Purpose: To measure the effect of environmental conditions on Daphnia heart rate.

Procedural overview: Students can observe individual Daphnia on well slides under the microscope and measure the water flea’s heart rate. Students can test the effect of ethanol on the heart rate of Daphnia. If time permits, students may design and conduct their own experiments with Daphnia, such as testing if pH affects the heart rate of Daphnia.

Approximate class time: About one class period.

Materials:

• Low-power stereo microscope (10x/30x stereo microscope from Home Science Tools)
• Microscope well slides (well slides from Home Science Tools)
• Live culture of Daphnia magna (Daphnia magna live culture from Home Science Tools)
• Stopwatches or cell phone timers
• Cell phone cameras (optional)
• Ethanol 5 percent solution in spring water
• Spring water (tap water can affect Daphnia)
• Disposable plastic pipettes (Home Science Tools) and scissors to cut off the end
• Stain Daphnia with eosin, methylene blue or iodine and mount on flat slides with coverslips
• Optional supplies for student-designed experiments on Daphnia, such as bottled carbonated water, pH strips, caffeine energy drinks, digital thermometers and supplies to make solutions of different salt, glucose or acidic/basic concentrations.

Background information: Daphnia, a genus of small planktonic crustaceans, are similar to shrimp and lobsters but much smaller. Daphnia live in freshwater. Daphnia are so small that a microscope is required to get a good look at them, but their exoskeletons are clear, so one can see their hearts beating and observe their other internal organs. A Daphnia’s heart is in its upper back — see the diagram of the functional anatomy of Daphnia below.

Advice and facts about Daphnia are given here by Carolina Biological. Scroll down on the page to view an instructional video about the care and handling of Daphnia.

Notes to the teacher: A small drop of water with one or more Daphnia can be placed on a well slide and observed under a microscope. The microscope light should be turned on only while briefly observing the Daphnia, and then turned back off. Otherwise the heat from the microscope light, and gradual evaporation of the water drop by that heat, will impact the Daphnia and interfere with their reactions to other conditions you are trying to test. If you hold a smartphone camera up to the microscope eyepiece, you can record video of the Daphnia, then turn off the light and play back the video as much as you want. It is easiest to coordinate the experiments if students work in pairs.

Procedure and student questions:

1. Using scissors, cut off about two inches of the bottom of a disposable pipette to create a larger opening. (You will use it to select and transport individual Daphnia magma to well slides.)

2. Spot a Daphnia moving around in the live culture container and use the plastic pipette to transfer it into the center of a well slide. Do not add a coverslip. Note that the smaller the drop in which the Daphnia is confined on the slide, the easier it will be to keep the Daphnia within the field of view of the microscope (but also the faster the drop evaporates). You can use the pipette to add or remove extra water from the well slide as necessary during the experiment. Be sure that the Daphnia is never left without water.

3. Take a photo or make a simple sketch of the Daphnia. Where is its heart?

A Daphnia typically appears to have a birdlike head and fat stomach (the shell). The heart is in the upper back and pulsates visibly.

4. Observe a Daphnia under the microscope while your lab partner times out 10 seconds, or video record the Daphnia for 10 seconds and then play it back. Or, determine another sound method for counting and recording heartbeats per 10 seconds. How many heartbeats does the Daphnia have in 10 seconds?

The heart rate depends on temperature and other conditions, but might typically be 30 or so beats within 10 seconds.

5. Multiply your measured number of beats for the 10-second observation by six. How many beats-per-minute (bpm) does that correspond to?

A typical value might be around 180 bpm.

6. Make two more measurements of the heart rate and record your results in bpm.

7. Calculate and record the average of your three heart rate measurements in bpm.

8. Use a clean pipette to add a drop of ethanol to the Daphnia on the well slide. Wait at least one minute, then make three measurements of the heart rate and record your results.

9. Calculate and record the average of the three heart rate measurements in bpm. Compare the two averages that you calculated. How did the addition of ethanol affect Daphnia’s average heart rate?

Ethanol slows the heart rate. Depending on the relative sizes of the original Daphnia droplet and the ethanol droplet, the ethanol might reduce the heart rate by as much as half or so (to around 90 bpm).

10. If humans are affected the same way as Daphnia, would ethanol be a stimulant or a depressant for the human nervous system?

Ethanol acts as a depressant to the human nervous system.

11. Add enough spring water to the Daphnia slide to dilute out the ethanol, or even place the Daphnia onto a new well slide filled with spring water. Make three more measurements of the Daphnia’s heart rate and calculate its average heart rate. How long does it take for the heart rate of the Daphnia to return to approximately normal?

It may take a few minutes in alcohol-free water for the heart rate to return to its normal higher value.

12. Compile each group’s results and analyze the class’ data. Are there trends in the data?

Compiling the class results will allow the students to examine multiple, individual Daphnia and try to determine trends given a larger population.

13. Research and draw a freshwater lake food web. Try to tailor it to a lake that is local to you. Explain how Daphnia fit into the food web and how changes in the population of Daphnia could affect the rest of the ecosystem.   

Possible activity extension: Once students are familiar with Daphnia from this initial experiment, allow them to design and perform their own experiment with Daphnia. You may want students to keep the same dependent variable, the number of Daphnia heartbeats, as used in the first experimental procedure below. Suggested steps and resources for helping students through the experimental design process are listed as a second procedure.

• Use the Research in Brief Educator Guide as an outline for the initial student experimental design process:

• Allow time for students to do additional research about Daphnia, and about other experiments that have involved Daphnia. Here are a few examples from Science News and Science News for Students:

Science News: “Zooplankton diet of mercury varies” and “Killer Cocktails: Drug mixes threaten aquatic ecosystems”

Science News for Students: “Common water pollutants hurt freshwater organisms,” Readability score: 6.8, and “Artificial sweetners pollute streams,” Readability score: 7.8

Additional experimental ideas: Students could test ethanol solutions of different concentrations and/or use more accurate pipettes to measure the amounts of water and ethanol added to the well slide. Students could also test other conditions: carbonated bottled water or distilled water with Tums or baking soda added (measure the pH with pH strips), salt water (measure the amount of salt dissolved to make the solution), glucose solution, caffeine energy drinks, detergent, etc. With a sensitive digital thermometer, water of different temperatures, and some patience, it may also be possible to measure the effect of temperature on the Daphnia, even with the complications of heat from the microscope light.

• Using the given procedure for Water flea circus, students should create a numbered, stepwise procedure before beginning their experimentation. A sound technique for reproducible data collection among trials should be defined. Students should also create a list of necessary materials and chemicals.
• Before beginning their procedure, students should create a data table. They should identify all measurements that need to be taken and should think about the number of trials that are needed and/or the number of individual Daphnia tested at every experimental condition. If students are defining a statistical test that they will use to analyze their results, they should make sure to align their sample size accordingly.
• Once students perform their experiments and collect data, ask them to analyze the data (provide appropriate options of statistical tests, if appropriate) and summarize the results.
• Students should share their hypotheses, experimental methods and results with their classmates. If there is time for students to reflect on their experimental designs, they should suggest procedural modifications they would make, if testing the same hypothesis.