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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

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 Scheffes 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...1418 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

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 persons 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.

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 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, thats 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 3month 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 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, 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 persons 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

bacteriums 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 bacteriums 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 endocarditisinflammation 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 heatresistant 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 persons 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 strainsup 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 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 normalappearing eggs, and if the eggs are eaten raw or undercooked, the 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. 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 DiskDiffusion 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.

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 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. MATERIALS

CLEANING

AND

PROPER

DISPOSAL

OF

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 Scheffes 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.) Replicates

Tot al

Mea n

Standa rd deviati on

1 8 11 8 D E F 9 9 11

2 7 10 9 9 8 9

3 8 1 4 8 1 0 1 0 8

4 8 9 1 0 1 0 9 9

5 9 1 0 8 8 8 1 0

6 1 0 9 9 9 1 0 9

7 8 1 1 8 9 1 1 1 0

8 1 1 8 1 0 1 0 1 0 1 1

9 1 1 1 2 1 0 9 1 2 7

1 0 1 0 9 8 1 1 1 1 8 90 103 88 94 98 92 9.0 10.3 8.8 9.4 9.8 9.2 1.41 1.77 0.92 0.84 1.32 1.32

A B C

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 Between Groups Within Groups Total

Degrees of Freedom 5

Sum of Squares

Mean Squares

F ratio (Comput ed)

Critical Value

15.28

3.056

54

91.30

1.691

1.807

2.37

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 Scheffes Test to find out on what treatments the difference lies. The results are presented in Table 3.

Table 3. Summary of the Scheffes Test Results for the ANOVA Results on the Zones of Inhibition of Ecolab on the four treatments

Significant Value Decision Treatment A vs. Treatment B Significant Treatment A vs. Treatment C Significant Treatment A vs. Treatment D Significant Treatment A vs. Treatment E Significant

Scheffes Value

2.22* 0.34* 0.69* 1.37*

Not Not Not Not

Treatment A vs. Treatment F Significant Treatment B vs. Treatment C Significant Treatment B vs. Treatment D Significant Treatment B vs. Treatment E Significant Treatment B vs. Treatment F Significant Treatment C vs. Treatment D Significant Treatment C vs. Treatment E Significant Treatment C vs. Treatment F Significant Treatment D vs. Treatment E Significant Treatment D vs. Treatment F Significant Treatment E vs. Treatment F Significant *F
critical

0.34* 2.57* 1.54* 0.86* 1.89* 1.02* 1.71* 0.69* 0.69* 0.34* 1.03*

Not

Not Not Not Not Not Not Not Not Not

of 2.37

21 The Scheffes 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 bacteria was investigated. The responses measured in the study were the zones of inhibition. The data are shown in Table 4.

Table 4. Zones of Inhibition of S. aureus (in mm.) Treatm ent Zone of Inhibition (mm.) Replicates Tot al Mea n Standa rd deviati on 1 2 3 4 5 6 7 8 9 1 0

2 11 10 9 11 8 11 9 1 9 1 2 9 8 1 1 1 0 8 1 0 1 2 1 0 8 9 9 1 1 1 0 9 1 4 8 2 1 0 1 0 9 9 1 0 1 0 8 1 1 1 1 1 0 1 1 9 1 1 9 1 0 9 1 2 1 1 1 0 1 4 1 0 1 2 8 1 0 95 110 97 96 101 94 9.5 11.0 9.8 9.6 10.1 9.4 1.08 1.83 1.14 1.26 1.97 1.17

A B C D E F

0 14 9 8 8 10 8 1 0 1 0 9 8

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. Table 5 shows the Analysis of Variance Test done on the Zones of Inhibition of S. aureus.
Table 5. ANOVA table for the Zones of Inhibition of S. aureus

Source of Variation Between Groups

Degrees of Freedom 5

Sum of Squares

Mean Squares

F ratio (Comput ed)

Critical Value

27.40

3.520

Within Groups Total 59 194.4 54 167.0 2.099

1.677

2.37

Based on the calculation performed, Table 5 shows that the F computed value is 1.677 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 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 Scheffes Test to find out on what treatments the difference lies. The results are presented in Table 6.

Table 6. Summary of the Scheffes Test Results for the ANOVA Results on the Zones of Inhibition of S. aureus on the six treatments.

Significant Value Decision Treatment A vs. Treatment B Significant Treatment A vs. Treatment C Significant Treatment A vs. Treatment D Significant Treatment A vs. Treatment E Significant Treatment A vs. Treatment F Significant Treatment B vs. Treatment C Significant Treatment B vs. Treatment D Significant Treatment B vs. Treatment E Significant Treatment B vs. Treatment F Significant Treatment C vs. Treatment D Significant Treatment C vs. Treatment E Significant Treatment C vs. Treatment F Significant Treatment D vs. Treatment E Significant Treatment D vs. Treatment F Significant

Scheffes Value

2.31* 0.46* 1.54* 0.92* 1.54* 1.85* 2.15* 1.38* 2.46* 0.31* 0.46* 0.62* 0.77* 0.31*

Not Not Not Not Not Not Not Not

Not Not Not Not Not

Treatment E vs. Treatment F Significant

1.08*

Not

*F

critical

value of 2.37

The Scheffes 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 othe pair oeaen And B (2.31<27), 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 bacteria was investigated. The responses measured in the study were the zones of inhibition. The data are shown in Table 4.

Table 4. Zones of Inhibition of S. enteritidis (in mm.) Treatm ent Zone of Inhibition (mm.) Replicates Tot al Mea n Standa rd deviati on 1 13 15 11 D E F 12 14 10 2 11 3 1 4 1 2 1 2 1 2 1 2 1 3 1 3 5 1 1 1 3 1 3 1 4 1 3 1 3 6 1 0 1 1 1 2 1 1 1 0 1 0 7 1 1 1 3 1 3 1 3 1 2 1 3 8 1 0 1 4 1 0 1 0 1 4 1 2 9 1 2 1 9 1 2 1 3 1 7 1 2 1 0 1 3 1 2 1 1 1 3 1 3 1 3 114 137 116 122 134 117 11.4 13.7 11.6 12.2 13.4 11.7 1.07 2.21 1.0 1.23 1.78 1.34

A B C

1 14 1 11 11 14 4 1 1 1 3 1

4 11 1 0

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 Table 5 shows the Analysis of Variance Test done on the Zones of Inhibition of S. enteritidis. Table 5. ANOVA table for the Zones of Inhibition of S. enteritidis Source of Variation Between Groups Within Groups Total 59 131.4 54 113.8 2.241 4.313 2.37 Degrees of Freedom 5 17.60 9.666 Sum of Squares Mean Squares F ratio (Comput ed) Critical Value

Based on the calculation performed, Table 5 shows that the F computed value is 4.313 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 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 Scheffes Test to find out on what treatments the difference lies. The results are presented in Table 6. 27 Table 6. Summary of the Scheffes Test Results for the ANOVA Results on the Zones of Inhibition of S. enteritidis on the four treatments.

Significant Value Decision Treatment A vs. Treatment B Significant Treatment A vs. Treatment C Significant Treatment A vs. Treatment D Significant Treatment A vs. Treatment E Significant Treatment A vs. Treatment F Significant Treatment B vs. Treatment C Significant

Scheffes Value

3.44* 0.30* 1.20* 2.99* 0.45* 3.14* Not Not Not

Treatment B vs. Treatment D Significant Treatment B vs. Treatment E Significant Treatment B vs. Treatment F Significant Treatment C vs. Treatment D Significant Treatment C vs. Treatment E Significant Treatment C vs. Treatment F Significant Treatment D vs. Treatment E Significant Treatment D vs. Treatment F Significant Treatment E vs. Treatment F Significant *F
critical

2.24* 0.45* 2.99* 0.90* 2.69* 0.15* 1.80* 0.75* 2.54*

Not Not

Not

Not Not Not

value of 2.37

The Scheffes 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 extracts 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 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. For Staphylococcus aureus 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. For Salmonella enteritidis 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).

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 to each other. For Escherichia coli. 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. For Staphylococcus aureus This implies that the effect of Treatment B (100% Kamias Leaves extract) is not statistically equal to Treatment F (50% Kamias fruit aureus and Salmonella enteritidis. Hence, the researchers concluded that all of the treatments are statistically equal

extract and 50% distilled water); and the remaining treatments are statistically equal to each other. For Salmonella enteritidis 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% 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. Dont 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. 14501461).

Case, C. L., Funke, B. R. & Tortora, G. J. Microbiology an Introduction 4th 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 6th 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.

36 APPENDICES

A. DEFINITION OF TERMS

Agarcomplex polysaccharide derived from marine alga and used as a solidifying agent in culture media. ANOVAAnalysis of Variance, a statistical test used to find out significant difference among 3 or more means. Antibacterial Agenta 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 Testa test for antibiotic sensitivity in bacteria; agar plates are inoculated with a standardized suspension of microorganism. Diureticmedication that increases urine output Endotoxins-- a toxin produce within certain bacteria that is released only when the bacteria disintegrate Enterotoxinsany 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. Endocarditisis an inflammation of the inside lining of the heart chambers and heart valves (endocardium) Extracta substance obtain from a compound by an industrial or chemical process Nutrientany substance that provide nourishment. Osmotic pressurethe pressure that must be applied to a solution to stop the inward diffusion of a solvent by osmosis through a semi permeable membrane. Pathogeniccausing diseases or able to cause disease Penicillinan 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, oxidasenegative, catalase-positive, Gram-negative rod-shaped bacteriait has an extraordinarily large number of serovars or strains. Staphylococcusa group of round looking bacteria that causes a multitude diseases Scheffes Testa 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