i

Comparison of Lemon and Green tea as Anti Bacterial Hand Sanitizer

____________________________________________

An Investigatory Project
Presented to the
Sacred Heart School Ateneo de Cebu
High School Department

_____________________________________________

In Partial Fulfillment
of the Requirements for the
SCIENCE and ENGLISH SUBJECTS

_____________________________________________
by
John Alessandro Chu
Peter Adrian Cambonga
Patricia Isabel Chua
Nicole David
RESEARCHERS


Grade 9 Southwell

Submitted to:
Mrs. Edgermi G. Gingoyon
Mrs. Cecilie A. Villacrucis
Research ADVISERS


March 2014


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ACKNOWLEDGMENT

The researchers would like to thank the following for their time and support to the
group in the said Investigatory Project.
Mdm. Villacrucis, for helping the researchers in various tasks, suggestions, and the
motivation to succeed in the Investigatory Project.
Mr. Araneta, for assisting the group in the laboratory, and for supplying the materials
needed for the said experiment.
Mdm. Gingoyon, for helping the researchers organize and editing the papers for our
Investigatory project.
The 3 volunteers who gave the researchers their time and effort.
Ms. Maluya, for lending the researchers her laptop and use the SmartScope
microscope.
Also Sacred Heart School Ateneo de Cebu for giving the opportunity to show,
enhance and discover themselves through science and accompanying the group
through the road to excellence through chemistry. AMDG
And lastly, God Almighty for always being there for the group, and showering the
group with his blessings. Thank God its done.






















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ABSTRACT

This research aims to promote different alternatives for anti bacterial hand
sanitizers such as the lemon and green tea extracts in a way that we try to innovate a
greener environment and in the organic way. This paper outlines the health benefits and
comparison for the effectiveness of anti bacterial growth of both variables especially in
the hands. With this kind of research, the researchers were able to gather data through
their hands-on experiments using the natural hands of the researchers and agar as to
culture the bacteria initiated. With the number of days awaited for the bacteria to grow,
they observed that:
a) that green tea possesses anti-bacterial properties which are more effective than
those of the lemon extracts.
b) lemon, expected to kill bacteria from its acidic properties, actually grew more
molds compared to green tea; But still showed signs of the decrease of bacteria. With this
data, the researchers have concluded that green tea is more effective in inhibiting hand
bacteria. Hence, the hypothesis is accepted. A recommendation is to use the standardized
procedure to culture the bacteria.































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Table of Contents

Content Page
I. INTRODUCTION v
Rationale of the Study .. v
Theoretical Background ... vi

II. THE PROBLEM .. x
Statement of the Problem ..... x
Scope and Limitation xi
Definition of Terms .. xi
Significance of the Study . xiii

III. RESEARCH METHODOLOGY .. xv
Research Instruments xv
Research Procedures . xvii

IV. PRESENTATION AND ANALYSIS OF DATA. xix

V. CONCLUSIONS AND RECOMMENDATION xxiii

VI. BIBLIOGRAPHY . xxv

VII. APPENDICES .. xxvi
Photos xxvi















v
CHAPTER I
INTRODUCTION
Rationale of the study
There are several uses for lemon and green tea in everyday life. Like, a chef who
accidentally cuts his finger and resorts to Lemon in order to avoid infection, a Chinese-
Filipino family who has too much green tea and if curious onto what else can green tea
benefit them, and four grade-nine students wondering what to do with their Investigatory
Project, and the list goes on. And it is for these reasons that the researchers chose this
topic.
The researchers seek to provide information about the anti-bacterial properties of
Lemon (Citrus Limon) and Green tea (Camelia sinensis). This experiment aims to
identify the several benefits of Lemon and Green tea on the inhabitation of bacteria on
human hands. The researchers seek to provide a highly effective and natural way to
prevent sicknesses of the students in Sacred Heart School- Ateneo de Cebu. This will
create a healthy and safe learning environment for the pupils, so they can concentrate
more on their studies. Students nowadays seem to neglect their hygiene more and more,
and it is very likely that harmful bacteria may have spread between students and may
spread infections and diseases, especially since they spend an average of more than 10
hours in school.
The researchers chose to perform this particular experiment with the goal of up keeping
the student's health and ensuring that they avoid these infections that may cause diseases
such as Diarrhea, the E. Coli bacteria, Campylobacter, Impetigo, and the Flu, and to not


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spread these germs to other students. Though this experiment aims to be qualitative and
focuses on the comparison of Lemon and tea, the researchers aim to find if these are
suitable to infuse with other commercial products.
In response to this problem, the study aims to compare Lemon and Green tea by
means of culturing the two on an agar and hand bacteria plate and as time passes, observe
whether Lemon or Green tea inhibits the bacteria, and which one is most effective. The
researchers would also like to consider other types of inexpensive, natural anti-bacterial
sources that may expand the research.
Theoretical Background
What if there isnt any available commercial product to disinfect ones hands when
you need it? According to the Centers for Disease Control and Prevention, keeping your
hands clean is one of the most important steps one can take to stay healthy and avoid
spreading germs to others. One can pick up germs when surfaces are touched like
elevator buttons or keyboards and then touch ones own eyes, nose or mouth before
washing his or her hands. Some viruses and bacteria can live two hours or longer on
surfaces like doorknobs, Michael Bell (2009) says. When it comes to safe food handling,
neglecting to wash up is the most common into an airport, a school or any public place.
One has to touch things. Also, one has to touch the railing on the stairs, the handle on
bags, the tabletop at the canteen. All of those things can be dirty, but if one washes ones
hands before you touch hiss own face, then you are not going to deliver the germs that
can cause an infection. (Johnson, 2009).
The lemon is both a small evergreen tree native to Asia and the trees oval yellow


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fruit. The fruit is used for culinary and nonculinary purposes throughout the world
primarily for its juice, though the pulp and zest are also used, mainly in cooking and
baking. Lemon juice is about 5% to 6% citric acid, which gives lemons a sour taste, and a
pH of 2 to 3 (Jazba, 2007). This makes lemon juice an inexpensive, readily available acid
for use in educational science experiments. Many lemon-flavored drinks and candies are
available, including lemonade and sherbet lemons. The distinctive sour taste that lemon
juice gives makes it a key ingredient in many dishes.
Green tea is full of anti-oxidants and if you are interested in beautiful skin, you
simply cannot ignore the green tea benefits for healthy skin. This type of tea is a light
green drink that originated from China and derived by boiling green tea leaves in water.
Numerous researchers have discovered the many green tea benefits for skin.
Green tea contains as many as 200 bioactive compounds - most important of these are
chemical compounds called polyphenols, which contain flavonoids. Flavonoids are a vital
class of anti-oxidants, and this is the major reason for the many green tea benefits for
skin.
Green tea also contains Vitamin B and Vitamin C. A generous amount of Vitamin B,
especially B12 is key for skin regeneration. Vitamin C is essential in the production of
collagen, a protein which enhances the firmness of skin and helps your skin repair itself..
(Stromme, 2012)
Hand bacteria consist of the following germs: E. Coli, Shigella, Klebsiella,
Staphylococcus, and the like. According to the Museum of science and industry, Chicago,
"These bacteria can reproduce every 20 minutes. The number of bacteria on your body


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right now is greater than the number of people in the United States. ... We call the
bacteria normally found on your skin resident bacteria. They exist on the skin of
normal, healthy people, and are usually not harmful. Other bacteria are transferred to
your skin in one way or another. These are called transient bacteria." (How clean are
your hands?, n.d.)
Products containing antimicrobial agents that kill, inhibit or reduce the number of
microorganisms on the skin are topical antiseptics (Jackson, 2005). Although normal
flora can display agonistic affects, where one organism forms a symbiotic relationship
with another organism, the flora may also serve as a source of infection for the host.
There are two types of normal flora on the skin: transient and resident flora. Resident
flora can be persistently found on the skin, while transient flora are contracted from the
external environment (Jackson, 2005). The current standards for antiseptic products only
require the elimination of transient microorganisms. (Syverson, 2006)
Based from research and extensive studies, the researchers have found that lemon
(Citrus Lemon) Green tea (Camelia Sinensis) both have anti-bacterial properties.
According to Acevedo, (n.d.) ascorbic acid, found in lemons, has been known to kill
Staphylococcus aureus, a bacteria found in the human mouth. (2011, p.3)
In addition, the fiber of citrus fruit also contains bioactive compounds, such as
polyphenols, the most important being vitamin C (or ascorbic acid), and they
certainly prevent and cure vitamin C deficiency-the cause of scurvy (Aronson, 2001).
Green tea extract is rich in polyphenols such as antioxidants like EGCG
(Epigallocatechin gallate. Based on Dr. Kathleen M. Zelman, EGCG is also known to be


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rich in vitamin C and E, which may include protection against immune system
deficiencies. It is also said from newsmax.com that vitamin E prevents the blood platelets
from clumping, is good for skin care and hair care as the epidermis layer of the skin
absorbs the vitamin E. It can be used for treating sunburn or protecting you from the sun,
scars or even wrinkles.
According to researchers at Purdue University, green tea with lemon improves the
health benefits of the tea. Citrus juice brings out the green tea's antioxidants making the
effect more abundant. Lemon can increase the amount of catechins or antioxidant
compounds your body extracts from green tea by up to six times.
According to ancient Egyptians they believed that lemons was an effective
protection against a variety of poisons, which is also confirmed by recent researches.
Lemons are known for its strong antibacterial, antiviral and immune-boosting powers.
Lemons contain many substances-- notably citric acid, calcium, magnesium, vitamin c,
bioflavonoids, pectin, and limeone--that promote immunity and fight infection. Lemon is
an important medicinal plant of the family Rutaceae. It is cultivated mainly for its
alkaloids, which are having anticancer activities and the antibacterial potential in crude
extracts of different parts (viz., leaves, stem, root and flower) of Lemon against clinically
significant bacterial strains has been reported (Kawaii et al., 2000). In addition,
the fiber of citrus fruit also contains bioactive compounds, such as polyphenols, the
most important being vitamin C (or ascorbic acid), and they certainly prevent and cure
vitamin C deficiency-the cause of scurvy (Aronson, 2001).



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CHAPTER II
STATEMENT OF THE PROBLEM
This study aims to compare the benefits of applying lemon or Green tea extracts to
human hands to inhibit bacterial growth.
Specifically, it seeks to answer the following questions:
Which of lemon or green tea is faster in inhibiting hand bacteria?
How effective are lemon and green tea extracts in inhibiting bacterial growth on
hands?
What are the visible signs of both variables in the bacterial growth?
Hypothesis
If Lemon is applied on hands, then there will be less bacterial growth.
If Green tea is applied on hands, then there will be less bacterial growth.
If Lemon is applied on hands, then there will be less bacterial growth compared to
green tea.
If Green tea is applied on hands, then there will be less bacterial growth compared to
lemon.




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Scope and limitations
The scope of this study is to find out whether Lemon and Green tea is capable of
inhibiting the growth of microorganisms on human hands, and comparing it to one
another. This study is limited only to the amount/number of microorganism colonies
cultured based on physical viewing and not the types of microorganisms. This
Investigatory project is mainly more of the quantitative side of the micro organism and
not much on the qualitative side.
The Investigatory Project was made in Sacred Heart School- Ateneo de Cebu
Biology and Physics laboratory and observed between the months of November 2013 to
December 2013. A different methodology was used (by means of glass slides and
observing it under the microscope) but then failed and a new methodology (by means of
culturing in gelatin plates) was formulated and used.
This study will not include the viewing of the cultured agar under the microscope,
the identification of the microorganisms. This study also did not use specialized agar
made specifically for culturing, and instead, improvised.

Definition of terms
Green Tea. This is also known as Camelia Sinesis. These are leaves that are not
fermented before being dried, least processed, and thus provides the most antioxidants,
which is believed to be responsible for most of the health benefits linked to green tea. It is
also known to be rich in vitamin C and E.


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Citrus Limon or lemon. This is a yellow sitrus fruit that has a sour taste which its juice
is about 5% to 6% citric acid. It is known for its strong antibacterial, antiviral and
immune-boosting powers.
Hand bacteria. This is a singular of bacterium group of microscopic single-celled
organisms that live in enormous numbers. A member of a large group of unicellular
microorganisms, may also cause diseases and infections in usual causes.
Agar Plate. This is a petri dish that contains a growth medium (typically agar plus
nutrients) used to culture microorganisms.
Culturing. Means to maintain microorganisms in conditions suitable for growth. It is to
multiply or increase as by natural reproduction.
Microorganisms. This is a single cell or multicellular organism which is also called
microbe. These are living organisms (such as a bacteria, fungi, viruses) too small to be
seen with naked eye but visible under a microscope.
Agar. This is a gelatinous substance derived from boiling and is also btained from algae
It has been chiefly used as an ingredient in desserts throughout Asia yet is used as a solid
substrate to contain culture media for microbiological work.






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Significance of the study
The study is beneficial to the following: to compare the effectiveness of lemon
and Green tea's antibacterial properties. The researchers aim to provide useful
information to the public by this investigatory project's results. This study will benefit our
society to use the effects of using lemon, tea or lemon and tea combined as a substitute
for antibacterial sanitizers. Through our research we can safely say that these two
substitutes are very effective.
This will also save and conserve our environment. with the help of these two
because they are organic. After using these two substances, we can easily place them into
a patch of soil to make it fertile.
This experiment aims to identify the several benefits of Lemon and Green tea on
the inhabitation of bacteria on human hands. The researchers seek to provide a highly
effective and natural way to prevent sicknesses of the students in SHS-AdC. This will
create a healthy and safe learning environment for the pupils, so they can concentrate
more on their studies. Students nowadays seem to neglect their hygiene more and more,
and it is very likely that harmful bacteria may have spread between students and may
spread infections and diseases, especially since they spend an average of more than 10
hours in school.
In response to this problem, the study aims to compare Lemon and Green tea by
means of culturing the two on an agar and hand bacteria plate and as time passes, observe
whether Lemon or Green tea inhibits the bacteria, and which one is most effective. The
researchers would also like to consider other types of inexpensive, natural anti-bacterial


xiv
sources that may expand the research.
This study will help us learn to be healthy and resourceful in the means that green
tea and lemon is a widely used beverage, which we may reuse again for further agendas
especially on our study.
















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CHAPTER III
RESEARCH METHODOLOGY
METHODOLOGY
Materials
Lemon - 3 pieces
Green tea leaves (Generic, dried) - 10 grams
Gelatin (Generic) - 30 grams
Distilled water - 900 mL
Beef bouillon - 2 pieces
Instruments, Apparatus and Equipment
Petri dishes - 12 pieces
Beakers - 3 pieces
Pot - 2 pieces
Stove - 1 piece
Thermometer - 1 piece
Wooden spoon - 1 piece
Ladle - 1 piece


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Digital weighing scale - 1 piece
Digital microscope - 1 piece
3 volunteers
Set-Ups
Set-Up Independent Variable Dependent Variable
A (Controlled) Controlled
Agar solution
Amount of sunlight
Size of Petri Dish
Growth in Petri Dish after 1
week
B (Lemon) Controlled
Agar solution
Amount of sunlight
Size of Petri Dish
Manipulated
Presence of Lemon
Growth in Petri Dish after 1
week
C (Green Tea) Controlled
Agar solution
Amount of sunlight
Size of Petri Dish
Growth in Petri Dish after 1
week


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Manipulated
Presence of Green
Tea

General Procedure
(Preparation) Using a pot, 800mL of distilled water was boiled with 30 grams of
gelatin and the 2 pieces of the beef bouillon until it was fully dissolved. The agar was set
aside to cool at 38-43 degrees Celsius. When the solution still felt warm but not too hot to
touch, 50ml of the gelatin was then poured to each of the twelve-labeled petri dishes. The
dishes were tilted back and forth gently until the agar coated the entire bottom of the dish.
The dish was then covered to ensure that there would be no contaminations. The lemon
juice from the 3 lemons was extracted and was set-aside in a beaker. Using the boiling set
up, the 10 grams of the generic green tea was placed in 100ml of distilled water. The
extract was set-aside in a beaker.
(Gathering of the bacteria and using of variables) Volunteer 1 then was asked to place
his unwashed fingertips and palm directly on the petri dish labeled A. The same volunteer
then would wash his right hand using 20mL of the lemon extract, then would have his left
hand washed thoroughly in warm water with the same time, and placed his finger tips and
palm on the labeled petri dish B. The volunteer repeats this procedure with his right hand,
using 20mL of the green tea extract, on the petri dish labeled C.
(Making of Duplicates) After having the prepared petri dished A, B and C, 3 more
trials were made with different volunteers using petri dishes (Trial 2) D, E, and F, (Trial


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2) G, H, and I, (Trial 3) J, K, L. For a total of 4 trials, with 3 petri dishes each. (Grand
total of petri dishes: 12)
(Observation, recording of data and disposal) The petri dishes were wrapped
individually with cling wrap, and were left in a cool, dry area, and were left for a period
of 5-7 days. This amount of time would determine the rate of bacteria and at the same
time it's type of colony for it would have different colors and textures at unstable
temperatures and other factors. The researchers then recorded observations. After use, the
specimens were disposed straight in a plastic bag and into the trashcan.













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CHAPTER IV
PRESENTATION AND ANALYSIS OF FINDINGS
The following tables interpret the data that we have gathered over the observation
period. This includes the timeline of its production and as well as the observations
recorded. This experiment was done in the Chemistry Laboratory (Table 1.1) and at a
local home (Table 1.2) Although it was concluded that the petri dishes from Table 1.1
was a failure, that is when Table 1.2 comes in to make up for the mistakes made in the
first trial.
Table 1.1 Descriptions of Set-Ups (Trial 1)
DATE CONTROLLED
SET UP
GREEN TEA
PETRI DISH
LEMON PETRI
DISH
December 11, 2013 Agar plates were made
December 12, 2013
(Day 1)
Green Tea + Hand
bacteria were placed
on the dish.
Lemon + Hand
bacteria were placed
on the dish.
December 13, 2013
(Day 2)
Smell was tolerable.
No visible culture
present.
A sharp, unpleasant
smell was present.
No visible culture
present.
A sharp, unpleasant
smell was present.
No visible culture
present.
December 16, 2013 Smell was tolerable. Smell was tolerable. Smell was tolerable.


xx
(Day 3) No visible culture
present.
Petri Dish turned
brown.
No visible culture
present.

Molds visible
December 17, 2013
(Day 4)
Smell was tolerable.
No visible culture
present.

Smell was tolerable.
No visible culture
present.
Smell was tolerable.
Molds continue to
grow.

If to analyze the results, it would seem that the controlled set up (without lemon
and tea) would be the most effective. The Green tea would come in second, and lemon
the last, the only variable with mold growth. What the researchers did not expect was the
biodegradability of the variables. Lemon, as the researchers know it, is capable of
degrading after a few days. This may have contributed to the molds that have appeared on
the petri dishes.
Table 1.2 Descriptions of Set-Ups (Trial 2)
DATE CONTROLLED
SET UP
GREEN TEA
PETRI DISH
LEMON PETRI
DISH


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January 2, 2014 Agar plates were made
January 6, 2014 Agar plates were put in the Chemistry laboratory
January 8, 2014 No visible growth
present.
No visible growth
present.
No visible growth
present.
January 10, 2014 Molds start to grow Molds start to grow. No visible growth
present.
January 13, 2014 Molds continue to
slowly grow by size
and number
No visible growth
present.
Molds continue to
slowly grow by size
and number
January 17, 2014 Molds continue to
slowly grow by size
and number
Molds continue to
slowly grow by size
and number
Molds continue to
slowly grow by size
and number
January 23, 2014 Molds continue to
slowly grow by size
and number
Molds continue to
slowly grow by size
and number
Molds continue to
slowly grow by size
and number
January 29, 2014 Molds continue to
slowly grow by size
and number
No visible growth
present.
No visible growth
present.
January 30, 2014 Molds continue to
slowly grow by size
No visible growth Molds continue to
slowly grow by size


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and number present. and number

As seen on the table above, it is quite clear that green tea is a more effective hand
sanitizing agent than lemon. Thus, the researchers concluded that green tea has more anti
bacterial properties compared to lemon. If you could observe, the growth of molds were
consistent for both the controlled-set up and the variables. But if you look closely you
could see that the growth of molds for the green tea set up lessens overtime. Thus, the
research team concluded that green tea extracts is a better hand sanitizer when compared
to lemon.












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CHAPTER V
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
The researchers decided to make an experiment about the effectiveness of lemon
and green tea as hand sanitizers, by using gelatin as a medium to culture the hand bacteria
collected from the 3 volunteers. After collecting the data over the span of 23 days, the
petri dishes presented mold growth. The researchers compared each of the variables
number of colonies. It was shown that the petri dishes, which contained the green tea,
exhibited less mold growth. Hence, it is concluded that green tea is more effective than
lemon in inhibiting hand bacteria.
Conclusion
The researchers therefore conclude that both lemon and green tea have qualities to
inhibit bacterial growth. According to the data gathered, Green tea has shown better
results compared to lemon. Therefore when applied to human hands, green tea has shown
to be a better and more effective hand sanitizer than lemon. By comparing the number of
colonies for each set up, Green tea has shown less growth than lemon. Hence, the
hypothesis is accepted and it is proved that the green tea, in fact has anti bacterial
properties.
Based on the findings and conclusions, the researchers recommend the following:
To use the standardized medium for culturing bacteria
To administer proper procedures for the culturing
To use fresh lemons when applied to hands


xxiv
To perform the experiment in a laboratory with the appropriate equipment and
materials to properly document the number of bacterial growth.































xxv
BIBLIOGRAPHY
Book, I. (1984). The World Book encyclopedia. Chicago: World Book.
Campbell, N. A. (2008). Biology (8th ed.). USA: Pearson Education, Inc..

Banaria, C., David, N. (2011-2012). Biological Control: Apergillus Niger VS
Cladisporium Cucumerinum.
Alcovendaz, J., Go, M., Pasco, P., & Reyes, K. (2012-2013). Inhibiting for E. Coli using
natural resources. (Manila, St. Paul College, Pasig).
Bacteria & Hand Washing. Home Science Tools. Retrieved from
http://www.hometrainingtools.com/bacteria-handwashing-newsletter/a/1678/
Human Hands and Fingernails (2010) Retrieved from
http://microbewiki.kenyon.edu/index.php/Human_Hands_and_Fingernails
Wikipedia. (January 20, 2014) Mold. Retrieved January 8, 2012,
from http://en.wikipedia.org/wiki/Mold












xxvi
APPENDICES


(First Set-Ups)


xxvii

(Second Set-Up)


AMDG