the prophylactic property of calabur on selected pathogenic bacteria (preliminaries)

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i DAGUPAN CITY NATIONAL HIGH SCHOOL

Science, Technology and Engineering (STE) Program

School Year 2013 - 2014

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



ii DAGUPAN CITY NATIONAL HIGH SCHOOL



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 the0mm 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.



iii DAGUPAN CITY NATIONAL HIGH SCHOOL



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



iv DAGUPAN CITY NATIONAL HIGH SCHOOL



Republic of the Philippines

Department of Education

Region I

Division of City Schools

DAGUPAN CITY NATIONAL HIGH SCHOOL

Dagupan City

RESEARCH PLAN (IA)



v DAGUPAN CITY NATIONAL HIGH SCHOOL



The Prophylactic Property of Calabur (Muntingia calabura ) onSelected 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



vi DAGUPAN CITY NATIONAL HIGH SCHOOL



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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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



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A

• Gathering of Laboratory Equipment and Collection of PlantMaterial

B• Extraction of Plant Material

C• Collection and Preparation of Culture Media

D• Sterilization of Materials

E

• Subculture of Pathogenic Bacteria and Incorporation in NutrientBroth

F• Antimicrobial Sensitivity Test

G

• Computation of Antimicrobial Indices of the Extract on thePathogenic Bacteria

H• Interpretation of Results

I• 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.



x DAGUPAN CITY NATIONAL HIGH SCHOOL



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



xi DAGUPAN CITY NATIONAL HIGH SCHOOL



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 =



xii DAGUPAN CITY NATIONAL HIGH SCHOOL



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; Todar’s 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)

http://www.stuartxchange.com/Aratiles.html




xiii DAGUPAN CITY NATIONAL HIGH SCHOOL



CONTENT

Title Page i

Abstract ii

Approval Sheet iiiResearch Plan ivContent xiii

CHAPTER 1 - INTRODUCTIONBackground of the Study 1

Statement of the Problem 2

Statement of Hypothesis 2

Significance of the Study 3Scope and Delimitations 3

Definition of Terms 4

CHAPTER 2 - REVIEW OF RELATED LITERATURE

Related Literature 5

Related Studies 8

Related Readings 10Relevance of the Study 12

CHAPTER 3 - METHODOLOGY

Materials Used in the Experiment 13

Procedures 14

CHAPTER 4 - RESULTS AND DISCUSSION 17

CHAPTER 5 - SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary of Findings 19

Conclusion 21Recommendations 21

Bibliography xivPlates xv

Appendices

Antimicrobial Computation xix

Acknowledgment xxv



xiv DAGUPAN CITY NATIONAL HIGH SCHOOL



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; Todar’s 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)






xv DAGUPAN CITY NATIONAL HIGH SCHOOL



PLATES

Preparation of Extract and Subculture of Pathogenic Bacteria

Materials used for the extraction of plantmaterial. Soaking of Calabur leaves in 95%ethanol in an Erlenmeyer flask

covered with parafilm.

Obtained extract is filtered using filter

paper.

Extract is subjected to water bathuntil all of the ethanol has

evaporated.

The obtained pure extract



xvi DAGUPAN CITY NATIONAL HIGH SCHOOL



Paper discs are soaked in the pure extractand placed in hot air oven at 00 C for 10

minutes to dry.

Agar is prepared to be placed in

petri dishes.

Petri dishes were labeled for the different bacteria.

Subculture of bacteria is inoculated into the

nutrient broth and is incubated overnight.

ot air oven at 00 C is used to drythe paper discs with pure extract for

10 minutes.



xvii DAGUPAN CITY NATIONAL HIGH SCHOOL



Standard antibiotic is prepared for each

bacteria. Vancomycin 30mcg andOxacillin 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 beaseptically swab on the agar plates usingsterile cotton swabs. The extract paper

discs and positive control will be

incorporated on the agar. The plates will

be incubated for 24 hours prior tointer retation of results.



xviii DAGUPAN CITY NATIONAL HIGH SCHOOL



Antimicrobial Susceptibility Test Results



xix DAGUPAN CITY NATIONAL HIGH SCHOOL





xx DAGUPAN CITY NATIONAL HIGH SCHOOL





xxi DAGUPAN CITY NATIONAL HIGH SCHOOL





xxii DAGUPAN CITY NATIONAL HIGH SCHOOL



APPENDICES

Computation of Data

Staphylococcus haemolyticus

Antimicrobial Index of Calabur leaves extract Zone of Inhibition: 11.7 mm

Against Staphylococcus haemolyticus


=

= 0.93

Antimicrobial Index of Vancomycin 30mcg Zone of Inhibition: 12.6 mm



=

= 1.00

Antimicrobial Index of Oxacillin 1mcg Zone of Inhibition: 0 mm



=

= 0



xxiii DAGUPAN CITY NATIONAL HIGH SCHOOL



Staphylococcus aureus

Antimicrobial Index of Calabur leaves extract Zone of Inhibition: 10.9 mm

Against Staphylococcus aureus


=

= 1.04

Antimicrobial Index of Vancomycin 30mcg Zone of Inhibition: 10.5 mm



=

= 1.00

Antimicrobial Index of Oxacillin 1mcg Zone of Inhibition: 0 mm



=

= 0



xxiv DAGUPAN CITY NATIONAL HIGH SCHOOL



Escheri chia coli

Antimicrobial Index of Calabur leaves extract Zone of Inhibition: 0 mm

Against Escherichia coli


=

= 0

Antimicrobial Index of Chloramphenicol 30mcg Zone of Inhibition: 10 mm

Against Escherichia coli


=

= 1.00



xxv DAGUPAN CITY NATIONAL HIGH SCHOOL

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 UPang-

PHINMA, 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.

the prophylactic property of calabur on selected pathogenic bacteria (preliminaries)

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