Practical 1. 1 Introduction: Caffeine belongs to a class of compounds called methylxanthines and can block a receptor on the surface of heart muscle cells for adenosine. In fact, it is caffeine’s blockade of the A1 adenosine receptor in the heart that causes the heart to pound after a significant caffeine dose. Caffeine and similar compounds also inhibit a class of enzymes known as cyclic nucleotide phosphodiesterases. These enzymes are, in part responsible for degrading a stimulatory signal produced when excitatory neurotransmitters activate different neurons in the central nervous system (CNS).
Thus, when they are inhibited by caffeine, the stimulatory signal remains active for a longer period of time resulting in a greater sense of alertness (a CNS effect) but also a higher heart rate, blood pressure and respiratory rate. Caffeine also acts as a stimulant drug, causing increased amounts of stimulatory neurotransmitters to be released. At high levels of consumption caffeine has been linked to restlessness, insomnia and anxiety, causing raised stress and blood pressure. This can lead to heart and circulation problems. Hypothesis:
Daphnia subjected to a caffeine solution will show a rise in heart rate as caffeine is a stimulant that speeds up the body affecting the nervous system and rate in which the nerve pulses are sent and received in humans. (Caffeine increases the heart rate of Daphnia. ) Null Hypothesis: There is no significant difference between the heart rate of Daphnia in distilled water compared to that in caffeine. Safety: Handle cavity slide carefully to prevent breakage. Ethics for the Daphnia: 1. Every possible attempt to keep Daphnia alive. 2. Use low concentration of caffeine so not to harm the Daphnia. . Place the Daphnia in the presence of pond water instead of distilled water. 4. Use the same Daphnia throughout the whole experiment and handle it with care. 5. Turn off the microscope light between observations to prevent the Daphnia from overheating while on the microscope. Materials: Culture of Daphnia (water fleas), cavity slides, dropping pipettes, distilled water or pond water, 0. 5% of caffeine solution, stopwatch, paper towels, microscope. Procedure: Independent variables: Type of treatment- distilled water/ pond water and 0. 5% caffeine solution.
Dependent variables: Number of heart beat per 20s Other variables: 1. Size of daphnia 2. Volume and concentration of caffeine 3. The time of Daphnia being immersed in the caffeine solution 4. Surrounding temperature 1. Have ready the 0. 5% of caffeine solution, the Daphnia culture, a stopwatch, cavity slides, pipettes and other materials. 2. Catch a daphnia with the pipette and place into well on a deep well cavity slide. 3. Use the microscope to get the daphnia in focus. If the Daphnia isn’t too large, use a cover slip to hold it in place. 4. Add two or three drops of the 0. % of caffeine solution. Leave the Daphnia immersed in the 0. 5% caffeine solution for 1 minute before removing the excess of the caffeine solution by using a pipette. 5. Using your stopwatch, count how many times the Daphnia’s heart beats in 20 seconds. Each heartbeat is recorded by tapping a pencil on a piece of paper and counting up the pencil marks after the specified time. 6. Repeat Step 5 for 2 times and record heart rates. Find the average heart rate of Daphnia with a 0. 5% caffeine solution. 7. Repeat all the above steps for the control (pond water). Results: Table
Treatment Heart beat rate/beat per 20sMean heart rate / beat per 20sMean heart rate / beat per min 1st repeat2nd repeat3rd repeat Distilled water/ Pond water94. 0095. 0096. 0095. 00285. 00 0. 5% caffeine solution110. 00100. 00100. 00103. 33310. 00 Graph: Discussion: 1. It is considered ethical to use invertebrates such as daphnia rather than large organisms e. g. monkey or human in scientific studies. Daphnia has reduced awareness of pain because of the lack of a well developed nervous system. It is transparent and its heart is visible without the need for dissection.
Daphnia is abundant in nature and there is no threat to it or its dependent species (food chains). Some people also feel that it is bred for fish food and will thus die anyway. Daphnia can reproduce asexually and may be clones, therefore there is no loss of genetic variation. At the other extreme, some research on animals involves suffering or loss of life. Animals that are kept in confinement on University premises are normally species that have been bred for many generations in captivity e. g. mice, rats, farm animals. You can obtain large numbers of invertebrates, including single-celled organisms, for study.
You can easily control the conditions under which they are grown and maintained. Many tissues from multicellular invertebrates do not require supplemental oxygen or complex media, unlike vertebrate tissues. Their central nervous systems are not as well-developed as those of vertebrates, therefore invertebrates are probably not self-aware. There are no ethical restrictions on their use, with the exception of concerns about pathogenicity or potential environmental hazards stemming from improper handling of potentially dangerous cultures. 2. The treatment with 0. % caffeine solution gave higher heart rate. This is because Daphnia subjected to a caffeine solution will show a rise in heart rate as caffeine is a stimulant that speeds up the body affecting the nervous system and rate in which the nerve pulses are sent and received in humans. 3. To make sure the results are reliable and valid, at least nine Daphnia are given to per student or pair. They should be unharmed by the activity, but may need too long to recover after the caffeine to use the same one in the same practical. Prepare a container for ‘used’ Daphnia.
This will stop them getting mixed up with the ‘fresh’ ones. Use lower power of microscope. Note that high concentrations of caffeine can be fatal for Daphnia. The Daphnia hearts are fairly easily seen but counting the number of beats can be difficult. Counting is easier if each heartbeat is recorded by tapping a pencil on a piece of paper and counting up the pencil marks after the specified time. In addition, cooling the Daphnia before the experiment may help slow their heart rate. A dissecting microscope with a light source under the stage works well for this experiment.
To prevent the Daphnia from overheating while on the microscope turn off the microscope light between observations and use a heat sink – a cavity tile filled with iced water placed on the microscope under the slide. Using pond water/Daphnia culture solution is recommended for both the control group and to dissolve the caffeine as this may give more valid results and be less stressful to the Daphnia. In distilled water the heart rate may rise due to lack of oxygen. 4. The possible sources of error are the miscalculating of the number of heart rate. Use of the stroboscope may overcome the problems of counting faster heart rates.
However, we would not recommend this method. Positioning the light sources and strobe is tricky. It is very difficult to freeze the motion and viewing with a strobe light can cause eye strain and dizziness. A simpler approach is to count the rate at which the legs beat. This rate is proportional to the rate at which the heart beats. Conclusion: Daphnia subjected to a caffeine solution will show a rise in heart rate as caffeine is a stimulant that speeds up the body affecting the nervous system and rate in which the nerve pulses are sent and received in humans. (Caffeine increases the heart rate of Daphnia). The hypothesis is accepted.
