Preparing Concentrations
For this experiment, a large volume of 0.5mol caffeine solution shall be provided. This shall be refered to as a “100%” concentration. Different concentrations of caffeine can be made by diluting the solution with distilled water using the following methods:
Pure distilled water can be referred to as a “0%” caffeine solution.
Variables
Method
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Set up apparatus. (see “Setting up a Microscope”)
- Select and extract a Daphnia organism using a dropping pipette. For each experiment, take care to ensure that no Daphnia organisms are used twice, as this may cause unnecessary stress for the invertebrate
- Prepare a cavity slide and “inject” a Daphnia into the concave dent with a few drops of water. Place a cover slide on top to prevent evaporation.
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Measure out 1cm3 of a caffeine solution using a 10cm3 measuring cylinder and add to the side of the slide. Place filter paper on the adjacent edge of the cover slide to ‘draw’ the solution into the cavity.
- Follow microscope instructions until specimen is viewed through the maximum magnification, and count its heartbeat for 30 seconds, using a stopwatch. Double the result to gain a value in beats per minute.
- Leave the Daphnia for 2 minutes for the caffeine to be used up in the environment and then repeat from step 4 using a different concentration of caffeine. Continue until samples of all solutions have been used.
- Repeat the experiment 3 times, using a different Daphnia each time.
Setting up a Microscope
(Adapted from SNAB website, Practical Support article 8)
- Check that the lens, eyepiece and stage are clean. If the lens needs cleaning, use a lens tissue.
- Illuminate the stage. If present, use the built-in light; otherwise use the mirror and the light from a window or a bench lamp placed 15–20 cm away to reflect light up through the hole in the stage. When using a lamp, check that the light is not shining onto the stage from above.
- Adjust the sub-stage condenser if present Place a slide on the stage and a pointed object such as a pencil on the light source. Adjust the condenser until both are in focus.
- Adjust the light intensity using the iris diaphragm or light control knob. Check the field of view is evenly illuminated and then reduce the light as much as possible without getting a shadow around the edge of the image.
- Place the slide in position. Do not tilt the stage, as there is liquid present in the slide.
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Check that the low-power objective lens is in position. Bring the lens as close to the slide as possible. Look through the eyepiece and use the coarse adjustment knob to move the lens away from the stage to bring the specimen into focus. This avoids the lens hitting the slide and damaging the microscope. To get a sharp focus, use the fine focus knob.
- Focus and search for the area you want using low power. If necessary, move the slide so it is in the middle of the field of view and then put a higher-power lens into position, watching from the side to check that it does not hit the slide. Use fine focusing for any fine adjustments. Adjust the condenser and light aperture to ensure the best illumination.
Hypothesis
I expect that the presence of caffeine will cause an increase in heart rate. This is because the caffeine will act as a stimulant and signal heat cells in the body to respire at a faster rate. I also predict that an increase in the concentration of caffeine will promote a subsequent increase in its abilities as a stimulant.
Prediction
I predict that the highest concentration of caffeine (100%) will produce the greatest heart rate. I also predict that the lowest concentration of caffeine (0%) will produce the lowest heartbeat.
Null Hypothesis
I predict that no relationship will be found between the concentration of caffeine and the heart rate of the Daphnia.
Results
Graph
The average result of each concentration and general trend of the investigation can be shown in this graph:
From the strong positive trend lines of the graphs, it is clear that there is a direct relationship between the concentration of caffeine and the heart rate of Daphnia.
Conclusion
My hypothesis and prediction were correct. From the results gathered, and the consistant trends on the graphs, it can be concluded that an increase in caffiene concentration produces an increase in heart rate. A sceientific explanation to this effect can be found in the molecular behavior of caffiene in the body.
The graphs also suggest that there could be a maxiumum potential heart rate, as the 100% caffeine results all produced results that were within 7 beats of eachother.
After caffiene is ingested, enters the blood stream after being absorbed through the stomach and small intestine. Here it travels throughout all tissue types, and can increase the levels of the enzyme cAMP in heart cells. This enzyme is responible for activating protein kinase A, which increases the responsiveness of cardiomyocytes to the calcium currents that control beating.
Caffeine can also increase the amounts of stimulatory neurotransmitters released by the brain, increasing cell metabolism and blood circulation.
Evaluation
Although the investigation was a success, there were major problems with the method and apparatus used that introduce doubts over the validity of the results gathered.
There are a few systematic errors that could have occurred due to faults in the method. For instance, the use of faulty or inaccurate measuring equipment when diluting the caffeine solution could have caused imprecise measurements of caffeine to be used. This could have lead to consistently inaccurate results.
Another source of error could have been the actual counting of the Daphnia’s heartbeats. Because the specimen had a rather unusual internal organ structure, it was difficult to distinguish the heat from the stomach at first glance. The lighting of the microscope was also inadequate and could have caused the recorder to initiate a “subconscious” counting rhythm, producing inaccurate results.
To improve the investigation, these measures could be taken
- A stroboscope could be used to record the Daphnia, and then play back the video at half speed. This would allow counting of the heartbeats to be calculated much more easily and accurately, increasing the validity of results.
- The Daphnia used could be raised from larvae to ensure that they are the same age. Different aged Daphnia may, like humans, experience variation in their average heartbeat, therefore the results gathered from specimens of the same age would be more reliable.