Objectives

The overall outcome of this long-term activity is for you to develop a simple, but accurate research model for testing the presence of cell toxins in environmental samples. You will be asked to use two existing cell toxicity strategies and a proposed bean beetle research model to measure endotoxins in water accurately, inexpensively, and rapidly.

• You will design a test that determines whether bean beetle larvae are susceptible to intestinal bacteria endotoxins.
• You will design a controlled experiment to evaluate how the Limulus amoebocyte lysate (LAL) test can be blended with the trypan blue test of cell viability to test for endotoxins.
• You will evaluate the feasibility of using the test you develop by evaluating its accuracy, cost, and simplicity.

 

Introduction

Biological monitoring, or biomonitoring, makes use of organisms to provide information about environmental quality. It is proving to be a reliable way of determining the presence many types of pollutants in air, soil, and water (CDC 2013). Biomonitoring can be done in the field on populations of organisms or can be performed in a laboratory on the biochemistry of an individual organism. Scientists are discovering that biomonitoring provides some of the best data for detecting minute amounts of a pollutant and for investigating the long-term effects of a pollutant on an organism. Biomonitoring is a rapidly growing field that makes use of microorganisms, animals, and plants as biomonitors.

 

Background

Water pollution is a major issue worldwide and prevents many people from having access to safe water (UN 2013). Three major types of water pollution are bacteria, dirt, and nutrients (EPA 2012). In developing nations water pollution decreases the availability of clean water needed for bathing, drinking, and cooking. In developed nations, water pollution greatly contributes to the cost of maintaining clean waterways used for commerce, consumption, and recreation. A disturbingly common pollutant is intestinal bacteria from animal and human sources. Most often, water needed for human consumption is contaminated with human intestinal bacteria (CDC 2012). Unlike chemical pollutants, human bacteria pollution can cause infectious diseases that spread from person to person. Infected people can also accidentally pollute clean water supplies. Much of the harm from the bacteria is due to a variety of enzymes and toxins released as the bacteria feed and replicate.

It is possible to detect and monitor intestinal bacterial pollution in water using a variety of techniques. However, there are few inexpensive, quick, and simple procedures for accurately determining the presence of intestinal bacteria in water (Deininger and Lee 2005). Even these procedures are not feasible in many situations where urgent water testing is needed, such as in developing nations with limited resources (Zakir Hossain et al 2012).

 

Problem

You have just learned that you were selected for an internship with a nonprofit group that studies global water issues. The group just formed a team that will investigate inexpensive and simple methods for determining the presence of intestinal bacteria in drinking water. This team is being led by a researcher, Dr. Erica Ojobi, who previously researched mysid shrimps as indicators or biomonitors of water pollution (Toussaint et al 1995). She did this by monitoring the health of the mysid shrimp in response to the different pollutants. Mysid shrimp are easy to grow and their response to pollutants is simple to measure. Plus, the method uses easy-to-find materials and does not need to be conducted in a laboratory.

After hearing about the natural history of bean beetles, Callosobruchus maculatus, at a conference, Dr. Ojobi was interested in using the beetle larvae as indicators of intestinal bacteria pollution in water supplies (Beck and Blumer 2007). She also remembered studying two procedures that could help determine the health of the beetles when exposed to intestinal bacteria pollution. One test, called the Limulus amoebocyte lysate (LAL) test, uses horseshoe crab blood cells as indicators of bacterial endotoxins (Lonza Group 2013, Prior 1990). Endotoxins are harmful molecules released by specific bacteria during death and replication. Certain endotoxins at very low levels are known to disrupt and sometimes lyse certain cells (Yu et al 1997, Svensson et al 2005).

Another test, called the trypan blue test, is used to investigate the health of cells (Sigma-Aldrich 2013). Trypan blue is a dye that is not taken up by healthy cells. However, trypan blue will pass through the membranes of dead and dying cells making the inside of these cells dark blue (Figure 1).

Figure 1: Trypan Blue Test Results

Dr. Ojobi has a hypothesis that the LAL tests and the trypan blue tests can be blended and modified in a way to test for intestinal bacteria in water. She is hoping to come up with a simple and low cost way to biomonitor the bacteria. Dr. Ojobi believes that bean beetles will be the ideal organism to use for developing this biomonitoring test.

 

Materials

Remember that Dr. Ojobi's work is focused on finding ways of monitoring pollutants using simple way and low cost strategies. Plus, she has limited funds and materials to carry out her work. Consequently, you are limited to the types of materials that are available in her work area for you to conduct your experiment.

Fortunately, Dr. Ojobi is prepared for the study and made an inventory of materials in her lab (that inventory list is available from your instructor). It is important that you be selective about the materials you use to conduct your experiment. Use only what is required to perform your specific experiment. Before requesting materials, please review the inventory list and evaluate whether each item on the list is useful for the success of your experiment. You may have the option of requesting other items by submitting a request to your instructor. In your request, you must justify why you want the item and what can be used in place of the item if it is not available. Keep in mind that you can substitute items in the inventory for less-expensive items that lower the cost of the experiment.

 

Experimental Design

The purpose of your experiment is to see whether bean beetle larvae can be used as biomonitors for intestinal bacteria in water. You will be using components of the LAL test and the trypan blue test to detect any effects of the endotoxins on the bean beetle larvae. In order to test Dr. Ojobi's hypothesis for this experiment, you need investigate the reasoning why she made the hypothesis. Use the references provided in this activity and the Internet to answer following questions:

Bean Beetle Questions:

1. Why did Dr. Ojobi think that bean beetles would make a good model for studying pollution?

2. What do you need to know about the bean beetle life cycle that might make them vulnerable to the endotoxins of intestinal bacteria?

3. How would endotoxins affect bean beetle cells based on what is known about other organisms?

Endotoxin Testing Questions:

1. What feature of the LAL test do think Dr. Ojobi finds valuable for indicating the presence of endotoxins?

2. What do you need to know about the bean beetle life cycle that might make them vulnerable to the endotoxins of intestinal bacteria?

3. How would you combine the two tests to come up with one test that could indicate the presence of endotoxin damage to cells?

Experimental Design:

1. What role would the bean beetles larvae play in the experimental design?

2. Identify the independent variable for your experiment.

3. Identify the dependent variable for your experiment.

4. What variables need to be constant your experiment.

5. Describe the experimental design for your experiment.

6. Describe how you would recognize if the bean beetle larvae are being affected by the endotoxins.

7. How would you set up the control group for this experiment?

8. How would you set up an experiment to determine if other pollutants and other types of bacteria give the same experimental results as the endotoxins?

9. What type of data would you have to collect to determine if the bean beetle larvae are responding to the endotoxins?

10. Describe any statistical analyses you need to compare your experimental group to your control group.

11. Explain if your experimental method can meet Dr. Ojobi's criteria of being accurate, inexpensive, and simple to do.

Come to class prepared to present your experimental design.

 

Literature Cited

Beck, C.W. and L.S. Blumer. 2007. Bean beetles, Callosobruchus maculatus, a model system for inquiry-based undergraduate laboratories. Pages 274-283, in Tested Studies for Laboratory Teaching, Volume 28 (M.A. O'Donnell, Editor). Proceedings of the 28th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 403 pages.

Centers for Disease Control and Prevention (CDC). Health Water. (Page last updated: July 9, 2012). http://www.cdc.gov/healthywater/wash_diseases.html.

Centers for Disease Control and Prevention (CDC). Fourth Report on Human Exposure to Environmental Chemicals. 2013. Atlanta, GA: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. http://www.cdc.gov/exposurereport/.

Deininger, R.A. and J. Lee. 2005. Rapid Detection of Bacteria in Drinking Water. Modern Tools and Methods of Water Treatment for Improving Living Standards.NATO Science Series, 48(4): 71-78.

Environmental Protection Agency (EPA). Three Big Pollutants. (Last updated on Tuesday, March 06, 2012). http://water.epa.gov/learn/resources/bigpollutants.cfm.

Lonza Group LTD. Limulus Amebocyte Lysate (LAL) QCL-1000 Manual. (Accessed 2013). http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_QCL-1000_Product_Insert.pdf.

Prior, R.B. 1990. Clinical Applications of the Limulus Amoebocyte Lysate Test. Boca Raton, Florida: CRC Press. pp. 28-30.

Sigma-Aldrich. 2013. Trypan Blue Product Information Sheet: Product Nos. T 8154, T 6146 and Z 35,962-9 (H7901). http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Usage/t8154use.pdf.

Svensson M., L. Han, G. Silfversparre, L. Häggström, S.O. Enfors. 2005. Control of endotoxin release in Escherichia coli fed-batch cultures. Bioprocess Biosyst Eng. 27(2): 91-97.

Toussaint, M.W., T.R. Shedd, W.H. van der Schalie, G.R. Leather. 1995. A comparison of standard acute toxicity tests with rapid-screening toxicity tests. Environmental Toxicology and Chemistry, 14(5): 907-915. Published online: 26 OCT 2009, DOI: 10.1002/etc.5620140524.

United Nations Department of Economic and Social Affairs (UN). International Decade for Action "Water for Life" 2005-2015. (Last updated: 12/27/2013). http://www.un.org/waterforlifedecade/quality.shtml.

Yu, C-G, M.A. Mullins, G.W. Warren, M.G. Koziel, J.J. Estruch. 1997. The Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Lyses Midgut Epithelium Cells of Susceptible Insects. Applied and Environmental Microbiology, 63(2): 532–536.

Zakir Hossain, S.M., C. Ozimok, C. Sicard, S.D. Aguirre, M. Monsur Ali, Y. Li, J.D. Brennan. 2012. Multiplexed paper test strip for quantitative bacterial detection. Analytical and Bioanalytical Chemistry. 403(6): 1567-1576. DOI: 10.1007/s00216-012-5975-x.

 

This experiment was written by Ms. Betsy Morgan and Dr. Brian R. Shmaefsky, 2014 (www.beanbeetles.org).  

Copyright © by Ms. Betsy Morgan and Dr. Brian R. Shmaefsky, 2014. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.

Background

There is ample evidence in the science education literature that engaging students in authentic experiments enhances science content comprehension and retention (Leonard 1991, Brickman et al 2009). One caveat is that the experiments should not be cookbook style and must be student-centered in order to reinforce the principles of experimental design (McClanahan and McClanahan 2002). Unfortunately, student-centered experiments can be an overwhelming task for faculty.

One difficulty is foreseeing, and having readily available, the materials and resources needed for the students to complete their open-ended study. However, much of this concern can be remedied by limiting the types of materials the students can work with. Another problem is restricting the student investigation to a certain body of literature and limiting their lines of reasoning to a targeted goal without giving too much instructor direction. Limitations can be placed on an investigation by putting it into an informed designed setting (Crismond and Adams 2012). An informed designed scenario requires the collection of a specific body of knowledge to resolve a well-defined problem.

It is important to stress to yourself and to your students the purpose of this activity. This activity was developed to analyze how students use biological principles to resolve a problem using an informed design scientific investigation process. The specific mission for students is to use two existing technologies [LAL (Bryans et al 2004) and trypan blue (Strober 2001) assays] and a proposed research model (bean beetles) to measure endotoxins in water accurately, inexpensively, and rapidly.

 

Purpose and Learning Objectives

This activity shows students how research models can be used to understand factors that affect homeostasis at the cellular level. It blends two cell study technologies: Limulus amoebocyte lysate assay and trypan blue vitality staining. The biology topics covered are:

• Apoptosis
• Cell membrane
• Cell structure
• Cell staining (vital stains)
• Experimental method
• Programmed cell death
• Research models

The activity's level-appropriateness is determined by the degree of detail expected in the complexity of the experimental design and in the explanation in the discussion section of student reports.

• Non-majors should be expected to design a proper controlled experiment comparing cell lysis with and without endotoxins based on the materials provided. Their level of explanation of the endotoxin effects on the insects should include that the endotoxins lyse the insect cells by disrupting the cell membrane. They should also be able to explain that trypan blue enters only damaged cells. Students should explain that damage to amoebocytes can be correlated to cells in other organism.

 

• Introductory majors should be expected to design a proper controlled experiment comparing cell lysis using a standard (a chemical known to cause cell lysis, a negative control, and an endotoxin. They should design the experiment based on the materials provided in addition to other relevant materials. Their explanation of the endotoxin effects on the insects should include susceptibility of the bean beetle cells to endotoxins and that endotoxins are molecules that attach to insect cells and cause organelles to release compounds that degrade the cell. Students should explain that damage to amoebocytes can be evolutionarily correlated to cells in other organism.

 

• Intermediate majors should be expected to design a proper controlled experiment comparing cell lysis using a standard (a chemical known to cause cell lysis, a negative control, and an endotoxin. The students should show evidence of the various environmental variables that should or should not be controlled based on how they would significantly affect the results. They should design in vitro and in vivo the experiment based on the materials provided in addition to other relevant materials. Their explanation of the endotoxin effects on the insects should include the role of natural selection in susceptibility of the bean beetle cells to endotoxins and that endotoxins are molecules that attach to insect cells and cause a degranulation event that degrades the cell. The students should recognize that not all cells are lysing; only the amoebocytes from the insect are lysing and the amoebocytes are present in early larval stages. Students should explain that damage to amoebocytes can be evolutionarily correlated to immune system cells in other organism.

 

Time Required to Complete this Activity

Planning and experimental design phase (out of class)	6 hours
Experimental phase
Practice removing the embryos	2 hours
Removing viable embryos for experiment	30 minutes
Endotoxin preparation	1 hours
Trypan blue testing	1 hours
Data Analysis and write-up (out of class)	6 hours

 

Experimental Design

The specific objective of the activity is to use bean beetles and a combination of two cytotoxicity assays to develop a research model for determining the presence of intestinal bacteria in water. This objective is set within the parameters that students will be asked to review the scientific literature on using insects as environmental pollution indicators, bacterial endotoxins, LAL testing, and trypan blue assays. In their informed designed project, students must be aware of the cost, rapidity, and simplicity of the procedure to assess its feasibility.

Students should be tackling the following questions when developing their procedure:

• How can bean beetle larvae be used as a model for environmental toxicology?
• Are bean beetle cells susceptible to bacterial endotoxins in water contaminated with intestinal bacteria?
• How can the Limulus lysate test and the trypan blue test be applied to study endotoxins?
• Are bean beetles an accurate model of testing the presence of environmental toxins such as endotoxins?
• Is the endotoxin test that is developed feasible for use in situations where inexpensive and rapid testing is needed
• Can the endotoxin test that is being developed be generalized to investigations on other environmental pollutants?

As the students are planning their experiments, it is important to remind students to keep their experiments simple. Additionally, remind the students the type of study they are doing must take in the following considerations:

1) Does the control group differ only in the independent variable from the experimental group?

2) Are the results of the experiment unique to intestinal bacterial pollution or can the results be explained by any type of common water pollutant or harmless bacteria?

3) If the experiment works, can it be done easily with reasonability low cost?

One major confounding issue with data collection is the harm caused to the larvae when they are removed from the bean. Students should be asked to justify if cell damage monitored in the trypan blue test is the result of the procedure or the result of the bacterial toxins.

Provide students with Figure 2 (a - f) from Page 4 of A Handbook on Bean Beetles, Callosobruchus maculatus as a reference when they are removing of the larvae.

 

Data Collection

Students typically come up and approve the following experimental protocol for this activity:

1. Set up microscopes with digital cameras

2. Collect beetle larvae

3. Place larvae in clear saline solution

4. Clean away all bean residue

5. Tease apart larvae to expose internal cells

6. Place cells on microscope slide with water (distilled, pond, saline, or seawater) and no coverslip

7. View cells under microscope and collect images. Note that the larvae cells are spherical and the bean cells are oblong (see Figure 1)

8. Add trypan blue to slide and collect images

a. Viable cells are clear or have background color

b. Dead or disrupted cells are dark blue (arrow)

9. Add treatment (E. coli, water, or pollutant) and collect images

a. Negative Control - water being used for study (distilled, pond, saline, or seawater)

b. Positive Control - disinfectants, pesticides (pyrethrins or mosquito agents), or some treatment that kills cells

c. Specificity - comparison to other common water pollutants such as sediment (soil), nutrient pollutants (fertilizers), and harmless bacteria.

10. Count relative number of cells disrupted or killed for each treatment

11. Record data in spreadsheet including summing and averaging the data for control and experimental groups

a. Data are typically recorded as number of cells killed out total count

b. Data are typically compared graphically and statistically as percentage of cells killed

12. Perform statistical analysis (Chi-Squared or T-test) to evaluate differences in the results

Figure 1. Experimental results of E. coli exposure to bean beetle larval cells. Arrow points to a larval bean beetle cell (spherical shape) whereas the oblong cells are of the host bean seed.

The classes mostly agreed upon a simple experiment comparing the experimental group to a negative control. However, the class should be encouraged to set up a positive control using any chemical or condition (for example, osmolarity) that causes cell lysis. Plus, students like to see if all the bacteria made available to them cause bean beetle cell lysis. Typically, students are aware of setting constant variables between the groups. They know to watch for the following variables:

• Temperature
• Water type being used
• Fluid volumes
• Amount of bacteria exposed to the beetles
• Amount of trypan blue added to experiment
• Mass or volume of beetle larvae being tested
• The isolation or clumping of larval cells being studied
• Growth conditions of beans (regular versus organic)
• Familial generation of the bean beetles

However, students should be encouraged to recognize that the developmental stage of the larvae may make a difference in the results.

 

Data analysis

By knowing the theory of the trypan blue tests, students are well-aware that they need to record their data as the percent of larval cells killed either on a field of isolated cells or cells found in small clumps. Students usually agree to collect data digitally by photographing the slides using digital cameras attached to the microscopes or by using the cameras on their cell phone through the microscope ocular. They then do cell counts from the digital images by recording the number of cells killed out total count of cells.

The data are typically compared graphically and statistically as the percentage of cells killed tallied for each group including any replicate studies. Most often students use spreadsheet graphing features to present their data. Simple data tallying is done with the spreadsheet formulas feature. However, the statistical analysis such as Chi-Squared and Student T-test are done using scientific calculators or statistics websites such as Stat Pages Org at http://statpages.org/. T-tests are conducted by comparing control to experimental group, for the simpler studies. Students who compare three of more groups run multiple T-tests and will compare the groups in the following way:

Variation 1

• Negative control group to experimental group
• Positive control group to experimental group
• Negative control group to positive control group

Variation 2

• Control group to E. coli experimental group
• Control group to Lactobacillus experimental group
• Control group to Staphylococcus experimental group
• E. coli experimental group to Lactobacillus experimental group
• E. coli experimental group to Staphylococcus experimental group

Students have never selected an analysis of variance (ANOVA) test to compare multiple data groups. ANOVA works best for comparing multiple groups, particularly for Variation 2.

 

Equipment and supplies

The strength of this activity is providing a limited amount of materials that students must make use of to successfully design their experiment. The informed design model of inquiry gives students a task and specific materials to resolve an issue. The issue in this activity is to biomonitor intestinal bacteria using a specific biomonitor organism and simple to interpret analytical methods.

Students are prompted to ask the instructor for the list of materials provided below. The items on this list are an inventory in a hypothetical laboratory where the students are interning. They must make use of only those materials needed to design the experiment according to the criteria set by the principle investigator in the study (Dr. Erica Ojobi). Students also are prompted to ask for inexpensive substitute materials. Students are instructed submit to the instructor a formal request for any items on the list knowing that that may not get their request fulfilled. Much of what they will request is likely commonly available in most teaching laboratories.

Inventory

Organisms

• Regular mung beans (Vigna radiata)
• Organic mung beans
• Regular mung beans containing bean beetle (Callosobruchus maculatus) eggs and larvae
• Organic mung beans containing bean beetle eggs and larvae
• Escherichia coli strains B or C broth cultures
• Lactobacillus acidophilus broth cultures
• Staphylococcus epidermidis broth cultures

Dissection Materials

• Lab scissors
• Forceps
• Straight teasing needles
• Dissecting trays

Reagents

• 0.9% (W/V) Saline solution
• Trypan Blue solution (0.4% W/V) (0.4 grams of trypan blue powder to 100ml of distilled water)
• Methylene blue solution
• Laboratory disinfectant solutions
• Distilled water
• Pond water mixture (1.0% W/V NaCl) (1.0 gram of sodium chloride to 100ml of distilled water)
• Seawater mixture (3.5% W/V NaCl) (3.5 grams of sodium chloride to 100ml of distilled water)
• Liquid houseplant fertilizer
• Pyrethrin based pesticide
• Mosquito Dunks® or Mosquito Bits®

Miscellaneous Materials

• Petri dishes (35mm)
• Well plates - 96 count
• Plastic lab droppers (pipettes)
• Test tubes with racks
• Microbiology transfer loops
• Bunsen burners
• Microscope slides
• Microscope slide coverslips
• Cell counting chambers
• Filter paper, 35mm #1
• Local soil sample

Laboratory Equipment

• Transmission light microscope
• Dissecting microscope or hand lens
• Digital camera for microscope
• Adjustable water bath
• Bench top balances
• Incubator
• Heating plates
• 50 ml graduated cylinders
• 100 ml beakers
• 250 ml beakers
• 500 ml beakers
• 250 ml Erlenmeyer flasks

Personal Protection Equipment

• Lab gloves
• Safety glasses or goggles
• Lab apron or lab coat

 

Literature Cited

Brickman, P., Gornally, C., Armstrong, N., and Hallar, B. 2009. Effects of inquiry-based learning on students' science literacy skills and confidence. International Journal for the Scholarship of Teaching and Learning, 2, http://hdl.handle.net/10518/4155.

Bryans T.D., Braithwaite C., Broad J., Cooper J.F., Darnell K.R., Hitchins V.M., Karren A.J., Lee P.S. 2004. Bacterial endotoxin testing: a report on the methods, background, data, and regulatory history of extraction recovery efficiency. Biomed Instrum Technol. 2004, 38(1):73-78.

Crismond, D., and R. Adams. 2012. The informed design teaching and learning matrix. Journal of Engineering Education, 101 (4): 738-797.

Leonard, W.H. 1991. Uncookbooking your laboratory investigations. Journal of College Science Teaching. 21, 84-87.

McClanahan, E.B., McClanahan, L.L. 2002. Active Learning in a Non-Majors Biology Class: Lessons Learned. College Teaching, 50(3): 92-97. http://www.personal.psu.edu/mfp131/pages/Reading_McClanahan_Active.pdf.

Strober W. 2001. Curr Protoc Immunol. Appendix 3B. doi: 10.1002/0471142735.ima03bs21.

 

This experiment was written by Ms. Betsy Morgan and Dr. Brian R. Shmaefsky, 2014 (www.beanbeetles.org).  

Copyright © by Ms. Betsy Morgan and Dr. Brian R. Shmaefsky, 2014. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.

Table 1. The percentage of killed cells found using trypan blue on bean beetle larval cells exposed to control and endotoxin treatments. Cell counts were done on specimens in which the bean cells were removed leaving behind only bean beetle larval cells. These data were collected by students in ENVR 1401 at Lone Star College, Kingwood, TX.

Endotoxin Cytotoxicity Study Data - Bean Beetle Larval Cells

	Field Count % Cells Killed (Field of 50 cells)
	Trial 1		Trial 2
	Control	Endotoxin	Control	Endotoxin
Group 1	10	56	6	44
Group 2	4	25	5	23
Group 3	8	40	7	31
Group 4	6	28	8	30
Group 5	12	60	9	45
Group 6	6	30	12	56
Group 7	3	22	5	25
Group 8	8	36	9	38
Sum	57	297	61	292
Mean	7.1	37.1	7.6	36.5

 

Mean Cytotoxicity of Endotoxin and Control Treatments

Figure 1. The mean percentage (mean±SE) of killed bean beetle (Callosobruchus maculatus) larval cells exposed to E. coli endotoxins were greater than those exposed to a control treatment of sterile pond water. There is a significant difference between the number of cells killed in the control compared to the endotoxin treatment (N=16 for each treatment, X²=154.7 df=1, p<0.0001).

 

This experiment was written by Ms. Betsy Morgan and Dr. Brian R. Shmaefsky, 2014 (www.beanbeetles.org).  

Copyright © by Ms. Betsy Morgan and Dr. Brian R. Shmaefsky, 2014. All rights reserved. The content of this site may be freely used for non-profit educational purposes, with proper acknowledgement of the source. All other uses are prohibited without prior written permission from the copyright holders.

Student Handout [pdf] [doc]

Student Handout slides [ppt]

Instructor's Notes [pdf] [doc]

Sample data [pdf] [doc]

Sample data figure slide [ppt]

Sample data spreadsheet [xls]