Method- Firstly hands need to be washed and a laboratory coat needs to be worn so that any substances from the hands are removed and that direct harm to the body is minimised. The working desk and area around should be clear of obstructions and loose hair clothing should be tied back to reduce the risk of an obstruction/accident.
All the required equipment then needs to be gathered and set up (e.g. microscope, electronic balances) so that there is no disruption and that the experiment can be carried out in a systematic and orderly manner. All apparatus should be then checked for cleanliness so that that no substance from prior experiments remain as this could affect the validity if the results. If any equipment is cleaned/rinsed out it should be thoroughly dry before using it again so that so that the volume of water does not affect the results. These measures will ensure more accurate results and reduce the chance of systematic errors (values differing from the true value by the same amount) as it will ensure the equipment is clean.
Beakers then need to be marked one-six so that there is no mix up between the concentrations. The results table should also be ready so that recording is easier so here is no carelessness and to ensure that the experiment is carried out systematically to reduce the risk of any random errors (lying equally above or below true value). The following concentration should be used as they are fairly small amounts and so it will be easier to distinguish trends and also so that the concentration does not prove fatal to the Daphnia
Start the experiment with the lowest concentration and work up to the highest, so that the procedure is systematic to follow and any trends will be more easily recognised. Normal distilled water could have been used as a control by monitoring the heart of a caffeine-free Daphnia, then monitoring the same Daphnia after caffeine had been added.
The 10ml measuring cylinder will then need to be obtained to measure the volume of distilled water needed in each beaker. Distilled water will be poured into the cylinder, but care must be taken that the cylinder is not tilted and is held vertically upright on a stable flat surface, so that the right amount of volume is collected. Also eyes should be kept at the same level as the meniscus. The water from the cylinder will then be transferred to a beaker, again making sure that the beaker is on a flat surface so none is spilt.
A pestle and mortar then needs to be obtained to crush the 50mg caffeine tablets. After the tablet is crushed they will be transferred to the container from the balance. However the tare button will first need to be presses so this zeros the balance with the container in place. The reading of the number will then come up and this is the mass of the substance. The balance will need to be set to zero using the tare button for each new container.
The caffeine form the container then needs to be transferred to the beaker and the beaker then needs to be shaken so that the solution is dispersed throughout the beaker.
Some cotton wool then needs to be placed on to a cavity slide and using the 10cm pipette the correct volume as indicated in the table will be obtained and dropped onto the piece of cotton wool.
The Daphnia will then be acquired using micro-pipettes and placed on to a cotton wool to keep them stable so it is easier to view and clearly observe the heart of the Daphnia and out its heart rate. The Petri-dish will then be placed on to the microscope and observed through the lens to record the heartbeat of The Daphnia every 10 seconds.
Whilst looking the microscope hands must be kept on the stop clock so that the clock can be started/ stopped when needed without having to look away, (makes ticking noise per second)as this would slow down the reaction time and maybe heartbeats could be miscounted and therefore alter the results.
After the experiment the results will be needed to be multiplied by the value by 6 to get the average beats for a minute. This procedure will be used as looking for too long could waver the concentration and consequently beats could be miscounted.
Carry out each concentration three times so that an average from the results can be obtained and that the precision and accuracy of the result can be evaluated. However carry out all the different concentrations once and then repeat the procedure, in case there is not enough time to repeat it. This same procedure must be followed for all the different concentrations.
The results obtained from the investigation will be firstly presented in a table so that the values can be systematically looked at and an average from the three trials can be worked out at the end, so that any tends/pattern or anomalies will be more easily distinguished. However a graph will then be produced as it will allow the result to be visualised better.
Fair testing- To ensure that the experiment is fair and reliable results are obtained the only variable that will be changed is the concentration of caffeine as this will be constantly getting higher (Independent variable)and have an effect on the heart rate. Distilled water with no concentration of caffeine will be used as a control. All other variables that could affect the results will need to be controlled to ensure that the results are reliable and that it is a fair test.
- All equipment will be cleaned so that any substances do not affect the accuracy of the results
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A flat surface will be worked upon so when transferring the water from the syringe to the test tube the right amount is transferred.
- The temperature of the water will be kept constant by using distilled water obtained from the same source.
- A pestle and mortar will be used to crush the caffeine tablets, instead of just breaking them up so that the same surface area is in contact with the water
- Because balances are sensitive enough to be affected by people leaning on the table or bench, so care must be taken Press
- Different cotton wool will be used for each concentration as otherwise their could be some caffeine solution from prior which would disrupt the independent variable and consequently not give reliable results.
- The same (1) number of Daphnia in each container will be ensured by using micro-pipette to obtain the Daphnia
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The time the Daphnia sit in the Petri-dish will be controlled by using a stop clock so that more beats are not recorded.
Safety-
- A laboratory coat should be worn at all times to reduce any risk of direct harm to the body
- Hands should be washed before the experiment so that any unwanted substances are not transferred during the experiment. Also they should be washed after the experiment to make sure that if their was contact with any substances they are removed.
- Any loose hair or clothing should be tied back so that it does not come in the way and cause a distraction / obstruction or a risk of an accident.
- No food/drink should be consumed as it is not very hygienic.
- Desktop should have only required equipment and otherwise should be clear with no obstructions. All stools should be tucked underneath the table so that there is no risk of tripping over or a spillage.
- All equipment, especially glassware should be handled with caution and familiarised with so that the handling is proper, so that any risk of spillages/breakages is minimised.
- If there are any accidents If equipment is broken or if there is a spillage they must be reported/ cleaned up straight away to avoid further accidents/disruption
- Daphnia are very fragile creatures so care must be taken to handle them with of caution. Also they should not be left without water longer than a few seconds as lack of water could kill the Daphnia.
- Precautions must be taken when handling the solutions so that water does not come in contact with the electrical equipment.
Ethical issues- Should be aware of moral issues arising form using living organisms in scientific experiments. Consequently the Daphnia should be respected and least harm should be done to them as possible. Consequently high concentration of caffeine will not be used as this could be fatal for the Daphnia. Also when obtaining the Daphnia through a pipette care must be taken to ensure that no harm is caused to them. As the natural habitat of Daphnia is ponds, lakes, and calm streams Distilled water will be used to minimise any reaction to the Daphnia. Also The Daphnia should not be left without water longer than a few seconds as lack of water could kill them. After the experiment is finished particular care must be taken in determining weather the Daphnia are alive or dead.
Results- The result obtained from the investigation is as follows:
Table recording the affect of different caffeine concentration on the heart beat of Daphnia for 3 trials
A mean from the data has been calculated as it descriptive statistic and describes a set of quantitative data and tells me the average of the data. Because the data is symmetrical, the mean was determined by adding all of the data values together for the 3 trails and then dividing the total by the number of data values
The results are fairly precise as the repeated values are quite close to each other, which do not show a lot of error. The values are overall systematic with an average difference in heart rate of 28 beats per minute. However the heart beat in between 0.6% and 0.8% concentration does not follow this pattern and increased the most with a difference of 86 beats per minute. This could have been because of some random error in the procedure or also as only the heartbeat for every 10 seconds were counted to get an average beats per minute and the time was calculated through personal interpretation. Also eyesight could have contributed to some inaccuracy.
A line graph has been presented below from this data. The data has then been presented in a scatter graph as it allows a more visual representation of data to be made and trends /patterns can be more easily identified. Also because of the line of best fit anomalies can be more easily seen, and it s tells us whether two variables are correlated, i.e. whether a change in one variable is accompanied by a change in the other variable. It will tell you whether the relationship is a positive correlation (both go up together) or a negative correlation (one goes up as the other goes down).
Conclusion- Although the graph does not show a direct proportional link, the results from the graph agree with the hypothesis and the link is positively correlated and overall there is a clear pattern. This indicates that as the caffeine concentration increases the heart rate increases. This is because the affect of Caffeine on Daphnia was the same as humans and stimulated the heart and nervous system in the daphnia. Higher concentrations meant more adenosine receptors in the brain were blocked, which slowed down the nerve cells. Therefore more adrenalin neurotransmitters were released which increased the activity of the receptors which increased the heart rate.
The waver in a complete direct proportional link can be as the heart beats were not the exact true value as they were only timed for ten seconds and multiplied to get the heart beat for a minute. Nevertheless the results are good enough to support a valid conclusion. The figures go up steadily which shows a systematic relationship between the concentration and the heart rate. Also because there are no extreme anomalies the results are more reliable.
Evaluation- The investigation gave the desired results however many improvements/changes could be made to the procedure to make the results more accurate.
The culture of daphnia used could affect the rate. This is because different cultures may have lived in different conditions with different Ph, organic matter, humidity, oxygen concentration and temperature and so have different lifestyles. To make the experiment reliable, subjectivity should be removed and a random sample of Daphnia could have been obtained from all places of the river e.g. upstream-downstream. However even with different cultures there is bound to be personal difference between the Daphnia.
When applying drops of the solution on the cotton wool the same pipette was used for each repeated concentration and for the different concentrations. This is not very accurate as some solution may have remained in the pipette and could have transferred some solution from one beaker to another. Also with dropping pipettes it was difficult to control the drops and sometimes several drops will come out. Also many times the end of the pipette was taken out of the solution before the bulb was released so a little air had got drawn into the end of the pipette.
Whilst observing the Daphnia under a microscope light was not used to aid the visibility and therefore the view could have been slightly obscured and eyesight could have contributed to some inaccuracy, a lamp could have been used to aid visibility. Also personal interpretation of the Daphnia’s heartbeats could not be 100% accurate. To overcome this problem of interpretation a video microscope could be used so that the beats could have been slowed down and counted more accurate, or a devise such as and ECG sensor would provide most accurate results.
Also through personal bias a systematic error could have been created. Because the effect of Caffeine on the heart rate was already known this could cause an expectation of higher results. The best way to avoid personal bias is to conduct a blind test.
Further more whilst start/stopping the clock the reaction time is bound to have been slightly slower than intended.
Although equipment was checked for cleanliness the microscope was not taken into account and could have also caused systematic error errors-(values differing from the true value by the same amount) as the lens, eyepiece and stage were not checked for cleanliness. Also the stage had not been illuminated and the sub-stage condenser was not adjusted.
To improve the reliability-large replicates of the results should be made using the same procedure will be used and results that vary considerably should be discounted or repeated. As it would allow seeing the degree of error as greater the variation of replicates the greater the degree of error
Further work
The heart rate was measured against a specific time scale, alternatively it could have been measured by calculating the time for a specific number of heart beats, or also calculated the heart beat against how many times the Daphnia moved its leg.
The experiment could have been carried out with more concentration of caffeine; to see what effect it had on the heart rate. Also different stimulants cold have been used to see if they all followed the same pattern as the results.
Bibliography-
www.daphnia.com
www.microscopy-uk.org.uk/mag/artjun99/wflea.html
www.naturegrid.org.uk/pondexplorer/gallery/daphnia.html -
www.energyfiend.com/death-by-caffeine/
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