Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Mix water and ice cubes to chill the water to 5 °C.
2. Mix 3.75 g of caffeine in 150 mL of distilled or deionized water. Stir until caffeine is dissolved.

Safety Precautions

Caffeine is a very toxic chemical; as little as 65 mg can be life threatening. Store caffeine in a Flinn Chem-Saf^™ bag. Ethyl alcohol is toxic by ingestion. Practice strict chemical hygiene—do not touch, taste or smell any chemicals in the lab. Avoid all contact of chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Caffeine water may be disposed of according to Flinn Scientific Suggested Disposal Method #5. Ethyl alcohol may be flushed down the drain with an excess of water according to Flinn Scientific Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. Both parts of this laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab, and the Post-Lab Questions may be completed the day after the lab.
• Once the experiment is complete Daphnia may be placed in an aquarium as fish food as well as to eat algae.
• Caffeine is a CNS stimulant. It is structurally similar to adenosine. Adenosine inhibits the CNS. The caffeine binds to the adenosine receptors on the heart competitively inhibiting adenosine’s ability to bind. Therefore it acts as a stimulant by blocking the CNS depressant adenosine.
• Caffeine is a very toxic chemical. Make sure to stress the importance of proper chemical hygiene.

Teacher Tips

• Daphnia require specific care. Please call Flinn Scientific at 1-800-452-1261 and request Culturing Daphnia (Publication No. 10580) for specific care instructions.

Answers to Prelab Questions

1. Based on the information in the Background section, what would happen if the Daphnia were exposed to water colder than 5 °C or warmer than 35 °C?

   If the Daphnia were exposed to either extreme, they would die. Below 5 °C they would freeze to death and above 35 °C is much too hot for them to survive.

2. What is the purpose of using a cotton ball before placing the Daphnia on the slide?

   The purpose is to hold them in place so their heart rate can be more easily observed.

Sample Data

Daphina Sketch

Answers to Questions

1. Explain how the heart rate of Daphnia changed between room temperature water and 5 °C water.

   Based on the results, the heart rate decreased at 5 °C. Therefore it suggest that the heart rate decreases at lower temperatures.

2. How do you think warm water would affect Daphnia’s heart rate?

   Since cold water decreased the heart rate by slowing metabolic activity, it would make sense that increased temperature would increase the heart rate. Increased temperature leads to increased metabolic activity the temperature increase causes enzymes to activate increasing heart rate.

3. What other chemicals or variables could be tested to determine their effect on heart rate? Note: Use additional textbooks or Internet resources.

   Daphnia are often exposed to adrenaline, nicotine and chloride salts.

4. What problems might occur in using Daphnia to test the effects of drugs on humans?

   Since it is inconclusive whether or not their heart is neurogenic or myogenic it cannot be directly compared to the human myogenic heart.

5. Examine the data table regarding the effect caffeine and alcohol have on Daphnia heart rate. Is this similar or different than the effect of these chemicals on the human heart rate?

   The caffeine increased the heart rate in Daphnia. It also increases the heart rate of humans. Alcohol decreases the heart rate of both Daphnia and humans.

6. Based on these results alone, does it suggest that Daphnia and humans have the same or different type of heart?

   These results indicate Daphnia and humans both have a myogenic heart because they respond the same to a given chemical. If either of the chemicals yielded the opposite effect, it might support the Daphnia having a neurogenic heart.

References

Morholt, E., Brandwein, P. A Sourcebook for the Biological Sciences; Harcourt Brace Javanovich: San Diego, CA; 1986; pp 267–8.

Introduction

Daphnia, also known as water fleas, are small, transparent crustaceans. They serve as ideal organisms to study physiological functions such as heartbeat. Determine the effect on heart rate when Daphnia are exposed to stimulants and depressants.

Concepts

• Heart rate
• Effects of stimulants

Background

Daphnia are characterized by a body enclosed in a transparent bivalve shell. Their flat, transparent bodies make them ideal organisms for introducing biology exercises and experiments.

Daphnia have a small heart located dorsally (see Figure 1). A ganglion cluster of neurons on the heart wall produces rhythmic motor output to the rest of the body. The heart rate of Daphnia depends on the species. It varies from 230 to 300 beats per minute. Counting the exact number of heartbeats is difficult because even the slowest output approaches four beats per second.

There is much debate as to whether Daphnia’s heart is neurogenic or myogenic. The distinguishing factor is in the means in which it depolarizes signaling contraction. A neurogenic heart requires stimulation by a nerve itself to depolarize and contract. A myogenic heart is capable of depolarization itself. This is not to say it is not affected by the nervous system but is instead capable of depolarization and contractility on its own. In humans, myogenic heart depolarization is initiated by the sinoatrial node (SA node). Most crustacean hearts are considered neurogenic. Although not unequivocally confirmed, the most recent electrophysiological studies indicate a myogenic heart in adult Daphnia.

Daphnia are ectothermic crustaceans. This means their body temperature is controlled by their external environment. As the environmental temperature increases, their rate of metabolism increases. This occurs because their cells are made up of many enzymes that increase in activity as the temperature increases. However, any temperature over 35 °C will result in death. Conversely, cold temperatures decrease heart rate as the enzymes are not activated at low temperatures.

Daphnia’s heart rate is affected by stimulants and depressants. Stimulants increase the activity of the central nervous system (CNS). Depressants decrease the activity of the CNS. The mechanism in which each chemical changes the heart rate varies. For example, stimulants can work in two ways. They can work by promoting excitatory neurotransmitters that result in alertness. They can also work by disrupting inhibitory neurotransmitters that signal the brain to sleep. In humans adenosine is considered an inhibitory neurotransmitter. It is released by the brain and binds to adenosine receptors, which decreases nerve activity and causes drowsiness. Caffeine works by binding to the adenosine receptors preventing adenosine from binding. If the adenosine cannot bind nerve activity will stay elevated promoting alertness.

Experiment Overview

Expose Daphnia to various conditions to determine the effect on their heart rate.

Materials

Prelab Questions

Read the Background and Procedure before beginning.

1. Based on the information in the Background section, what would happen if the Daphnia were exposed to water colder than 5 °C or warmer than 35 °C?
2. What is the purpose of using a cotton ball before placing the Daphnia on the slide?

Safety Precautions

Caffeine is a very toxic chemical; as little as 65 mg can be life-threatening. Ethyl alcohol is toxic by ingestion. When working with live cultures always make sure to exercise humane care to the experimental organisms. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

1. Obtain three Petri dishes and separate the bottoms from the lids.
2. Using a marker, label each of the three dishes as follows:
   • Water, 5 °C
   • Caffeine solution
   • Ethyl alcohol
3. Obtain 10 mL of each of the above solutions and place them in their respective dishes.
4. Place the thermometer in the dish labeled 5 °C. If the temperature begins to climb add an ice cube to the dish until it reaches the correct temperature.
5. Carefully separate a few fibers from the cotton ball. Place the fibers in the depression of the slide. The cotton ball helps contain the Daphnia in one location so they can be easily observed.
6. Using a clean pipet, carefully transfer 1–3 Daphnia from the main culture into the well of the depression slide.
7. Carefully observe the Daphnia using a compound microscope. Sketch the Daphnia on the Studying Heart Rate with Daphnia Worksheet. Label the identifiable features paying special attention to the location of the heart. Record observations concerning the heart rate and activity of the Daphnia.
8. Using a pipet, remove as much of the culture water as possible from the slide. Use the edge of a paper towel to absorb any excess water by capillary action.
9. Add about 10 drops of the 5 °C water onto the Daphnia.
10. Observe the Daphnia in the cool water and compare their relative activity/heart rate. Has the heart rate increased, decreased or not changed from the original room-temperature water rate? Record observations on the worksheet.
11. Return the Daphnia to a separate container so that the same Daphnia are not used twice.
12. Using a clean pipet, repeat steps 5 and 6 to transfer fresh Daphnia to the slide.
13. Repeat steps 9–12 using the caffeine water solution. Record observations on the worksheet.
14. Repeat steps 9–12 using 10% ethyl alcohol. Record observations concerning the heart rate and activity of the Daphnia.

Student Worksheet PDF

11049_Student1.pdf