bagong kamias

Kamias (Averrhoa bilimbi) LEAVES AND

FRUIT EXTRACT:

ITS ANTIBACTERIAL EFFECT ON

Escherichia coli,

Staphylococcus aureus AND Salmonella

enteritidis

A Science Investigatory Project as

an Entry for the 2008 School

Level Science Fair

Ellaine M. Andales

Angelica Mae J. Flores

Hannah C. Mendoza

Research Proponents

Mrs. Noreen T. Catis

Project Adviser

REGIONAL SCIENCE HIGH SCHOOL

Malasiga, San Roque, Zamboanga City

http://rds.yahoo.com/_ylt=A9G_bHInF.ZISGUBk2ujzbkF/SIG=128sfc0tj/EXP=1223125159/**http%3A/toptropicals.com/pics/garden/2004/1/1883s.jpg

October, 2008

ABSTRACT

This research study aims to prevent diseases caused by

Escherichia coli, Staphylococcus aureus and Salmonella enteritidis. The

study makes use of kamias (Averrhoa bilimbi) leaves and fruit extract

as an antibacterial agent that is more or equally effective than the

commercialized one and at the same time, available in our

environment. Ten Replicates on ten Petri dishes were prepared for

each bacteria. Pure cultures of the three bacteria were spread on the

replicates. After extracting the needed substances from the kamias

leaves and fruit extract, the researchers were able to begin the

experimentation. Six treatments were made in the set-up-Treatment A

as 100% water served as the negative control; Treatment B as 100%

Kamias leaves extract; Treatment C as 50% Distilled water and 50%

Kamias leaves extract; Treatment D as 100% antibiotics was used as

the positive control because of its known antibacterial properties;

Treatment E as 100% Kamas fruit extract and Treatment F as 50%

Distilled water and 50% Kamias fruit extract. The disk-diffusion test

was admonished to the replicates and the data were recorded after 24

hours of incubation at 35.5 degrees Centigrade of all the treated

dishes. The data gathered were subjected to Analysis of Variance

(ANOVA) and Scheffe’s Test. Results of statistical analyses show that

Treatment B and Treatment E is equally effective with Treatment D for

Escherichia coli, Staphylococcus aureus and Salmonella enteritidis.

This study led to the conclusion that Kamias leaves and fruit extract is

an effective antibacterial against Escherichia coli, Staphylococcus

aureus and Salmonella enteritidis.

ACKNOWLEDGMENT

The Researchers would like to acknowledge and express their

deepest sincerity to the following that had contributed a lot to the

success of this Research Study.

Their beloved and supportive parents who are always ready to

support them financially and morally for the completion of this

study.

R.S.H.S. principal, Mrs. Melanie A. Minez, their class adviser, Mrs.

Collin Ceneciro and the faculty members for their moral support

and consideration to the Researchers’ hectic schedule.

The Department of Science and Technology for providing them

vital information and the materials needed for the actual

experimentation.

Mrs. Jovita Amparo and Ms. Arlene S. Herbieto and the personnel

of the Department of Science and Technology for sharing their

knowledge concerning our research study and for guiding the

researchers to conduct their actual experimentation.

Ms. Felsa Fae C. Mendoza for helping the group to gather more

information about the study.

The IV-Zara class for their moral support in accomplishing this

research proposal.

Their amiable Research Adviser, Mrs. Noreen T. Catis for her

guidance, motivation and for the wisdom he had imparted to us.

And above all, to our Almighty God who always provides us with

knowledge and understanding, strength, guidance, protection,

and inspiration as well as sending the Holy Spirit, which granted

the success of this research study.

TABLE OF CONTENTS

Title Page

Abstract

Acknowledgement

Table of Contents

I. Introduction

A. Background of the Study……………………………………………..

……………..……..…...….1

B. Statement of the

Problem…………………………………………………….…….........…....…

2

C. Significance of the

Study……………………………………………………………………….…..2

D. Scope and Limitations…………………………………………….

…………...3

II. Review of Related Literature…………………………………...

……………….....4-13

III.

Methodology……………………………………………………………………...14-

18

IV. Results and Discussion……………………………………….

…………………..19-29

V. Summary and Conclusion………………………………………………….

….......30-33

VI.

Recommendation……………………………………………………………….....34

Appendices…………………………………………………………………........

35-36

A. Definition of

terms…………………………………………………...37-38

B. Pictures and Illustrations………………………………………...

…..39-40

INTRODUCTION

A. BACKGROUND OF THE STUDY

Disease is an abnormal condition of an organism that impairs

bodily functions and can be deadly. In human beings, “disease" is often

used to refer to any condition that causes extreme pain, dysfunction,

distress, social problems, and death to the person afflicted, or similar

problems for those in contact with the person. Throughout the years

health is one of the main concerns of the country.

In some parts of the country, kamias tree parts are used as

treatment for diseases and for other purposes such as swellings of

mumps and rheumatism, and on skin eruptions, as well as seasoning

for sweets and pickling. Kamias fruit is use to remove stains from

clothing and for washing hands.

Antibacterial agents are big help in the society by preventing the

growth of bacteria. Since commercialize antibacterial agents are

costly; people tend to find alternative ways of preventing these

diseases by utilizing natural and effective, yet cheaper antibacterial

agent.

E. coli (Escherichia coli), gram-negative bacteria, normally

inhabit the intestine of humans and animals, which commonly cause

diarrhea and urinary tract infection.

S. aureus (Staphylococcus aureus), gram- positive bacteria,

frequently living in a healthy person’s nose and skin, also present in

raw food and in certain food with high osmotic pressure that commonly

cause food poisoning and other skin infection.

http://en.wikipedia.org/wiki/Death

http://en.wikipedia.org/wiki/Social_problems

http://en.wikipedia.org/wiki/Distress

http://en.wikipedia.org/wiki/Dysfunction

http://en.wikipedia.org/wiki/Pain

1

S. enteritidis (Salmonella enteritidis), gram-negative

enterobacteria that causes typhoid fever, paratyphoid fever, and food

borne illness.

Thus, by the used of common plant parts such as Averrhoa

bilimbi (kamias) Leaves and Fruit Extract as an antibacterial agent

against E. coli, S. aureus and S. enteritidis, will help in lessening the

rampant spreading of diseases in the society.

B. STATEMENT OF THE PROBLEM

The main objective of this study is to determine the potential of

Averrhoa bilimbi (kamias) leaves and fruit extract as an antibacterial

agent against Escherichia coli, Staphylococcus aureus and Salmonella

enteritidis. Specifically, it aims to answer the following questions:

1. Which among the different concentrations of the kamias leaves

and fruit extract is the most effective antibacterial agent against

E. coli, S. aureus and S. enteritidis?

2. Which among the three bacteria does the kamias leaves and fruit

extract prove to be the most effective?

3. Which among the treatments of the three bacteria has the

greatest significant difference of the mean zones of inhibition?

C. SIGNIFICANCE OF THE STUDY

http://en.wikipedia.org/wiki/Foodborne_illness

http://en.wikipedia.org/wiki/Foodborne_illness

http://en.wikipedia.org/wiki/Paratyphoid_fever

http://en.wikipedia.org/wiki/Typhoid_fever

http://en.wikipedia.org/wiki/Enterobacteriaceae

http://en.wikipedia.org/wiki/Gram-negative

Health is one of the main concerns of the country nowadays.Food

poisoning and diseases are common to all which is caused by these

pathogenic bacteria. Bacteria can be

2

found anywhere, in sponges, raw foods, soil, bathrooms and

laboratories, that’s why diseases caused by bacteria are common in

the country. Since commercialize antibacterial agents are synthetic,

costly and can post health hazards, effective and cheap antibacterial

agent can be utilize which derives from environmental friendly

materials and can easily be found in the community.

This study that uses kamias leaves and fruit focuses on the

prevention of these bacteria, namely Escherichia coli, Staphylococcus

aureus and Salmonella enteritidis. The discovery of the potential of

kamias leaves and fruit as a solution as shown by its effect on the

three bacteria will be a breakthrough and will contribute additional

knowledge in the field of medicine or even in microbiology.

This research study will give way for an immediate solution and

response to the problems produce by these harmful bacteria.

Indirectly, this study will provide great knowledge to some researchers

or even companies that deal in microbiological studies to focus on the

effectiveness of this plant as an effective antibacterial agent against

pathogenic bacteria.

D. SCOPE AND LIMITATIONS

This study focused on the effectiveness of the kamias leaves and

fruit extract as an antibacterial agent in terms of its inhibitory effect on

E. coli, S. aureus and S. enteritidis. The researchers conducted the

study including all laboratory works and research work for one month.

They use different techniques in conducting this research, which deals

with microbiology.

This study would be more comprehensive and meaningful if it

covers other pathogenic bacteria, such as Lactobacillus and Bacillus

subtilis, which are some of the common laboratory bacteria.

3

REVIEW OF RELATED LITERATURE

KAMIAS TREE

The bilimbi, Averrhoa bilimbi, L., (Oxalidaceae), is closely allied

to the carambola but quite different in appearance, manner of fruiting,

flavor and uses. The only strictly English names are "cucumber tree"

and "tree sorrel", bestowed by the British in colonial times. "Bilimbi" is

the common name in India and has become widely used.

Bilimbis are all much the same wherever they are grown, but P.J.

Wester reported that a form with sweet fruits had been discovered in

the Philippines. The bilimbi is a tropical species, more sensitive to cold

than the carambola, especially when very young. In Florida, it needs

protection from cold and wind. Ideally, rainfall should be rather evenly

distributed throughout most of the year but there should be a 2- to 3-

month dry season. The bilimbi is not found in the wettest zones of

Malaya. The tree makes slow growth in shady or semi-shady situations.

It should be in full sun. While the bilimbi does best in rich, moist, but

http://en.wikipedia.org/wiki/Lactobacillus

well-drained soil, it grows and fruits quite well on sand or limestone

(Morton, 1987).

Some of the folkloric uses of kamias are in skin diseases,

especially with pruritus, reduce the leaves to a paste and apply

tolerably warm to areas of affected skin. It is used as a post-partum

and rectal inflammation while in infusion of leaves it is used in mumps,

acne, and localized rheumatic complaints. Paste of leaves applied to

affected areas. Warm paste of leaves also used for pruritus. In Fever,

Fruit can be a cooling drink and it can be also used for a variety of

maladies: beriberi, cough, prevention of scurvy. The Infusion of leaves

also drank as a protective tonic after childbirth.

In Java, the fruits combined with pepper are eaten to cause

sweating when people

5

are feeling "under the weather". A paste of pickled bilimbis is smeared

all over the body to hasten recovery after a fever. The fruit conserve is

administered as a treatment for coughs, beri-beri and biliousness. A

sirup prepared from the fruit is taken as a cure for fever and

inflammation and to stop rectal bleeding and alleviate internal

hemorrhoids. Fruit used to remove stains from clothing and for

washing hands.

Very acid bilimbis are employed to clean the blade of a kris

(dagger), and they serve as mordant in the preparation of an orange

dye for silk fabrics. Bilimbi juice, because of its oxalic acid content, is

useful for bleaching stains from the hands and rust from white cloth,

and also tarnish from brass.

The bilimbi is generally regarded as too acid for eating raw.

Mainly, the bilimbi is used in place of mango to make chutney, and it is

much preserved. The flowers are sometimes preserved with sugar.

Very acid bilimbis are employed to clean the blade of a kris (dagger),

and they serve as mordant in the preparation of an orange dye for silk

fabrics. Bilimbi juice, because of its oxalic acid content, is useful for

bleaching stains from the hands and rust from white cloth, and also

tarnish from brass

(http://www.hort.purdue.edu/newcrop/morton/bilimbi.html)

Bacteria

Bacteria are small and simple in structure when compared with

eukaryotes, yet they often have characteristics shapes and sizes.

Although they have a plasma membrane which is required by all living

cells, bacteria generally lack extensive complex, internal membrane

systems. Some bacteria form resistant endospores to survive harsh

environmental conditions in a dormant stale. Bacterial species may

differ in their patterns of flagella distribution (Harley, Klein & Prescott,

2005).

6

Escherichia coli

E. coli Infection, potentially fatal form of food poisoning caused

by certain strains of the bacterium Escherichia coli. Some 5 million E.

coli bacteria normally inhabit the human and animal intestinal tract,

and are vital to processing vitamins in the diet. However, a number of

strains are pathogenic, and cause gastroenteritis. Some strains, known

as entero-pathogenic strains, are associated with undercooked meat,

http://www.hort.purdue.edu/newcrop/morton/bilimbi.html

and are a common cause of diarrhea in infants, but rarely produce

gastroenteritis in adults. Other “entero-toxicogenic” strains are the

main cause of “travelers' diarrhea”.

A relatively large number of organisms (100 million or more) are

normally required to cause such infections, which are generally

associated with food and water contaminated by feces. Entero-invasive

strains of the bacterium invade cells of the intestines, causing

dysentery, with bloody diarrhea. These are highly virulent strains, and

ingestion of just a few organisms may cause infection. Outbreaks of

such infection have been associated with undercooked hamburgers

and unpasteurized milk. The entero-hemorrhagic strains are also highly

virulent, causing both bloody diarrhea and possibly fatal systemic

infection.

Ingestion of as few as 10 organisms may cause intestinal

hemorrhaging and possible kidney failure. The fatality rate from the

infection is 50 per cent in children and the elderly. The main source of

infection is undercooked beef, which has been contaminated, often in

abattoirs, with feces containing the bacterium. Infection through

nursing of victims has also occurred. Once infected, people in confined

areas can pass on the infection (Microsoft Encarta Premium Suite

2005, 1993-1994).

Gram-negative bacteria such as E. coli, enter the blood from a

focus of infection in the body. As you may recall, the cell walls of many

gram-negative bacteria contain endotoxins that are released upon the

lysis of the cell. It is the endotoxin that actually

7

causes the symptoms. Once released, the endotoxin damages blood

vessels, this damage causes the low blood pressure and subsequent

shock (Tortora, Funke, & Case, 1992).

Escherichia coli are now recognized as an important food-borne

disease organism. This bacterium circulates in the resident population,

typically without causing symptoms due to the immunity afforded by

previous exposure. Because many of these bacteria are needed to

initiate infection, contaminated food and water are the major means by

which bacteria are spread (Harley, Klein & Prescott, 2005).

According to Dr. Vicente Iturriaga, a medical specialist at Jose

Locsin Memorial Provincial Hospital in Silay City said “Tuesday E. Coli

bacteria is predominant in meat but could be present in seawaters due

to some form of wastes and pollutants thrown into the bodies of

water.”

The bacteria could be present not only seawaters could prove

harmful but also everyone's favorites, the oysters locally known as

“talaba” and hamburgers that are not well cooked.

An E. coli attack causes dehydration, severe vomiting and other

forms of gastro-intestinal diseases. But Iturriaga, however, said that

Ecolab, though a highly poisonous bacteria, could be easily killed by

any antibiotic.

"Ecolab is treatable. But if one will ignore it, it becomes

dangerous," said Iturriaga. Health Secretary Manuel Dayrit cautioned

people against buying noodles or pasta dishes sold by ambulant

vendors. He said that such foods are prone to bacterial contamination.

Dayrit said spaghetti and "pansit" are included among those foods that

could get spoiled and could be contaminated with E. Coli bacteria

(http://www.sunstar.com.ph/static/bac/2005/04/27/news/public.warned.

v..deadly.bacteria.html, 2005, April 7).

8

There are other diseases that are caused by E. coli bacteria.

These are Urinary tract infection, peritonitis, pneumonia, septicemia,

neonatal (in new born), meningitis and many more (Tortora, Funke,

and Case, 1992).

Staphylococcus aureus

Staphylococcus aureus, gram-positive bacteria, frequently living

in a healthy person’s nose and skin, also present in raw food and in

certain food with high osmotic pressure that commonly cause food

poisoning and other skin infection. The most important Staphylococcus

species is Staphylococcus aureus, named for its yellow-pigmented

colonies (aureus means golden). They grow comparatively well under

conditions of high osmotic pressure and low moisture, which partially

explains why they can grow and survive in nasal secretions (many of

us carry the bacteria in our noses and skin). This ability also explains

how S. aureus can grow in certain food with high osmotic pressure

(such as ham and other cured meat) or in low-moisture food that tend

to inhibit the growth of other organism.

S. aureus produces many toxins that contribute to the

bacterium’s pathogenicity by increasing its ability to invade the body

or damage tissues. The infection of surgical wounds by S. aureus is a

common problem in hospitals. The bacterium’s ability to develop

resistance quickly to such antibiotics as penicillin contributes to its

danger in hospitals.

S. aureus is the agent of toxic shock syndrome, a severe

infection causing high fever and vomiting and some times death. S.

aureus also produces an enterotoxin that causes vomiting and nausea

when ingested, one of the most common causes of food poisoning. S.

aureus can also cause serious infections such as osteomyelitis,

septicemia

9

and acute bacterial endocarditis—inflammation of the lining of

the heart. These bacteria can also cause other urinary and respiratory

tract infections (Tortora, Funke, & Case, 1992).

Staphylococcus aureus is found on the skin and in the nostrils of

many healthy individuals. These bacteria often give rise to minor

superficial diseases, for example, the formation of pustules or boils in

hair follicles. Much more rarely Staphylococcus aureus can give rise to

more serious infections; these normally occur when the resistance of a

tissue or the host is reduced. Staphylococcus aureus infections are

characterized by the presence of pus and formation of abscesses. This

form of staphylococcus is responsible for skin pustules, boils and

carbuncles, impetigo, infections of wounds and burns, breast

abscesses, whitlow, osteomyelitis, bronchopneumonia, septicemia,

acute endocarditis, food poisoning, and scalded skin syndrome (Lewis,

1993-2004).

Most Staphylococcus aureus strains staphylococcal enteritis

related to the synthesis of extra cellular toxins. These are heat-

resistant proteins, and heating will not usually render the food safe.

The effects of the toxins are quickly felt, with disease symptoms

occurring within 2 to 6 hours. The main reservoir of S. aureus is the

human nasal cavity. Frequently S. aureus is transmitted to a person’s

hands and then is introduced into food during preparation. Growth and

enterotoxin production usually occur when contaminated foods are

held at room temperature for several hours Escherichia coli are now

recognized as an important food-borne disease organism. This

bacterium circulates in the resident population, typically without

causing symptoms due to the immunity afforded by previous exposure.

Because many of these bacteria are needed to initiate infection,

contaminated food and water are the major means by which bacteria

are spread (Harley, Klein & Prescott, 2005).

10

Salmonella enteritidis

S. enterica has an extraordinarily large number of serovars or

strains—up to 2000 have been described. Salmonella enterica Serovar

Typhi (historically elevated to species status as S. typhi) is the disease

agent in typhoid fever. Other serovars such as Typhimurium (also

known as S. typhimurium) can lead to a form of human gastroenteritis

sometimes referred to as salmonellosis.

Salmonella Typhi is a serovar of Salmonella enterica (formerly

known as Salmonella choleraesuis) and the cause of the disease

http://en.wikipedia.org/wiki/Serovar

http://en.wikipedia.org/wiki/Salmonellosis

http://en.wikipedia.org/wiki/Gastroenteritis


http://en.wikipedia.org/wiki/Disease_agent

http://en.wikipedia.org/wiki/Disease_agent

http://en.wikipedia.org/wiki/Species


typhoid fever. The organism can be transmitted by the fecal-oral route

—it is excreted by humans in feces and may be transmitted by

contaminated water, food, or by person-to-person contact (with

inadequate attention to personal hygiene).

Most cases of salmonellosis are caused by food infected with S.

enterica, which often infects cattle and poultry, though also other

animals such as domestic cats and hamsters[6] have also been shown

to be sources of infection to humans. However, investigations of

vacuum cleaner bags have shown that households can act as a

reservoir of the bacterium; this is more likely if the household has

contact with an infection source.

Raw chicken and goose eggs can harbor salmonella enterica,

initially in the whites of the eggs, although most eggs are not infected.

As the egg ages at room temperature, the yolk membrane begins to

break down and salmonella enterica can spread into the yolk.

Refrigeration and freezing do not kill all the bacteria, but substantially

slow or halt their growth. Pasteurizing (briefly heating to a specific

temperature) and irradiation are used to kill salmonella for

commercially produced foodstuffs containing raw eggs such as ice

cream. Foods prepared in the home from raw eggs such as

mayonnaises, cakes and cookies can spread salmonella if not properly

cooked before consumption

(http://en.wikipedia.org/wiki/Salmonella_enteritidis).

11

Egg-associated salmonellosis is an important public health

problem in the United States and several European countries. A

bacterium, Salmonella enteritidis, can be inside perfectly normal-

appearing eggs, and if the eggs are eaten raw or undercooked, the

http://en.wikipedia.org/wiki/Salmonella_enteritidis

http://en.wikipedia.org/wiki/Vacuum_cleaner

http://en.wikipedia.org/wiki/Salmonella_enteritidis#cite_note-5%23cite_note-5

http://en.wikipedia.org/wiki/Hamster

http://en.wikipedia.org/wiki/Cat

http://en.wikipedia.org/wiki/Cattle

http://en.wikipedia.org/wiki/Personal_hygiene

http://en.wikipedia.org/wiki/Feces

http://en.wikipedia.org/wiki/Fecal-oral_route


bacterium can cause illness. Consumers should be aware of the

disease and learn how to minimize the chances of becoming ill.

A person infected with the Salmonella enteritidis bacterium

usually has fever, abdominal cramps, and diarrhea beginning 12 to 72

hours after consuming a contaminated food or beverage. The illness

usually lasts 4 to 7 days, and most persons recover without antibiotic

treatment. However, the diarrhea can be severe, and the person may

be ill enough to require hospitalization.

The elderly, infants, and those with impaired immune systems

may have a more severe illness. In these patients, the infection may

spread from the intestines to the blood stream, and then to other body

sites and can cause death unless the person is treated with antibiotics

(http://www.cdc.gov/ncidod/dbmd/diseaseinfo/salment, 2005, October

13).

Microbiological Assay

The appropriate bacterium is grown in a series of culture vessels,

each containing medium with an excess amount of all required

component except the growth factor to be assayed. A different amount

of growth factor is added to each vessel. The standard curve is

prepared by plotting the growth factor quantity or concentration

against the total extent of bacterium growth. Ideally the amount of

growth resulting is directly proportional to the quantity of growth factor

present; if the growth factor concentration doubles, the final extent of

bacterium growth also doubles. The quantity of the growth factor in a

test sample is determined by comparing the extent of growth cause by

the unknown sample with the resulting from the standards.

http://www.cdc.gov/ncidod/dbmd/diseaseinfo/salment

12

Microbiological assay is specific, sensitive and simple. They still

are used in the assay of substances like vitamin B12 and biotin. The

observation that many organisms can synthesize large quantities of

vitamins has led to their use of industry. Several water-soluble, and

fat- soluble vitamins are produced partly or completely using industrial

fermentation (Prescott, Harley & Klein, 1996).

13

METHODOLOGY

STUDY SITE

This study was conducted at the Department of Science and

Technology, Petit Barracks, Zamboanga City. The specimens of E. coli,

S. aureus and S. enteritidis were obtained at the Department of

Science and Technology. The specimens were subjected to Disk-

Diffusion Test.

MATERIALS AND METHODS

I. SOURCE AND GATHERING OF DATA

The data for this study were gathered through selected

encyclopedia, journals, books, Internet and previous related

microbiological study that gave background information. But the most

relevant information was gathered from the experiment proper.

II. STERILIZATION OF MATERIALS

All laboratory materials for this study were covered with foil and

were sterilized in an autoclave at 15 psi (pounds per square inch) at

121 degrees centigrade for 15 minutes. After sterilization the

researchers waited for 15 minutes to allow the temperature to cool

down.

14

III. PREPARATION OF THE TEST ORGANISM

The test organism namely Escherichia coli, Staphylococcus

aureus and Salmonella enteritidis were taken from pure cultures using

spread method, and were provided by the DOST, Petit Barracks,

Zamboanga City. The laboratory in-charge assisted the researchers in

spreading the bacteria from pure cultures to two test tubes filled with

nutrient agar. These test tubes were incubated at 35.5 degrees

centigrade for 24 hours.

IV. PREPARATION OF THE KAMIAS LEAVES EXTRACT

Kamias leaves and fruits were gathered at the EAAB (Edwin

Andrews Air Base), Sta. Maria, Zamboanga City. The leaves were then

wash with water and dried. Using a pair of scissors, the leaves were cut

into tiny pieces. Using a blender, the leaves were grinded and

extracted leaving behind residues. The residues were then extracted

once more with the use of cheesecloth. The remaining residues were

thrown out. The Kamias leaves extract were then poured and sealed in

a sterilized bottle and stored in the refrigerator at 4 degrees

Centigrade for 24 hours.

V. PREPARATION OF THE KAMIAS FRUIT EXTRACT

Kamias fruit were gathered at the EAAB (Edwin Andrews Air

Base), Sta. Maria, Zamboanga City. The fruits were then wash with

water and dried. Using a blender, the fruit were grinded and extracted

leaving behind residues. The residues were then extracted once more

with the use of cheesecloth. The remaining residues were thrown out.

The Kamias fruit extract were then poured and sealed in a sterilized

bottle and stored in the refrigerator at 4 degrees Centigrade for 24

hours.

15

VI. MICROBIOLOGICAL ASSAY

Twenty- four (24) grams of PCA agar was dissolved in one liter of

distilled water by mixing inside an Erlenmeyer flask with the use of a

stirring rod. The agar was then boiled on a hot plate and was

constantly stirred using the stirring rod. The agar was then sterilized in

an autoclave at 15 psi (pounds per inches) at 121 degrees Centigrade

for 15 minutes.

After sterilization, 15 mL of agar was poured in each of the 60

petri dishes. The petri dishes were covered and allowed to solidify for

30 minutes. All petri dishes were divided into six parts and each part

was labeled as A, B, C, D, E and F respectively using a marker to

represent the six treatments used. Ten petri dishes were labeled as E.

coli, ten as S. aureus and ten as S. enteritidis.

Afterwards, the incubated pure cultures were prepared to be

distributed in 60 petri dishes. Using a stirring rod ( L- shaped), an

amount of bacteria is transferred from the incubated test tube to a

new test tube with Normal Saline Solution (NSS) in it and was stirred.

The L- shaped stirring rod was exposed to flame from the alcohol lamp

each time it was used. This process was done to the three types of

bacteria producing two test tubes with bacterial suspensions in it.

With the use of syringe, 0.5 mL of each bacteria specimen in the

test tube with NSS was transferred to its corresponding 10 petri dishes.

The specimen was spread evenly using cotton buds. The cotton buds

were exposed to flame from the alcohol lamp after they were used.

They were then placed in a beaker with Lysol Antibacterial Solution in

it for disinfection.

16

VII. PREPARATION OF THE TREATMENTS

The different concentrations of the leaves and fruits extracts

were prepared namely Treatment A as 100% water served as the

negative control; Treatment B as 100% Kamias leaves extract;

Treatment C as 50% Distilled water and 50% Kamias leaves extract;

Treatment D as 100% antibiotics was used as the positive control

because of its known antibacterial properties; Treatment E as 100%

Kamas fruit extract and Treatment F as 50% Distilled water and 50%

Kamias fruit extract.

VIII. DISK-DIFFUSION TEST

To administer the disk-diffusion test, the 180 paper discs were

prepared using filter paper and a puncher. These filter papers were

sterilized in an autoclave beforehand. Then, the researchers identified

that each paper discs has a diameter of 6 millimeters.

Afterwards, the plates were treated by using forceps in

transferring one paper disc which was soaked in its corresponding

treatment to each of the corresponding division in each petri dish.

Then, the treated plates were incubated at 35.5 degrees Centigrade for

24 hours in inverted position.

After 24 hours, the petri dishes were found to have clear zones

already. With that, the researchers proceeded with the quantitative

observation for the zones of inhibition. The results gathered were

subjected to Statistical analyses.

IX. CLEANING AND PROPER DISPOSAL OF

MATERIALS

After the experimentation, all the materials and Laboratory

apparatuseslike Petri

17

dishes and forceps used in the study were placed inside an autoclave

and were sterilized

at 15 psi at 121 degrees Centigrade for 30 minutes. Then they were

washed using an antibacterial soap and with flowing water. After

washing, the unnecessary materials were disposed by the researchers

with the help of the laboratory-in-charge.

X. STATISTICAL ANALYSES

This study utilized both descriptive and inferential statistics in

analyzing and interpreting the data. Mean was chosen as the measure

of central tendency. Analysis of Variance (ANOVA) and Scheffe’s Test

were conducted to analyze and interpret the results.

18

RESULTS AND DISCUSSION

Escherichia coli

The effect of Kamias leaves and fruit extract on the E. coli

bacteria was investigated. The responses measured in the study were

the zones of inhibition. The data are shown in table 1

Table 1. Zones of Inhibition of E. coli (in mm.)

Treatm

ent

Zone of Inhibition (mm.) Tot

al

Mea

n

Standa

rd

deviati

on

Replicates

1 2 3 4 5 6 7 8 9 1

0

A8 7 8 8 9 1

0

8 1

1

1

1

1

0

90 9.0 1.41

B11 10 1

4

9 1

0

9 1

1

8 1

2

9 103 10.3 1.77

C8 9 8 1

0

8 9 8 1

0

1

0

8 88 8.8 0.92

D 9 9 1

0

1

0

8 9 9 1

0

9 1

1

94 9.4 0.84

E 9 8 1

0

9 8 1

0

1

1

1

0

1

2

1

1

98 9.8 1.32

F 11 9 8 9 1

0

9 1

0

1

1

7 8 92 9.2 1.32

Table 1 shows the comparison of the zones of inhibition of E. coli

bacteria around the paper discs soaked in the six treatments used. As

shown in Table 1, Treatment B has

19

the greatest zone of inhibition of the E. coli bacteria while Treatment C

has the least.

Table 2 shows the Analysis of Variance Test done on the Zones of

Inhibition of E. coli.

Table 2. ANOVA table for the Zones of Inhibition of E. coli

Source

of

Variation

Degrees

of

Freedom

Sum of

Squares

Mean

Squares

F ratio

(Comput

ed)

Critical

Value

Between

Groups 5 15.28 3.056

1.807 2.37

Within

Groups 54 91.30 1.691

Total

59 106.6

Based on the calculation performed, Table 2 shows that the F

computed value is 1.807 while the critical value of F with alpha level

equals 0.05 with degrees of freedom 5 and 54 is 2.37. Since the

computed F value is less than the critical value, then the null

hypothesis is accepted. There is no significant difference among the

zones of inhibition of E. coli on the six treatments used.

Since ANOVA test shows that there is no significant difference

among the zones

20

of inhibition of E. coli on the six treatments used, it is proper to

proceed to Scheffe’s Test to find out on what treatments the difference

lies. The results are presented in Table 3.

Table 3. Summary of the Scheffe’s Test Results for the ANOVA Results on the Zones

of Inhibition of Ecolab on the four treatments

Significant Value Scheffe’s Value

Decision

Treatment A vs. Treatment B 2.22* Not

Significant

Treatment A vs. Treatment C 0.34* Not

Significant

Treatment A vs. Treatment D 0.69* Not

Significant

Treatment A vs. Treatment E 1.37* Not

Significant

Treatment A vs. Treatment F 0.34* Not

Significant

Treatment B vs. Treatment C 2.57*

Significant

Treatment B vs. Treatment D 1.54* Not

Significant

Treatment B vs. Treatment E 0.86* Not

Significant

Treatment B vs. Treatment F 1.89* Not

Significant

Treatment C vs. Treatment D 1.02* Not

Significant

Treatment C vs. Treatment E 1.71* Not

Significant

Treatment C vs. Treatment F 0.69* Not

Significant

Treatment D vs. Treatment E 0.69* Not

Significant

Treatment D vs. Treatment F 0.34* Not

Significant

Treatment E vs. Treatment F 1.03* Not

Significant

*F critical of 2.37

21

The Scheffe’s Test was done to determine which mean zone of

inhibition of E. coli significantly differs from other mean zone of

inhibition of E. coli. Table 3 shows that there is no significant difference

between the mean of zone of inhibition of E. coli on the pair of

treatments A and B (2.22<2.37), A and C (0.34<2.37), A and

D(0.69<2.37), A and E (1.37<2.37), A and F (0.34<2.37)B and C

(2.57>2.37), B and D (1.54<2.37), B and E (0.86<2.37), B and F (1.89),

C and D (1.02<2.37), C and E (1.71<2.37), C and F (0.69<2.37), D and

E (0.69<2.37), D and F (0.34<2.37) and E and F (1.03<2.37).

This implies that the effect of Treatment B (100% Kamias leaves

extract) is not statistically equal to the Treatment C (50% Distilled

water and 50% Kamias leaves extract); and the remaining treatments

are statistically equal to each other.

Staphylococcus aureus

The effect of Kamias leaves and fruit extract on the S. aureus


the zones of inhibition. The data are shown in Table 4.

Table 4. Zones of Inhibition of S. aureus (in mm.)

Treatm

ent


al

Mea

n

Standa

rd

deviati

on

Replicates

1 2 3 4 5 6 7 8 9 1

0

2

2

A11 9 1

0

9 8 9 1

0

8 1

1

1

0

95 9.5 1.08

B10 14 9 1

2

1

0

1

1

1

0

1

1

9 1

4

110 11.0 1.83

C9 8 1

0

9 1

2

1

0

9 1

1

1

0

1

0

97 9.8 1.14

D 11 8 1

0

8 1

0

9 9 1

0

9 1

2

96 9.6 1.26

E 8 10 9 1

1

8 1

4

1

0

1

1

1

2

8 101 10.1 1.97

F 11 8 8 1

0

9 8 1

0

9 1

1

1

0

94 9.4 1.17

Table 4 shows the comparison of the zones of inhibition of S. aureus

bacteria around the paper discs soaked in the six treatments used. As

shown in Table 4, Treatment B has the greatest zone of inhibition of

the S. aureus bacteria and Treatment F has the least.


Inhibition of S. aureus.

Table 5. ANOVA table for the Zones of Inhibition of S. aureus

Source

of

Degrees

of

Sum of

Squares

Mean

Squares

F ratio

(Comput

Critical

Value

Variation Freedom ed)

Between

Groups 5 27.40 3.520

1.677 2.37Within

Groups 54 167.0 2.099

Total

59 194.4




computed F value is less than the critical value, then the null

hypothesis is accepted. There is no significant difference among the

zones of inhibition of S. aureus on the six treatments used.

Since ANOVA test shows that there is a significant difference

among the zones of inhibition of S. aureus on the six treatments used,

it is proper to proceed to Scheffe’s Test to find out on what treatments

the difference lies. The results are presented in Table 6.

Table 6. Summary of the Scheffe’s Test Results for the ANOVA

Results on the Zones of Inhibition of S. aureus on the six treatments.


Decision

Treatment A vs. Treatment B 2.31* Not

Significant


Significant


Significant

Treatment A vs. Treatment E 0.92* Not

Significant


Significant

Treatment B vs. Treatment C 1.85* Not

Significant


Significant


Significant

Treatment B vs. Treatment F 2.46*

Significant


Significant

Treatment C vs. Treatment E 0.46* Not

Significant


Significant


Significant


Significant

Treatment E vs. Treatment F 1.08* Not

Significant

*F critical value of 2.37

The Scheffe’s Test was done to determine which mean zone of

inhibition of S. aureus significantly differs from other mean zone of

inhibition of S. aureus. Table 6 shows that there is no significant

difference between the mean of zone of inhibition of S. aureus oÿÿthe

pair oÿÿÿÿeaÿÿenÿÿ Aÿÿnd B (2.31<2ÿÿ7), A and C (0.46<2.37), A and

D (1.54<2.37), A and E (0.92<2.37), A and F (1.54<2.37)B and C

(1.85<2.37), B and D (2.15<2.37), B and E (1.38<2.37), B and F

(2.46>2.37), C and D (0.31<2.37), C and E (0.46<2.37), C and F

(0.62<2.37), D and E (0.77<2.37), D and F (0.31<2.37) and E and

F (1.08<2.37).

This implies that the effect of Treatment B (100% Kamias Leaves

extract) is not statistically equal to Treatment F (50% Kamias fruit

extract and 50% distilled water); and the remaining treatments are

statistically equal to each other.

25

Salmonella enteritidis

The effect of Kamias leaves and fruit extract on the S. enteritidis


the zones of inhibition. The data are shown in Table 4.

Table 4. Zones of Inhibition of S. enteritidis (in mm.)

Treatm

ent


al

Mea

n

Standa

rd

deviati

on

Replicates

1 2 3 4 5 6 7 8 9 1

0

A13 11 1

1

1

2

1

1

1

0

1

1

1

0

1

2

1

3

114 11.4 1.07

B15 14 1

4

1

2

1

3

1

1

1

3

1

4

1

9

1

2

137 13.7 2.21

C11 11 1

1

1

2

1

3

1

2

1

3

1

0

1

2

1

1

116 11.6 1.0

D 12 11 1

3

1

2

1

4

1

1

1

3

1

0

1

3

1

3

122 12.2 1.23

E 14 14 1

4

1

3

1

3

1

0

1

2

1

4

1

7

1

3

134 13.4 1.78

F 10 11 1

0

1

3

1

3

1

0

1

3

1

2

1

2

1

3

117 11.7 1.34

Table 4 shows the comparison of the zones of inhibition of S.

enteritidis bacteria around the paper discs soaked in the six

treatments used. As shown in Table 4, Treatment B has the greatest

zone of inhibition of the S. aureus bacteria and Treatment F has the

least.

26


Inhibition of S. enteritidis.

Table 5. ANOVA table for the Zones of Inhibition of S.

enteritidis

Source

of

Variation

Degrees

of

Freedom

Sum of

Squares

Mean

Squares

F ratio

(Comput

ed)

Critical

Value

Between

Groups 5 17.60 9.666

4.313 2.37

Within

Groups 54 113.8 2.241

Total

59 131.4




computed F value is greater than the critical value, then the null

hypothesis is rejected. There is a significant difference among the

zones of inhibition of S. enteritidis on the six treatments used.

Since ANOVA test shows that there is a significant difference

among the zones of inhibition of S. enteritidis on the four treatments

used, it is proper to proceed to Scheffe’s Test to find out on what

treatments the difference lies. The results are presented in Table 6.

27

Table 6. Summary of the Scheffe’s Test Results for the ANOVA

Results on the Zones of Inhibition of S. enteritidis on the four

treatments.


Decision

Treatment A vs. Treatment B 3.44*

Significant


Significant


Significant

Treatment A vs. Treatment E 2.99*

Significant


Significant

Treatment B vs. Treatment C 3.14*

Significant


Significant


Significant

Treatment B vs. Treatment F 2.99*

Significant


Significant

Treatment C vs. Treatment E 2.69*

Significant


Significant


Significant


Significant

Treatment E vs. Treatment F 2.54*

Significant

*F critical value of 2.37

The Scheffe’s Test was done to determine which mean of zone of

inhibition of S. enteritidis significantly differ from which other mean of

zone of inhibition of S. enteritidis. Table 6 shows that there is no

significant difference between the mean of zone

28

of inhibition of S. enteritidis on the pair of treatments A and B

(3.44>2.37), A and C (0.30<2.37), A and D (1.20<2.37), A and E

(2.99>2.37), A and F (0.45<2.37)B and C (3.14>2.37), B and D

(2.24<2.37), B and E (0.45<2.37), B and F (2.99>2.37), C and D

(0.90<2.37), C and E (2.69>2.37), C and F (0.15<2.37), D and E

(1.80A<2.37), D and F (0.75<2.37) and E and F (2.5>2.37).

This implies that the effect of Treatment A (100% Distilled water)

is statistically equal to Treatment C (50% Kamias Fruit Extract and 50%

distilled water); Treatment A (100% Distilled water) is statistically

equal to Treatment D (Antibiotics); Treatment A (100% Distilled water)

is statistically equal to Treatment F (50% Kamias Fruit extract and 50%

distilled water); Treatment B (100% Kamias Leaves extract) is

statistically equal to Treatment D (Antibiotics); Treatment B (100%

Kamias Leaves extract) is statistically equal to Treatment E (100%

Kamias Fruit Extract); Treatment C (50% Kamias Fruit Extract and 50%

distilled water) is statistically equal to Treatment D (Antibiotics);

Treatment C (50% Kamias Fruit Extract and 50% distilled water) is

statistically equal to Treatment F (50% Kamias Fruit extract and 50%

distilled water); Treatment D (Antibiotics) is statistically equal to

Treatment E (100% Kamias Fruit Extract); and Treatment D

(Antibiotics) is statistically equal to Treatment F (50% Kamias Fruit

extract and 50% distilled water).

29

SUMMARY AND CONCLUSION

SUMMARY

This study is all about the effectiveness of Kamias leaves and

fruit extract as an antibacterial agent against Escherichia coli,

Staphylococcus aureus and Salmonella enteritidis. The researchers

observed that there is really a need of an effective, cheaper and more

environment-friendly antibacterial agent since commercialized one is

costly

hazardous to our body as well as to our environment. Thus, the Kamias

leaves and fruit

extract’s potential of being an effective antibacterial agent was

explored.

The methodology involved in the investigation was divided into

two major phases namely: the Study Site and Materials and Methods.

Specific methods were: Source and Gathering Data, Sterilization of

Materials, Preparation of Test Organisms, Preparation of Kamias

Leaves and Fruit Extract, Microbiological assay, Preparation of the

Treatments, and Disk-Diffusion Test.

Data were collected and recorded for each series of experiments.

In general, after the data obtained on the experiment were subjected

to further statistical tests and analyses, the Kamias leaves and fruit

extract was proven to be an effective antibacterial agent.

SUMMARY OF FINDINGS

For Escherichia coli

30





For Staphylococcus aureus





For Salmonella enteritidis









Kamias Fruit Extract); Treatment C (50% Kamias Fruit Extract and 50%

distilled water) is statistically equal to Treatment D (Antibiotics);

Treatment C (50% Kamias Fruit Extract and 50% distilled water) is

statistically equal to Treatment F (50% Kamias Fruit extract and 50%

distilled water); Treatment D (Antibiotics) is statistically equal to

Treatment E (100% Kamias Fruit Extract); and Treatment D

(Antibiotics) is statistically equal to Treatment F (50% Kamias Fruit

extract and 50% distilled water).

31

CONCLUSIONS

The following conclusions are stated based on the findings.

This research study shows the Antibacterial effect of Kamias

(Averrhoa bilimbi) Leaves and Fruit extract on Escherichia coli,

Staphylococcus aureus and Salmonella enteritidis. Hence, the

researchers concluded that all of the treatments are statistically equal

to each other.

For Escherichia coli.





For Staphylococcus aureus





For Salmonella enteritidis









Kamias Fruit Extract); Treatment C (50%

32

Kamias Fruit Extract and 50% distilled water) is statistically equal to

Treatment D (Antibiotics); Treatment C (50% Kamias Fruit Extract and

50% distilled water) is statistically equal to Treatment F (50% Kamias

Fruit extract and 50% distilled water); Treatment D (Antibiotics) is

statistically equal to Treatment E (100% Kamias Fruit Extract); and

Treatment D (Antibiotics) is statistically equal to Treatment F (50%

Kamias Fruit extract and 50% distilled water).

33

RECOMMENDATION

For a more wide-ranging investigation, the researchers recommend the

following:

1. Further studies should be conducted utilizing other parts of

the kamias tree.

2. It would be better if it covers a wider range of pathogenic

bacteria to better prove its anti-bacterial property.

3. The researchers also recommend the Phytochemical Analyses

of the kamias leaves and fruit to be able to explore more of its

anti-bacterial properties.

4. Don’t hesitate to look and gather information and help at the

DOST (Department of Science and Technology) for better

guidance especially to those performing first time in

antibacterial effect.

34

BIBLIOGRAPHY

BOOKS

Adkins J.N. et al (2006). "Analysis of the Salmonella typhimurium

Proteome through Environmental Response toward Infectious

Conditions". Molecular and Cellular Proteomics 5 . (pp. 1450–1461).

Case, C. L., Funke, B. R. & Tortora, G. J. Microbiology an

Introduction 4 th edition. 390 Bridge Parkway, Redwood city, California

94065, the Benjamin/ummings Publishing Company, Inc. (1992).

Morton, J. (1987). In Fruits of Warm Climates. Miami, FL (pp. 128–

129).

Prescott, L. M., Harley, J. P. & Klein, D. A. Microbiology 6 th edition,

International Edition. 1221 avenue of the Americas New York, YK, MC

Graw-Hill publishing (2005).

ENCYCLOPEDIA

Grolier Encyclopedia of Knowledge. Volume 2, “Bacteria”, pp. 280.

Grolier Encyclopedia of Knowledge. Volume 2, “Diseases cause by E.

coli”, pp. 362.

Grolier Encyclopedia of Knowledge. Volume 2, “E. coli”, pp.330.

Grolier Encyclopedia of Knowledge. Volume 2, “Principles of Diseases

and Eepidemiology”, p.380.

35

INTERNET

Role of S. aureus on diseases. September 1, 2008,

http://en.wikipedia.org/wiki/Staphylococcus_aureus

E. coli. Retrieved September 3, 2008,

http://www.sunstar.com.ph/static/bac/2005/04/27/news/public.warned.

v..deadly.bacteria.html,2005, April 7.

S. enteritidis. Retrieved September 3, 2008,

http://www.cdc.gov/ncidod/dbmd/diseaseinfo/salment, 2003, October

13.

Uses of Kamias, Retrieved September 4, 2008,

(http://www.hort.purdue.edu/newcrop/morton/bilimbi.html)

E. coli Retrieved September 5, 2008, Microsoft Encarta Premium Suite

2005, 1993-1994.

S. aureus. Retrieved September 5, 2008, http://en.wikipedia.org/wiki/S.

aureus

PAST RESEARCH STUDY

Uro and Prias (2006). Zingiber officinale (Ginger) Rhizome Extract:

Its Antibacterial Effect on Ecolab, S. enteritidis and S. aureus.

Unpublished Science Investigatory Project. Regional Science High

School for Region IX, San Roque, Zamboanga City.

http://en.wikipedia.org/wiki/S

http://www.hort.purdue.edu/newcrop/morton/bilimbi.html

http://www.sunstar.com.ph/static/bac/2005/04/27/news/public.warned.v..deadly.bacteria.html,2005

http://www.sunstar.com.ph/static/bac/2005/04/27/news/public.warned.v..deadly.bacteria.html,2005

36

APPENDICES

A. DEFINITION OF TERMS

Agar—complex polysaccharide derived from marine alga and used as

a solidifying agent in culture media.

ANOVA—Analysis of Variance, a statistical test used to find out

significant difference among 3 or more means.

Antibacterial Agent—a substance that prevents, kills or reduces the

growth bacteria.

Autoclave— A strong steel vessel that is used for steam sterilization

of equipment or materials.

Bacteria— a single- celled, often parasitic microorganism without

distinct nuclei or organized cell structures.

Disk-diffusion Test—a test for antibiotic sensitivity in bacteria; agar

plates are inoculated with a standardized suspension of

microorganism.

Diuretic—medication that increases urine output

Endotoxins-- a toxin produce within certain bacteria that is released

only when the bacteria disintegrate

Enterotoxins—any toxin produces by bacteria that cause vomiting

and diarrhea associated with food poisoning.

37

Escherichia— is one of the main species of bacteria living in the lower

intestines of mammals, known as gut flora.

Endocarditis—is an inflammation of the inside lining of the heart

chambers and heart valves (endocardium)

Extract—a substance obtain from a compound by an industrial or

chemical process

Nutrient—any substance that provide nourishment.

Osmotic pressure—the pressure that must be applied to a solution to

stop the inward diffusion of a solvent by osmosis through a semi

permeable membrane.

Pathogenic—causing diseases or able to cause disease

Penicillin—an antibiotic originally derived from mould but also

produced synthetically which is used to treat wide range of bacterial

infections.

Salmonella--- is comprised mostly of facultatively anaerobic, oxidase-

negative, catalase-positive, Gram-negative rod-shaped bacteriait has

an extraordinarily large number of serovars or strains.

Staphylococcus—a group of round looking bacteria that causes a

multitude diseases


Scheffe’s Test—a test that will analyze a pair of mens to se if there

is a difference.

38

B. PICTURES AND ILLUSTRATIONS

GATHERING OF THE MATERIALS WASHING OF KAMIAS LEAVES

BLENDING OF THE LEAVES KAMIAS LEAVES AND FRUIT EXTRACT

STERILIZATION OF MATERIALS TEST ORGANISM

39

INCUBATION OF THE SPECIMENS MICROBIOLOGICAL ASSAY

DISK-DIFFUSION TEST TREATMENTS

Zzzz

INCUBATION OF THE 30 REPLICATES CLEANING AND PROPER

DISPOSAL OF MATERIALS

STATISTICAL ANALYSES

40

bagong kamias

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