- Petri Dish
- Small Piece of cotton wool
- Pasteur pipette (for water from the Daphnia culture tank)
- Caffeine of different concentrations
- Culture of Daphnia
- Concentration of caffeine given to individual daphnia; 0, 0.001, 0.01, 0.1, 1, 10, 100 (%). This is a logarithmic scale as concentration increases by a factor of 10 each time
- Heartbeat of the Daphnia per minute
- Temperature of the Daphnia. All solutions were cooled to a room temperature of about 20c, it was necessary to keep temperature constant as at different temperatures the Daphnia’s metabolic rate may change due to processes that are enzyme controlled. This results in varying demands of oxygen intake and carbon dioxide removal. As Daphnia are ectotherms they do not thermoregulation so the effect altering the temperature of the solution will a greater effect on the heart rate.
- The lamps of the microscopes also output a significant amount of heat for a small organism. To alleviate this the Daphnia were put in the caffeinated solutions for a period of time to allow them to acclimatise. They were then put on under the microscope and heart beat recorded immediately. This reduces the inaccuracies that could arise due to the microscope lamp
- Size of Daphnia. A larger Daphnia will likely have larger muscles and organs all requiring a greater volume oxygen to ensure they have sufficient oxygen to meet an increased demand. There will be a higher output of carbon dioxide from respiration within cells which will need to be taken from the cells. These reasons result in heart rate being proportional to size of organism. To alleviate potential inaccuracy experiments were measured in change in heart rate from a control measurement. All experiments done by an individual were done using the same Daphnia
- This table indicated potential risks that may arise from this experiment. Both risk level and risk likelihood being on a scale of 1-3 with 3 being dangerous and likely to occur. The risk index is risk level x risk likelihood. By using control measures the aim is to reduce the risk index of a particular risk.
- Take a small piece of cotton wool and place it in the middle of a small petri dish
- Select a Daphnia, then use a Pasteur pipette to transfer the organism to the cotton wool
- Add pond water to the petri dish until the animal is covered by the water
- Place petri dish under the microscope and identify the position of the heart
- Record the heartbeat of the Daphnia over 20 seconds. Multiply result by x3.
- 20 seconds is used as it is difficult to keep count over the minute as the heart is beating multiple times a second. By multiplying by 3 we introduce inaccuracy by assuming the heart beat is constant over the minute
- Take the Daphnia off of the microscope to avoid heating it up, place the Daphnia into another petri dish but this time using different concentrations of caffeine solution; 0.001, 0.01, 0.1, 1, 10, 100 %
- After allowing the Daphnia time to acclimatise to the new solution place under the microscope and record the heart beat
- Repeat for different concentrations of caffeine
Analysis of Results
The first result, at 0% concentration showed an increase of 40 beats per minute. This may initially seem strange as the water is pure and free of any physiologically-active compounds and so, compared to the pond water which may contain many minerals and substances not present in distilled water, you may even expect a decrease in heart rate. The reason there was still an increase in heart rate was due to the stress of moving the Daphnia from the pond water and to and from the microscope. Many studies have observed that levels of stress are proportional to an increase in heart rate. This means that for many of our results the same observed stress related increase will be included. Up until 10% caffeine concentration we can see a clear correlation between heart rate and caffeine concentration. However, after this point there is a cessation of the consistently increasing heart rate. One study into concentrations of caffeine and the effect on the heart rate of embryonic chicks theorised that this was due to the high concentrations bringing about an excess of potassium ions. However, contrasting articles and studies suggest that caffeine actually makes the organism susceptible to becoming potassium deficient, a condition known as Hypokalemia, this commonly presents with bradycardia, a slowing of the heart rate due to muscle weakness due to an initial, uncontrolled increase in heart rate. In our experiment we allowed our Daphnia to acclimatise to each concentration so an initial spike in heart rate would not have been measured. This phenomenon is known as Coffee-induced Hypokalemia, although the name may be misleading it is no exclusive to coffee but the effects of caffeine