BIO300/NOV 2013

UNIVERSITI TEKNOLOGI MARA

NEGERI SEMBILAN
FACULTY OF APPLIED SCIENCES
BIOLOGICAL TECHNIQUES AND SKILLS
BIO300
PROJECT PROPOSAL
AS1205A2
TITLE : The Effect of Caffeine and Green
tea on the heart rate of Daphnia sp.

BIO300/NOV 2013

PART A:

i.

ii.

RESEARCHER BACKGROUND

PROJECT LEADER/ MATRIC NO. /CONTACT NO.:

Muhammad Hafiz bin Shahrin
2011346605
0133798620

PROJECT MEMBERS:

NO.

NAME

MATRIC NUMBER

1)
2)

Alissa Azureen bt Norehan

Amirul Hazimi bin Abu Bakar

2011923445
2011591655

CONTACT
NUMBER
0136863120
0172256687

3)

Mohamad Ridzuwan bin Mohd Rahimi

2011728527

0142302305

iii.

PROJECT SUPERVISOR:
Miss NurAzimah Bt Osman

iv.

CO-SUPERVISOR (if any)

None

v.

LECTURER (Lecture/Laboratory)
Madam Izzati Adilah Bt Azmir

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PART B:
i)

RESEARCH PROJECT PROPOSAL

OBJECTIVES:

ii)

To investigate the effect of caffeine on the heartbeat of Daphnia sp.

To create an awareness on excessive consumption of coffee and green
tea.
SIGNIFICANCE OF PROJECT:
Nowadays, coffee and green tea consumption had become
trending to society. This research will indicate how coffee and green tea
consumption effect on beating of heart rates on living organism such as
Daphnia sp. and also human. The result of this research will provide to
society the benefits and side effects to compare between these two
ingredients. This research will also show the effect of high level of
consumption and long term effect of coffee and green tea in body
system. Thus, it will give awareness and guidance to society on which
between these two drinks they should take and better for their health.

iii)

DEFINITION OF TERMS/CONCEPTS:

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iv)

LITERATURE REVIEW:
Heart is a specialised organ that beats with an intrinsic rhythm
which can be easily observed. The normal heart rate of human is 70
beats per minute which means that the cardiac cycle of a heart takes
about 0.8 seconds for every heart beat (Solomon, 2008). Daphnia sp. in
comparison has 180 beats per minute (Princeton) that is far higher than
the Homo sapien. The translucent exoskeleton of this crustacean able to
provides a clear picture of how its organ systems work even for
observing any responds toward a stimuli.
Not only that, Daphnia sp. can tolerate the high intensity of light and
the significant amount of heat produced from the microscope during
observation process.
Heart rate can increases or decreases depending on several
factors which can alter the cardiac output, hence affecting the blood
pressure (Solomon, 2008). Cardiac output can be calculated by
multiplication of stroke volume by the number of times the left ventricle
beats per minute. Blood pressure can be defined as the force exerted by
the blood toward the blood vessels inner walls.
1,3,7-trimethylxanthine or caffeine is an organic compound that
exists in a form of white powder which has a distinctive bitter taste.
Caffeine is a type of alkaloid in a group of amines that occur naturally in
plants (Crowe, 2006). Caffeine can be found in several plants either in
their leaves or fruits. The compound can be extracted by simple
distillation process from tea leaves, cocoa, coffee beans and many more.
Caffeine is classified as stimulants of psychoactive substances due to its
effect in raising heart rate of some animals. Caffeine is also classified as
a legal safe drug to be taken by the U.S Food and Drug Administration
(FDA).
Caffeine has certain effects on animals when ingested. The
compound hold some benefits but also it does has some negative effect
that mostly depends on the the intake amount. When caffeine is
consumed, it can cause a person to feel jittery and shaky, raise blood
pressure, feel dehydrated, causes headaches or nervousness, faster
heart beat and others.
Caffiene abused has been reported as one of the culprit that has
claimed number of lives either in a long term effect(health conditions) or

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in a short period (usually due to overdose). Albeit the classification of the

caffeine as safe by the FDA, the organic compound is now under study in
persuing people to limit their or at least control the amount of caffeine
consumed.
Green tea contains about 40-80 mg of caffeine which is lower than
how much the compound contains in coffee which is 60-150 mg. (U.S.
Food and Drug Administration, 2007)

v)

REFERENCES:
http://www.scribd.com/doc/174459475/Bio-Lab-Rep-Daphnia-docx
Cofee Recent Development, R.J Clarke
http://www.ncbi.nlm.nih.gov/pubmed/7994635
http://onlinelibrary.wiley.com/doi/10.1111/j.1476-5381.1965.tb02069.x/pdf
http://circ.ahajournals.org/content/28/5/932.full.pdf
http://hyper.ahajournals.org/content/33/1/586.full
http://www.nhlbi.nih.gov/health/health-topics/topics/smo/

vi)

RESEARCH METHODS:
Preparation of Sample Extracting Caffeine form Green Tea
Obtain 4 tea bags and note the net mass of tea in each bag. In a
250 ml beaker, heat 100 ml of distilled water to just below boiling point.
Swirl the tea bags several minutes. Allow the tea solution to cool at room
temperature. Pour the tea solution into a 250 ml of extraction funnel. Add
20 ml of methylene chloride(dichloromethane) and using the swirling
technique. Separate the layers, carefully storing the organic layer in a
covered Erlenmeyer. Re-extract the aqueous layer two more times with
20 ml portions of methylene chloride. Collect all organic layers in the
same vessel. Take the combined organic layers and wash it with two
successive 20 ml portions of NaOH and then on 20 ml portion of distilled
water. Take the combined layer and add anhydrous sodium sulphate to
dry the solution so that the solution looks transparent. Decant the
solution into a tared, 100 ml round bottomed flask and rotary evaporator
the solution to dryness. Measure the mass of this flask and determine

BIO300/NOV 2013

the mass of crude caffeine. Put it in desiccant until pure solid caffeine
obtain.
Preparation of Sample Extracting Caffeine form Coffee
Obtain coffee and note the net mass of coffee. In a 250 ml beaker,
heat 100 ml of distilled water to just below boiling point. Swirl the coffee
several minutes. Allow the coffee solution to cool at room temperature.
Pour the coffee solution into a 250 ml of extraction funnel. Add 20 ml of
methylene chloride (dichloromethane) and using the swirling technique.
Separate the layers, carefully storing the organic layer in a covered
Erlenmeyer. Re-extract the aqueous layer two more times with 20 ml
portions of methylene chloride. Collect all organic layers in the same
vessel. Take the combined organic layers and wash it with two
successive 20 ml portions of NaOH and then on 20 ml portion of distilled
water. Take the combined layer and add anhydrous sodium sulphate to
dry the solution so that the solution looks transparent. Decant the
solution into a tared, 100 ml round bottomed flask and rotary evaporator
the solution to dryness. Measure the mass of this flask and determine
the mass of crude caffeine. Put it in desiccant until pure solid caffeine
obtain.
Testing the Caffeine using Daphnia sp.
A light microscope is set up. A low power objective of 40x
magnification is used. One single Daphnia sp. is selected from the
Daphnia sp. culture. By using a pipette, the selected Daphnia sp. is
carefully sucked out of the culture and then transferred to a slide along
with an appropriate amount of pond water. By using a filter paper, the
excess water on the slide is absorbed so that the Daphnia sp. lies on its
side and has limited movement so that it can be viewed under the
microscope. A small amount of water is left so that the Daphnia sp. can
survive. The slide is then placed on the microscope stage and held in
position using stage clips. The microscope is adjusted by first adjusting
the coarse focusing knob and the fine focusing knob until a fine image of
the Daphnia sp. is observed. The position of the slide is adjusted until the
heart of the Daphnia sp. can be seen clearly. One member of the group
is assigned to observe the Daphnia sp. using a microscope and count
the number of heart beat made by the Daphnia sp. within 15 seconds.
Another member will record the time. These steps will be repeated to
obtain another 2 set of data. The average reading is worked out all the
data is recorded. The Daphnia sp. is then returned to a separate beaker.
Then another Daphnia sp. is tested and placed into that the separate
beaker. Then a few drops of caffeine solution 0.2% is added to the
Daphnia sp. . The Daphnia sp. is left in the caffeine solution for 1 minute.

BIO300/NOV 2013

After that, its heart beat is counted by the same member and same
method. These steps was repeated by using solutions of
0.4%,0.6%,0.8%,1.0% and 1.2% respectively. Each average value of
heart beat of Daphnia sp. is multiplied by four to obtain the value of heart
beat of Daphnia sp. in unit beats per minute. The resulted are tabulated.
A bar graph showing the comparisons of Daphnia s. with treatments and
without treatments is plotted. A line graph of the heartbeat of Daphnia sp.
per minute against the concentration of caffeine solution is drawn.

vii)

LIST OF EQUIPMENTS/FACILITIES:
Beaker
Erlenmeyer flask
Extraction funnel
Round bottomed flask
Rotary evaporator
Light microscope
Pipette
Glass slide
Filter paper
Trinocular microscope with video
Petri dish

viii)

LIST OF CHEMICALS/MEDIA:
Distilled water
Methylene chloride
Sodium chloride
Anhydrous sodium sulphate
Daphnia sp.
Coffee
Green tea
Dichloromethane

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ix)

DECLARATION AND SIGNATURE

We are hereby declaring that the project is based on our original

work except for quotations and citations which have been duly
acknowledge. We are conducting our investigations with honesty
and integrity

SIGNATURE OF PROJECT LEADER: ________________________

( Muhammad Hafiz bin Shahrin )
SIGNATURE OF PROJECT MEMBER:
1) Alissa Azureen bt Norehan :
2) Amirul Hazimi bin Abu Bakar :
3) Mohamad Ridzuwan bin Mohd Rahimi :

Signature of Supervisor: _________________________________

Received date: ____________________________
Returned date: ____________________________

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