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TEACHING IN THE LABORATORY
Department of Biology, Swarthmore College, Swarthmore, Pennsylvania
Address for reprint requests and other correspondence: S. M. Hiebert, Dept. of Biology, Swarthmore College, Swarthmore, PA 19081-1390 (e-mail: shieber1{at}swarthmore.edu)
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Abstract |
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 TOP Abstract IntroductionREFERENCES |
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Key words: endothermy; ectothermy; respirometry
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Introduction |
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TOPAbstractIntroductionREFERENCES |
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1/5 to 1/10 of the basal metabolic rate of endotherms) even when they are maintaining comparable Tbs (2). Thus, the endotherm lifestyle, while affording a relatively constant Tb at which physiological systems can function at their best, is an energetically costly one. Ectotherms are more limited in the habitats, seasons, and times of day at which they can be active because they rely on heat provided by the environment, but they gain the advantage of requiring much less energy overall.
General Principles That Can Be Taught With This Exercise
In the fact-laden realm of biology, it is important for students to understand that specific examples, such as the response of chick embryo metabolism to Ta, are less important in their own right than as illustrations of larger, recurring themes that can help them understand the lives of all organisms. Students may find it challenging but ultimately very useful to generate a list of general principles related to the chicken embryo metabolism experiment. The following are some important concepts that can be taught in this laboratory exercise; the ones you include will depend on the content of your course, the background knowledge of your students, and the time your course devotes to this exercise.
O2) is an indirect measure of metabolic rate. O2 to metabolic rate requires either knowing which metabolic fuel (carbohydrate, protein, or fat) is being metabolized, making an educated guess, or using an intermediate conversion constant likely to yield the lowest maximum error.
Experiment Design
Experimental designs, suggestions for classroom implementation of a student-centered experiment design process, and specific content pertaining to the chick embryo metabolism experiment are described in detail in the companion article "Teaching simple experimental design to undergraduates: do your students understand the basics?" (19). Briefly, either of the two following designs is recommended. In the first design, the O2 of all embryos is measured at the normal incubation temperature (38°C), and the results are used to create two treatment groups balanced for O2. The experimental group is then placed at 23°C (room temperature), whereas the control group remains at 38°C. After 90 min of temperature equilibration, the O2 of all embryos is measured again, and the final O2 of the two treatment groups is compared with an unpaired t-test. In the second design, each embryo is subjected to a measurement of O2 following 90 min of equilibration at each temperature, but half of the embryos are measured first at 38°C, whereas the other half are measured first at 23°C. A paired t-test is then used to compare all O2 at 38°C with all O2 at 23°C. For both experimental designs, a total of 12 eggs (16 days old) is sufficient for clear, easily interpretable results using any of the methods described in this article.
Instructions for Students: Measurements and Calculations
This section contains instructions for the collection and analysis of air samples, calculations, statistical analysis, and interpretation of results. These methods are to be used in conjunction with an experimental design (see Experimental Design above) that has been designed by students or chosen by the instructor. At a minimum, this design requires one incubator that is large enough to 1) maintain the eggs when they are not being used in the experiment and 2) house respirometers when O2 is being measured at 38°C. The second temperature treatment (23°C) can be achieved by either placing the eggs at room temperature for 90 min or placing them in an incubator at 23°C. After a suitable incubation period in the respirometer, air samples are drawn into a syringe and injected into an oxygen analyzer. Instructions are for respirometers consisting of 500-ml jars, the lids of which have been fitted with a two-way stopcock (Fig. 2). Several different respirometer designs and low-cost alternatives for measuring O2 without specialized equipment are described in Equipment: Descriptions and Alternatives. Students should practice critical aspects of the protocol, such as obtaining air samples and injecting them into the oxygen analyzer, before they begin an experiment with live embryos.
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O2 is measured the second time. 15 min, follow the instructions below to draw a 20-ml air sample from the respirometer into a syringe. Be sure to record the time at which you withdrew the air sample; you will need it for your calculation of O2.
II. Measuring the oxygen content of the air sample.
III. Calculating the volume of air in the respirometer.
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IV. Calculating O2.
Use the following equation to convert percent oxygen in the air sample you measured to O2 at standard temperature (273 K) and pressure (760 mmHg) in dry conditions (STPD) (20). Be sure to enter each of the values in the correct number format as shown below:
 | (2) |
where O2 is measured in ml/h; V is the volume of air in the respirometer (in ml); Ta is the temperature of air in the respirometer at the beginning of the trial (in K; K = °C + 273); Pb is measured in mmHg; WVP is the water vapor pressure in fully saturated air at Ta [in mmHg; e.g., 19.83; values can be found in a table entitled the Vapor Pressure of Water in the CRC Handbook (23a)]; RH is the relative humidity of air in the respirometer at the beginning of the trial (e.g., 72% would be entered as 0.72); FI is the initial fractional concentration of oxygen [the fractional concentration of oxygen in the room air (e.g., 0.2096)]; FE is the ending fractional concentration of oxygen (e.g., 0.2074); and t is the amount of time the egg was closed in the respirometer (in min; e.g., 15.5).
V. Calculating mass-specific O2.
In many cases, it is useful to standardize O2 between individuals of different masses by expressing O2 per gram of tissue. In the case of the chicken embryo, however, using the mass of the whole egg to compute mass-specific O2 is probably not very meaningful because not all of the egg mass consists of embryonic tissue. This is especially true at the earlier stages of development, when much of the egg's mass is made up of the shell, albumen, and unmetabolized yolk.
O2 of your embryos, divide the O2 you calculated in step IV by this embryo mass. O2 you calculated fall within the range expected for an endotherm or for an ectotherm?
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O2.O2 (expressed as the volume of oxygen consumed per unit time) is not a direct measure of metabolic rate (expressed as energy turnover per unit time), because the energy equivalent for a given O2 depends on which fuel (carbohydrate, protein, or fat) is being metabolized. In the case of chicken embryos, the primary energy source is the fatty yolk. When fats are broken down for energy, 19.8 kJ is liberated for every liter of oxygen consumed (21). Thus, for chicken embryos,
 | (3) |
The conversion factor for carbohydrates is 21.1 kJ/l oxygen and for proteins is 18.7 kJ/l O2 (21). When the metabolic fuel is unknown, a conversion factor of intermediate value, 20.2 kJ/l O2, is used. Note that calculating the metabolic rate from O2 is not needed to answer the question posed in this exercise, because the metabolic rate and O2 are always related by the same constant (19.8 kJ/l O2). Many published studies have reported O2 instead of metabolic rate unless the authors wanted to quantify energy use per se.
VII. Comparing O2 at the two experimental temperatures.
For the first experimental design, use an unpaired (two-sample) t-test to compare the O2 of the embryos at 23°C with the O2 of the embryos at 38°C. For the second experimental design, use a paired t-test to compare O2 at 23 and 38°C for all embryos. Is there a significant difference between the O2s measured at the two temperatures? If so, do your embryos show a positive or negative relation between Ta and O2? On the basis of this finding, do you conclude that your embryos are endotherms or ectotherms? If your conclusions in steps V and VII are different, which do you think provides the most conclusive answer to the question of whether chicken embryos are endotherms or ectotherms? Why?
VIII. Calculating Q10.
Q10 is a standardized way of describing how reaction rates change with temperature. It can be calculated not only for simple chemical reactions but also for more complex physiological processes such as metabolic rate or running speed. Q10 is the number of times the reaction rate increases when the temperature is increased by 10°C. For example, if the reaction rate doubles when the temperature is increased from 20 to 30°C, then Q10 = 2 for this temperature range. However, Q10 can be calculated even when reaction rates are measured at temperatures that are not exactly 10°C apart using the following formula:
 | (4) |
where R1 is the reaction rate at the first temperature (T1) and R2 is the reaction rate at the second temperature (T2). If you have determined that your embryo is an ectotherm, calculate Q10 from the O2s that you measured for the whole embryo. Typically, Q10 for biological reactions ranges from 2 to 3. Does the Q10 you calculated fall within this range?
IX. Presenting your results.
Primates, including the readers of your report, are especially skilled at interpreting effective visual images. Present a summary of the data you have collected in a single graph that shows all of the important findings you have made in this experiment. Remember to show all means with some measure of variability (SD, SE, or 95% confidence interval) so that your reader can determine whether any differences between means are meaningful or simply a result of random differences among individuals.
Sample Data
For both experimental designs, measurements of healthy embryos should yield a significantly higher O2 at 38°C than at 23°C, leading to the conclusion that embryos behave as ectotherms (Fig. 3). In over a decade of using this laboratory exercise, our students have always found significant differences in the expected direction for ectotherms with a total sample size of 12 eggs regardless of which experimental design they chose to use.
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O2, whereas, at other times, the rate of energy expenditure is expressed as metabolic rate. O2 is expressed in units of volume of oxygen consumed per unit time (e.g., ml O2/h), whereas metabolic rate is expressed as energy turnover per unit time (e.g., J/h). O2 to metabolic rate? Answer: the fuel (carbohydrate, protein, or fat) being metabolized is required, because the relation between O2 and metabolic rate differs for each of these metabolic substrates. O2 is sufficient to answer the question. Give an example of an experiment in which converting O2 to metabolic rate is necessary to answer the question. Answer: expressing rates of energy expenditure as O2 is generally suitable for any experiment in which one wants to compare the energy cost of activities (e.g., an animal walking versus running, a hibernating versus normothermic animal, or an animal thermoregulating at different temperatures). This is especially true if the metabolic fuel is 1) the same for each activity or 2) not known for any of the activities, since one would have to use the same conversion constant for each calculation. In these cases, a conversion to metabolic rate would not change the result qualitatively. Expressing rates of energy expenditure as metabolic rate is necessary when one wants to construct an energy budget (e.g., how many kJ does this animal burn per day or year?) or when comparing energy intake and expenditure (e.g., how many acorns are needed to fuel a squirrel's daily activity? or How many additional acorns are needed to fuel a dominant male's territorial and aggressive activities compared with the activities of a subordinate male?). In the latter examples, it would also be necessary to determine the amount of energy available per acorn by bomb calorimetry, a procedure in which one measures the amount of heat released when a known mass of a substance is burned. O2) increases because the body must generate more heat to maintain Tb in a cold environment. In this case, a calculation of Q10 would tell us not about the direct effect of tissue temperature on metabolic rate but about the active thermoregulatory responses of the animal to cold.
Classroom Implementation: Practice and Philosophy
Possible laboratory schedules (based on one 3-h laboratory period/day).
3-DAY SCHEDULE.
On day 1, students learn the principles behind different methods for measuring metabolic rate and learn how to measure O2, perform calculations, and handle the eggs. On day 2, students plan their experiment and discuss the content of a strong-inference protocol. On day 3, students turn in their strong-inference protocol for grading, perform their experiment, and collect data. Note that if there is 1 laboratory session/wk, this schedule requires two sets of experimental embryos: one set for week 1 and another set for week 3.
2-DAY SCHEDULE.
On day 1, students plan their experiment and discuss the content of a strong-inference protocol. On day 2, students turn in the strong-inference protocol for grading, perform their experiment, and collect data.
1-DAY SCHEDULE.
Students perform their experiment and collect data.
Rationale.
When this laboratory exercise is taught as part of our intermediate-level Animal Physiology course, we use the 3-wk schedule for the chick metabolism experiment because it fulfills a variety of pedagogical and content functions. In relation to content, the exercise bridges the topics of metabolism and temperature relations. The exercise also prepares students for the independent project that they will conduct later in the semester, by guiding them through the process that begins with a question, follows with the design of an experiment to address the question and the formulation of a strong-inference protocol, and concludes with the collection of data, statistical analysis, and preparation of a report in the style of a scientific paper. The student-centered experiment design process and the use of strong-inference protocols are described in detail in the companion articles "Teaching simple experimental design to undergraduates: do your students understand the basics?" (19) and "The strong-inference protocol: not just for grant proposals" (18).
The need for practical instruction in the scientific method has been highlighted in a recent study (33) of secondary science education by the National Academies of Science. According to this report, first-year undergraduates are arriving in the science classroom with little if any practical experience in using the scientific method and sparse experience with any kind of investigative laboratory exercises in which they do not know the outcome before they start the experiment. Even though instruction focusing on the scientific process has been articulated as a major goal of science education, teachers and administrations tend to favor content coverage in the interest of preparing students for standardized examinations (33). Until significant changes are made in high school curricula, your course may be the first (and possibly the last) chance a future well-educated citizen has to acquire this fundamental aspect of science literacy.
Because prior experience in the experimental method is lacking, most of my students are surprised to learn that planning an experiment could fill a 3-h laboratory session. This schedule effectively makes the point that designing a good experiment is at least as important as actually performing it. Instructors may raise the concern that extensive planning will initiate the "expectancy effect," which can color students' interpretation of results, or that it will take away from the enjoyment that students derive from a laboratory exercise when they don't know the outcome in advance (14, 24). Despite the extensive thought, planning, and discussion that precede the experiment, however, most students in our course are surprised by the data they obtain in the chicken embryo metabolism experiment. In addition, the increased clarity regarding data analysis and interpretation offered by the strong-inference approach (28) sets the stage for a more satisfying and rigorous laboratory experience. It is well worth noting that instructors can also benefit from the 3-wk schedule because a different laboratory setup is not required each week, thus reducing preparation time. Sometimes, a better educational experience for students is also less work for teachers.
Educators feeling the constraint of content coverage may be relieved to learn that this laboratory module can be taught in fewer than 3 wk. Each instructor must, of course, respond to the particular needs of his or her own course and/or department, but we urge instructors to consider which skills (specific content knowledge versus the ability to design, execute, and evaluate an experiment) will prove more universally applicable in the longer view. We cannot teach our students everything there is to know; therefore, we might as well teach what we teach in a way that will help students to learn it deeply so that they will retain the information after the exam is over. Some educators may argue that it is the students who demand a new laboratory exercise each week, but the feedback we have received regarding the 3-wk laboratory modules in the Animal Physiology course is extremely favorable; the student who complains about lack of variety is extremely rare.
Experiment Variations
The basic experiment described above may be expanded or modified in a variety of ways. Some possibilities include the following:
Technical Notes for Instructors
Temperature treatments.
MAINTENANCE INCUBATION.
For this exercise, we use 16-day-old chicken eggs. Until used in the experiment, eggs should be incubated at 38°C in a humidified incubator. Any 38°C chamber can be adequately humidified by placing a tray filled with water at the bottom of the incubator; check this tray at least once per day and refill it as necessary. It is easiest to order the eggs so that they will arrive close to the time of use. If you plan to keep the eggs in the laboratory for a day or more, place them in an automatic egg turner or turn them gently by hand 4 times/day. Turning is required to prevent developmental abnormalities (13, 27).
CHOOSING TEST TEMPERATURES.
The temperatures stipulated in the procedure for students are known to provide statistically significant differences in O2 and a qualitative result that is easy for students to interpret. You may, however, modify the experiment by choosing different temperatures. Ultimately, this decision will be informed by the precise question students want to answer, the equipment available, the amount of time available for collecting data, the range of temperature tolerance by avian embryos, and a general knowledge of temperature-metabolism relations for endotherms and ectotherms. The goal is to choose physiologically tolerable temperatures that will result in either a positive (ectotherms) or negative (endotherms) relation between Ta and O2. For ectotherms, any two temperatures in the normal range experienced by the animals in nature will show a negative relation between Ta and O2. For endotherms, the higher Ta should be either within the TNZ or below the LCT and the lower Ta should be below the LCT (see Fig. 4 for a graphical explanation).
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32°C at hatching and 23°C 4 wk later, by which time the birds have increased substantially in both body mass and insulation (38). At least some students will know that it is common to keep newly hatched chicks under heat lamps for the first few days of life and will use this fact in their reasoning about how low the LCT might be in a late-stage embryo if it were endothermic. An additional general principle that students can discuss is that the LCT is typically lower (and the TNZ wider) as animals increase in size or become cold adapted (5, 7). For a 15- to 20-g bird (the approximate mass of the embryo between 16 and 18 days of incubation) descended from a warm-zone species and raised commercially in warm conditions, one would not expect to find a LCT as low as 23°C (see also Fig. 1).
EQUILIBRATION AT DESIRED TEST TEMPERATURES.
The egg cooling curve (Fig. 5) provides approximate times required for an unfertilized chicken egg, which contains no embryo, to reach a particular internal temperature when it is removed from a 38°C incubator and placed at room temperature (23°C). According to this curve, an egg undergoing Newtonian cooling should reach an internal temperature of 23°C within 90 min and 30°C within 25 min. Note that if the eggs were placed in a 30°C environment to cool, the internal temperature would take >25 min to reach 30°C because the temperature differential between the center of the egg and the surrounding air would be smaller; thus, to speed up cooling from 38°C to temperatures higher than room temperature, place the eggs at room temperature for the time indicated on the cooling curve and then place them in an incubator at the desired temperature. Because eggs containing living embryos tend to cool more quickly than unfertilized eggs (37), the cooling rates shown in Fig. 5 are likely to be overestimates, especially for eggs weighing <58 g.
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Respirometry.
It is recommended that the FE be within the range of 20.00–20.90%. Keeping values in this range will prevent any stress due to low oxygen availability while ensuring that oxygen content will be sufficiently different from ambient to be accurately measurable. Decreasing the size of the respirometer or increasing the incubation time in the respirometer will decrease FE, whereas increasing the size of and/or decreasing the egg's time in the respirometer will have the opposite effect. Ideally, incubation times and chamber volumes should be chosen so that the same chamber size and incubation time produce usable results at both temperatures. To minimize the influence of any transient effects of handling on the embryos, it is recommended that the eggs remain in the respirometer for a minimum of 10 min. Note that some oxygen analyzers require up to 24 h of equilibration after being turned on before they will provide accurate measurements. A sample airflow circuit is shown in Fig. 6. Consult the manufacturer's instructions for the analyzer warm-up times, calibration instructions, and details of airflow circuitry.
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O2, students will find it very helpful to use a spreadsheet or other calculating tool that has been preprogrammed to make their calculations as soon as they have entered their measurements. This will hasten the process of collecting class data and, in the case of experiment design 1, assigning eggs to treatment groups so that the experiment can be completed within the 3-h laboratory period. Students using hand calculators frequently make errors; mistakes of this kind can be especially critical if erroneous calculations are used to divide the eggs into balanced treatment groups, as in experiment design 1.
Eggs.
Fertilized chicken eggs of a specified age can be purchased from a commercial supplier. Although 15- to 16-day-old embryos are recommended because the O2 differentials at the recommended test temperatures are large, students have obtained data with clear results from embryos that were mistakenly sent to us by the vendor at 6 days rather than 16 days after fertilization. Thus, a wide range of embryo ages appears to be suitable for this experiment.
At the end of the experiment, eggs can be frozen and disposed of according to your institution's guidelines. Small cracks that develop in the eggs during the experiment can be sealed with veterinary-grade cyanoacrylate adhesive (Vetbond or Nexaband), which differs in composition from and has lower toxicity than over-the-counter adhesives such as Krazy Glue (30).
Embryo masses.
Instructors may want the students to dissect and weigh their embryos so that the masses used in the calculations of mass-specific rates are empirically determined. If they want to do so, embryos can be killed by placing the eggs in a jar containing halothane vapors (4). We generally do not ask students to dissect the embryos because we reuse them in later laboratory sections to reduce the total numbers of eggs required and because a single 3-h laboratory period is already very full without this extra step. In this case, use the information shown in Table 1 for calculations.
Regulations.
Current federal animal use guidelines do not govern vertebrate embryos. Before beginning this experiment, however, you should consult your Institutional Animal Care and Use Committee or other research ethics committee for policies at your institution. Hatching the eggs is permissible only with an approved animal use protocol, as stipulated in the Guide for the Care and Use of Laboratory Animals (17). Eggs of all native species of vertebrates are protected by law and may not be collected without the applicable federal and state research permits.
Background information on avian incubation and embryo development.
The stage at which birds become fully endothermic depends on whether birds are altricial (helpless, featherless, and sightless at hatching) or precocial (ambulatory, sighted, and nearly or completely endothermic at hatching) (40). As is common in ground-dwelling birds, chickens are precocial, but their thermoregulatory capacity is not fully developed until several days after they hatch, which takes place on day 21 of incubation. If challenged with small (
3°C) reductions in ambient temperature, an 18-day chicken embryo responds not by increasing O2 but by failing to decrease O2, and, by 19 days, embryos respond with a small, transient increase in respiration rate (for a review, see Ref. 42). At this stage, the ability of the embryo to thermoregulate fully seems to be limited by two factors. First, O2 is constrained by the ability of oxygen to diffuse through the eggshell. Evidence that embryos are operating under an oxygen limitation comes from the observation that internal pipping (when the chick's beak breaks into the air cell inside the egg at around day 19) and exposure to an artificial high-oxygen atmosphere result in an increase in O2. Second, the metabolic capacity of the embryo to produce heat is limited, even in experiments where the oxygen availability is experimentally increased. As a consequence of low heat production, the embryo temperature begins to drop at Ta < 38°C. Arrhenius (Q10) effects then cause the metabolic rate to drop, thus further decreasing heat production capacity and further lowering embryo temperature. Thus, even embryos with some thermogenic capacity would appear ectothermic by the major criterion used in this laboratory exercise: a positive relation between O2 and Ta. However, at the age (16 days) and temperatures (23 and 38°C) suggested for this experiment, the embryos should be fully ectothermic.
During incubation in many species, the incubating parent(s) may leave the eggs unattended for varying periods of time (17) to search for food or fend off predators. An incubating parent energetically challenged by a continuous lack of food due to low temperatures, bad weather, or lack of prey may even abandon the eggs so that it can search farther from its nest for food; if conditions improve before too long, the parent may be able to resume incubation with good hatching success, especially if the interruption in incubation has occurred during the less vulnerable early stages of incubation (13). In some species, the eggs are not incubated until the entire clutch has been laid, with the result that the eggs hatch synchronously (13). Thus, it is normal for avian eggs in nature to experience periods of cooling. However, the Arrhenius relation reminds us that although low temperatures may not kill a developing embryo, we would expect developmental processes to slow as eggs cool. The temperature below which developmental progress becomes unmeasurable [the "physiological zero temperature" (13, 17)] is close to 23°C, substantially higher than some of the temperatures from which embryos have been known to recover (35). Eggs kept for long periods of time at temperatures above the physiological zero temperature but below the optimum incubation temperature may develop abnormally. Embryos are much more capable, however, of surviving the state of nearly suspended animation at temperatures below the physiological zero temperature (13). For example, fertilized chicken eggs may be kept at 11–13°C for several weeks without apparent harm to the embryo (13). Nevertheless, it is generally to an incubating parent's advantage to keep egg temperature close to the optimum incubation temperature once incubation has begun.
Because students are measuring rates of gas exchange, they will be curious about how oxygen and carbon dioxide are exchanged across the eggshell (see Ref. 31 for further details). Under a dissecting microscope or with a hand lens, students can see pores in the shell, through which three important gasses pass: oxygen (inward), carbon dioxide (outward), and water vapor (outward). Because they are focusing on the growth of the embryo, students will initially be surprised to learn that the mass of the egg decreases steadily during incubation (31). This decrease in mass is due to the steady loss of water vapor throughout incubation, which would proceed at a lethally high pace if the incubator were not humidified (13). This general trade off between water loss and gas exchange is a central problem for plants, in which open stomata facilitate the gas exchange (primarily carbon dioxide uptake) necessary for photosynthesis but hasten desiccation by permitting increased water loss. In a discussion of gas exchange by avian embryos, it is also worth mentioning that the moist membranes under the eggshell are similar to other gas exchange organs such as gills, lungs, and amphibian skin. If sufficient oxygen is present, all that is required for gas exchange to take place at a physiologically useful rate is a relatively large, thin, and moist exchange surface and a blood supply that carries oxygen and carbon dioxide convectively between the exchange surface and tissues.
Equipment: Descriptions and Alternatives
Incubators.
A humidified incubator at 38°C is needed to house the eggs when they are not in use for the experiment. If this incubator is also large enough to hold the respirometers during test incubations, it is the only incubator required. Room temperature is completely adequate for the second test temperature, although students will recognize that using two incubators of similar type for the two temperature treatments results in a better-controlled experiment. Temperature-regulated water baths, which are relatively small, inexpensive, and commonly available, work well as treatment incubators; weights, such as large stones, pieces of brick, or flexible plastic bags filled with sand, can be placed on the lid of the respirometer to keep it submerged in the water bath during incubation.
Respirometers.
Respirometers may be of several different designs. The minimum requirements are that 1) the chamber must be air tight; 2) if air samples are removed from the respirometer for measurement by an oxygen analyzer, they must be under neutral or positive pressure when the sampling device is disconnected from the respirometer; and 3) removed samples must be of sufficient volume for the oxygen analyzer to make accurate measurements.
JAR-TYPE RESPIROMETERS FROM WHICH AIR SAMPLES ARE WITHDRAWN.
A jar-type respirometer with a single port and a two-way stopcock is inexpensive and easy to make (Fig. 2), and disposable plastic syringes can be used for air sample collection. Directions for their use are detailed above in Instructions for Students: Measurements and Calculations. Three-way stopcocks are not recommended because students frequently make mistakes when using them, leading to the loss or contamination of air samples.
SYRINGE-TYPE RESPIROMETERS FROM WHICH AIR SAMPLES ARE EXPELLED.
Large syringes can serve as respirometers. At the end of the respirometry incubation period, the air sample to be analyzed can be expelled into a sample syringe by pressing on the plunger of the respirometer, but care must be taken not to damage the egg in the process. For insects or very small eggs, commercially available plastic syringes (e.g., 60 ml) can be used as respirometers. Larger syringe-type respirometers, modeled after those used by Vleck and Kenagy (39), can be constructed from large-diameter acrylic tubing; note, however, that manufacturing syringe-type respirometers requires a skilled fabricator, specialized materials and tools, and regular maintenance.
Manometry chamber.
A simple manometry chamber can be made by turning a jar on its side and closing the opening with a one-hole stopper into which the tip of a 1-ml pipet has been inserted (Fig. 7). A false floor for the chamber can be fashioned from wire mesh to separate the egg or animal from soda lime on the bottom of the chamber. As the animal respires, the exhaled carbon dioxide is absorbed by the soda lime. Thus, changes in the volume of the gas space in the chamber, recorded by the movement of a soap bubble in the pipet, are due to the removal of oxygen by the animal in the chamber. This apparatus requires no oxygen analyzer, is extremely cheap and easy to make, and, because it so graphically makes the point that respiring animals consume oxygen, is especially suitable for beginning physiology students. A colleague uses such an apparatus to demonstrate the effect of thyroid hormones on the metabolic rates of rats to fourth graders. This design can also be scaled down for use with very small animals; as an undergraduate, one of us (S. M. Hiebert) used a miniature version of this apparatus to measure O2 values by an individual Drosophila. Where extremely small chamber volumes are needed, a concentrated solution of KOH can be substituted as a carbon dioxide absorbant, but care must be taken to prevent this caustic solution from coming into contacting with either the students or the animals.
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O2 at STPD by the following equation:
 | (5) |
where all abbreviations are as described in Instructions for Students: Measurements and Calculations, IV. Calculating O2.
To ensure success, check the air tightness of all fittings and use a dilute solution of soap and water for the soap bubble; wet the inside of the pipet before the animal is placed into the chamber so that the bubble does not break as it moves toward the stopper. After the animal has been placed in the chamber, make a bubble by squeezing the dilute soap solution (e.g., from children's bubble toy) or create bubbles in dishwashing liquid by opening the cap and rapidly squeezing to create bubbles above the liquid. Use an eyedropper or fingers to transfer a bubble (or bubbles) to the horizontal pipet.
Students should record the position of the soap bubble at frequent intervals so that if the soap bubble bursts prematurely, they will still have data to analyze. Note that a warm animal (endotherm or warm ectotherm) will heat the air inside the chamber at first, causing the volume inside the chamber to expand and the bubble to move outward. Once the temperature has equilibrated, the bubble will start to move in the direction of the stopper as a consequence of oxygen consumption. Asking students to plot the position of the soap bubble beginning immediately after a warm animal has been placed inside the chamber is a good way to find out whether they are observing the apparatus carefully and recording their data accurately! Instructors may want to ask students to identify potential shortcomings of this method; however, the effect is sufficiently robust to allow students to draw clear conclusions from the relative O2 values at the suggested temperatures.
The design shown in Fig. 7 works best when the entire apparatus can be placed inside an incubator. If a water bath is used to warm or cool the respirometry chamber, or for better control of the effects of changing ambient air pressure on the manometric apparatus, a U-shaped manometer tube filled with fluid can be used to monitor the O2 in the chamber, as illustrated in box 5.4 of Ref. 21 and as described in Ref. 37. For student use, a calibrated buret containing water can be inserted into the stopper in place of the syringe containing pure oxygen. At the end of the measurement period, water is released from the buret until the manometer fluid level returns to the position marked at the beginning of the trial. The volume of water required is the difference between the initial and final readings on the buret, and this value is then substituted for V in Eq. 5. Flexible tubing (attached to the tip of the buret that protrudes through the stopper into the respirometer) can be used to direct water away from the egg and desiccant as it is released into the chamber.
Syringes for air samples.
Each student or group will need a syringe for removing air samples from any jar- or syringe-type respirometer. For classroom use, plastic disposable syringes are preferable as long as students understand the potential problem of creating a low-pressure air space in the syringe when an air sample is removed; unless students follow the instructions carefully, they may cause room air to be sucked into, and thus contaminate, the air sample. Glass syringes, when held horizontal, do not have this problem because of the nearly frictionless fit between the plunger and barrel. When an air sample is expelled into a glass syringe, air pressure alone pushes outward on the plunger. However, glass syringes require extra care in handling, break more easily, and are more expensive to replace. Students will need to practice using a glass syringe before they use it in an experiment.
Syringe caps for friction-fit or Luer-type syringe tips can be purchased. However, students keen to create an airtight seal tend to strip the threads from Luer-type syringe caps in fairly short order. We prefer to make our own airtight syringe caps by cutting off the needle (flush with the plastic base) of a 27-gauge or smaller (i.e., higher gauge) disposable syringe needle. Cutting the needle crimps the needle opening shut, and the plastic base of the needle has a Luer fitting that conveniently screws into or slides onto the syringe tip.
Oxygen analyzers and sensors.
An oxygen analyzer or sensor is needed for jar- and syringe-type respirometers. Air is pulled through the sensor by a pump that runs continuously (Fig. 5). An air sample is injected into the continuously flowing air stream (follow instructions provided with the oxygen analyzer; see Fig. 8 for sample injection ports). As the sample air is pulled through the analyzer, the leading and trailing edges of the bolus mix with the room air in the sample line. Thus, the concentration of oxygen drops until the middle of the sample has entered the oxygen analyzer and remains low as this region of undiluted sample passes through the analyzer; thereafter, the concentration again rises until the sample has completely cleared the analyzer. The lowest concentration registered by the analyzer is an entirely serviceable measure of FE for the purposes of this experiment.
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Barometers, hygrometers, and thermometers.
A barometer, hygrometer, and thermometer should be available in the room where the experiment is taking place to measure Pb, RH, and Ta values, respectively.
Selected Articles for Students and Instructors
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Acknowledgments |
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Footnotes |
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Received for publication May 13, 2006. Accepted for publication November 29, 2006.
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; n = 6) remained at 38°C for 90 min, before a second measurement (measurement B) was taken. 
