Hypothesis
The heart rate of daphnia will increase as the concentration of caffeine is increased. This is because the caffeine actually blocks the adenosine (form in which broken down glucose energy is stored) receptive sites on the heart muscle cells causing an increase in heart rate.
Another reason is that caffeine inhibits the production of an enzyme called cyclic nucleotide. Cyclic nucleotide’s purpose is to neutralise a stimulatory signal produced when excitatory neurotransmitters activate different neurons in the central nervous system. Therefore, when cyclic nucleotide is inhibited (by caffeine) the stimulatory signal can’t be “turned off” meaning, not only a greater sense of alertness but also a higher heart rate, blood pressure and respiratory rate.
Plan
I intend to use caffeine of 0.01 mol because this is the most concentrate that I am allowed to use. It will gradually be diluted down with distilled water. I will use similar sized daphnia because this is the closest indicator of age. Different aged daphnia will have even more varied metabolic rates and heart rates.
I will keep the body temperature of the daphnia constant because at different temperatures cell metabolism will occur at different rates. All chemical reactions are at a faster rate at a higher temperature because the molecules have more energy, move faster and therefore collide more often. It will be easy to maintain a constant body temperature because daphnia are cold-blooded and don’t thermoregulate. It will simply mean monitoring the external water temperature.
The pH will loosely be set at as the pH of the tap water I am using. PH doesn’t really need to be controlled because a change in pH won’t actually affect the daphnia, only the amount of reproduction that can occur.
The salinity of the water will be kept constant by using tap water each time. The only variable I will be changing is the concentration of caffeine. Each caffeine concentration will be used 3 times in order to obtain more reliable results.
When counting the heart beat under the microscope I will count for a minute using a clicker counter so that I don’t lose count.
I will take every care not to stress the daphnia by being gentle as possible with them and not disturbing their habitat. If they became sufficiently stressed their heart pressure would rise and caffeine would again rise this amount giving an anomalous result.
My preliminary research informs me that daphnia die at about 0.02 mol caffeine concentration when the heart rate is about 340 bpm. I will therefore go no higher than 0.01 caffeine concentration. Preliminary work also informed me that the most efficient and least stressful method of retrieving the daphnia is to use a medicine dropper and to then place it in the well of a depression slide. The caffeine solution will be constructed using distilled water and caffeine mixed to give a total volume of 10cm3. The daphnia will then be immersed in the solution for a set period. The heart rate will then be monitored under a microscope for a minute whilst using a clicker timer to keep count.
Following my preliminary work I found it difficult to distinguish between separate heartbeats. I decreased the temperature of the water using an ice bath to decrease the heart rate of the daphnia and make it easier to count. It worked and so for my final experiments the water will be 5°C.
Safety
The only safety concern is that of the caffeine. I will not be using particularly concentrate caffeine because the school is forbidden to allow me. However, the 0.01-mol solution I am using is still quite dangerous.
Goggles will be worn to prevent any touching my eyes, which can cause blindness. If any caffeine does touch skin or eyes it must be cleaned with copious amounts of water for at least 15 minutes. Contaminated clothing must be removed. Do not inhale caffeine, if done inadvertently get fresh air and seek medical advice.
Ethics and Care for the Daphnia
Every possible attempt will be made to keep the daphnia alive. I must be careful when immersing the daphnia into liquids because if they are released above the surface air will block the respiratory vents and death will occur.
The 0.01-mol caffeine solution is not strong enough to harm the daphnia. Following the experiments every daphnia will be returned to its colony.
I will take every care not to stress the daphnia by being gentle as possible with them and not disturbing their habitat.
Apparatus
- Light microscope - easy to use and gives a good view of the heart beating
- Caffeine solution (0.1 mol) – not as dangerous as pure caffeine and isn’t strong enough to kill the daphnia
- Daphnia fleas (30)
- Medicine dropper – easiest and kindest way to extract the daphnia from the solution
- Depression slides with slip covers – daphnia will not be crushed by cover slips
- Ice bath – easiest way to reduce heart rate by cooling the daphnia
- Thermometer
- Goggles – keep eyes safe from caffeine
- Clicker counter – easiest way to keep track of the heart rate.
Method
To begin with placed the daphnia filled beaker within a larger beaker and surrounded with ice to reach a water temperature of 5°C. I then measured the heart rate of a daphnia with no caffeine to obtain a starting point. I used the medicine dropper to extract the daphnia fly. I put it in the depression slide and covered with a cover slip. I counted the heartbeats for a 20 seconds using a clicker counter and then multiplied the result by 3 to gain the reading in beats per minute. I then mixed 1cm3 of caffeine with 9 cm3 of distilled water. I put the daphnia in the solution for 1 minute. I then had 30 seconds to transfer the daphnia to a depression slide and cover it with a cover slip before counting the heart beat for 20 seconds using a clicker counter and multiplying by 3 to obtain a result in bpm. I repeated each concentration variant 5 times and took an average to obtain a more reliable result.
Results
Conclusion
From my results and graph I can conclude that the heart rate of daphnia increases as the concentration of caffeine increases. The reason for this is that caffeine is a stimulant. The caffeine actually blocks the adenosine (form in which broken down glucose energy is stored) receptive sites on the heart muscle cells causing an increase in heart rate.
Another reason is that caffeine inhibits the production of an enzyme called cyclic nucleotide. Cyclic nucleotide’s purpose is to neutralise a stimulatory signal produced when excitatory neurotransmitters activate different neurons in the central nervous system. Therefore, when cyclic nucleotide is inhibited (by caffeine) the stimulatory signal can’t be “turned off” meaning, not only a greater sense of alertness but also a higher heart rate, blood pressure and respiratory rate.
The heart rate always increased positively (except one occasion) but the extent at which it increased ranged from 2.66 to 16.7. I have included a graph to show the extent of variation. The reasons that could be to blame for the wide range of increase include variety in the individual daphnia. Certain daphnia might have been more stressed than others, which would have caused an increase in heart rate. Other Daphnia might have been of a differing age meaning a lower or higher resting heart rate to begin with. Another cause may have been the inaccuracy of my counting. Despite using a clicker counter to keep count and cooling the water with ice to lower the resting heart rate it was still a very fast and difficult to count heart rate. This meant that there was great variety in the results. This was almost definitely the cause for the anomalous negative heart rate increase. All three attempts were relatively low and the relatively high results of the previous concentration meant that the average heart rate of the higher concentration was actually lower than the average heart rate of the lower concentration. If these problems could feasibly be precisely controlled the graph should actually be a horizontal line as opposed to a graph that shows no correlation what so ever.
