstances of its persistence.

QUESTIONS FOR SECTION 4: Case Study 4 (Genetic Drift)1) Explain how the final p and q frequencies compare with the prior simulations.2) What do your results indicate about the importance of population size as an evolutionary force?3) Give an example of a real life situation where genetic drift has caused alleles to shift

dramatically in two different locations. Explain the mechanism behind this.

QUESTIONS FOR SECTION 5: Practice Problems

LAB 10: Artificial Selection in Brassica Plants1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Background 1) What vegetables are all derived from a single species of Brassica?2) Why is genetic variability useful in agriculture?3) What is artificial selection?4) Discuss several other varieties of genetically pliable animals or plants that have been

domesticated and give examples of how their traits have been selected in different directions.5) Which variable traits did you target for selection?6) How did you decide to quantitatively measure the differences between generations 1 & 2?7) What were your independent and dependent variables?8) What control variables were necessary in this experiment?9) How might a lack of control obscure whether or not traits were genetically or environmentally

influenced? Give an example of how this might happen.

QUESTIONS FOR SECTION 2: Data Collection 1) Provide data tables for the plants in generation 1 and generation 2.2) Provide appropriate graphs for the data you chose to measure in each generation.3) Statistical analysis. Prove whether or not your trait was successfully artificially selected. You

may choose to do a Chi-squared or a t-test.4) Are the plants in generations I and II genetically distinct?5) What natural mode of genetic selection might result in a similar population distribution to the

one you created artificially?

LAB 11: Comparative Anatomy Part I 1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Cat Digestive & Excretory Systems1) Follow the prescribed dissection procedure. Check off the following organs and parts as you find

them in the cat. REMEMBER…..ANY and ALL of this is fair game for a lab practical quiz, so you better be sure what you’re looking at before you cheat check.

Masseter Muscle

Parotid Gland

Sublingual Gland

Submaxillary Gland

Pharynx Gingiva Vestibule

Hard Palate Soft Palate Epiglottis Esophagus Falciform Ligament

Coronary Ligament

RT Lateral Lobe of Liver

RT Medial Lobe of Liver

LT Lateral Lobe of Liver

LT Medial Lobe of Liver

Caudate Lobe

Gall Bladder

Hepatic Duct

Cystic Duct

Common Bile Duct

Fundus of Stomach

Greater Omentum of Stomach

Rugae of Stomach

Cardiac Sphincter

Pyloric Sphincter

Duodenum

Jejunum Ileum Villi of Small Intestine

Mesentery Hepatic Portal Vein

Caecum Ileocecal Valve

Ascending Colon

Transverse Colon

Descending Colon

Rectum & Anus

Pancreas Pancreatic Duct

Spleen

Gastrosplenic Ligament

Kidney Renal Pyramids

Renal Pelvis Cortex of Kidney

Medulla of Kidney

Ureters

Urinary Bladder

Urachus Urethra Adrenal Glands

Celiac Trunk

Thoracic Aorta

Inferior Vena Cava

Gastric Artery & Vein

Splenic Artery & Vein

Hepatic Artery & Vein

Renal Artery & Vein

Hepatic Artery & Vein

Colic Arteries & Veins

Lacteals

Describe how form fits function in each of the following digestive organs. IMPORTANT: In your answer, describe the types of tissues that make up the structure of each organ, and how they contribute to that function.

2) Pancreas3) Small Intestine4) Kidney5) Stomach6) What components found in the human digestive system are absent in the system of the cat?7) Why do you think the intestine of a cat is relatively short, when compared to the intestine of a

human, or shorter still, with regard to a rabbit.8) Why is the caecum in a cat and the appendix in a human, largely a vestigial structure?9) How does the location of the adrenal glands in a cat differ from the location in humans?10) List at least 5 functions of the liver.11) How is the circulatory system in the thorax designed for efficiency?

12) Identify the regions responsible for the processing of each of these biomolecules. Name the responsible enzymes or hormone secretions produced by these locations. In some cases, there will be multiple answers for a type of biomolecule. Biomolecule Organ of Processing Secretions ResponsibleStarches

Sucrose

Proteins

Smaller Peptide Chains

Cholesterol

Glucose

Triglycerides

Fatty Acids

QUESTIONS FOR SECTION 2: Cat Circulatory & Respiratory System & Sheep Heart1) Follow the prescribed dissection procedure. NOTE: The cat’s heart is too small to be of great

value for dissection, when the sheep heart is available. Please leave the cat heart intact and do your best to trace circulatory structures from the heart as a central point. Check off the following organs you find them in the cat. REMEMBER…..ANY and ALL of this is fair game for a lab practical quiz, so you better be sure what you’re looking at before you cheat check.

CAT CIRCULATORY SYSTEM

Anterior Vena Cava

Posterior Vena Cava

Brachiocephalic Artery & Vein

Jugular Veins Carotid Arteries

Subclavian Artery & Vein

Mammary Artery & Vein

Posterior Auricular A/V

Posterior Facial A/V

Anterior Auricular A/V

Anterior Facial A/V

Internal Maxillary A/V

Subscapular A/V

Brachial A/V Axillary A/V Umbilical A/V

Celiac Trunk Ascending Aorta

Descending Aorta

Iliolumbar A/V

Hepatic Artery & Vein

Renal Artery & Vein

Hepatic A/V Colic Arteries & Veins

Gastric A/V Splenic A/V Common Iliac A/V

Internal Iliac A/V

External Iliac A/V

Femoral A/V

Popliteal A/V Superior Articular A/V

Greater Saphenous A/V

Muscularis A/V

CAT RESPIRATORY SYSTEMDiaphragm Pleura Pleural Cavity Pulmonary

ArteryPulmonary Vein

Larynx

Thyroid Cartilage

Cricoid Cartilage

Arytenoid Cartilage

Epiglottis Vocal cords Glottis

LT LungAnterior Lobe

LT Lung Medial Lobe

LT LungPosterior Lobe

RT LungAnterior Lobe

RT Lung Medial Lobe

RT LungPosterior Lobe

Trachea Bronchi Bronchioles Alveoli

SHEEP HEARTRight Auricle Left Auricle Right Atrium Left Atrium AortaPulmonary Artery

Pulmonary Vein Right Ventricle Left Ventricle Coronary Artery

Inferior Vena Cava

Superior Vena Cava

Tricuspid Valve Bicuspid Valve Semilunar Valves

Papillary Muscles

Chordae Tendineae

Septum Fossa Ovalis Purkinje Fibers

A/V Node S/A Node Myocardium Pericardium

2) Why is it necessary for blood vessels to branch all the way into capillaries in order to serve all the homeostatic functions needed to stay alive?

3) What do you notice about the difference in the structure of arteries and veins (BESIDES the fact that one is dyed blue and the other is dyed red!)?

4) How do you think the naming system for arteries and veins was derived?5) How does form fit function, with regard to the following structures of the heart?

a. Auriclesb. Atriac. Ventricles

6) How is the circulatory system that you observed different from that of a fish? What evolutionary advances do you see that necessitate extra structures?

7) How is the circulatory system that you observed different from that of a frog? What evolutionary advances do you see? What changes have been made?

8) Stick with the frog. Now explain how the respiratory structures that you noted are an advance over the rudimentary amphibian respiratory system? What changes have been made?

9) Why is it incorrect to say that arteries always carry oxygenated blood and veins always carry deoxygenated blood?

10) How does form fit function, with regard to the lungs. IMPORTANT: Discuss the types of tissue present in the lungs and how they facilitate the respiratory function.

LAB 12: Comparative Anatomy Part II 1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Sheep Brain1) Follow the prescribed dissection procedure. Check off the following parts of the brain as you

find them. REMEMBER…..ANY and ALL of this is fair game for a lab practical quiz, so you better be sure what you’re looking at before you cheat check.

Cerebrum Cerebellum Medulla Oblongata

Longitudinal Fissure

Sulci Gyri

Frontal Lobe Parietal Lobe Occipital Lobe Olfactory Bulb Circle of Willis Pituitary GlandOptic Nerve Optic Chiasma Optic Tract Mammilary

BodyPons Abducens

NerveVagus Nerve Trigeminal

NerveOculomotor Nerve

Hypophysis First Spinal Nerve

Corpus Callosum

Fornix Arbor Vitae Transverse Fissure

Superior Colliculus

Fourth Ventricle

Thalamus

Limbic System Hypothalamus Cerebral Aqueduct

Cerebral Peduncle

Hippocampus Meninges

2) List the parts of the brain present in each of the five ventricles.3) How is the sheep’s brain evolved, in comparison to a lower vertebrate? Which part of the brain,

in particular, has been modified and why?4) List the structures in the brain that are critical to homeostasis and describe the general functions

that each does. This should NOT be a two sentence answer.5) Identify the functions of each of the cranial nerves that you found.6) What is the function of the corpus callosum?7) Identify the functions of each of the major lobes of the brain.8) What is the difference between white matter and gray matter?

QUESTIONS FOR SECTION 2: Sheep Eye1) Follow the prescribed dissection procedure. Check off the following parts of the eye as you find

them. REMEMBER…..ANY and ALL of this is fair game for a lab practical quiz, so you better be sure what you’re looking at before you cheat check.Cornea Sclera Ciliary Body Lens Aqueous HumorSuspensory Ligament

Iris Vitreous Humor Choroid Coat Scleroid Coat

Optic Nerve Macula Lutea Retina Rectus Muscles Oblique Muscles

2) How is the sheep eye distinctly different from that of an insect? Be sure to detail how the structure of the lens and design of the eye are completely different.

3) Why is the mucula lutea pigmented?

4) Explain why near-sightedness and far-sightedness develop. How might a dissection of such an eye look slightly different than the one you are seeing?

5) Why is it incorrect to say that the eye is the only organ of sight? What specific parts of the brain are involved in the integration and processing of visual information?

QUESTIONS FOR SECTION 3: Cat Muscles (Time Permitting ONLY)1) Follow the prescribed dissection procedure. Check off the following muscles as you find them.

REMEMBER…..ANY and ALL of this is fair game for a lab practical quiz, so you better be sure what you’re looking at before you cheat check.

Sternomastoid Clavobrachialis

Masseter Digastric Frontalis Myohyloid

Zygomaticus Temporalis Occipitalis Sternothyroid Pectoanebrachialis Pectoralis MajorFlexors of Forearm

Extensors of Forearm

Biceps Brachiii Triceps Brachii

Flexor Carpi Ulnaris

Palmaris Longus

Brachioradialis Epitrochlearis Pectoralis Minor Intercostal Muscles

Latissimus Dorsi Acromiodeltoid

Spinodeltoid Spinotrapezius Acromiotrapezius External Obliques

Internal Obliques Rectus Abdominus

Linea Alba Adductor Femoris

Adductor Longus Achilles Tendon

Soleus Tibialis Anterior

Sartorius Gracilis Gastrocnemius Tensor Fasciae Latae

Biceps Femoris Semimembranosus

Semitendonosus Vastus Medialis

Vastus Lateralis

2) List 3 anatomical differences between the muscles of the cat and the muscles of a human do you see? Suggest anatomical and evolutionary reasons for this.

3) All the muscles that you see are skeletal muscles. How do these differ from cardiac muscle and smooth muscle?

4) Describe the structure of a sarcomere and the steps required for a muscle to contract.

LAB 13: Transpiration in Coleus 1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Background 1) Describe how transpiration occurs in plants. Describe the role of the following factors in moving

water up from the roots to the leaves…..a. Cohesion and adhesionb. Properties of xylemc. Water potentiald. Pressure gradient

2) How does transpiration also help guide the transduction of sugars?3) Why is it necessary to remove all air bubbles from the potometer?4) How will the rate of photosynthesis affect the rate of transpiration? Explain the relationship by

discussing the inputs and outputs of photosynthesis.5) Why is transpiration greater in rainforests and highly productive ecosystems?

QUESTIONS FOR SECTION 1: Background 1) What was the total mass of the leaves on your cutting?2) Cut five 1 cm2 squares out of one of your leaves. Take the mass of each section and determine

the average mass for a 1 cm3 section of a coleus leaf.Section 1 2 3 4 5 AVERAGEMass

3) Now determine the leaf mass for a square meter of coleus tissue. Multiply your average leaf

mass in #3 by 10,000. Report this value and show your calculations.4) Divide the mass of the leaves in your cutting (#2) by the value for a square meter of tissue mass

in #4. This will tell you the total surface area of your plant. Report this value and show your calculations.

5) Record your potometer readings here. Then divide your potometer readings by the surface area of your plant to determine water loss in mL/m2.

Time 0 3 6 9 12 15 18 21 24 27 30Reading (mL)

Water Loss (mL/m2)

6) Predict how the following factors will affect the rate of transpiration.a. Exposure to wind (fan)b. Rainfall (mist)c. Intense sunlight (lamp)d. Whether or not the plant is a C3 plant or a C4 plant (coleus vs. wandering Jew)

7) Record class data in the grid belowTreatment 0 3 6 9 12 15 18 21 24 27 30Room

Light

Fan

Mist

C4 Plant

8) Graph the data in the table above, being certain to include all necessary components. As long as the scale of the graph will suffice for each treatment, it is not necessary to plot more than one graph. You may simply plot 5 lines on the same graph.

9) Using the slope of the graph, calculate and report the mean water loss for each treatment.10) Explain why each condition caused a change in transpiration rate. How did this compare to your

answers for question #6?11) What are the advantages and disadvantages to a closed stomata, if water is in short supply?12) How do CAM plants get around needing to perform transpiration to photosynthesize?13) Why will even a CAM plant eventually need to open its stomata?14) Discuss three adaptations that leaves have developed in order to minimize water loss. Give an

example of a species of plant that employs each strategy.15) Why is it necessary to tabulate leaf surface area when reporting results?

QUESTIONS FOR SECTION 3: Plant Vascular Systems 1) Sketch and define the functions of each of the following types of plant tissues.

Tissue Picture FunctionTracheids

Vessel Elements

Sieve Tube Members

Guard Cells

Parenchyma

Collenchyma

Sclerenchyma

2) Examine each cross-section under a microscope listed below. Provide a high-quality sketch and identify all the plant tissue types in the cross-sections.

a. Stem cross-section from a monocotb. Stem cross-section from a dicotc. Root cross-section from a monocotd. Root cross-section from a dicote. Leaf cross-section from a monocotf. Leaf cross-section from a dicot

LAB 14: Food Web Energetics1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Background1) What factors determine the primary productivity of an ecosystem?2) Explain why plants are not able to incorporate all available light energy into

photosynthesis and the formation of chemical bonds in biomolecules.3) What is the efficiency of cellular respiration? Hint: find out the amount of energy in a

mole of glucose and compare this to the amount of moles of ATP made. 4) Explain why cellular respiration has limited efficiency.5) What is the 10% rule in ecology?6) What factors contribute to the 10% rule? 7) How is energy allocated from plant matter into animals? To what activities and uses is

the energy in the food budgeted?8) Why is it important to only compare DRY masses and not to use the raw mass when

comparing the tissues of plants and animals?9) List some of the challenges that this lab presents, in terms of keeping an accurate

account of the energy balance in the plants and grasshoppers.

QUESTION FOR SECTION 2: Biomass Determination

1) Collect data on the biomass of your plants through the first 15 days of development (before introducing your grasshopper collection. Allow the plants to sprout before beginning your day count.

Day Mass of 10 Individual Plants

Dry Mass of 10 Individual Plants

Number of Plants

Estimated Dry Mass of Culture

3691215

2) Graph the development of biomass in your plant specimens vs. time.3) Before introducing grasshoppers, take the wet mass and dry mass of 3 individuals (try to

select a good range of sizes to represent your specimen sample) and extrapolate the values to the remainder of the population.

Mass of 3 bugs Dry Mass of 3 bugs Population Size Biomass of population

Dry Mass of Population

4) Complete the energy budget table below after your grasshoppers have had a week to feed and get acclimated. Repeat the measurement the following week.

Age of Grasshoppers One Week Two Weeks One Week GrowthWet Mass of Plants at MeasurementDry Mass of Plants at MeasurementPlant Percent Biomass (Dry/Wet)Plant Energy(Wet*Percent*4.35 kCal/g)Wet Mass of 3 grasshoppersAverage Wet MassDry Mass of 3 grasshoppersAverage Dry MassGrasshopper Percent Biomass (Dry/Wet)Energy per Individual(Wet*Percent*5.5 kCal/g)Grasshopper Energy (For All Bugs)Percent of Plant Mass AssimilatedDry Mass of All Grasshopper PooPoo per IndividualPercent of Plant Mass Lost as PooEnergy Excreted per Individual(Individual poo mass)(4.76 kCal/g)Respiration Estimate(Grass consumed – Poop)

5) How many calories are in a typical gram of……a. Carbohydratesb. Proteinsc. Lipids

6) Why is there a different estimated energy value for a gram of plants versus a gram of grasshopper flesh? Estimate the percent composition of each organism.

7) Why is grasshopper poo lower in calories than grasshopper flesh?8) Why must you look at the difference between Week 1 and Week 2 to get an accurate

estimate of energy budget? Why will one data point not suffice?9) Suppose that a frog weighing 10 grams was introduced to your little world. Could this

ecosystem support the frog long term? Give numerical reasons why or why not.

LAB 15: Primary Productivity & Dissolved Oxygen 1 point value each unless otherwise noted.

QUESTIONS FOR SECTION 1: Dissolved Oxygen and Water Temperature1) Record your values for dissolved oxygen in the 3 water sources. Use the nomogram to

determine the level of oxygen saturation at each temperature.

Temperature Dissolved Oxygen Saturation %

2) How does temperature affect oxygen saturation? How does this fit with the predicted

behavior of gases?

QUESTIONS FOR SECTION 2: Primary Productivity

3) Record your data for the intital light-dark bottle set.Individual Data Class Mean

Initial DO

Dark Bottle DO

Respiration Rate(Initial – Dark)

4) Record your primary productivity data for the screened bottles.Screens Light % DO Gross Productivity

(Light – Dark)Net Productivity (Light – Initial)

0 100

1 65

3 25

5 10

8 2

5) What were your independent and dependent variables here?6) On the same graph, plot the net and gross productivity as a function of light level.7) What are three ways to measure primary productivity?8) What is the relationship between oxygen production and the assimilation of carbon?9) Examining your graph, predict the points where gross productivity and net productivity

would zero out.10) What causes productivity to zero out, even if algae and cyanobacteria in the water are

actually still photosynthesizing?11) What will eventually happen to a body of water if the primary productivity remains close

to zero for long enough?12) A mammal uses about 1-2% of its metabolic energy on ventilation, while a fish uses

upwards of 15% of its energy moving water over the gills. Explain this huge discrepancy.13) It is possible for a eutrophic lake full of algae and plants to become super-saturated with

oxygen. Explain what you would see as a result of this.14) What is eutrophication? How does it indirectly cause dissolved oxygen levels to drop?15) What are the roles of phosphates and/or nitrates in eutrophication?

16) Predict the relative amount of dissolved oxygen in each of the following situations…..a. At the base of a waterfall in a trout streamb. The same stream in a deep poolc. The same deep pool after a torrential rainfalld. In a stagnant puddle in a salt marshe. At the top of the water column at Lake Hartwell during winterf. At the top of the water column at Lake Hartwell during summer at 7:00 AMg. At the top of the water column at Lake Hartwell during summer at 5:00 PMh. Below the thermocline in Lake Hartwell during the summer

QUESTIONS FOR SECTION 3: Macroinvertebrate Stream Survey

1) Why does the local macroinvertebrate population useful in predicting stream quality?2) Give examples of three macroinvertebrates that are highly sensitive to pollution.3) Give three examples of macroinvertebrates that are highly tolerant of pollution.4) What is the relationship between dissolved oxygen levels and the species make-up of the

macroinvertebrate community in a stream?5) Use the macroinvertebrate table below to index the section of the creek you are studying.

Check the Dendy sampler in your location, but also look under rocks and leaf piles and scour the stream bed looking for bugs. Do not count fish and other vertebrates.

TABLE OF COMMON STREAM MACROINVERTEBRATESPicture Specimen Description Score # Found TOTALS

Mayfly Small antennae on round head6 legs on thoraxGills on abdomen terminating in 3 tail bristlesWhite, brown, or greenish in colorUsually around ½ inch longFound under rocks, on leaf packs

10

Caddisfly Body segmented, worm-like, curled. 6 legs near head.May live in protective ‘case’; found under rocks.Light body, dark head.Size: ½ to ¾ inch.Gills protrude out of tail.

10

Dragonfly Large stout body 1-2 inches longLarge compound eyes, short antennaeAbdomen segmented, soft, one point at tipUsually found lying directly on the bottomOlder larvae may have wing buds

6

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Stonefly Prominent antennae 6 legs on thorax, 2 tail bristlesThinner and less ‘armored’ than mayflyFound underneath rocksPronounced segmented abdomenGills not obvious

10

Black fly Maggot-like segmented body. About ¼ inch.White with black edges to segmentsStouter posteriorHead ends in breathing tubeFound on debris sticking head out.

6

Midge Very small, white, worm-likeSkinner, paler body than blackflyBreathing tube at head of body

5

Mosquito Typically found in still polluted water.Float suspended beneath surface, jerky movementBreathing tube, hairy siphons on body¼ to ½ inch long worm-like body.Body often striped at segments

1

Picture Specimen Description Score # Found TOTALSDobsonfly Very large, segmented body 2-3

inches long.Large biting mouthparts, round headMultiple false legs on abdomenArmored, segmented shell.Usually black or dark brown in color.

10

Fishfly Look like dobsonflies, but no visible gillsLarge biting mandiblesNumerous straight abdominal appendages

8

Damselfly Two prominent feather-like gills at tailThinner and more elongated than stonefliesWhite or gray bodyUsually somewhat large ½ or more inchesSmall antennae

6

Sowbug Aquatic crustaceans resemble land pillbugsSegmented thorax with 7 pairs of legsHard gray or brown shell, long antennaeAbout ½ inch in length. Plate-like

7

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abdomenCranefly Large, worm-like larvae (up to 4

inches)No legs, head can be retracted into bodyWhitish color, transparent. Heart and gut visible

6

Oligochaete(aquatic worm)

Small segmented worms, no appendages or eyesRed, tan, brown in colorOften found in polluted areas. Scavengers.Very small: ¼ to ½ inch. Very active and mobile.

0

Planaria Flatworm: compressed body, move by slidingProminent head with 2 eyespotsNo appendages, body may regenerateWhite, gray, brown or pink colorScavengers; usually under ½ inch,

3

Scud Freshwater crustacean known as amphipodsShrimplike appearance, hard exoskeletonWhite or clear body with 8-10 pairs of legs

6

Crawfish Large invertebrates: 1-6 inches longSwim with backward strokesProminent claws and tail, 8 walking legsFound under rocks and in burrowsHead and thorax fusedProminent antennae and mouthparts

6

Riffle beetle larvae

Resembles small mealwormHard, segmented shell6 legs on thorax, 2 tail bristlesWhitish in color

8

Riffle beetle adult

More sensitive to pollution than larvaeSmall beetle about ladybug size ( ¼ inch)Found on bottom, does NOT skim surfaceUsually black

10

Aquatic snails

One shelled mollusk with muscular footGraze organic material off of stones, branchesScraping mouthparts (radula) and eyes on stalksWide variety of sizes, colors, species

4

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Bivalves Shellfish with 2 shells and muscular footFound in mud burrows or crusted to surfacesMany species. Size: ¼ inch to 4 inches.Black, brown, or whiteFilter feed on algae and bacteria

6

Water penny Beetle larvae; 6 legs and gills underneathHard, plate-lake segmented shell

10

Leeches Large segmented worms found in polluted areasFeed primarily on blood from vertebrates.Brown, red, black in colorAble to stretch 3-5 times body length (1-6 inches)

2

6) Add up your total insects and your total scores

Insects ScorePage 1Page 2Page 3Page 4GRAND TOTALCLASS TOTAL

Score Conversion Scale

80-100 Exceptional Clean natural oligotrophic stream.70 –79 Excellent Very clean. If polluted, only trace amounts.60-69 Very Good Minor pollution, but most organisms survive.50-69 Good Minor pollution. Very sensitive organisms disappear.40-49 Fair Somewhat polluted. Varied mix of organisms.30-39 Poor Significant pollution. Sensitive organisms die off.0- 30 Bad Only hardy organisms survive. Very polluted.

7) Grade out the stream.GRAND TOTAL SCORE = SCORE/INSECTS x 10 ________________CLASS TOTAL SCORE = SCORE/INSECTS x 10 ________________STREAM RATING ________________

8) Sketch a relief map of the area you surveyed and provide a description of foliage, slope, etc.9) How did the pollution index and species distribution in your area of the stream compare to the

data from other groups in the class? What factors do you think were responsible for this?

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