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The Prophylactic Property of Calabur (Muntingia calabura) Extract on

Selected Pathogenic Bacteria

A Science Investigatory Project


Presented to
The Faculty of Science and Technology Department
Dagupan City National High School
Fourth Year

Proponent:
April Antonette F. Velasco

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

ABSTRACT

Title: The Prophylactic Property of Calabur (Muntingia calabura) Extract on


Selected Pathogenic Bacteria
Proponent: April Antonette F. Velasco
School: Science, Technology and Engineering (STE) Program Dagupan City National
High School

The study entitled, The Prophylactic Property of Calabur (Muntingia calabura)


Extract on Selected Pathogenic Bacteria was conducted to determine if Calabur exhibits
a prophylactic property on Staphylococcus haemolyticus, Staphylococcus aureus and
Escherichia coli. Specifically, the study aimed to determine the effect of Calabur on the
selected pathogenic bacteria based on the zone of inhibition (mm).
Specifically, the study sought to determine the prophylactic property of Calabur
leaves extract on the selected pathogenic bacteria based on their zones of inhibition. It
also sought to determine whether there is a significant difference between the effect of
Calabur leaves extract and the standard antibiotics used.
One hundred grams of Calabur leaves were gathered, washed with water and
soaked in 300ml ethyl alcohol for 24 hours. Then, the plant material was separated with
the extract. The obtained extracts were subjected to evaporation until the ethyl alcohol
has completely evaporated. The obtained pure extract undergone Antimicrobial
Sensitivity Testing.
Results revealed that Staphylococcus haemolyticus is moderately susceptible to
Calabur extract; susceptible to Vancomycin; and resistant to Oxacillin based on the
11.7mm, 12.6mm, and 0mm zones of inhibition respectively. Staphylococcus aureus is
intermediate to Calabur extract; intermediate to Vancomycin; and resistant to Oxacillin
based on the 10.9mm, 10.5mm and 0mm zones of inhibition respectively. Escherichia
coli is resistant to Calabur extract; and intermediate to Chloramphenicol based on the
0mm and 10.0mm zones of inhibition respectively.
The researcher had concluded that Calabur leaves extract exhibits a prophylactic
property on Staphylococcus haemolyticus and Staphylococcus aureus. It was also found
out that there is no significant difference between the effect of Calabur leaves compared
with the effect of standard antibiotics used.
DAGUPAN CITY NATIONAL HIGH SCHOOL
Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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APPROVAL SHEET
The investigatory project entitled The Prophylactic Property of Calabur
(Muntingia calabura) Extract on Selected Pathogenic Bacteria prepared by April
Antonette F. Velasco in partial fulfillment of the requirements, has been examined and
approved by the Scientific Review Committee (SRC) on Oral Examination.

Adviser:

______________________

Critic Reader:

______________________

SRC:

______________________

______________________

Chairperson:

______________________

Accepted in partial fulfillment of the requirements of Research II.

Cherry A. Cayabyab

______________________

Head Teacher VI, Science

Erly G. Datario

______________________

Principal IV

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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Republic of the Philippines


Department of Education
Region I
Division of City Schools
DAGUPAN CITY NATIONAL HIGH SCHOOL
Dagupan City

RESEARCH PLAN (IA)

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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The Prophylactic Property of Calabur (Muntingia calabura) on


Selected Pathogenic Bacteria

A Research Plan Presented to the


Scientific Research Committee of the Science, Technology and Engineering Program
Dagupan City National High School

By:
April Antonette F. Velasco

June 24, 2013

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

Statement of the Problem


This study aims to determine the prophylactic property of Calabur extract on
selected pathogenic bacteria Staphylococcus haemolyticus, Staphylococcus aureus
and Escherichia coli. Specifically, it will seek to answer the following questions:
a) Does Calabur (Muntingia calabura) exhibit a prophylactic property based on
the zone of inhibition of the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli)?
b) Is there a significant difference between the effect of Calabur (Muntingia
calabura) leaves extract on the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli) compared to the
effect of the standard?
c) Among the selected pathogenic bacteria, on which of the following is Calabur
extract most effective based on their zones of inhibition?

Hypothesis
The hypothesis will be tested at 0.1 level of significance.
Ho Calabur (Muntingi calabura) does not exhibit a prophylactic property based
on the zone of inhibition of the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli).
Ho There is no significant difference between the effect of Calabur (Muntingia
calabura) leaves extract on the selected pathogenic bacteria (Staphylococcus
haemolyticus, Staphylococcus aureus and Escherichia coli) compared to the
effect of the standard.
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Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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I. Materials and Equipment


Plant Collection and Extraction

Calabur leaves

beaker

graduated cylinder

funnel

weighing scale

300 ml 95% ethanol

filter paper

Erlenmeyer flask

parafilm

thin white cloth

Preparation of Culture Media and Sensitivity Test

Staphylococcus aureus

Escherichia coli

Staphylococcus haemolyticus

Mueller Hinton Agar plates and Nutrient Broth (NB)

forceps

cotton swabs

inoculating loop

standard (commercial antibiotic)

petri dish

alcohol lamp
DAGUPAN CITY NATIONAL HIGH SCHOOL
Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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Gathering of Laboratory Equipment and Collection of Plant


Material

Extraction of Plant Material

Collection and Preparation of Culture Media

Sterilization of Materials

Subculture of Pathogenic Bacteria and Incorporation in Nutrient


Broth

Antimicrobial Sensitivity Test

Computation of Antimicrobial Indices of the Extract on the


Pathogenic Bacteria

Interpretation of Results

Summary of Findings and Conclusion

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II. Extraction of Plant Material


The plant extract will be obtained by maceration method. The Calabur leaves
will be cut into smaller portions and will be placed inside an Erlenmeyer flask.
Ninety-five percent ethanol will be measured to 300 ml using the graduated cylinder
and is to be placed in the Erlenmeyer flask containing the Calabur leaves. The flask
will be covered with parafilm to prevent the evaporation of ethanol. The botanical
material will be soaked in ethanol for 24 hours at room temperature. The plant extract
will be obtained by separating the botanical material using a clean white cloth. Then,
the mixture will be filtered using the filter paper. The obtained extracts will then be
subjected to evaporation of ethanol to obtain the pure extract.

III. Collection and Preparation of Culture Media


The test organisms: Staphylococcus haemolyticus, Staphylococcus aureus and
Escherichia coli will be obtained and cultured at the medical laboratory science of
University of Pangasinan PHINMA.
First, the inoculating loop and the opening of the screw cap tube containing
the nutrient broth will flame-sterilized using alcohol lamp to avoid contamination and
spread of fungus. The bacteria in nutrient broth will be inoculated across the surface
of the agar with the use of sterile inoculating loop. After this, the bacteria in the plate
will incubated for about 24 hours at 38 degree Celsius. The process will be done on
each of the selected pathogenic bacteria.

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Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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IV. Sterilization of Materials


The treatments, forceps, paper discs, and cotton swabs will be wrapped in
coupon bond and will be sterilized using an autoclave machine; the Mueller Hinton
Agar plates, Nutrient Broth (NB) will be refrigerated to avoid contamination and
growth of other microorganisms.

V. Subculture of Pathogenic Bacteria


Inoculating loop, alcohol lamp and Nutrient Broth (NB) will be prepared in
subculturing the test organisms. The inoculating loop and the opening of the screw
scrap tube containing the Nutrient Broth (NB) will be flame sterilized by the use of
alcohol lamp. The bacteria on the plate after incubation will be incorporated on the
Nutrient Broth (NB) with the aid of the inoculating loop. Right after this, subculture
of bacteria will be incubated prior to the sensitivity test.

VI. Antimicrobial Sensitivity Test


Paper disc method will be followed in conducting the sensitivity test. The
subculture of bacteria will be applied using sterile cotton swabs on the agar plates.
The plates will be incubated overnight and will be observed thereafter.
After incubation, the absence or presence of circular inhibition zones will be
used as bases in determining the activity of the sample being tested. The length of
zones of inhibition will be recorded and interpreted using The Interpretative
Guidelines for Non-fastidious Aerobic bacteria Zone Diameter Interpretative

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

Standards published in the book Introduction to Diagnostic Microbiology by Maria


Damnessa Delost. The results were given descriptive equivalents of Susceptible,
Moderately Susceptible, Intermediate and Resistant.

VII. ANTIMICROBIAL INDEX OF THE EXTRACT


For the antimicrobial index of the extract, the following formula will be used:
Antimicrobial Index =

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Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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Bibliography

Madrid, Domingo A., A Dictionary of Philippine Plant Names; pg. 58

Delost, Maria D., Introduction to Diagnostic Microbiology; pg. 72-73

Rummel, Dietmar J., Useful Plants of the Philippines Vol. 1

Philippine Medicinal Plants Aratiles/Calabur;


www.stuartxchange.com/Aratiles.html

Staphylococcus aureus, www. wikipedia.com

Zone of Inhibition test: Antimicrobial activity screening;


www.antimicrobialtestlaboratories.com

Staphylococcus aureus, Escherichia coli; Todars Online Textbook of


Bacteriology; 2011

Staphylococcus haemolyticus, www.microbewiki.kenyon.edu (July 15, 2013)

Staph infections: Treatments and drugs, www.mayoclinic.com (July 3, 2013)

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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CONTENT

Title Page
Abstract
Approval Sheet
Research Plan
Content

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ii
iii
iv
xiii

CHAPTER 1 - INTRODUCTION
Background of the Study
Statement of the Problem
Statement of Hypothesis
Significance of the Study
Scope and Delimitations
Definition of Terms

1
2
2
3
3
4

CHAPTER 2 - REVIEW OF RELATED LITERATURE


Related Literature
Related Studies
Related Readings
Relevance of the Study

5
8
10
12

CHAPTER 3 - METHODOLOGY
Materials Used in the Experiment
Procedures

13
14

CHAPTER 4 - RESULTS AND DISCUSSION

17

CHAPTER 5 - SUMMARY, CONCLUSION AND RECOMMENDATIONS


Summary of Findings
Conclusion
Recommendations

19
21
21

Bibliography
Plates
Appendices
Antimicrobial Computation
Acknowledgment

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Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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BIBLIOGRAPHY

Books

Madrid, Domingo A., A Dictionary of Philippine Plant Names; pg. 58

Rummel, Dietmar J., Useful Plants of the Philippines Vol. 1

Internet Sources

Philippine Medicinal Plants Aratiles/Calabur;


www.stuartxchange.com/Aratiles.html

Republic Act No. 9502 Universally Accessible Cheaper and Quality Medicines
Act of 2008; www.lawphil.net

Staphylococcus aureus, www. wikipedia.com

Zone of Inhibition test: Antimicrobial activity screening;


www.antimicrobialtestlaboratories.com

Staphylococcus aureus, Escherichia coli; Todars Online Textbook of


Bacteriology; 2011

Staphylococcus haemolyticus, www.microbewiki.kenyon.edu (July 15, 2013)

Staph infections: Treatments and drugs, www.mayoclinic.com (July 3, 2013)

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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PLATES
Preparation of Extract and Subculture of Pathogenic Bacteria

Materials used for the extraction of plant


material.

Obtained extract is filtered using filter


paper.

Soaking of Calabur leaves in 95%


ethanol in an Erlenmeyer flask
covered with parafilm.

Extract is subjected to water bath


until all of the ethanol has
evaporated.

The obtained pure extract


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Paper discs are soaked in the pure extract


and placed in hot air oven at 00 C for 10
minutes to dry.

Petri dishes were labeled for the different


bacteria.

Agar is prepared to be placed in


petri dishes.

ot air oven at 00 C is used to dry


the paper discs with pure extract for
10 minutes.

Subculture of bacteria is inoculated into the


nutrient broth and is incubated overnight.
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Standard antibiotic is prepared for each


bacteria. Vancomycin 30mcg and
Oxacillin 1mcg for Staph. aureus and
Staph. haemolyticus; Chloramphenicol
30mcg for E. coli.

The nutrient broth containing


the bacteria after incubation.

The bacteria in the nutrient broth will be


aseptically swab on the agar plates using
sterile cotton swabs. The extract paper
discs and positive control will be
incorporated on the agar. The plates will
be incubated for 24 hours prior to
interpretation of results.

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Antimicrobial Susceptibility Test Results

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School Year 2013 - 2014

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School Year 2013 - 2014

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APPENDICES

Computation of Data
Staphylococcus haemolyticus
Antimicrobial Index of Calabur leaves extract

Zone of Inhibition: 11.7 mm

Against Staphylococcus haemolyticus

Antimicrobial Index =
=
= 0.93
Antimicrobial Index of Vancomycin 30mcg

Zone of Inhibition: 12.6 mm

Against Staphylococcus haemolyticus

Antimicrobial Index =
=
= 1.00

Antimicrobial Index of Oxacillin 1mcg

Zone of Inhibition: 0 mm

Against Staphylococcus haemolyticus

Antimicrobial Index =
=
=0

DAGUPAN CITY NATIONAL HIGH SCHOOL


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School Year 2013 - 2014

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Staphylococcus aureus

Antimicrobial Index of Calabur leaves extract

Zone of Inhibition: 10.9 mm

Against Staphylococcus aureus

Antimicrobial Index =
=
= 1.04
Antimicrobial Index of Vancomycin 30mcg

Zone of Inhibition: 10.5 mm

Against Staphylococcus aureus

Antimicrobial Index =
=
= 1.00

Antimicrobial Index of Oxacillin 1mcg

Zone of Inhibition: 0 mm

Against Staphylococcus aureus

Antimicrobial Index =
=
=0

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
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Escherichia coli

Antimicrobial Index of Calabur leaves extract

Zone of Inhibition: 0 mm

Against Escherichia coli

Antimicrobial Index =
=
=0
Antimicrobial Index of Chloramphenicol 30mcg

Zone of Inhibition: 10 mm

Against Escherichia coli

Antimicrobial Index =
=
= 1.00

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

xxiv

ACKNOWLEDGMENT

I would like to extend my profound gratitude and appreciation to the following


who extended their generous support and contribution allowing the completion of this
study:
Mr. and Mrs. Pilar F. Velasco, my parents, for all their unwavering concern,
financial and moral support, and understanding;
Mrs. Monica E. Cayabyab, my mentor, who guided me throughout the research
process, unselfishly imparted her ideas, time and knowledge and for all her valuable
suggestions, comments and contributions in every aspect of the study which inspired me
to make this study as successful as it can be;
Mrs. Cherry A. Cayabyab, science department head of DCNHS, for letting me
borrow laboratory materials for the extraction process of my project;
Dr. Hannah A. Balanon, head of the medical technology department of UPangPHINMA, for allowing me to use their laboratory and facilities in the conduct of my
experiment.
Mr. David Contad, for his assistance in sub-culturing the bacteria and the
experiment in general;
The Almighty God, for the wisdom, strength, and vision he has given me to be
able to create this masterpiece;
And for those whose names were not mentioned who had helped me in a way or
another in making this science investigatory project possible.

DAGUPAN CITY NATIONAL HIGH SCHOOL


Science, Technology and Engineering (STE) Program
School Year 2013 - 2014

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