The American Physiological Society

DRAFT VERSION 11/29/09

FrontiersinPhysiology/ExplorationsinBiomedicine ProfessionalDevelopmentFellowship

Activity Cover Sheet

Title of Activity Grade Level(s)

WhatMakesaWormsPulseRace? AnexplorationintopulseratesofLumbriculusvariegatus 912

InquiryLevel

Studentswillfirstlearnhowtomeasurethepulserateofthe blackwormsandreinforceconceptsofexperimentaldesign, includingthepotentialpitfallsofexperimentalbiasand appropriateuseofanimalsinexperiments.Oncethis backgroundisestablishedstudentswilldoanInternet TreasureHuntonblindanddoubleblindclinicaltrialsand oneonLumbriculusvariegatusandpotential stimulants/depressants/toxinsthatcouldbeusedfor experimentation.Studentswillthenworkcooperativelyto Oneparagraph designanexperimenttotesttheimpactoftheirchosen description substanceonthewormspulserate.Theteacherwillprovide thestudentswithcodedsolutionssothestudentswillnot knowduringexperimentationanddataanalysiswhichsolution usedintheexperimentistheirtestsolutionandwhichisthe placebo/control.Oncestudentshavedonetheirbasicdata analysistheteacherwillrevealthesolutionidentitiestothe studentsandsothestudentscanwritetheirconclusionsand developaposterpresentationtopresenttheirexperimentto theclass. blackworms,Lumbriculusvariegatus,pulse,stimulants, Key Words depressants,toxins,blind,doubleblind,experimentalbias
Teachers Name Teachers School SchoolCity,State ResearchHost Institution InstitutionCity,State FellowshipYear

PaulaDonham OlatheEastHighSchool Olathe,Kansas NorbertoGonzalez,M.D. UniversityofKansasMedicalSchool KansasCity,Kansas 2009

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What Makes a Worms Heart Race? Teachers

Suggestions for

Purpose: This is a series of labs and activities to kick off the school year with in which students will explore the impact of treatments (stimulants, depressants, toxins) on the pulse rate of the worm Lumbriculus variegatus while: 1. Refreshing and refining their understanding of experimental design;

2. Focusing on recognizing and minimizing experimental bias; and

3. Reinforcing graphing and communication skills. Objectives: Upon completion of this activity, students will be able to: Count pulse rate in blackworms (Lumbriculus variegatus).

Demonstrate the ability to design and perform an experiment using the strong inference model and multiple hypotheses with attention to the minimization of experimental bias. Demonstrate an understanding of graphing and the use of simple statistical data analysis to evaluate the impact of an experimental treatment.

Explain the relationship between dependent and independent variables (in this particular case the pulse rate of the blackworms and the treatment with a stimulant/depressant/toxin). Grade Level: 9-12 Prior Knowledge: This series of labs is designed for the beginning of the year to develop student interest and refresh and expand their understanding of experimental design. The content knowledge students should bring to this lesson is the general understanding of the purpose of animal circulation that usually has been acquired by the time students reach high school. Thus, with high school students this lab can be used as an exploration that begins with the first day of class. With younger students the lab would likely need to be preceded by background discussions on experimental design and animal circulation. Time Required: The labs and presentations will require 5-7 class days depending on the level of the students, length of class periods, and whether everything is done in class or some portions (Internet Treasure Hunt and/or experiment design and hypothesis writing) are done as homework. Including All Students (Diversity and Equity Strategies): All the activities can be done in small groups designed by the instructor. The groups should be designed to include a diversity of cultures, learning styles, skills, and/or abilities as appropriate for the particular class. A camera and projection system can be used in the microscope portion of the experiment to make gathering data for students unable to use a traditional light microscope. The lesson includes 5-7 days worth of activities with hands-on experimentation with live animals, a guided Internet search, and the development and presentation of posters by the students. The unit is designed to stimulate and encourage the participation of diverse students with varied skill sets, learning styles, and abilities. Questions to Ask Along the Way: Following are some of the questions you may want to ask as students proceed through this unit: o What is the dependent variable? In all phases of this experiment the pulse of the worms is the dependent variable. This is both what the students are measuring and what they expect may change as they expose the worms to different conditions. What is the purpose of a control? The control is a set of trials where the organism is exposed to the same conditions as the experimental group except for the experimental treatment. The control is also what you compare your results to in order to judge whether the experimental treatment had an impact. In general for these experiments the control

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trials have the worms in plain spring water; the independent or experimental variable being the various potential stimulants, depressants, and toxins. o What is experimental bias? According to Wikipedia (10/04/2009): In experimental science, experimenters bias is bias towards a result expected by the human experimenter. . . The inability of a human being to remain completely objective is the ultimate source of this bias. It occurs more often in sociological and medical sciences, for which reason double blind techniques are often employed to combat the bias. o What is a stimulant? Would you expect something which stimulates a human to necessarily stimulate a tiny aquatic worm? Do stimulants work the same on all humans? Here you can emphasize both the diversity and the commonalities of living organisms. You can mention the toxicity of Tylenol in dogs as an example of how seemingly similar organisms (humans and dogs) can react very differently to some chemicals. Ask the students if all humans have the same reaction to medicines? In the future we will likely do genetic analysis before prescribing many medicines in order to head off potentially serious side effects. It can also be interesting to discuss the use of stimulants to calm and focus people with attention deficit disorder. o What makes the blackworms a good experimental organism? There are many appropriate answers: Theyre small. Its easy to see the pulsing of their ventral vessel. Theyre hardy even cutting them in two doesnt kill the two segments. Because theyre aquatic a treatment to their skin is readily absorbed giving timely results in the experiments. (4) Pedagogy Employed in This Lesson: Six Star Science Lesson Components Student-centered Instruction Students will do two experiments: 1st they develop their own hypotheses relating to conditions outlined by the teacher and perform the experiment and 2nd they research possible stimulants/depressants/toxins and then design a double blind study to test the impact of their solution on blackworms. Between the two above experiments, students will do an Internet Treasure Hunt to gain knowledge on the research organism, to select their test chemical, and to guide their development of their experimental design. See Internet research outlined in above Technology description. This activity meets the following 9-12 NSES Standards: Science as Inquiry Life Science: Matter, energy, and organization in living systems This activity meets the following 8-12 Kansas Standards: Benchmark 1: Scientific inquiry Benchmark 7: Understanding the diversity of structure and function in organisms Valuing Diversity Addresses different learning styles via the hands-on experiment, Internet research, and the development and presentation of their poster summary of the experiment.

Technology

Current Content

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 Authentic Assessment

Lab reports and class presentations graded with a rubric. Class presentation grades will be a weighted average of peer review rubric scores and teacher evaluation scores. After the instructor should complete a Lab/Lesson Debrief and reflect on the effectiveness of the entire unit with particular focus on the procedures for the double blind study. Then the instructor should revise the unit as needed for the following years use within their particular classes and student populations.

Reflecting on Teaching & Learning

Materials: For every 2 students: o Petri dish with 2-3 blackworms (Lumbriculus variegatus) in spring water (from the class worm container) (6,7,11) o piece of plastic micro-hematocrit capillary tube (8) o Thin stem pipette o Compound microscope or dissecting/stereoscope o Stopwatch o Coded chemical solutions (for second set of experiments only) (2) For my coding I had the first letter represent the solution and the second letter told whether it was the chemical or the control and then I added a number to represent which set it was (so I could have multiple sets of solutions in small bottles ready to hand out to the students). So, I had AF1 and AO1, AF2 and AO2, etc., with AF representing a full strength alcohol solution and AO the control (pure spring water), the series with CF and CO were caffeine solutions made from and the companion control, and the series labeled BF and BO were generic Benadryl solutions. (I considered using more sophisticated codes that would have more completely hidden the controls but was concerned about making it too complex for easy monitoring by the instructor.) For the alcohol solutions, I left the test solution and the control both clear, however, the other test solutions always had a color (imparted by the dyes used in the tablets) so I used food coloring to make the test solution and control the same color. Safety: Safe, appropriate, and humane use of animals, including invertebrates, should be discussed and the form Use of Animals in the Classroom (in student handouts) read and signed by all students.

The teacher should evaluate and monitor all chemicals used in the second set of experiments and read and follow the MSDS for all chemicals. For chemicals that are health hazards, including nicotine, wear gloves and minimize contact. Preparation and Procedure: DAY ONE: Exploration Students will need basic instruction on how to load worms into the capillary tubes and may require reminders on how to use microscopes. Its fairly easy to draw the worms up into the thin stem pipette then squirt them gently into the capillary tube. It takes a bit of practice but can easily be mastered with patience. There should also be a discussion of the safe, appropriate, and humane use of animals (including invertebrates) in the laboratory. Students should be provided the Use of Animals in the Classroom and they should read form then sign and date it and submit it to the teacher prior to any experimentation.

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Students should practice loading worms and counting the pulse rate before doing any experiments. Be sure they looking at the dorsal vessel. The ventral vessel looks nearly the same but has no pulsation wave.
Image by Charles D. Drewes, Ph.D. Permission to use image granted by the Iowa State Universitys Department of Ecology, Evolution, and Organismal Biology

Once students are comfortable with the techniques, they will write their hypotheses and identify their variables for the first experiment (to be completed on day two). The intent of using multiple hypotheses is for students to think through all the possible outcomes of their experiment. The strong inference model (5) guides them to include in each prediction what it means if the data supports that hypothesis. If students tested all three containers (they are only being asked to compare two sets of worms) one hypothesis might be: I predict the worms in plain water will have the same pulse rate as the worms from the other two containers which would mean that the worms are not impacted by the differences in water.

DAY TWO and THREE: First cycle of experimenting NOTE: Although for this first experiment students should be told: The worms will be in 3 containers: one labeled plain water, one labeled plain water with stimulant, and one labeled plain water with depressant, all three containers will only have plain spring water (or dechlorinated water like used in aquariums). This experiment is intended to show students how experimental bias can creep into an experiment without them being aware of it. Some students will get data they believe to be wrong (no difference in pulse rates between worms from two different containers). If they question you while they are still working on the lab, you can use the opportunity to ask if they were very careful in their technique. If they assure you they were, tell them they should trust their data and seek to understand what it means.

Upon completion of the lab students should analyze their data then create a poster presenting the hypothesis that was supported by the data, their data analysis, and conclusions to the class. Box and whiskers graphs (aka box plots) are good tools for this analysis. (1) In the following example, although the means and medians for the two sets of data might indicate the two sets of data are different, the fact that the inner quartile ranges (the boxes) overlap so much indicates there is not a statistically significant difference in the sets of data. This graphing style has become common since the 1970s when it was developed by John Tukey of Princeton. The median (by convention) is the center vertical line, the left end of the whisker is the lowest value (if there are no outliers), the left vertical line (left side of the box) is the first quartile, the right vertical line (right side of the box) is the 3rd quartile, and the right side of the whisker (again if there are no outliers) is the highest value.
Comparison of Blackworms in Plain Water versus Blackworms in Water with Stimulant
Worms in water w/stimulant

Example Graph:

Worms in plain water

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pulses per minute

DAY FOUR: Presenting results and hanging the posters around the room grouping them by type of results (stimulant water higher pulse, stimulant water lower pulse, etc.) After all students have presented their results and the class discusses whether there is a general consensus, the teacher discloses the secret that all the waters were the same. This can lead into a discussion of how some of the greatest discoveries have come about when scientists got unexpected results and strove to understand and uncover what had really happened. It also leads to one of the topics of the Internet Treasure Hunt (blind and doubleblind studies) which can be assigned as homework or completed in class. DAY FOUR homework (can be done in-class adding a day to the timeline): Searching the Internet Students are guided to answer specific questions and are guided to websites where they can find the answers. The Internet Treasure Hunts of the students will be the background for their design of the second set of experiments. DAY FIVE and SIX: Designing and Performing an Inquiry Lab Based on Internet Research From their Internet research students should have information on chemicals which can impact pulse/heart rates of animals and an understanding of the purpose and design of doubleblind studies. They now should design a double-blind experiment (3,10) based on the basic experiment carried out earlier to test the impact of one of those chemicals on the blackworms.

The teacher can choose how much freedom to give the students in the solutions to be tested. In order to make management of the lab practical, limiting the students to a selection of 5 (or fewer) solutions could be considered. If two or three groups use the same solutions their results can then be compared to judge the reliability of the conclusions.

The instructor can also decide how much latitude to give the students in their experimental design (beyond the selection of the solution being tested). For instance, are they going to simply test whether the chemical impacts the pulse rate or are they going to design a more complex experiment such as whether the dose changes the intensity of the response (does a stronger solution give a stronger response), whether the length of the exposure changes the intensity of the response, etc. If students are given greater latitude in the design, the instructor may need to allow greater time to plan and complete the experiments. Following is an example of three hypotheses that cover the possible outcomes for an experiment where students are only testing whether the chemical impacts the pulse rate: Hypothesis 1: The worms in nicotine will have a higher pulse rate per minute than those in plain water indicating that nicotine increases the pulse of the blackworms. Hypothesis 2: The worms in nicotine will have a lower pulse rate per minute than those in plain water indicating that nicotine decreases the pulse of the blackworms. Hypothesis 3: The worms in nicotine will have the same pulse rate per minute as those in plain water indicating that nicotine has no impact on the pulse of the blackworms.

Upon completion of the lab students should analyze their data and discuss it with the teacher who will then reveal the identities of the solutions (which coded solution is the experimental solution and which is the control or in a more complex experiment what are the relative strengths of the solutions).

Once students know the identity of the solutions used they can proceed with writing their conclusions and creating their poster for presenting their experiment to the class much as they did with the first experiment. DAY SEVEN: Students present their lab results for discussion and peer and teacher evaluation.

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Where to Go From Here: This unit lays the groundwork for a variety of open-ended labs on topics such as respiration, photosynthesis, animal behavior, etc. Students should be encouraged to utilize the multiple hypotheses approach whenever possible. (9) Suggestions for Assessment: Students can be assessed on their class discussion and Internet Treasure Hunt notes. In addition, both labs can be assessed by the class and by the teacher using the rubrics provided. Grades can then be a weighted average of the class and teacher rubric scores. References and Resources: 1. Box and Whisker graphing Additional information on box and whisker graphing can be found in a lab found on the Kansas Biology Teachers website: http://www.kabt.org/wp-content/uploads/2008/04/water-drops-on-a-penny-black.pdf Accessed on 11/28/09 2. Chemical recipes Chemical recipes and additional ideas for experiment design can be found in: Effects of Drugs on Pulsation Rate of Lumbriculus variegatus (Blackworms) http://www.ableweb.org/volumes/vol-27/07_Bohrer.pdf Accessed on 11/28/09 Biological Smoke Detectors: A Toxicology Primer for Student Inquiry http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/Toxweb3.PDF Accessed on 11/28/09 Toxicity Testing with California Blackworms: Nicotine http://www.niehs.nih.gov/health/docs/blackworms-nicotine.pdf Accessed on 11/28/09 3. Double-Blind, Placebo-Controlled Trial http://chronicfatigue.about.com/od/fmsglossary/g/doubleblind.htm Accessed on 11/28/09 This is a definition for double-studies provided by About.com under their general description of fibromyalgia and chronic fatigue. 4. Experimental organisms For more information on guidelines for choosing experimental/model organisms see http://www.dnalc.org/resources/animations/model_organisms.html Accessed on 11/29/09 http://www.biologyreference.com/Ma-Mo/Model-Organisms-Physiology-and-Medicine.html Accessed on 11/29/09 5. Strong Inference The following two articles by John R. Platt and Sara M. Hiebert explain the value of the strong inference model in writing hypotheses and how it can be used in the classroom. http://pages.cs.wisc.edu/~markhill/science64_strong_inference.pdf Accessed on 11/29/09 http://www.the-aps.org/education/ugsrf/pdf/Sara%20Hiebert%20-%20Strong%20Inference.pdf Accessed on 11/29/09 6. Invertebrate Biology http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/ Accessed on 11/28/09 This website was originally created by Charles D. Drewes, Ph.D. and has been maintained since his death by Iowa State Universitys Department of Ecology, Evolution, and Organismal Biology. It has extensive information on where to acquire the worms, how to maintain cultures, and how to do labs and activities with invertebrates.

7. Lumbriculus variegatus aka blackworms

One source for these is Carolina Biological, 2700 York Road, Burlington, NC 27215, 800-3345551, http://www.carolina.com (catalog # 141720, approximately $8 + live shipping charge for enough for 2-3 classes depending on how you organize the lab). Accessed on 11/28/09

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8. Non-heparinized plastic micro-hematocrit capillary tubes

One source for these is Fisher Scientific Education, 4500 Turnberry Drive, Hanover Park, IL, 60133, 800-766-7000, http://www.fishersci.com (catalog # 22-315791, $12 to $17 per 100 depending on your schools discount and the number ordered). Accessed on 11/28/09

9. Open-ended Labs Sources

http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/ Accessed on 11/28/09 This website, mentioned above (6), has many ideas for using invertebrates in open-ended labs, especially for behavior studies. http://www.exploratorium.edu/IFI/resources/workshops/lab_activities.html Accessed on 11/28/09 This document on the website maintained by the Exploratorium in San Francisco gives some ideas on how to modify labs to make them more open-ended. http://www.kabt.org Accessed on 11/28/09 This website, maintained by the Kansas Association of Biology Teachers, has a variety of labs exploring properties of water, photosynthesis and respiration, and other topics in biology.

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10.What is a clinical trial? http://www.antigenics.com/trials/about/ Accessed on 11/28/09 This site is provided by Antigenics, Inc. to help people decide whether to participate in a clinical trial. It contains descriptions of clinical trials, blind and double-blind studies, and placebos. 11.Worms that keep the Beat http://www.kabt.org/wp-content/uploads/2008/08/worm_lab.pdf Accessed on 11/28/09 This lab by Randy Dix of Olathe North High School in Olathe, KS, a daphnia lab by Sandy Collins of West Junior High in Lawrence, KS, and the website (and workshops) by Charles D. Drewes, Ph.D. (listed above) provided the inspiration for the lab portions of this activity.

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