Daphnia Caffeine Experiment
A-LEVEL
BIOLOGY LAB REPORT
NAME: NURUL AYUNI BT SABRI
GROUP: 11SC4
TITLE: THE EFFECT OF CAFFEINE ON HEART RATE
LECTURER : MDM ZAKIAH BINTI ZAKARIA
TITLE
The effect of caffeine on heart rate OBJECTIVE
To determine the effect of caffeine on heart rate of daphnia
INTRODUCTION
Caffeine, medically known as trimethylzanthine. Its chemical formula is C8H10N4O2. When in pure form, caffeine is a white crystalline powder that taste very bitter. 1The most common way of acquiring pure caffeine is the process of decaffeinating coffee and tea. Caffeine is found in varying quantities in the seeds, leaves, and fruit of some plants, where it acts as a natural pesticide that paralyzes and kills certain insects …show more content…
feeding on the plants, as well as enhancing the reward memory of pollinators. It is most commonly consumed by humans in infusions extracted from the seed of the coffee plant and the leaves of the tea bush, as well as from various foods and drinks containing products derived from the kola nut. Other sources include yerba maté, guarana berries,guayusa, and the yaupon holly.
In humans, caffeine acts as a central nervous system stimulant, temporarily warding off drowsiness and restoring alertness. It is the world 's most widely consumed psychoactive drug, but unlike many other psychoactive substances, it is legal and unregulated in nearly all parts of the world. Beverages containing caffeine, such as coffee, tea, soft drinks, and energy drinks, enjoy great popularity. In North America, 90% of adults consume caffeine daily. Part of the reason caffeine is classified by the Food and Drug Administration as GRAS (generally recognized as safe) is that toxic doses (over 1 gram for an average adult) are much higher than typically used doses (less than 500 milligrams). Ordinary consumption can have low health risks, even when carried on for years – there may be a modest protective effect against some diseases, including Parkinsons Disease, and certain types of cancer. Caffeine can have both positive and negative effects on anxiety disorder. Some people experience sleep disruption if they consume caffeine, especially during the evening hours, but others show little disturbance and the effect of caffeine on sleep is highly variable.
Evidence of a risk to pregnancy is equivocal, but some authorities have concluded that prudent advice is for pregnant women to limit consumption to the equivalent of two cups of coffee per day or less. The American Congress of Obstetricians and Gynecologists (ACOG) concluded in 2010 that caffeine consumption is safe up to 200 mg per day in pregnant women. Caffeine has pressor and mild diuretic effects when administered to people who are not used to it, but regular users develop a tolerance to this effect, and studies have generally failed to support the common notion that ordinary consumption contributes significantly to dehydration. With heavy use, tolerance develops rapidly to autonomic effects such as elevated heart rate and muscle twitching, but not to the cognitive or arousal effects of caffeine. The degree to which caffeine can produce clinically significant dependency and addiction remains a subject of controversy in the medical literature.2
Excessive intake of caffeine can result in restlessness, insomnia, and heart irregularities. The effects of caffeine vary from person to person, as people excrete it at different rates. Physical dependence and unpleasant symptoms upon withdrawal (headache, fatigue and depression) are common in regular caffeine users.
A study of effect of caffeine in human heart rate will be very valuable. However,there are many difficulties in conducting an experiment that involves human. Thus, an alternative is to use a model organism to represent human. The results of the experiment can then be used as a reference to the effect of caffeine on human.Daphnia are selected as the model as they can be easily handled in many ways in the investigation. Daphnia sp. are small, planktonic crustaceans.
They, between 0.2 to 5mm in length, are common freshwater cladocerans, often classified with other tiny crustaceans as “microcrustaceans.” Cladocerans are commonly known as water fleas not only due to their salutatory swimming style, butalso because of their resemblance to real fleas, though real fleas are insects and share only an extremely distant common ancestry with Daphnia, since both crustaceans and insects are arthropods. Most species in the Order Cladocera are freshwater species, although there are some marine species. The classification of Cladocera is as an order within the Subclass Diplostraca within the ClassBranchiopoda within the Subphylum Crustacea.
All species of Daphnia occur indifferent strains - sometimes the same species can look completely different, both interms of size and shape, depending on its origin, and environmental factors at that location. There are approximately 150 known species in North America, and a similar number in Europe (many of these species are found on both continents, either through accidental introduction by man, or nature). Many foreign species have been introduced to America and Europe from Asia and Africa (the most notorious of which is Daphnialumholtzi, which is native to Africa). It is not uncommon to collect 20 or more species in one small area of lake bottom.3
Daphnia sp. live in various aquatic environment sranging from acidic swamps to freshwater lakes, ponds, streams and rivers. The body of Daphnia is usually 1–5 millimetres (0.04–0.20 in) long, and is divided into segments, although this division is not visible. The head is fused, and is generally bent down towards the body with a visible notch separating the two. In most species, the rest of the body is covered by a carapace, with a ventral gap in which the five or six pairs of legs lie. The most prominent features are the compound eyes, the second antennae, and a pair of abdominal setae. In many species, the carapace is translucent or nearly so and as a result they make excellent subjects for the microscope as one can observe the beating heart.
Even under relatively low-power microscopy, the feeding mechanism can be observed, with immature young moving in the brood pouch; moreover, the eye being moved by the ciliary muscles can be seen, as well as blood blood cells being pumped around the circulatory system by the simple heart. The heart is at the top of the back, just behind the head, and the average heart rate is approximately 180 bpm under normal conditions.
Daphnia, like many animals, are prone to alcohol intoxication, and make excellent subjects for studying the effects of the depressant on the nervous system due to the translucent exoskeleton and the visibly altered heart rate. They are tolerant of being observed live under a cover slip and appear to suffer no harm when returned to open water. This experiment can also be performed using caffeine, nicotine or adrenaline, each producing an increase in the heart rate.4
PROBLEM STATEMENT
In humans, caffeine 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. An experiment is conducted to prove the effect of caffeine on heart beat of human model, Daphnia sp.
HYPOTHESIS
The higher the concentration of caffeine, the higher the heart rate of daphnia VARIABLES VARIABLES
WAYS TO CONTROL VARIABLE
Manipulated variable:
Concentration of caffeine
Using different amount of caffeine powder to prepare five caffeine solutions of different concentration, that is 0.1%, 0.2%, 0.3%, 0.4% and 0.5% concentration of caffeine solution.
Responding variable :
Heart rate of Daphnia
Count and record the beating of heart of Daphnia in every 15 seconds
Controlled variable :
Temperature of caffeine solution
Use distilled water at fixed room temperature
APPARATUS
Cavity slides, dropping pipettes, cotton wool, dropper, standard glassware (beakers, measuring cylinders, etc), stopclock, filter paper, spatula, microscope
MATERIAL
Culture of Daphnia (water fleas), distilled water, caffeine solution
TECHNIQUE
1. Placing Daphnia on a cavity slide and immobilising it by using cotton wool.
2. Observe the beating hearts of Daphnia under microscope
3. Counting the beat of the heart of Daphnia in every 15 seconds to identify the heart beat rate
4. Measure the time recorded by using stopwatch
5. Calculate the mean heart rate by using calculator
PROCEDURE
1. Caffeine solutions of concentrations of 0.1%, 0.2%, 0.3%, 0.4% and 0.5% are prepared using caffeine powder and distilled water. To prepare 0.1% caffeine solution, 0.1g of caffeine powder is dissolved in 100ml of distilled water. The amount of caffeine powder is altered accordingly to obtain the caffeine solutions of respective concentration.
2. A light microscope with microscope light is set up. A low power objective lens of 40x magnification is selected.
3. One Daphnia is selected and scooped cautiously from the container using a petri dish along with moderate amount of water.
4. The Daphnia is transferred from the petri dish onto a cavity slide, by using a spatula, along with a small amount of pond water.
5. A piece of cotton wool are placed on the Daphnia, so that to make sure that it is held in position and does not move while it is being observed under the microscope.
6. Some water is inevitablely absorbed. One or two extra drops of water is be added to prevent the Daphnia from dying.
7. The cavity slide is placed onto the microscope stage and held in position using stage clips.
8. The microscope is focused by adjusting the coarse focusing knob and fine focusing knob until a clear image of Daphnia is observed. The position of cavity slide is adjusted until the heart of Daphnia can be observed clearly
9. Count the number of heart beat or leg beat made by the Daphnia within 15 seconds.
10. This step is repeated again to obtain a second reading. The average of value of the readings are worked out.
11. .Steps 4 to 10 are repeated for five times by adding caffeine solution of concentrations 0.1%, 0.2%, 0.3%, 0.4% and 0.5% to the cavity slide respectively.
12.
RESULT
CONCENTRATION OF CAFFEINE SOLUTION (%)
HEART RATE (BEAT /15 S )
MEAN HEART RATE (BEATS/MIN)
1
2
3
4
AVERAGE
0.0
11
15
13
14
13.25
53
0.1
21
19
17
21
19.50
78
0.2
23
26
26
30
26.25
105
0.3
39
32
37
39
36.75
147
0.4
50
47
42
42
47.00
188
0.5
56
53
52
52
52.25
209
DISCUSSION Based on the graph obtained, it shows that the higher the concentration of caffeine, the higher the mean heart rate. Like human, a daphnia’s heart will beat faster when it receives a dose of caffeine. 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.5
Caffeine also mimics some of the effects of adrenaline and noradrenaline in the heart. By a different mechanism not involving beta-1 adrenoceptors, caffeine also increases the amount of cAMP in the sinoatrial node. Then cAMP levels increase and this increases the electrical activity of the sinoatrial node, making it depolarize and 'beat ' faster. Caffeine has additional effects on the heart. Like adrenaline and noradrenaline, it can affect the main pumping chambers (ventricles), leading to an increase in the rate of contraction and relaxation of each heart beat. This means that, as well as beating faster, the heart 's individual beats are associated with an increased volume of blood ejected into the circulation per unit time. This is called increasing cardiac output. Two or three cups of strong coffee or tea contain enough caffeine (and a similar acting compound called theobromine) to cause an increase in human heart rate of 5-20 beats/min.6 It is known that the chemicals absorbed by the Daphnia will be stayed in the body in half an hour. Therefore, the caffeine solution could be introduced to Daphnia 5 minutes prior before the experiment. Daphnia is suitable for the experiment because it has no selection in absorbing chemical substances from the surrounding environment. Any chemicals will have the physiological effect on the Daphnia immediately. Besides, Daphnia body is translucent which allows the observation of heart beat of Daphnia to be made without cutting them open. The experiment should be made as quickly as possible because Daphnia may not survive in the new environment.
Furthermore, large Daphnia may show different heart rate compared to smaller Daphnia due to the discrepancy in concentration of caffeine in the system between the large and the small Daphnia. Slower heart beat rate may be shown by larger Daphnia. Therefore, it is important to use the same Daphnia throughout the experiment without injured or kill the Daphnia.
The temperature could be easily controlled before the experiment by placing Daphnia in a water bath. The temperature should be controlled because an increase in temperature of 10 degree Celsius will increase the rate of reaction, and thus the heart rate of Daphnia. The amount of water on the slide could be controlled by using a dropper that only allows a drop of water on the slide.
In order to observe Daphnia more effectively, the Daphnia has to be propped up by cotton wool to show the Daphnia’s ventral view. The heart is at the top of the back, just behind the head. At the beginning of the experiment, the Daphnia may struggle in the cavity slide, making the observation of the heart beat rate more difficult. It is important that only a drop of solution is placed on the cavity slide which is smeared thinly with grease in order to secure Daphnia in place. Small pieces of cotton wool may be added to restrict the movement of Daphnia.
The concentration of oxygen and other gases in the solution could be controlled by using an aeration pump that can dissolve the maximum amount of oxygen gas in the solution before the experiment. pH of the solution could be controlled by using a buffer solution. The concentration of caffeine in the solution should be carefully monitored to by measuring out the exact mass of caffeine during the preparation of the standard solution by using a volumetric flask.
During the experiment, a natural light is preferred over the artificial light because artificial light generates more heat than natural light. Alternatively, a cooling system on the slide or a thermostat may be installed.
A video camera may be used to increase the accuracy of the readings. This is because human reflexes is not enough to count the fast beating rate of Daphnia’s heart especially at high concentration of caffeine solution. A video may be taken and the recordings can be played at a slower pace the heart rate to be counted more accurately.7
In addition, cooling the Daphnia before the experiment may help slow their heart rate. Use of the stroboscope may overcome the problems of counting faster heart rates. A simpler approach is to count the rate at which the legs beat. This rate is proportional to the rate at which the heartbeats. 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.8
FURTHER DISCUSSION
Other than caffeine, there are also a few chemicals that might affect our heart beat without us noticing it. Firstly is acetylcholine. In humans and many other animals, heart rate is slowed by the parasympathetic nervous system (neurotransmitter: acetylcholine) via activation of cell surface receptors in the sinoatrial node (pacemaker) called acetylcholine muscarinic receptors. This occurs after feeding, during sleep, and during breath-holding and swimming underwater. A slowed heart rate and the associated fall in the rate of ejection of blood from the heart is sufficient to maintain body function during rest, and conserves energy in the heart under conditions where its supply (and the supply of oxygen in the blood) are diminished. A drug that slows heart rate is called a negative chronotrope; this is demonstrated in this experiment, where acetylcholine is used to slow the rate of the Daphnia 's heart.
Next is noradrenaline and adrenaline.
In contrast, heart rate is increased by the sympathetic nervous system (neurotransmitter: noradrenaline) and the hormone adrenaline circulating in the blood via activation of cell surface receptors in the sinoatrial node - pacemaker) (called beta-1 adrenoceptors). This occurs during exercise or fear. The effect is to increase the rate of ejection of blood by the heart. This means that there will be more blood flow to skeletal muscle (in which exercise causes dilatation of blood vessels), so the skeletal muscle cells are supplied with more oxygen and respiratory substrates used to generate energy in respiration where it is needed. A drug that increases heart rate is called a positive chronotrope, and this is demonstrated in this experiment when adrenaline is used to increase heart rate in Daphnia.
One of the ways adrenaline increases heart rate is through the action of what is known as a 'second messenger ' or 'transduction component ', in this case it is a chemical made in the cell known as cyclic adenosine monophosphate (cAMP). Transduction is the process that follows the action of a drug, hormone or neurotransmitter at a receptor. Thus, when adrenaline activates the beta-1 adrenoceptor in the sinoatrial node, this leads to an increase in cAMP in the sinoatrial node and the result is an increase in heart
rate.
Ethanol slows heart rate. At the concentrations used in this experiment, ethanol depresses the nervous system by acting as what is known as a non-selective neurodepressant. The amounts of ethanol necessary to achieve this effect in humans would also be sufficient to depress the respiratory centres of the brain, rather like the effect of an overdose of general anaesthetic, resulting in death.
Aspirin has no effect on heart rate. Despite this, aspirin has beneficial effects in the heart. By reducing the ability of platelets to adhere to damaged blood vessel walls, aspirin reduces the chance of coronary artery thrombosis, the event that precipitates a heart attack. People who are take aspirin long-term for medical reasons (because they have cardiovascular disease or diabetes) may have a lower heart rate than controls, simply because they experience less coronary and peripheral thrombosis and thus have a better lubricated cardiovascular system.9
SAFETY PRECAUTION
i) INDIVIDUAL
1. Wear lab coat
2. Take care when using electrical equipment like microscope light near to water to prevent from being electrocuted
3. The microscope is fragile and the light bulbs can get hot so handle with care.10
ii) DURING EXPERIMENT
1. The caffeine solution is ensured not too concentrated so that the Daphnia does not die of it.
2. Each experiment of different concentration of caffeine solution ought to be conducted as fast as possible for there is a risk for the Daphnia to die in a new environment
3. The Daphnia used should be handled with care to avoid it from injury and thus affect the results of the experiment.
4. Repeat the experiment four times to get the average value and accurate value of heart beat of Daphnia
CONCLUSION
It is shown that the amount of caffeine in the solution will determine the heart rate of the daphnia. It will be directly proportional to each other so the increase of the concentration of caffeine will also increase the heart rate. Hypothesis is accepted.
REFERENCES
1. http://www.herndonmagnetschool.com/samplescifair10.PDF
2. http://en.wikipedia.org/wiki/Caffeine
3. http://www.scribd.com/doc/29218203/Heart-Rate-of-Daphnia
4. http://en.wikipedia.org/wiki/Daphnia
5. http://www.madsci.org/posts/archives/jul99/931925101.Zo.r.html
6. http://www.nuffieldfoundation.org/practical-biology/investigating-factors-affecting-heart-rate-daphnia
7. http://www.markedbyteachers.com/as-and-a-level/science/purpose-to-investigate-the-effect-of-caffeine-on-the-heart-rate-of-daphnia-water-fleas.html
8. http://essay911.org/17053-effect-of-concentration-of-caffeine-on-the-heart-beat-rate-of-daphnia.html
9. http://www.ukessays.co.uk/essays/biology/concentration-of-caffein.php
BIOLOGY LAB REPORT
NAME: NURUL AYUNI BT SABRI
GROUP: 11SC4
TITLE: THE EFFECT OF CAFFEINE ON HEART RATE
LECTURER : MDM ZAKIAH BINTI ZAKARIA
TITLE
The effect of caffeine on heart rate OBJECTIVE
To determine the effect of caffeine on heart rate of daphnia
INTRODUCTION
Caffeine, medically known as trimethylzanthine. Its chemical formula is C8H10N4O2. When in pure form, caffeine is a white crystalline powder that taste very bitter. 1The most common way of acquiring pure caffeine is the process of decaffeinating coffee and tea. Caffeine is found in varying quantities in the seeds, leaves, and fruit of some plants, where it acts as a natural pesticide that paralyzes and kills certain insects …show more content…
feeding on the plants, as well as enhancing the reward memory of pollinators. It is most commonly consumed by humans in infusions extracted from the seed of the coffee plant and the leaves of the tea bush, as well as from various foods and drinks containing products derived from the kola nut. Other sources include yerba maté, guarana berries,guayusa, and the yaupon holly.
In humans, caffeine acts as a central nervous system stimulant, temporarily warding off drowsiness and restoring alertness. It is the world 's most widely consumed psychoactive drug, but unlike many other psychoactive substances, it is legal and unregulated in nearly all parts of the world. Beverages containing caffeine, such as coffee, tea, soft drinks, and energy drinks, enjoy great popularity. In North America, 90% of adults consume caffeine daily. Part of the reason caffeine is classified by the Food and Drug Administration as GRAS (generally recognized as safe) is that toxic doses (over 1 gram for an average adult) are much higher than typically used doses (less than 500 milligrams). Ordinary consumption can have low health risks, even when carried on for years – there may be a modest protective effect against some diseases, including Parkinsons Disease, and certain types of cancer. Caffeine can have both positive and negative effects on anxiety disorder. Some people experience sleep disruption if they consume caffeine, especially during the evening hours, but others show little disturbance and the effect of caffeine on sleep is highly variable.
Evidence of a risk to pregnancy is equivocal, but some authorities have concluded that prudent advice is for pregnant women to limit consumption to the equivalent of two cups of coffee per day or less. The American Congress of Obstetricians and Gynecologists (ACOG) concluded in 2010 that caffeine consumption is safe up to 200 mg per day in pregnant women. Caffeine has pressor and mild diuretic effects when administered to people who are not used to it, but regular users develop a tolerance to this effect, and studies have generally failed to support the common notion that ordinary consumption contributes significantly to dehydration. With heavy use, tolerance develops rapidly to autonomic effects such as elevated heart rate and muscle twitching, but not to the cognitive or arousal effects of caffeine. The degree to which caffeine can produce clinically significant dependency and addiction remains a subject of controversy in the medical literature.2
Excessive intake of caffeine can result in restlessness, insomnia, and heart irregularities. The effects of caffeine vary from person to person, as people excrete it at different rates. Physical dependence and unpleasant symptoms upon withdrawal (headache, fatigue and depression) are common in regular caffeine users.
A study of effect of caffeine in human heart rate will be very valuable. However,there are many difficulties in conducting an experiment that involves human. Thus, an alternative is to use a model organism to represent human. The results of the experiment can then be used as a reference to the effect of caffeine on human.Daphnia are selected as the model as they can be easily handled in many ways in the investigation. Daphnia sp. are small, planktonic crustaceans.
They, between 0.2 to 5mm in length, are common freshwater cladocerans, often classified with other tiny crustaceans as “microcrustaceans.” Cladocerans are commonly known as water fleas not only due to their salutatory swimming style, butalso because of their resemblance to real fleas, though real fleas are insects and share only an extremely distant common ancestry with Daphnia, since both crustaceans and insects are arthropods. Most species in the Order Cladocera are freshwater species, although there are some marine species. The classification of Cladocera is as an order within the Subclass Diplostraca within the ClassBranchiopoda within the Subphylum Crustacea.
All species of Daphnia occur indifferent strains - sometimes the same species can look completely different, both interms of size and shape, depending on its origin, and environmental factors at that location. There are approximately 150 known species in North America, and a similar number in Europe (many of these species are found on both continents, either through accidental introduction by man, or nature). Many foreign species have been introduced to America and Europe from Asia and Africa (the most notorious of which is Daphnialumholtzi, which is native to Africa). It is not uncommon to collect 20 or more species in one small area of lake bottom.3
Daphnia sp. live in various aquatic environment sranging from acidic swamps to freshwater lakes, ponds, streams and rivers. The body of Daphnia is usually 1–5 millimetres (0.04–0.20 in) long, and is divided into segments, although this division is not visible. The head is fused, and is generally bent down towards the body with a visible notch separating the two. In most species, the rest of the body is covered by a carapace, with a ventral gap in which the five or six pairs of legs lie. The most prominent features are the compound eyes, the second antennae, and a pair of abdominal setae. In many species, the carapace is translucent or nearly so and as a result they make excellent subjects for the microscope as one can observe the beating heart.
Even under relatively low-power microscopy, the feeding mechanism can be observed, with immature young moving in the brood pouch; moreover, the eye being moved by the ciliary muscles can be seen, as well as blood blood cells being pumped around the circulatory system by the simple heart. The heart is at the top of the back, just behind the head, and the average heart rate is approximately 180 bpm under normal conditions.
Daphnia, like many animals, are prone to alcohol intoxication, and make excellent subjects for studying the effects of the depressant on the nervous system due to the translucent exoskeleton and the visibly altered heart rate. They are tolerant of being observed live under a cover slip and appear to suffer no harm when returned to open water. This experiment can also be performed using caffeine, nicotine or adrenaline, each producing an increase in the heart rate.4
PROBLEM STATEMENT
In humans, caffeine 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. An experiment is conducted to prove the effect of caffeine on heart beat of human model, Daphnia sp.
HYPOTHESIS
The higher the concentration of caffeine, the higher the heart rate of daphnia VARIABLES VARIABLES
WAYS TO CONTROL VARIABLE
Manipulated variable:
Concentration of caffeine
Using different amount of caffeine powder to prepare five caffeine solutions of different concentration, that is 0.1%, 0.2%, 0.3%, 0.4% and 0.5% concentration of caffeine solution.
Responding variable :
Heart rate of Daphnia
Count and record the beating of heart of Daphnia in every 15 seconds
Controlled variable :
Temperature of caffeine solution
Use distilled water at fixed room temperature
APPARATUS
Cavity slides, dropping pipettes, cotton wool, dropper, standard glassware (beakers, measuring cylinders, etc), stopclock, filter paper, spatula, microscope
MATERIAL
Culture of Daphnia (water fleas), distilled water, caffeine solution
TECHNIQUE
1. Placing Daphnia on a cavity slide and immobilising it by using cotton wool.
2. Observe the beating hearts of Daphnia under microscope
3. Counting the beat of the heart of Daphnia in every 15 seconds to identify the heart beat rate
4. Measure the time recorded by using stopwatch
5. Calculate the mean heart rate by using calculator
PROCEDURE
1. Caffeine solutions of concentrations of 0.1%, 0.2%, 0.3%, 0.4% and 0.5% are prepared using caffeine powder and distilled water. To prepare 0.1% caffeine solution, 0.1g of caffeine powder is dissolved in 100ml of distilled water. The amount of caffeine powder is altered accordingly to obtain the caffeine solutions of respective concentration.
2. A light microscope with microscope light is set up. A low power objective lens of 40x magnification is selected.
3. One Daphnia is selected and scooped cautiously from the container using a petri dish along with moderate amount of water.
4. The Daphnia is transferred from the petri dish onto a cavity slide, by using a spatula, along with a small amount of pond water.
5. A piece of cotton wool are placed on the Daphnia, so that to make sure that it is held in position and does not move while it is being observed under the microscope.
6. Some water is inevitablely absorbed. One or two extra drops of water is be added to prevent the Daphnia from dying.
7. The cavity slide is placed onto the microscope stage and held in position using stage clips.
8. The microscope is focused by adjusting the coarse focusing knob and fine focusing knob until a clear image of Daphnia is observed. The position of cavity slide is adjusted until the heart of Daphnia can be observed clearly
9. Count the number of heart beat or leg beat made by the Daphnia within 15 seconds.
10. This step is repeated again to obtain a second reading. The average of value of the readings are worked out.
11. .Steps 4 to 10 are repeated for five times by adding caffeine solution of concentrations 0.1%, 0.2%, 0.3%, 0.4% and 0.5% to the cavity slide respectively.
12.
RESULT
CONCENTRATION OF CAFFEINE SOLUTION (%)
HEART RATE (BEAT /15 S )
MEAN HEART RATE (BEATS/MIN)
1
2
3
4
AVERAGE
0.0
11
15
13
14
13.25
53
0.1
21
19
17
21
19.50
78
0.2
23
26
26
30
26.25
105
0.3
39
32
37
39
36.75
147
0.4
50
47
42
42
47.00
188
0.5
56
53
52
52
52.25
209
DISCUSSION Based on the graph obtained, it shows that the higher the concentration of caffeine, the higher the mean heart rate. Like human, a daphnia’s heart will beat faster when it receives a dose of caffeine. 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.5
Caffeine also mimics some of the effects of adrenaline and noradrenaline in the heart. By a different mechanism not involving beta-1 adrenoceptors, caffeine also increases the amount of cAMP in the sinoatrial node. Then cAMP levels increase and this increases the electrical activity of the sinoatrial node, making it depolarize and 'beat ' faster. Caffeine has additional effects on the heart. Like adrenaline and noradrenaline, it can affect the main pumping chambers (ventricles), leading to an increase in the rate of contraction and relaxation of each heart beat. This means that, as well as beating faster, the heart 's individual beats are associated with an increased volume of blood ejected into the circulation per unit time. This is called increasing cardiac output. Two or three cups of strong coffee or tea contain enough caffeine (and a similar acting compound called theobromine) to cause an increase in human heart rate of 5-20 beats/min.6 It is known that the chemicals absorbed by the Daphnia will be stayed in the body in half an hour. Therefore, the caffeine solution could be introduced to Daphnia 5 minutes prior before the experiment. Daphnia is suitable for the experiment because it has no selection in absorbing chemical substances from the surrounding environment. Any chemicals will have the physiological effect on the Daphnia immediately. Besides, Daphnia body is translucent which allows the observation of heart beat of Daphnia to be made without cutting them open. The experiment should be made as quickly as possible because Daphnia may not survive in the new environment.
Furthermore, large Daphnia may show different heart rate compared to smaller Daphnia due to the discrepancy in concentration of caffeine in the system between the large and the small Daphnia. Slower heart beat rate may be shown by larger Daphnia. Therefore, it is important to use the same Daphnia throughout the experiment without injured or kill the Daphnia.
The temperature could be easily controlled before the experiment by placing Daphnia in a water bath. The temperature should be controlled because an increase in temperature of 10 degree Celsius will increase the rate of reaction, and thus the heart rate of Daphnia. The amount of water on the slide could be controlled by using a dropper that only allows a drop of water on the slide.
In order to observe Daphnia more effectively, the Daphnia has to be propped up by cotton wool to show the Daphnia’s ventral view. The heart is at the top of the back, just behind the head. At the beginning of the experiment, the Daphnia may struggle in the cavity slide, making the observation of the heart beat rate more difficult. It is important that only a drop of solution is placed on the cavity slide which is smeared thinly with grease in order to secure Daphnia in place. Small pieces of cotton wool may be added to restrict the movement of Daphnia.
The concentration of oxygen and other gases in the solution could be controlled by using an aeration pump that can dissolve the maximum amount of oxygen gas in the solution before the experiment. pH of the solution could be controlled by using a buffer solution. The concentration of caffeine in the solution should be carefully monitored to by measuring out the exact mass of caffeine during the preparation of the standard solution by using a volumetric flask.
During the experiment, a natural light is preferred over the artificial light because artificial light generates more heat than natural light. Alternatively, a cooling system on the slide or a thermostat may be installed.
A video camera may be used to increase the accuracy of the readings. This is because human reflexes is not enough to count the fast beating rate of Daphnia’s heart especially at high concentration of caffeine solution. A video may be taken and the recordings can be played at a slower pace the heart rate to be counted more accurately.7
In addition, cooling the Daphnia before the experiment may help slow their heart rate. Use of the stroboscope may overcome the problems of counting faster heart rates. A simpler approach is to count the rate at which the legs beat. This rate is proportional to the rate at which the heartbeats. 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.8
FURTHER DISCUSSION
Other than caffeine, there are also a few chemicals that might affect our heart beat without us noticing it. Firstly is acetylcholine. In humans and many other animals, heart rate is slowed by the parasympathetic nervous system (neurotransmitter: acetylcholine) via activation of cell surface receptors in the sinoatrial node (pacemaker) called acetylcholine muscarinic receptors. This occurs after feeding, during sleep, and during breath-holding and swimming underwater. A slowed heart rate and the associated fall in the rate of ejection of blood from the heart is sufficient to maintain body function during rest, and conserves energy in the heart under conditions where its supply (and the supply of oxygen in the blood) are diminished. A drug that slows heart rate is called a negative chronotrope; this is demonstrated in this experiment, where acetylcholine is used to slow the rate of the Daphnia 's heart.
Next is noradrenaline and adrenaline.
In contrast, heart rate is increased by the sympathetic nervous system (neurotransmitter: noradrenaline) and the hormone adrenaline circulating in the blood via activation of cell surface receptors in the sinoatrial node - pacemaker) (called beta-1 adrenoceptors). This occurs during exercise or fear. The effect is to increase the rate of ejection of blood by the heart. This means that there will be more blood flow to skeletal muscle (in which exercise causes dilatation of blood vessels), so the skeletal muscle cells are supplied with more oxygen and respiratory substrates used to generate energy in respiration where it is needed. A drug that increases heart rate is called a positive chronotrope, and this is demonstrated in this experiment when adrenaline is used to increase heart rate in Daphnia.
One of the ways adrenaline increases heart rate is through the action of what is known as a 'second messenger ' or 'transduction component ', in this case it is a chemical made in the cell known as cyclic adenosine monophosphate (cAMP). Transduction is the process that follows the action of a drug, hormone or neurotransmitter at a receptor. Thus, when adrenaline activates the beta-1 adrenoceptor in the sinoatrial node, this leads to an increase in cAMP in the sinoatrial node and the result is an increase in heart
rate.
Ethanol slows heart rate. At the concentrations used in this experiment, ethanol depresses the nervous system by acting as what is known as a non-selective neurodepressant. The amounts of ethanol necessary to achieve this effect in humans would also be sufficient to depress the respiratory centres of the brain, rather like the effect of an overdose of general anaesthetic, resulting in death.
Aspirin has no effect on heart rate. Despite this, aspirin has beneficial effects in the heart. By reducing the ability of platelets to adhere to damaged blood vessel walls, aspirin reduces the chance of coronary artery thrombosis, the event that precipitates a heart attack. People who are take aspirin long-term for medical reasons (because they have cardiovascular disease or diabetes) may have a lower heart rate than controls, simply because they experience less coronary and peripheral thrombosis and thus have a better lubricated cardiovascular system.9
SAFETY PRECAUTION
i) INDIVIDUAL
1. Wear lab coat
2. Take care when using electrical equipment like microscope light near to water to prevent from being electrocuted
3. The microscope is fragile and the light bulbs can get hot so handle with care.10
ii) DURING EXPERIMENT
1. The caffeine solution is ensured not too concentrated so that the Daphnia does not die of it.
2. Each experiment of different concentration of caffeine solution ought to be conducted as fast as possible for there is a risk for the Daphnia to die in a new environment
3. The Daphnia used should be handled with care to avoid it from injury and thus affect the results of the experiment.
4. Repeat the experiment four times to get the average value and accurate value of heart beat of Daphnia
CONCLUSION
It is shown that the amount of caffeine in the solution will determine the heart rate of the daphnia. It will be directly proportional to each other so the increase of the concentration of caffeine will also increase the heart rate. Hypothesis is accepted.
REFERENCES
1. http://www.herndonmagnetschool.com/samplescifair10.PDF
2. http://en.wikipedia.org/wiki/Caffeine
3. http://www.scribd.com/doc/29218203/Heart-Rate-of-Daphnia
4. http://en.wikipedia.org/wiki/Daphnia
5. http://www.madsci.org/posts/archives/jul99/931925101.Zo.r.html
6. http://www.nuffieldfoundation.org/practical-biology/investigating-factors-affecting-heart-rate-daphnia
7. http://www.markedbyteachers.com/as-and-a-level/science/purpose-to-investigate-the-effect-of-caffeine-on-the-heart-rate-of-daphnia-water-fleas.html
8. http://essay911.org/17053-effect-of-concentration-of-caffeine-on-the-heart-beat-rate-of-daphnia.html
9. http://www.ukessays.co.uk/essays/biology/concentration-of-caffein.php