The lab presented in this paper utilizes a proven four-step pedagogicalframework (McLaughlin & Coyle, 2016) to redesign a classic Association ofBiology Laboratory Education (ABLE) undergraduate lab (McLaughlin &McCain, 1999) into an authentic research experience on vertebrate four-chambered heart development and physiology. The model system is thechicken embryo. Through their research, students are also exposed to theembryonic anatomy and physiology of the vertebrate heart, the electricalcircuitry of the developing heart, and the effects of pharmacological drugs onheart rate and contractility. Classical embryological micro-techniques,explantation of the embryo, surgical removal of the beating heart, isolationof the heart chambers, and more advanced tissue culture methods are alsoconducted. In this laboratory paradigm, students work in pairs to ask theirown questions concerning the effects of two human cardiovascular drugs,denopamine™ and acebutolol™ on both in vivo and in vitro chickenembryonic heart rate and contractility, develop testable hypotheses based oninformation gathered from relevant scientific literature, devise and carry outa controlled experiment, and present the data in a professional scientificmanner pertaining to a topic of clinical significance.
IntroductionCourse-based undergraduate research experi-ences (CUREs) provide a way to increaseand broaden participation of students inauthentic research (Auchincloss et al., 2014;Bangera & Brownell, 2014), and train themin the essential elements of authenticresearch, which include reading scientific lit-erature, generating questions (that don’t cur-rently have answers), forming hypotheses, designing experiments,collecting and analyzing data, working toward significant findings,and presenting results in both oral and written forms (Spell et al.,
2014; Lopatto, 2003; Seago, 1992). A four-step pedagogical frame-work that includes all essential elements of authentic research hasbeen developed that can serve to simplify and streamline the develop-ment and implementation process in an introductory biology labora-tory setting (McLaughlin & Coyle, 2016). This framework has beenshown to improve student perceptions of science and research, skillsand knowledge levels in scientific research practices set forth by theNational Research Council (2000), and confidence to effectivelyengage in scientific research practices including the ability to writestrong scientific lab reports and read scientific papers (McLaughlinet al., 2017). These results are consistent with other studies whichmeasured students’ perceptions and attitudes (Brownell et al., 2012;Harrison et al., 2011; Caruso et al., 2009; Weaver et al., 2008;Howard&Miskowski, 2005), ability to design experiments and inter-pret data (Myers & Burgess, 2003), and ability to develop informationfluency (Gehring & Eastman, 2008) in laboratory courses that inte-
The chicken embryo is a classic modelorganism used to illustrate the principles ofbasic vertebrate embryology primarily becauseit is easily accessible, inexpensive, and directobservation of a living embryo is possible evenat the early stages of embryogenesis. With theaid of a dissecting microscope, the key stagesof heart development and the circulatory pat-terns can be observed in vivo, even when thechicken embryo is still attached to the yolk.Another advantage to using the live chickenembryo to demonstrate the principles of heartdevelopment is that the embryo can beremoved from the shell and maintained in vitroin a temperature controlled environment for
several hours while the beating of the heart, flow of blood, andthe developmental stages accompanying the formation of the fourchambers are even more clearly visible. In addition, the chicken
Course-basedundergraduate
research experiences(CUREs) provide away to increase and
THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 645
I N Q U I R Y &I N V E S T I G A T I O N
An Authentic Research Experiencefor Undergraduates in theDevelopmental Biology andPhysiology Laboratory Usingthe Chick Embryonic Heart
• JACQUELINE S. McLAUGHLIN, MIT A.PATEL
embryonic heart is large enough for properly trainedstudents to surgically isolate it from the embryo.Thus, students who work with this particularembryo in their development and/or physiology lab-oratories not only learn principles of vertebrate mor-phogenesis, organogenesis, and cardiac physiology,but are also exposed to simple, yet useful micro-manipulative techniques. These particular dexterousmanipulations allow many of those who aspire tobecome physicians to try their hands (no punintended) at surgical technique.
We thoroughly redesigned a classic cookbookAssociation of Biology Laboratory Education (ABLE)undergraduate lab on vertebrate heart development(McLaughlin and McCain, 1999) to incorporate theabove highlighted four-step laboratory pedagogicalframework. This allows students to carry-out a higherlevel inquiry-based, authentic research experience(McLaughlin & Coyle, 2016; Goedhart & McLaugh-lin, 2016; McLaughlin et al., 2017) using present-day cardiovasculardrugs and updated tissue culture techniques and equipment. Stu-dents also master more advanced developmental biology, cardio-vascular physiology, and pharmacology concepts.
Lab Protocol
Developmental and Physiological Aspects of theChicken Embryonic Heart: Chronotropic andInotropic Effects of Select Drugs
ObjectivesThe key objectives in this lab are three-fold: (1) to think criticallyabout, and understand, how the four-chambered vertebrate heartdevelops using the chicken embryo as a model system; (2) to thinkcritically about specific pharmacological agent(s) and their modesof action(s) at both the cellular and organ (heart) levels; and (3)to carry out a scientific research experiment. To accomplish theseobjectives, you will have eight weeks to carry out a four-step pro-cess wherein you will design and execute an authentic researchproject. This experimental framework is consistent with the man-ner in which professional research scientists design, execute, inter-pret, and communicate their experimental results. The over-archingexperimental question that you will be attempting to answerthrough your research is the following: How do the cardiovasculardrugs, denopamine™ and acebutolol™, affect the in vitro heart rate(chronotropic effect) and force of contraction (inotropic effect) ofthe developing isolated chicken heart?
Background KnowledgeThe development of the four-chambered vertebrate heart involves aseries of cellular migrations, fusions, and specific differentiations—that is, a multitude of morphogenetic events. Although gestation ofthe human and chick differ, their four-chambered hearts develop ina very similar fashion, and because of this the chicken embryo is
used as a model system for human heart development. The heartsof the human and chicken embryos develop from the fusion ofpaired precardiac mesodermal tubes (aka “endocardial tubes”)located on either side of the developing foregut, on the ventral sur-face. These paired tubes begin to fuse at the anterior end (head)and continue to fuse posteriorly to form one continuous tubeknown as the tubular heart (Figure 1a). Once formed, the tubularheart is completely ventral to the foregut, and it has five distinctregions that can be identified from anterior to posterior: truncusarteriosus, bulbus cordis, primitive ventricle, primitive atrium, andsinus venosus (Figure 1b). Blood flows anteriorly, from the sinusvenosus to the truncus arteriosus (future ascending aorta and pul-monary trunk). The heart begins to beat just after the paired hearttubes begin to fuse. It is the sinus venosus that becomes the futureembryonic pacemaker.
The tubular heart then elongates on the right side, looping andbending to form an S shape, with the prominent ventricle bulgingto the right (Figures 1b and 2). The heart continues to fold uponitself, moving the sinus venosus and atrium to a position anterior
THE AMERICAN BIOLOGY TEACHER VOLUME. 79, NO. 8, OCTOBER 2017646
and dorsal to the ventricle and the bulbus cordis (Figure 3). The ven-tricle is now U-shaped and in the medial ventral position. The bloodflows posteriorly and then makes a sharp turn to flow anteriorly.
The atrium then expands to the left in preparation for its divi-sion into the right and left atria (Figure 3). Although the heart stillhas two chambers at this time, communication between the sinusvenosus and the atrium is via the right side of the atrium. This isthe first step toward the sinus venosus becoming part of the futureright atrium (SA node). The bulbus cordis will eventually give riseto parts of the ventricles, while the truncus arteriosus will becomethe future ascending aorta and pulmonary trunk. Eventually, whenthe atrium and ventricle have each divided into a pair of chambers,a typical four-chambered heart is present (Figure 3).
Laboratory AssignmentBelow is a description of the four steps youwill be following to carry out authenticresearch and to address the question athand (Figure 4). Each step will require atleast two weeks.
Step 1. Learn Essential LabTechniquesDuring step one, you will learn essential andrelevant in vivo and in vitro experimentaltechniques associated with both chickenembryo and heart manipulation via instruc-tor(s) demonstrations and hands-on train-ing. This step will include a small-scale andinstructor-led exercise (use Appendix 1worksheet).
Step 2. Design an ExperimentYou will work with your lab partner to research, select, and readprimary literature articles (at least three) related to the aboveguided question in order to devise a more specific, self-directedresearch question. You will then formulate a hypothesis directedtoward your specific question that is substantiated by the referenceliterature you select. Your next step is to design an experiment thatattempts to test your hypotheses and incorporates the knowledgeyou have gained on your selected drug(s), using an appropriatecontrol. Your experimental design must be written up as a tradi-tional research protocol, including detailed procedures and datainterpretation. You should consider the following when writingyour protocol: (a) how you will administer the drug to an isolatedchicken heart; (b) what concentrations you will use (you must have
Figure 3. The developmental stages of a human heart beginning with the fusion of two endocardial tubes up to specificcompartmentalization. Illustration of development of heart by OpenStax is licensed under CC by 3.0.
Figure 4. The four-step pedagogical framework for authentic scientific research(McLaughlin & Coyle, 2016).
THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 647
at least three different concentrations); (c) what will be your con-trol; (d) what kind of data you will collect; (e) how you will collectthe data; and (f) how you will represent these data in their finalform. Your instructor(s) must review your protocol to ensure thatit is adequately written, relevant, referenced, and executable withinthe allotted time frame before you are allowed to proceed with yourexperiment.
Step 3. Conduct the ExperimentConduct this experiment using learned techniques while gatheringdata in your notebook. This will require coordinated work withyour lab partner, and you will be expected to make good use ofopen laboratory time, in addition to scheduled class time. Yourinstructor(s) will be available for assistance or questions, but theexpectation is that your work will be self-directed and pacedappropriately.
Step 4. Interpret Data & Communicate ResultsReview your results and interpret your data. If your data are puz-zling or unclear, your instructor(s) can help you make sense of it.Once you are able to draw the conclusions of your experiment,you will present it to your instructor(s) in the form of a formalresearch paper or an oral presentation.
Techniques
Materials and Equipment (per student pairs)• chicken eggs: 3, 4, or 5 days old (half dozen eggs per week)*
• chicken egg humidified incubator
• CMRL (Connaught Medical Research Laboratories) media 1066[Ward’s Science]
• glass dish (110 mm diameter) lined with cotton (2)
• stock solution of R(–) denopamine™ [0.1mg/ml] Sigma AldrichD7815†
• stock solution of acebutolol HCl™ [0.1mg/ml] Sigma AldrichA3669†
• Falcon centrifuge tubes, 15 mL (6)
• water bath
• incubator for glass dishes and drugs
• dissecting microscope (illumination from above and below thespecimen) (2)
• gooseneck lamp with 100 W bulb (2)
• Syracuse culture dishes, 65 mm (2)
• Delta T-EDU Culture Dish Control System [Bioptechs]‡
• 20 G needle attached to a syringe
• iris fine scissors (2)
• iris micro-dissecting scissors
• fine forceps (2)
• micro-knife (hatchet)§
• embryo spoon§
• filter paper doughnuts§
• transfer (beral) pipettes, 1.5 mL (5)
• Beaker for egg waste (400 mL)
• DI water squirt bottle for rinsing off embryological tools
• Scotch Magic tape
• stopwatch (digital or cell phone)
• biohazard waste disposal
Notes for Instructors*Fertilized eggs at specific days of development can be ordered andshipped in-state or out-of-state through Moyer’s Chick Farm, Qua-kertown, PA (215-536-3155).†Any cardiovascular drugs with agonist/antagonist effect can beused in this experiment.‡Contact Bioptechs, Butler, PA (420-050-4212) directly, if youwould like to try an EDU system using chick embryos in yourclassroom and to discuss pricing.§See Appendix 2 for detailed instructions for making micro-knives,embryo spoons, and filter paper doughnuts.
I. Preparation of Chicken Egg and Bioptechs™ Equipment
1. Order eggs (in advance) from a chicken farm to receive fer-tilized eggs of the appropriate day(s) (i.e., ages). Followingpick-up, store eggs in a chicken egg incubator at 41°C untiluse.
2. Place CMRL media in a 45°C water bath two hours prior touse. (CMRL media has been shown to be the optimal mediafor in vitro chicken heart experimentation in our lab.)
3. Place all the glassware in incubator at 41°C two hours priorto use.
4. Set up Bioptechs EDU Culture Dish Control System on oneof the two dissecting scopes. Verify that EDU dish isinserted correctly and system is attached to the powersupply.
5. Add a small amount of warm CMRL media to the Delta Tculture dish. Allow the culture dish about two minutes toreach selected 41°C (avian) temperature. Close the lid ofthe dish.
II. Windowing an Egg (modified from Cruz, 1993)
1. Obtain an egg (day 3, 4, or 5) from the incubator and placehorizontally on a glass dish lined with cotton (under agooseneck lamp).
2. Use Scotch Magic tape to tape along the center of the longaxis of the egg, covering most of the top surface of theegg. Place two more pieces of tape on either side of the cen-ter piece (Figure 5a).
3. Puncture the rounded end of the egg using a 20 G needle.Insert the needle pointing down into the egg. Withdraw1–2 mL of albumen. This allows the embryo to move awayfrom the upper surface of the egg, where you will be cuttingout the window. Discard the albumen and rinse syringewith water.
4. CAREFULLY puncture the tape-covered surface of the eggwith the tip of your fine scissors. The location of the punc-ture should be about half an inch off-center (Figure 5a).
5. Cut out an oval opening while pulling up with your scissorsto keep them as far away as possible from the embryo and
THE AMERICAN BIOLOGY TEACHER VOLUME. 79, NO. 8, OCTOBER 2017648
Figure 5. The isolation of heart from a 5-day-old chicken embryo illustrated in a series of six steps: (A) preparing to “window anegg” using three vertical strips of tape placed across the long axis of an egg; (B) cutting an oval opening using fine scissors toexpose the embryo; (C) “explantation of the embryo” within its amniotic membrane using embryo spoon; (D) removal of amnioticand allantois membranes with fine forceps and/or micro-dissecting scissors in Bioptechs equipment; (E) surgical “isolation of theheart” from the embryo using fine forceps and micro-dissecting scissors/micro-knife; and, (F) “administration of drug” to theisolated heart using plastic transfer pipette.
THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 649
vitelline membrane (membrane around yolk). The size ofthe opening depends on the size of the egg, but it shouldbe about 1.5–2.0 inches in diameter. Remove the shell capwith forceps, exposing the window (Figure 5b).
6. If you are going to observe the embryo for more than acouple minutes while it is still in the egg, add several dropsof CMRL media to the surface of the embryo to preventdehydration. Do not add any CMRL media if you are goingto immediately explant the embryo. This will prevent thefilter paper doughnut from adhering to the vitellinemembrane.
7. Determine the in vivo heart rate of the embryo three timesat 15-second intervals. Record the heart rate in beats perminute (bpm) in your lab notebook. [It is recommendedthat one student record the time using a stop-watch whileanother student counts heart beats of the embryo lookingthrough the microscope.]
III. Explantation of a Chicken Embryo (modified from Cruz, 1993)
1. Explantation refers to removal of an embryo from its nor-mal in vivo environment and placement in a new location;in this case the embryo is transferred to a Bioptechs EDUCulture Dish. This is a useful method because it is mucheasier to manipulate or operate on an embryo when it isin a dish, rather than in an egg. To begin this process,add about quarter inch of warm CMRL media into apre-warmed Syracuse dish. Then, place the dish on thestage of your first dissecting microscope and angle thegooseneck lamp as close to the dish as possible in orderto keep the media warm. This is a critical step since thechicken embryo’s normal body temperature needs to remainat 41°C.
2. *Using forceps, gently place a filter paper doughnut aroundthe embryo such that it frames the embryo. The filter paperwill stick to the vitelline membrane, and the embryo will beexposed through the hole.
3. Using surgical scissors, cut along the edges of the filterpaper (vitelline membrane) off the surface of the egg whileholding it with forceps. As you release the filter paper, theembryo will remain attached to it.
4. Quickly transfer the filter paper doughnut along withattached embryo into a Syracuse culture dish already placedon the microscope.
5. If the embryo doesn’t stay attached to the filter paper, useyour embryo spoon to collect it from the underlying yolk.Remember to replace the yolk that transferred over from thisprocess with fresh CMRL media several times (Figure 5c).
6. Remove the embryo from all attached extra-embryonic mem-branes and extraneous yolk using fine forceps (Figure 5d).
7. Then, carefully transfer the embryo from the Syracuse dishinto the Bioptechs EDU dish placed on your second dissect-ing microscope using fine forceps. Check to make sure thetemperature is set to 41°C (avian). Add warm CMRL media,if needed.
8. Determine the in vitro heart rate of the explanted embryothree times at 15-second intervals. Record the heart rate in
bpm in your lab notebook. How does this heart rate com-pare with the in vivo heart rate?
9. Use Atlas of Descriptive Embryology to identify the exact stage ofdevelopment of your embryo. Record this in your laboratorynotebook.
IV. Isolation of the Heart (modified from McLaughlin & McCain,1999)
1. The explanted chicken embryo should be oriented ventralside up; this allows you to access the beating heart.
2. Surgically remove the beating heart by cutting it above thebulbus cordis and below the sinus venosus of the atrium(Figure 5e and 1b). Take a picture of the isolated heart.
3. Determine the in vitro heart rate of the isolated heart threetimes at 15-second intervals. Record the heart rate in bpmin your lab notebook.
V. Administration of Cardiovascular Drugs†
1. Prepare the serial dilutions of the drug(s) that you chose towork with. Make each dilution in a separate Falcon centri-fuge tube using the warm CMRL media as your solvent.Store your labeled tubes in the rack at 45°C water bath orincubator.
2. Design table(s) similar to the ones in Appendix 1, whereinyou adequately record all of your heart rate data per yourexperimental design. Remember to include both in vivoand in vitro embryonic heart rates (control baseline heartrates) before you exogenously expose your embryos to anydrugs.
3. Remove as much CMRL media bathing the isolated heart aspossible using transfer pipette, and add 1 mL of the lowestconcentration of the drug to the isolated heart in the dish (Fig-ure 5e). Wait at least 30 seconds for it to equilibrate, thenrecord the heart rate (three times at 15-second intervals), not-ing any arrhythmias (such as tachycardia, bradycardia, atrialflutter, fibrillation, etc.) or changes in heart contractility (vari-ation in force of contraction in any or all chambers). Removethe drug as best as you can from the Bioptechs EDU dish usinga transfer pipette, then immediately add the next higher dilu-tion of the drug. Repeat the procedures in this step to obtainin vitro heart rate for all your drug dilutions.
4. It is imperative that you work up at least five embryos per theage group(s) selected. For example, if you chose to observe theeffect of acebutolol™ on the 4- and 5-day old isolated chickenhearts, you will need to work up five 4-day-old eggs and five5-day-old eggs. Work up your embryos one at a time, repeat-ing Steps II–VI and recording the control heart rates at allstages.
5. Carry out your experiment!
Notes for Students*Use a filter paper donut on 3- and 4-day-old embryos; use anembryo spoon only on 5-day-old embryos.†This step is preformed following the approval of your lab protocolby your instructor(s).Important: Discard egg shells, embryonic waste and embryos inappropriate biohazard containers.
THE AMERICAN BIOLOGY TEACHER VOLUME. 79, NO. 8, OCTOBER 2017650
ConclusionThe simplicity and flexibility involved in the four-step pedagogicallaboratory framework allows it to be easily adopted for use withinthe unique infrastructure and resourceful environments at a varietyof institutions and at different levels of biological study, effectivelyincreasing student access to authentic scientific research. For exam-ple, this pedagogical framework was used to successfully transforma sophomore-level, introductory, cell biology laboratory for majorsat a 4-year college branch campus (McLaughlin & Coyle, 2016)and an honors, introductory biology laboratory for non-majors ata 2-year college (Goedhart & McLaughlin, 2016) into authenticresearch experiences. For the former, students investigated variousfactors affecting the growth and viability of a mammalian cell cul-ture line, Vero cells, and related their findings to a current issuein the field of cell biology, nutritional and/or physical stress of cells.For the latter, students selected and investigated factors affectingmicroalgae cell growth and related their findings to a real-life appli-cation of social significance. A short video documenting the use ofthe pedagogical framework from both instructor and student pers-pectives, From Cookbook to Critical Thinking (https://vimeo.com/118326855), was created by Citrus College and published on Vimeo,Inc. In both laboratory paradigms, students worked within groups tolearn modern cellular biology techniques, ask their own questions,develop testable hypotheses based on information gathered from rele-vant scientific literature, devise and carry out a controlled experiment,and present the data in a professional scientific manner.
The lab presented in this paper also utilizes the four-step ped-agogical framework, but this time to transform a sophomore-level,introductory, developmental biology laboratory for biology majors.Students investigate the development of the vertebrate four-chambered heart using the chicken embryo as a model system,while simultaneously being exposed to the embryonic anatomyand physiology of vertebrate heart, the electrical circuitry of thedeveloping heart, and the effects of pharmacological drugs on heartrate and contractility. Classical embryological micro-techniques andprocedures are conducted as well. Student, working in pairs, devisea hypothesis concerning the effects of two human cardiovasculardrugs, denopamine™ and acebutolol™, on in vitro heart rate andheart contractility. These drugs were selected for their known ago-nist or antagonist effects on the human heart rate. Other drugs thathave been successfully used in this lab to substitute these drugsinclude dobutamine, verapamil, ractopamine, epinephrine, nico-tine, and pseudoephedrine. Indeed, the list of drug substitutionsis endless, and as such, allows the instructor(s) semester-to-semesterversatility while maintaining the authentic research nature and, assuch, enhanced performance and engagement of students (lack ofplagiarism). To best exemplify the authentic research nature andenhanced performance of students who have experienced thechicken lab in particular is this reality: Many students have pre-sented their work at peer-reviewed conferences or have publishedtheir research (e.g., Gonzalez et al., 2015).
It is important to point out the following when utilizing theframework: Instructors should act as research chaperones, guidingstudent scientists through each step, providing constant feedbackand environments that allow time for student self-reflection, mis-takes, and dialogue over assignments before their final submissionfor grading (i.e., protocol, notebook, scientific paper, poster, etc.).
Additionally, the four steps outlined herein should scaffold the sci-entific process, allowing students who are novices to the scientificprocess to progressively gain familiarity and comfort with theessential elements of scientific inquiry. Lastly, it is recommendedthat instructor(s) read how this framework was used by otherinstructors to rework the semester with regard to time and work-load (McLaughlin & Coyle, 2016; Goedhart & McLaughlin, 2016).
AcknowledgementWe would like to extend our gratitude to Michelle Lynn for sketch-ing a realistic illustrations of the chicken heart isolation procedure.We would also thank Dr. Elizabeth McCain, who worked tirelesslywith Dr. Jacqueline McLaughlin on publishing the original version(ABLE) of this lab.
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Hanauer, D. I., … Dolan, E. L. (2014). Assessment of course-basedundergraduate research experiences: A meeting report. CBE-LifeSciences Education, 13, 29–40.
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Cruz, Y. P. (1993). Laboratory exercises in developmental biology. SanDiego, CA: Academic Press.
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Goedhart, C. M., & McLaughlin, J. S. (2016). Student scientists: Transformingthe undergraduate research laboratory into a research environment.American Biology Teacher, 78(6), 502–508.
Gray, H. (1918/2000). Anatomy of the Human Body. Philadelphia: Lea &Febiger; Bartleby.com, 2000.
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McLaughlin, J. S., Favre, D. E., Weinstein, S., & Goedhart, C. M. (2017). Theimpact of a four-step laboratory pedagogical framework on biologystudents’ perceptions of laboratory skills, knowledge and interest inresearch. Journal of College Science Teaching, 47(1), 83–91.
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McLaughlin, J. S., & McCain, E. R. (1999). Developmental and physiologicalaspects of the chicken embryonic heart. In C. A. Goldman (Ed.), TestedStudies for Laboratory Teaching. Proceedings of the 20th Workshop/Conference of the Association for Biology Laboratory Education (ABLE)(vol. 20, pp. 85–100).
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JACQUELINE S. MCLAUGHLIN ([email protected]) is an Associate Professorof Biology in the Department of Biology at the Pennsylvania StateUniversity—Lehigh Valley, 2809 Saucon Valley Rd., Center Valley, PA18034. MIT A. PATEL ([email protected]) is a Postbac IRTA at NationalInstitute of Health in the Department of National Institute of Allergy andInfectious Diseases, 9000 Rockville Pike, Bethesda, MD 20892.
APPENDIX 1 Examining the Heart Rate of the Embryonic Heart
Table 1. Summary of in vivo and in vitro heart rates of embryo and isolated heart.
Table 2. Summary of in vitro heart rates in each separated region of heart.
In vitro
Bulbus Cordis Sinus Venosus Atrium Ventricle
HR(15 sec)
HR(bpm)
HR(15 sec)
HR(bpm)
HR(15 sec) HR (bpm)
HR(15 sec)
HR(bpm)
Trial 1
Trial 2
Trial 3
Average
1. In your lab notebook, using an embryological atlas like Atlas of Descriptive Embryology, diagram and thoroughly label3-day-old and 4-day-old chicken hearts (whole mounts).
2. Window a 3- or 4-day-old egg, then determine and record the in vivo heart rate three times at 15-second intervals in Table 1.
3. Explant the embryo out of the egg, then determine and record the in vitro heart rate of the embryo three times at15-second intervals in Table 1.
• How does this heart rate compare to that of in vivo heart rate?4. Surgically isolate the heart. Observe the beating of the isolated heart, then determine and record the in vitro heart rate of
the heart three times at 15-second intervals in Table 1.
• Where does the beating begin and end? Be precise.
• Is the isolated heart rate similar or different from the heart beat of the explanted, in vitro embryo?
• Draw a picture of the isolated heart and label each region.
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5. Use the micro-knife or iris micro-dissecting scissors to separate the four regions of the isolated heart (bulbus cordis,sinus venosus, atrium, and ventricle). Observe each isolated region of the heart, then determine and record the in vitroheart rate of each region three times at 15-second intervals in Table 2.• Does each region have an intrinsic heart beat? If so, are they synchronous?
• Does each region beat at the same rate as the in vivo or in vitro explanted heart?
• Draw a picture of the isolated regions of heart and appropriate labels.
APPENDIX 2 Directions for Making Embryological Tools
Micro-knives (modified from Tyler, 1994)
1. Break a single-edge razor blade into small fragments with the pliers or tin snips, making surethat each fragment has the cutting edge on it. Avoid damaging this edge. The fragment shouldbe between 5 and 10 mm long.
2. Soak one end of the wooden applicator stick (2 mm diameter; 100–150 mm long) in waterfor about 5 minutes to soften the wood. Make a 5-mm split down the center of the moistenedend of the stick with a fresh razor blade. Do not splay out the two sides of the split;just create a crack in the stick large enough for the razor blade fragment to fit in.
3. Carefully insert the rough edge of the razor blade fragment into the split at 45° angle to theaxis of the stick. You can’t use the pliers to do this because they will damage the cutting edge.You must use your fingers—be careful! The cutting edge of the blade should not be embeddedin the wood at all, and you should be able to comfortably hold the stick like a pencil and havethe razor’s edge almost flat with a desk surface.
4. Let the wood completely dry. Paint the split edges with super glue to firm the placement of the razor blade fragment.
5. Store the micro-knives such that their edges are protected. One method is to stick Styrofoam peanuts on the razor edge.
Note: Micro-knives can only be used for one lab period; they dull quickly.
Embryo Spoon (modified from Tyler, 1994)
1. Heat an insect or dissecting pin over a flame and puncture 8–10 holes in the bowl area of theplastic spoon (ice cream spoon), as shown in the picture.
2. Use the sandpaper (220 or 400 grit) to remove the sharp plastic edges.
Filter Paper Doughnuts
1. Fold a piece of filter paper (Whatman #1, 3.2 cm diameter) in half, then cut out a semi-circlein center of the filter paper, keeping 0.5 cm distance between two circles.
2. The end result should be an oval-shaped doughnut.
THE AMERICAN BIOLOGY TEACHER CHICKEN EMBRYONIC HEART 653
