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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 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.
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, 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
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 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
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
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 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
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.) Tot
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.
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
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
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 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.
Significant Value Scheffe’s Value
Decision
Treatment A vs. Treatment B 2.31* Not
Significant
Treatment A vs. Treatment C 0.46* Not
Significant
Treatment A vs. Treatment D 1.54* Not
Significant
Treatment A vs. Treatment E 0.92* Not
Significant
Treatment A vs. Treatment F 1.54* Not
Significant
Treatment B vs. Treatment C 1.85* Not
Significant
Treatment B vs. Treatment D 2.15* Not
Significant
Treatment B vs. Treatment E 1.38* Not
Significant
Treatment B vs. Treatment F 2.46*
Significant
Treatment C vs. Treatment D 0.31* Not
Significant
Treatment C vs. Treatment E 0.46* Not
Significant
Treatment C vs. Treatment F 0.62* Not
Significant
Treatment D vs. Treatment E 0.77* Not
Significant
Treatment D vs. Treatment F 0.31* Not
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
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.) Tot
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
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
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
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 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.
Significant Value Scheffe’s Value
Decision
Treatment A vs. Treatment B 3.44*
Significant
Treatment A vs. Treatment C 0.30* Not
Significant
Treatment A vs. Treatment D 1.20* Not
Significant
Treatment A vs. Treatment E 2.99*
Significant
Treatment A vs. Treatment F 0.45* Not
Significant
Treatment B vs. Treatment C 3.14*
Significant
Treatment B vs. Treatment D 2.24* Not
Significant
Treatment B vs. Treatment E 0.45* Not
Significant
Treatment B vs. Treatment F 2.99*
Significant
Treatment C vs. Treatment D 0.90* Not
Significant
Treatment C vs. Treatment E 2.69*
Significant
Treatment C vs. Treatment F 0.15* Not
Significant
Treatment D vs. Treatment E 1.80* Not
Significant
Treatment D vs. Treatment F 0.75* Not
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
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 aureus and Salmonella enteritidis. Hence, the
researchers concluded that all of the treatments are statistically equal
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
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. 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.
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